WO2022105914A1

WO2022105914A1 - Antibody binding to cd70 and application thereof

Info

Publication number: WO2022105914A1
Application number: PCT/CN2021/132079
Authority: WO
Inventors: 马晓丽; 曹卓晓; 葛虎; 唐任宏; 任晋生
Original assignee: 江苏先声药业有限公司
Priority date: 2020-11-23
Filing date: 2021-11-22
Publication date: 2022-05-27
Also published as: CN116490210A

Abstract

An antibody binding to CD70 and an application thereof. Specifically, disclosed are an antibody or antigen-binding fragment capable of binding to CD70, a corresponding multispecific antigen-binding molecule, a chimeric antigen receptor and an immune effector cell thereof, a nucleic acid fragment, a vector, a cell, a composition, a preparation method, a pharmaceutical use, and a method for treatment of cancers or tumors, autoimmune diseases or viral infections, which have important significance for treatment of tumors, autoimmune diseases, viral infections and the like.

Description

CD70 antibody and its application

The present invention claims the priority of the prior application with the patent application number 202011318991.X and the invention title "CD70 antibody and its application" submitted to the State Intellectual Property Office of China on November 23, 2020. The entire contents of the aforementioned prior application are incorporated herein by reference.

technical field

The present invention relates to the field of antibodies, in particular to CD70 antibodies and applications thereof.

Background technique

CD70 is a type II transmembrane glycoprotein belonging to the tumor necrosis factor (TNF) superfamily. It consists of 193 amino acids and exists in the form of homotrimers. CD70 is not expressed in normal tissues and hematopoietic cells, and is transiently expressed in activated immune cells. However, CD70 is expressed in a variety of solid tumors and non-Hodgkin's lymphomas, and its expression is often associated with poorer prognosis. After its ligands CD27 and CD70 are combined, the intracellular end will bind to TRAFs, such as TRAF2 and TRAF5, thereby activating the NFκB and c-jun kinase pathways, which play an important role in cell proliferation and differentiation, and can regulate T cells and B cells. function. These make CD70 an important target for tumors, autoimmune diseases and viral infections.

The antibody in the present invention is combined with CD70 protein, can block the combination of CD70 and CD27, and block the downstream signaling pathway, which is of great significance in the treatment of tumors, autoimmune diseases and viral infections.

SUMMARY OF THE INVENTION

The present invention discloses and provides an antibody or antigen-binding fragment that specifically binds to CD70, a corresponding multispecific antigen-binding molecule, a chimeric antigen receptor, an immune effector cell, a nucleic acid fragment, a carrier, a cell, a composition, and a preparation method thereof , pharmaceutical uses and methods of treatment.

In a first aspect, the present invention discloses an antibody or antigen-binding fragment that specifically binds to CD70, the antibody or antigen-binding fragment comprising:

(a) SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 23, 26, 27, 29, 30, 32, 34, 35, 38, 39, 41 , HCDR1, HCDR2 and HCDR3 of the VH of any one of 43, 45 or 46;

and/or, (b) SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 21, 24, 25, 28, 31, 33, 36, 37, 40, 42, 44 or 47 LCDR1, LCDR2 and LCDR3 of any of the indicated VLs;

Preferably, the HCDR1-3 and/or the LCDR1-3 are determined according to the Kabat numbering system, the Chothia numbering system or the IMGT numbering system, more preferably, the HCDR1-3 and/or the LCDR1-3 are selected from the table 1.

In some specific embodiments, the antibody or antigen-binding fragment comprises:

(1) HCDR1-3 of VH represented by SEQ ID NO: 3, 15, 17 or 19, and LCDR1-3 of VL represented by SEQ ID NO: 4, 16 or 18;

Or, (2) HCDR1-3 of VH shown in SEQ ID NO: 5, 20, 22, 23 or 26, and LCDR1-3 of VL shown in SEQ ID NO: 6, 21, 24, 25;

Or, (3) HCDR1-3 of VH shown in SEQ ID NO: 7, 27, 29 or 30, and LCDR1-3 of VL shown in SEQ ID NO: 8, 28, 31;

Or, (4) HCDR1-3 of VH shown in SEQ ID NO: 9, 32, 34, 35 or 38, and LCDR1-3 of VL shown in SEQ ID NO: 10, 33, 36 or 37;

Or, (5) HCDR1-3 of VH shown in SEQ ID NO: 11, 39 or 41, and LCDR1-3 of VL shown in SEQ ID NO: 12, 40 or 42;

Or, (6) HCDR1-3 of VH shown in SEQ ID NO: 13, 43, 45 or 46, and LCDR1-3 of VL shown in SEQ ID NO: 14, 44 or 47.

In some specific embodiments, the HCDRs 1-3 of the VH set forth in SEQ ID NO: 3 are according to the Kabat, Chothia or IMGT numbering system, having as SEQ ID NO: 66-68, SEQ ID NO: 69-71 or SEQ ID NO : the sequence shown in 72 to 74;

HCDRs 1-3 of the VH shown in SEQ ID NO: 15 have the sequence shown in SEQ ID NO: 75-77, SEQ ID NO: 78-80 or SEQ ID NO: 81-83 according to the Kabat, Chothia or IMGT numbering system ;

HCDRs 1-3 of the VH shown in SEQ ID NO: 17 have the sequence shown in SEQ ID NO: 84-86, SEQ ID NO: 87-89 or SEQ ID NO: 90-92 according to the Kabat, Chothia or IMGT numbering system ;

HCDRs 1-3 of the VH shown in SEQ ID NO: 19 have the sequence shown in SEQ ID NO: 93-95, SEQ ID NO: 96-98 or SEQ ID NO: 99-101 according to the Kabat, Chothia or IMGT numbering system ;

HCDRs 1-3 of the VHs shown in SEQ ID NO: 5, 20, 22 or 23 are according to the Kabat, Chothia or IMGT numbering system, with eg SEQ ID NO: 112-114, SEQ ID NO: 115-117 or SEQ ID NO: 118 The sequence shown in ~120;

HCDRs 1-3 of the VH shown in SEQ ID NO: 26 have the sequence shown in SEQ ID NO: 121-123, SEQ ID NO: 124-126 or SEQ ID NO: 127-129 according to the Kabat, Chothia or IMGT numbering system ;

HCDRs 1-3 of the VH shown in SEQ ID NO: 7 have the sequence shown in SEQ ID NO: 140-142, SEQ ID NO: 143-145 or SEQ ID NO: 146-148 according to the Kabat, Chothia or IMGT numbering system ;

HCDRs 1-3 of the VH shown in SEQ ID NO: 27 have the sequence shown in SEQ ID NO: 149-151, SEQ ID NO: 152-154 or SEQ ID NO: 155-157 according to the Kabat, Chothia or IMGT numbering system ;

The HCDRs 1-3 of the VH shown in SEQ ID NO: 29 or 30 are according to the Kabat, Chothia or IMGT numbering system and have as shown in SEQ ID NO: 158-160, SEQ ID NO: 161-163 or SEQ ID NO: 164-166 the sequence of;

HCDRs 1-3 of the VHs shown in SEQ ID NO: 9, 32, 34 or 35 are according to the Kabat, Chothia or IMGT numbering system, with eg SEQ ID NO: 177-179, SEQ ID NO: 180-182 or SEQ ID NO: 183 The sequence shown in ~185;

HCDRs 1-3 of the VH shown in SEQ ID NO: 38 have the sequence shown in SEQ ID NO: 186-188, SEQ ID NO: 189-191 or SEQ ID NO: 192-194 according to the Kabat, Chothia or IMGT numbering system ;

HCDRs 1-3 of the VH shown in SEQ ID NO: 11 have the sequences shown in SEQ ID NO: 205-207, SEQ ID NO: 208-210 or SEQ ID NO: 211-213 according to the Kabat, Chothia or IMGT numbering system ;

HCDRs 1-3 of the VH shown in SEQ ID NO: 39 have the sequence shown in SEQ ID NO: 214-216, SEQ ID NO: 217-219 or SEQ ID NO: 220-223 according to the Kabat, Chothia or IMGT numbering system ;

HCDRs 1-3 of the VH shown in SEQ ID NO: 41 have the sequence shown in SEQ ID NO: 224-226, SEQ ID NO: 227-229 or SEQ ID NO: 230-233 according to the Kabat, Chothia or IMGT numbering system ;

HCDRs 1-3 of the VHs shown in SEQ ID NO: 13, 43, 45 or 46 are according to the Kabat, Chothia or IMGT numbering system, with eg SEQ ID NO: 244-246, SEQ ID NO: 247-249 or SEQ ID NO: 250 The sequence shown in ~252;

LCDRs 1 to 3 of the VL shown in SEQ ID NO: 4, 16 or 18 are according to the Kabat, Chothia or IMGT numbering system, with eg SEQ ID NO: 102 to 104, SEQ ID NO: 105 to 107 or SEQ ID NO: 108 to 111 the sequence shown;

LCDRs 1 to 3 of the VL shown in SEQ ID NO: 6, 21, 24 or 25 are according to the Kabat, Chothia or IMGT numbering system and have as SEQ ID NO: 130 to 132, SEQ ID NO: 133 to 135 or SEQ ID NO: 136 The sequence shown in ~139;

LCDRs 1 to 3 of the VL shown in SEQ ID NO: 8, 28 or 31 are according to the Kabat, Chothia or IMGT numbering system, with eg SEQ ID NO: 167-169, SEQ ID NO: 170-172 or SEQ ID NO: 173-176 the sequence shown;

LCDRs 1 to 3 of the VL shown in SEQ ID NO: 10, 33, 36 or 37 are according to the Kabat, Chothia or IMGT numbering system and have as SEQ ID NO: 195 to 197, SEQ ID NO: 198 to 200 or SEQ ID NO: 201 The sequence shown in ~204;

LCDRs 1 to 3 of the VL shown in SEQ ID NO: 12, 40 or 42 are according to the Kabat, Chothia or IMGT numbering system, with eg SEQ ID NO: 234 to 236, SEQ ID NO: 237 to 239 or SEQ ID NO: 240 to 243 the sequence shown;

LCDRs 1 to 3 of the VL shown in SEQ ID NO: 14, 44 or 47 are according to the Kabat, Chothia or IMGT numbering system, with eg SEQ ID NO: 253-255, SEQ ID NO: 256-258 or SEQ ID NO: 259-262 the sequence shown.

In some specific embodiments, the antibody or antigen-binding fragment is contained in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 23, 26, 27, 29 , 30, 32, 34, 35, 38, 39, 41, 43, 45, or 46 of any one of the sequences of up to 6 mutations in HCDR1, HCDR2 and/or HCDR3 of the VH; and/or,

The antibody or antigen-binding fragment is contained in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 21, 24, 25, 28, 31, 33, 36, 37, 40, 42, 44 or a sequence of up to 6 mutations on LCDR1, LCDR2 and/or LCDR3 of the VL shown in any one of 47;

Preferably, the mutation is selected from substitution, deletion or insertion mutation; more preferably, the substitution is a conservative amino acid substitution;

Preferably, the number of mutations may be selected from 1, 2, 3, 4, 5 or 6;

Preferably, the mutation comprises a G55 mutation occurring on HCDR2 of SEQ ID NO: 3, 5, 15, 17, 20, 22 or 23, numbered according to the Kabat numbering system, more preferably, the G55 mutation is a G55A mutation or, according to the Kabat numbering system, the mutation comprises a D61 mutation occurring on HCDR2 of SEQ ID NO: 9, 32, 34 or 35, more preferably, the D61 mutation is a D61Q mutation.

In some specific embodiments, the antibody or antigen-binding fragment comprises SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 23, 26, 27, 29 , 30, 32, 34, 35, 38, 39, 41, 43, 45 or 46 of the VH of any one of the HCDR1, HCDR2 and/or HCDR3 having at least 80%, 85%, 90%, 91%, 92% %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical sequences;

And/or, the antibody or antigen-binding fragment comprises SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 21, 24, 25, 38, 31, 33, 36, 37, 40 , LCDR1, LCDR2 and/or LCDR3 in the VL of any one of 42, 44 or 47 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Sequences of 97%, 98%, 99%, 100% identity.

(a) a heavy chain variable region comprising said VH CDR1, VH CDR2 and VH CDR3; and/or,

(b) a light chain variable region comprising the VL CDR1, VL CDR2 and VL CDR3.

(a) SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 23, 26, 27, 29, 30, 32, 34, 35, 38, 39, 41 The sequence shown in any one of , 43, 45 or 46; and/or, (b) SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 21, 24, 25, 28, 31 , 33, 36, 37, 40, 42, 44 or 47 any one of the sequences shown;

Preferably, the antibody or antigen-binding fragment comprises:

(1) the sequence shown in any one of SEQ ID NO: 3, 15, 17 or 19 and the sequence shown in any one of SEQ ID NO: 4, 16 or 18;

(2) the sequence shown in any one of SEQ ID NO:5, 20, 22, 23 or 26 and the sequence shown in any one of SEQ ID NO:6, 21, 24 or 25;

(3) the sequence shown in any one of SEQ ID NO: 7, 27, 29 or 30 and the sequence shown in any one of SEQ ID NO: 8, 28 or 31;

(4) the sequence shown in any one of SEQ ID NO: 9, 32, 34, 35 or 38 and the sequence shown in any one of SEQ ID NO: 10, 33, 36 or 37;

(5) the sequence shown in any one of SEQ ID NO: 11, 39 or 41 and the sequence shown in any one of SEQ ID NO: 12, 40 or 42;

(6) the sequence shown in any one of SEQ ID NO: 13, 43, 45 or 46 and the sequence shown in any one of SEQ ID NO: 14, 44 or 47.

In some specific embodiments, the antibody or antigen-binding fragment comprises SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 23, 26, 27, 29 , 30, 32, 34, 35, 38, 39, 41, 43, 45, or 46 have at least 80%, 85%, 90%, 91%, 92%, 93%, 94% %, 95%, 96%, 97%, 98%, 99%, 100% identical framework region sequences; and/or,

VL set forth with any one of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 21, 24, 25, 28, 31, 33, 36, 37, 40, 42, 44, or 47 The framework regions have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical framework region sequences.

In some specific embodiments, the antibody or antigen-binding fragment comprises SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 23, 26, 27, 29 , 30, 32, 34, 35, 38, 39, 41, 43, 45 or 46, compared to the framework region of the VH shown in any one of the sequences, having at most 15 amino acid mutations; and/or, comprising the same sequence as SEQ ID NO. : the framework phase of the VL shown in any one of 4, 6, 8, 10, 12, 14, 16, 18, 21, 24, 25, 28, 31, 33, 36, 37, 40, 42, 44 or 47 ratio, a sequence with up to 15 amino acid mutations;

Preferably, the number of mutations may be selected from 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, T28N, F29I, T30E compared to the framework region of the VH shown in SEQ ID NO: 15 , V37L, M69I or R71A; more preferably, at least T28N, F29I, T30E, V37L, M69I and R71A mutations;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, Q43K, R71V or T73K compared to the framework region of the VH shown in SEQ ID NO: 20 Mutations, more preferably, at least R71V and T73K mutations or at least Q43K, R71V and T73K mutations;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, G26D, I37V or R94K compared to the framework region of the VH shown in SEQ ID NO: 27 Mutations, more preferably, at least G26D and R94K mutations or at least G26D, I37V and R94K mutations;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, V2I, D72E or V75A compared to the framework region of VH shown in SEQ ID NO: 32 Mutations, more preferably, at least the V2I mutation or at least the D72E and V75A mutations;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, F24V or G26D mutation, compared to the framework region of the VH shown in SEQ ID NO: 39, More preferably, at least the F24V and G26D mutations;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, G42E, G44R or N73I compared to the framework region of the VH shown in SEQ ID NO: 43 Mutations, more preferably, at least G42E, G44R and N73I mutations, or at least G44R and N73I mutations;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, Y49S, G57E or I58F compared to the framework region of VL shown in SEQ ID NO: 16 Mutations, more preferably, at least the Y49S, G57E and I58F mutations;

Preferably, the antibody or antigen-binding fragment comprises, compared with the framework region of VL shown in SEQ ID NO: 21, at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, Y36L, P44F, L46G , F71Y or V85D mutations, more preferably, at least F71Y and V85D mutations, or at least Y36L, P44F, L46G and F71Y mutations;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, V3Q, Q38R or D60A compared to the framework region of VL shown in SEQ ID NO: 28 Mutations, more preferably, at least V3Q, Q38R and D60A mutations;

Preferably, the antibody or antigen-binding fragment comprises a framework region sequence having at least one or more mutations selected from the group consisting of: numbering according to the Kabat numbering system, A43G compared to the framework region of VL shown in SEQ ID NO: 33 , Y49H, T69R or F71Y mutation, more preferably, at least A43G and Y49H mutation, or at least A43G, Y49H, T69R and F71Y mutation;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, M4L or N22S mutation, compared to the framework region of VL shown in SEQ ID NO: 40, More preferably, at least M4L and N22S mutations;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, M4L or N22S mutation, compared to the framework region of VL shown in SEQ ID NO: 44, More preferably, at least the M4L and N22S mutations are present.

In some specific embodiments, the antibody or antigen-binding fragment comprises or does not comprise a heavy chain constant region and/or a light chain constant region;

Preferably, the heavy chain constant region comprises a full-length heavy chain constant region or a heavy chain constant region fragment, which may be selected from a CH1, Fc or CH3 domain;

Preferably, the heavy chain constant region and/or light chain constant region is a human heavy chain constant region and/or a human light chain constant region;

Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgGl heavy chain constant region, an IgG2 heavy chain constant region, an IgG3 heavy chain constant region or an IgG4 heavy chain constant region;

Preferably, the heavy chain constant region is a human IgG1 heavy chain constant region, a human IgG2 heavy chain constant region, a human IgG3 heavy chain constant region or a human IgG4 heavy chain constant region;

Preferably, the heavy chain constant region has the amino acid sequence shown in SEQ ID NO:48, and the light chain constant region has the amino acid sequence shown in SEQ ID NO:49;

Preferably, the antibody or antigen-binding fragment has a heavy chain as set forth in any one of SEQ ID NOs: 50, 52, 54, 56, 58 or 60, and/or, the antibody or antigen-binding fragment has a heavy chain as set forth in any of SEQ ID NOs: 50, 52, 54, 56, 58 or 60 ID NO: light chain shown in 51, 53, 55, 57, 59 or 61;

More preferably, the antibody or antigenic fragment has a heavy chain as shown in SEQ ID NO:50 and a light chain as shown in SEQ ID NO:51, or has a heavy chain as shown in SEQ ID NO:52 and SEQ ID The light chain set forth in NO:53, or with the heavy chain set forth in SEQ ID NO:54 and the light chain set forth in SEQ ID NO:55, or with the heavy chain set forth in SEQ ID NO:56 and SEQ ID The light chain set forth in NO: 57, or with the heavy chain set forth in SEQ ID NO: 58 and the light chain set forth in SEQ ID NO: 59, or with the heavy chain set forth in SEQ ID NO: 60 and SEQ ID NO: light chain shown in 61;

Preferably, the antibody lacks fucosylation.

In some specific embodiments, the antibody or antigen-binding fragment is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (eg, bispecific antibodies), monovalent antibodies Antibodies, Multivalent Antibodies, Whole Antibodies, Fragments of Whole Antibodies, Naked Antibodies, Conjugated Antibodies, Chimeric Antibodies, Humanized Antibodies, Fully Human Antibodies, Fab, Fab', Fab'-SH, F(ab') ₂ , Fd, Fv, scFv, diabody or single domain antibody.

In some specific embodiments, the antibody or antigen-binding fragment specifically binds human CD70 and/or monkey CD70; preferably, the antibody binds human CD70 and/or monkey CD70 with a KD of less than 1.00-8EM, 1.00 E-9M, 1.00E-10M, 2.00E-10M, 3.00E-10M, 4.00E-10M, 5.00E-10M, 6.00E-10M, 7.00E-10M, 8.00E-10M, 9.00E-10M, 1.00 E-11M, 2.00E-11M, 3.00E-11M, 4.00E-11M, 5.00E-11M, 6.00E-11M, 7.00E-11M, 8.00E-11M, 9.00E-11M or 1.00E-12M.

In some specific embodiments, the antibody or antigen-binding fragment inhibits and/or blocks the binding of CD70 to its ligand CD27; preferably, the CD70 is human CD70 and/or monkey CD70.

In some specific embodiments, the antibody or antigen-binding fragment exhibits one or more effector functions selected from the group consisting of antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP).

In some specific embodiments, the antibody or antigen-binding fragment is further coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioisotopes, chemotherapeutic agents or immunomodulatory agents, and the tracer Selected from radiographic contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers.

In another aspect, the present invention discloses an antibody or antigen-binding fragment that specifically binds to CD70, and the antibody or antigen-binding fragment is:

(1) directly obtained by immunizing mice with CHO cells overexpressing the antigen shown in SEQ ID NO: 1, or

(2) The antibody obtained in (1) is obtained by further humanization and improvement.

In some specific embodiments, the antibody or antigen-binding fragment comprises some or all of the technical features of the antibody or antigen-binding fragment disclosed in the first aspect of the present invention.

In a second aspect, the present invention discloses a multispecific antigen-binding molecule comprising a first antigen-binding moiety comprising the aforementioned antibody or antigen-binding fragment; and a second antigen-binding moiety a module, the second antigen-binding module specifically binds to other antigens other than CD70, or binds to a different CD70 antigenic epitope from the first antigen-binding module;

Preferably, the other antigen is selected from CD3, CD3ε, CD16, CD16A, CD28, CD20, CD19, CD47 or CD40L;

Preferably, the multispecific antigen binding molecule is bispecific, trispecific or tetraspecific.

In a third aspect, the present invention discloses a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, the extracellular The antigen-binding domain comprises the aforementioned antibodies or antigen-binding fragments.

In a fourth aspect, the present invention discloses an immune effector cell, the immune effector cell expresses the aforementioned CAR or comprises a nucleic acid fragment encoding the aforementioned CAR; preferably, the immune effector cell is selected from T cells, NK cells, (natural killer) cell), NKT cells (natural killer T cells), monocytes, macrophages, dendritic cells or mast cells;

Preferably, the immune effector cells are autoimmune effector cells or allogeneic immune effector cells;

Preferably, the T cells are selected from cytotoxic T cells, regulatory T cells or helper T cells.

In a fifth aspect, the present invention discloses an isolated nucleic acid fragment encoding the aforementioned antibody or antigen-binding fragment, multispecific antigen-binding molecule or chimeric antigen receptor.

In a sixth aspect, the present invention discloses a vector comprising the aforementioned nucleic acid fragment.

In a seventh aspect, the present invention discloses a host cell comprising the aforementioned vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (E. coli), fungi (yeast), insect cells or mammalian cells Animal cells (CHO cell line or 293T cell line); preferably, the cells lack a fucosyltransferase such as FUT8.

In an eighth aspect, the present invention discloses a method for preparing the aforementioned antibodies or antigen-binding fragments and multispecific antigen-binding molecules, the method comprising culturing the aforementioned cells, and isolating the antibodies or antigen-binding fragments expressed by the cells, or separating The multispecific antigen-binding molecule expressed by the cell.

In a ninth aspect, the present invention discloses a method for preparing the aforementioned immune effector cells, the method comprising: introducing a nucleic acid fragment comprising the aforementioned CAR into the immune effector cells, optionally, the method further comprises activating the aforementioned CAR The immune effector cells express the aforementioned CAR.

In a tenth aspect, the present invention discloses a pharmaceutical composition comprising the aforementioned antibody or antigen-binding fragment, multispecific antigen-binding molecule, chimeric antigen receptor, immune effector cell, nucleic acid fragment, vector or cell; Preferably, the composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; preferably, the composition comprises the antibody or antigen in an amount capable of administering 0.1 to 50 mpk to a subject The binding fragment is preferably 1 to 20 mpk, more preferably 10 to 20 mpk.

In an eleventh aspect, the present invention discloses that the aforementioned antibodies or antigen-binding fragments, multispecific antigen-binding molecules, chimeric antibody receptors, immune effector cells, nucleic acid fragments, vectors or cells are prepared for the treatment of cancer or tumor, autoimmune Use in medicines for diseases or viral infections; preferably, the cancer or tumor can be selected from renal cell carcinoma, melanoma, myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia ( CLL), non-Hodgkin's lymphoma, T-cell lymphoma, mantle cell lymphoma, or cutaneous T-cell lymphoma; preferably, the medicament comprises the antibody or antigen-binding agent in an amount capable of administering 0.1 to 50 mpk to a subject Fragment, preferably 1 to 20 mpk, more preferably 10 to 20 mpk.

In a twelfth aspect, the present invention discloses a method of treating cancer or tumor, autoimmune disease or viral infection, the method comprising administering to a subject an effective amount of the aforementioned antibody or antigen-binding fragment, multispecific antigen Binding molecule, chimeric antibody receptor, immune effector cell, nucleic acid fragment, vector or cell; preferably, the cancer or tumor can be selected from renal cell carcinoma, melanoma, myelodysplastic syndrome (MDS), acute myeloid Leukemia (AML), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma, T-cell lymphoma, mantle cell lymphoma or cutaneous T-cell lymphoma; preferably, the effective amount of the antibody or antigen-binding fragment is 0.1 to 50 mpk, preferably 1 to 20 mpk, more preferably 10 to 20 mpk.

In a thirteenth aspect, the present invention discloses the aforementioned antibodies or antigen-binding fragments, multispecific antigen-binding molecules, chimeric antigen receptors, immune effector cells, nucleic acid fragments, nucleic acid vectors or host cells for use in the treatment of cancer or tumor, Autoimmune disease or viral infection; preferably, the cancer or tumor can be selected from renal cell carcinoma, melanoma, myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) , non-Hodgkin's lymphoma, T-cell lymphoma, mantle cell lymphoma or cutaneous T-cell lymphoma; preferably, the effective amount of the antibody or antigen-binding fragment to the subject is 0.1-50 mpk, preferably 1-20 mpk , more preferably 10 to 20 mpk.

Definition and Explanation of Terms

Unless otherwise defined herein, all terms herein have the meaning as commonly understood by one of ordinary skill in the art.

Furthermore, unless otherwise indicated herein, terms in the singular shall include the plural and terms in the plural shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a" and "the" include plural referents unless the content clearly dictates otherwise.

The terms "comprising,""comprising," and "having" are used interchangeably herein to denote an inclusive scheme, meaning that elements other than the listed elements may be present in the scheme. It is also to be understood that the use of "comprising", "comprising" and "having" descriptions herein also provides for "consisting of" aspects. Exemplarily, "a composition comprising A and B" should be understood as the following technical solutions: a composition consisting of A and B, and a composition containing other components in addition to A and B , all of which are within the scope of the aforementioned "a composition".

The term "and/or" as used herein includes the meanings of "and", "or" and "all or any other combination of the elements linked by the associated term."

The term "CD70" herein, also known as "TNFSF7" or "CD27L", is a member of the TNF ligand family and is a ligand for CD27 (also known as TNFRSF27). "CD70" herein includes mature or immature full-length wild-type CD70 protein or mutants thereof (eg, point mutations, insertion mutations or deletion mutations), splice variants, orthologs (Orthologs) and the foregoing Fragment of CD70. "CD70" herein can be derived from humans, primates, such as monkeys (eg, rhesus monkeys, cynomolgus monkeys), and rodents, such as mice and rats. Exemplarily, the amino acid sequence of human CD70 can be found in UniProt number: P32970, and the amino acid sequence of rhesus monkey CD70 can be found in UniProt number: F7GPA5.

The term "specifically binds" herein refers to an antigen-binding molecule (eg, an antibody) that specifically binds an antigen and a substantially identical antigen, usually with high affinity, but does not bind with high affinity to an unrelated antigen. Affinity is usually reflected in the equilibrium dissociation constant (KD), where lower KD indicates higher affinity. Taking an antibody as an example, high affinity generally refers to having about ^10-7 M or less, about ^10-8 M or less, about 1× ^10-9 M or less, about 1× ^10-10 M or less, KD of 1× ^10-11 M or lower or 1× ^10-12 M or lower. KD is calculated as follows: KD=Kd/Ka, where Kd represents the dissociation rate and Ka represents the association rate. Equilibrium dissociation constant KD can be measured using methods well known in the art, such as surface plasmon resonance (eg Biacore) or equilibrium dialysis method, for example, see the method for obtaining KD value shown in Example 5 herein.

The term "antigen-binding molecule" is used herein in the broadest sense to refer to a molecule that specifically binds an antigen. Illustratively, antigen binding molecules include, but are not limited to, antibodies or antibody mimetics. "Antibody mimetic" refers to an organic compound or binding domain that can specifically bind to an antigen, but is unrelated to the structure of an antibody. Exemplarily, antibody mimetics include, but are not limited to, affibody, affitin, affilin, designed ankyrin repeat proteins (DARPin), nucleic acid aptamer or Kunitz-type domain peptide.

The term "antibody" is used herein in the broadest sense to refer to a polypeptide comprising sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to enable specific binding to an antigen or peptide combination. "Antibody" herein encompasses various forms and various structures so long as they exhibit the desired antigen-binding activity. "Antibody" herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antibody, and fully synthetic scaffolds comprising, eg, biocompatible polymers. See, eg, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

"Antibody" herein includes a typical "quad-chain antibody", which is an immunoglobulin consisting of two heavy chains (HC) and two light chains (LC); heavy chain refers to a polypeptide chain that is The N-terminal to C-terminal direction consists of the heavy chain variable region (VH), the heavy chain constant region CH1 domain, the hinge region (HR), the heavy chain constant region CH2 domain, the heavy chain constant region CH3 domain; and, When the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain; the light chain is composed of a light chain variable region (VL) and a light chain constant in the N-terminal to C-terminal direction The polypeptide chain composed of the region (CL); the heavy chain and the heavy chain, and the heavy chain and the light chain are connected by disulfide bonds to form a "Y"-shaped structure. Due to the different amino acid composition and arrangement sequence of the constant region of immunoglobulin heavy chain, its antigenicity is also different. Accordingly, the "immunoglobulins" herein can be divided into five classes, or isotypes called immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ and δ chains, respectively. , γ chain, α chain and ε chain. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are classified into kappa chains or lambda chains by the difference in the constant region. Each of the five classes of Ig can have a kappa chain or a lambda chain.

"Antibody" herein also includes antibodies that do not contain a light chain, such as those produced by Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe, and alpaca ( Vicugna pacos) and other heavy-chain antibodies (heavy-chain antibodies, HCAbs) and sharks and other cartilaginous fish found in the new immunoglobulin antigen receptors (Ig new antigen receptor, IgNAR).

An "antibody" herein can be derived from any animal, including, but not limited to, humans and non-human animals, which can be selected from primates, mammals, rodents, and vertebrates, such as camelid, llama , ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fishes (eg sharks).

"Antibody" herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (eg, bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies , conjugated antibodies, chimeric antibodies, humanized antibodies or fully human antibodies.

The term "monoclonal antibody" herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., except for possible variants (e.g., containing naturally occurring mutations or produced during the manufacture of a preparation, such variants typically defined as except that the individual antibodies comprising the population are identical and/or bind the same epitope. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. The modifier "monoclonal" herein should not be construed as requiring the production of the antibody or antigen-binding molecule by any particular method. For example, monoclonal antibodies can be made by a variety of techniques including, but not limited to, hybridoma technology, recombinant DNA methods, phage library display technology, and the use of transgenic animals that contain all or part of the human immunoglobulin loci method and other methods known in the art.

The term "natural antibody" herein refers to an antibody that is produced and paired by the immune system of a multicellular organism. The antibody of the term "engineered antibody" herein refers to a non-natural antibody obtained by genetic engineering, antibody engineering and other techniques. Exemplarily, "engineered antibody" includes humanized antibody, small molecule antibody (such as scFv, etc.), dual specific antibodies, etc.

The term "monospecific" herein refers to having one or more binding sites, wherein each binding site binds the same epitope of the same antigen.

The term "multispecific antibody" herein refers to having at least two antigen-binding sites, each of which is associated with a different epitope of the same antigen or a different epitope of a different antigen. Bit binding. Thus, terms such as "bispecific", "trispecific", "tetraspecific" etc. refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.

The term "valency" herein refers to the presence of a defined number of binding sites in an antibody/antigen binding molecule. Thus, the terms "monovalent", "bivalent", "tetravalent" and "hexavalent" refer to one binding site, two binding sites, four binding sites and six binding sites, respectively, in an antibody/antigen binding molecule the existence of points.

Herein, "full-length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to mean having a structure that is substantially similar to that of a native antibody.

"Antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not possess the full structure of an intact antibody, but only include partial or partial variants of the intact antibody that have the ability to bind antigenic capacity. "Antigen-binding fragments" or "antibody fragments" herein include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fd, Fv, scFv, diabodies, and single domain antibodies.

Papain digestion of intact antibodies produces two identical antigen-binding fragments, termed "Fab" fragments, each containing the heavy and light chain variable domains, as well as the light chain constant domain and the heavy chain first constant domain (CH1 ). As such, the term "Fab fragment" herein refers to a light chain fragment comprising the VL domain and constant domain (CL) of the light chain, and an antibody fragment comprising the VH domain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by adding a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine residues of the constant domains carry free thiol groups. Pepsin treatment produces an F(ab') ₂ fragment with two antigen binding sites (two Fab fragments) and a portion of the Fc region.

The term "Fd" herein refers to an antibody consisting of VH and CH1 domains. The term "Fv" herein refers to antibody fragments consisting of one-armed VL and VH domains. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (eg, an Fd fragment, which contains only three CDRs specific for the antigen) is able to recognize and bind the antigen, albeit probably with lower affinity than the intact binding site.

The term "scFv" (single-chain variable fragment) herein refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, eds. Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker with the amino acid sequence (GGGGS) ₄ can be used, but also variants thereof (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between the VH and VL of the scFv, forming a disulfide-linked Fv (dsFv).

The term herein is a "diabody" whose VH and VL domains are expressed on a single polypeptide chain, but use a linker that is too short to allow pairing between the two domains of the same chain, forcing the domains to interact with The complementary domains of the other chain pair and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2: 1121-1123 (1994)).

The terms "single domain antibody" (sdAb), "VHH" and "nanobody" have the same meaning and are used interchangeably herein and refer to the variable region of a cloned antibody heavy chain, constructed from only one A single-domain antibody composed of the variable region of the heavy chain, which is the smallest fully functional antigen-binding fragment. Usually, an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1) is obtained first, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region. Single domain antibodies can be derived from camelid heavy chain antibodies or from cartilaginous IgNARs.

The term "naked antibody" herein refers to an antibody that is not conjugated to a therapeutic agent or tracer; the term "conjugated antibody" refers to an antibody that is conjugated to a therapeutic agent or tracer.

The term "chimeric antibody" herein refers to an antibody in which a portion of its light or/and heavy chain is derived from an antibody (which may be derived from a particular species or belong to a particular antibody class or subclass). class), and the other part of the light chain or/and the heavy chain is derived from another antibody (which may be derived from the same or a different species or belong to the same or different antibody class or subclass), but which nevertheless retains the Binding activity of target antigen (U.S.P 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)). For example, the term "chimeric antibody" can include antibodies (eg, human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from a primary antibody (eg, a murine antibody) and the heavy and The light chain constant region is derived from a second antibody (eg, a human antibody).

The term "humanized antibody" herein refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase homology to the sequence of a human antibody. Generally, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (eg, variable FR and/or constant regions) are derived from human Immunoglobulins (receptor antibodies). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, and the like.

The term "fully human antibody" herein refers to an antibody having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibodies" herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.

The term "variable region" herein refers to the region of an antibody heavy or light chain that is involved in binding an antibody to an antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR". " is used interchangeably with "VL", "LCVR". The variable domains (VH and VL, respectively) of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, eg, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p.91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. The terms "complementarity determining region" and "CDR" are used interchangeably herein, and generally refer to the variable region of the heavy chain (VH) or the hypervariable region (HVR) of the light chain variable region (VL), which is spatially structured It can form precise complementarity with the antigenic epitope, so it is also called the complementarity determining region. Among them, the heavy chain variable region CDR can be abbreviated as HCDR, and the light chain variable region CDR can be abbreviated as LCDR. The terms "framework region" or "FR region" are used interchangeably and refer to those amino acid residues other than the CDRs in the variable region of the heavy or light chain of an antibody. Usually a typical antibody variable region consists of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

For a further description of CDRs, see Kabat et al, J. Biol. Chem., 252:6609-6616 (1977); Kabat et al, U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al, J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al, J. Mol. Biol., 273:927-948 (1997); MacCallum et al, J. Mol . Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc M.P. et al., Dev. Comp. Immunol., 27:55-77 (2003) ; and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001). The "CDRs" herein may be labeled and defined by means known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using tool websites including, but not limited to, the AbRSA website (http://cao.labshare. cn/AbRSA/cdrs.php), abYsis website (www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi) and IMGT website (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign. cgi#results). The CDRs herein include overlaps and subsets of amino acid residues differently defined.

The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

The term "Chothia numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying CDR region boundaries based on the position of structural loop regions (see, eg, Chothia & Lesk (1987) J. Mol. Biol 196:901-917; Chothia et al. (1989) Nature 342:878-883).

The term "IMGT numbering system" herein generally refers to the numbering system based on The International ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

Illustratively, annotating and identifying SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 23, 26, 27, 29, 30, 32, 34, 35, 38 , 39, 41, 43, 45, 46 and SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 21, 24, 25, 28, 31, 33, 36, 37, 40, 42 The CDRs of the VH or VL sequences shown in , 44 and 47, the specific results are shown in the following table:

Table 1 Anti-CD70 antibody heavy chain and light chain CDR region amino acid sequence list

The term "heavy chain constant region" herein refers to the carboxy-terminal portion of an antibody heavy chain that is not directly involved in the binding of the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, relative to the availability of the antibody The variable domains have more conserved amino acid sequences. A "heavy chain constant region" comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof. "Heavy chain constant region" includes "full-length heavy chain constant region" and "heavy chain constant region fragment", the former has a substantially similar structure to that of natural antibody constant region, while the latter includes only "full-length heavy chain constant region" part". Exemplarily, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain In the case of an antibody, it does not include the CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" can be selected from a CH1, Fc or CH3 domain.

The term "light chain constant region" herein refers to the carboxy-terminal portion of an antibody light chain that is not directly involved in binding the antibody to an antigen, which light chain constant region may be selected from a constant kappa domain or a constant lambda domain.

The term "Fc" herein refers to the papain hydrolyzed carboxy-terminal portion of an antibody from an intact antibody, which typically comprises the CH3 and CH2 domains of the antibody. Fc regions include, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus. The C-terminal lysine of the Fc region (residue 447 according to the Kabat numbering system) can be removed, for example, during production or purification of the antibody, or by recombinant engineering of nucleic acid encoding the antibody heavy chain, thus, the Fc region can include or excluding Lys447.

Unless otherwise stated, the numbering of amino acid residues in "antibodies" or "antigen-binding fragments" described herein is determined by the Kabat numbering system, see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). The description will be given below in conjunction with amino acid residue mutations. For example, the heavy chain variable region T28N mutation means that the amino acid residue at position 28 of the heavy chain determined according to the aforementioned Kabat numbering system is mutated from T to N.

The term "conserved amino acids" herein generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity). Illustratively, the amino acids within each of the following groups belong to each other's conserved amino acid residues, and substitutions of amino acid residues within a group belong to conservative amino acid substitutions:

Illustratively, the following six groups are examples of amino acids that are considered conservative substitutions for each other:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K), Histidine (H);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

The term "identity" herein can be calculated by aligning the sequences for optimal comparison purposes in order to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences (eg, it may be optimal The alignment may introduce gaps in either or both of the first and second amino acid sequences or nucleic acid sequences or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

Taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap, the percent identity between the two sequences varies with the identical positions shared by the sequences.

Sequence comparisons and calculation of percent identity between two sequences can be accomplished using mathematical algorithms. For example, using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at www.gcg.com), which has been integrated into the GAP program of the GCG software package, using the Blossum 62 matrix or The PAM250 matrix and

gap weights

16, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5 or 6 determine the percent identity between two amino acid sequences. As another example, using the GAP program in the GCG software package (available at www.gcg.com), using the NWSgapdna.CMP matrix and

gap weights

40, 50, 60, 70 or 80 and length weights 1, 2, 3, 4, 5 or 6, determine the percent identity between the two nucleotide sequences. A particularly preferred set of parameters (and one that should be used unless otherwise stated) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

It is also possible to use the PAM120 weighted remainder table, a gap length penalty of 12, a gap penalty of 4, using the E. Meyers and W. Miller algorithm that has been incorporated into the ALIGN program (version 2.0), ((1989) CABIOS, 4:11-17 ) determines the percent identity between two amino acid sequences or nucleotide sequences.

Additionally or alternatively, the nucleic acid and protein sequences described herein can be further used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences. Such searches can be performed, for example, using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid (SEQ ID NO: 1) molecule of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the corresponding programs (eg, XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

The term "antigen chimeric receptor (CAR)" herein refers to an artificial cell surface receptor engineered to be expressed on immune effector cells and to specifically bind an antigen, comprising at least (1) an extracellular antigen binding domain, eg, an antibody The variable heavy or light chain, (2) the transmembrane domain that anchors the CAR into immune effector cells, and (3) the intracellular signaling domain. CARs can utilize extracellular antigen-binding domains to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC-restricted manner.

The term "nucleic acid" herein includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), sugar (ie deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is generally represented as 5' to 3'. In this context, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, as well as synthetic forms of DNA or RNA. A mixed polymer of one or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms. Furthermore, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for the direct expression of the antibodies of the invention in vitro and/or in vivo, eg, in a host or patient. Such DNA (eg, cDNA) or RNA (eg, mRNA) vectors can be unmodified or modified. For example, the mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to generate antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi: 10.1038/nm.4356 or EP 2 101 823 B1). An "isolated" nucleic acid herein refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

The term "vector" herein refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of the host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

The term "host cell" herein refers to a cell into which exogenous nucleic acid has been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be identical in nucleic acid content to the parental cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the initially transformed cell are included herein.

The term "pharmaceutical composition" herein refers to a formulation that is in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain unacceptable toxicity to the subject to whom the pharmaceutical composition is administered of additional ingredients.

The term "treatment" herein refers to surgical or therapeutic treatment for the purpose of preventing, slowing (reducing) undesired physiological changes or pathologies, such as cancer, autoimmune diseases and viral infections, in the subject being treated. progress. Beneficial or desirable clinical outcomes include, but are not limited to, reduction of symptoms, reduction in disease severity, stable disease state (ie, no worsening), delayed or slowed disease progression, improvement or alleviation of disease state, and remission (whether partial remission or complete remission), whether detectable or undetectable. Those in need of treatment include those already suffering from the disorder or disease as well as those prone to develop the disorder or disease or for whom the disorder or disease is to be prevented. When referring to terms such as alleviation, alleviation, weakening, alleviation, alleviation, etc., the meanings also include elimination, disappearance, non-occurrence, etc.

The term "subject" herein refers to an organism receiving treatment for a particular disease or disorder as described herein. Examples of subjects and patients include mammals, such as humans, primates (eg, monkeys) or non-primate mammals, receiving treatment for a disease or disorder.

The term "effective amount" herein refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent, to a cell, tissue, or subject, is effective to prevent or alleviate a disease condition or progression of the disease. "Effective amount" also refers to an amount of the compound sufficient to relieve symptoms, eg, treat, cure, prevent or alleviate related medical conditions, or an increased rate of treatment, cure, prevention or alleviation of such conditions. When an active ingredient is administered to a subject alone, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, consecutively or simultaneously.

The term "autoimmune disease" herein refers to a disorder in which cells, tissues and/or organs are damaged by a subject's immune response to its own cells, tissues and/or organs.

The term "cancer" herein refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Benign and malignant cancers are included in this definition. The term "tumor" or "neoplastic" herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when referred to herein.

The term "EC50" herein refers to the half-maximal effective concentration, which includes the concentration of antibody that induces a half-way response between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of the antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art.

beneficial effect

Compared with the prior art, the present invention provides a novel CD70 antibody, which has at least one of the following beneficial effects:

1. Good CD70 affinity;

2. Binds human/or monkey CD70 protein, and/or binds or expresses human/or monkey CD70 protein cells (including low-expressing cells);

3. Block the binding of CD70 to CD27 or the CD70-CD27 signaling pathway;

4. Mediating ADCC, CDC and/or ADCP killing effect of CD70-expressing cells (including low-expressing cells);

5. In terms of the aforementioned effects, the CD70 antibody of the present invention is superior to or substantially equivalent to the positive control antibody 41D12.

Description of drawings

Figure 1 shows the ELISA Binding results of CD70 antibodies Hab003.22a, Hab035.31a, Hab055.13, Hab077.12, Hab095.21 and huCD70.ECD-TNC.his;

Figure 2 shows the ELISA Binding results of CD70 antibody Hab058.21a and huCD70.ECD-TNC.his;

Figure 3 shows the ELISA Binding results of CD70 antibodies Hab003.22a, Hab035.31a, Hab055.13, Hab077.12, Hab095.21 and macaca CD70.ECD-TNC.his;

Figure 4 is the ELISA Binding result of CD70 antibody Hab058.21a and macaca CD70.ECD-TNC.his;

Figure 5 shows the ELISA binding results of CD70 antibodies Hab003.22a, Hab035.31a, Hab055.13, Hab058.21a, Hab077.12, Hab095.21 and huCD70-CHO-K1 cells;

Figure 6 is the FACS Binding results of CD70 antibody Hab003.22a, Hab035.31a, Hab055.13, Hab077.12, Hab095.21 and 786-O cells;

Figure 7 shows the results of FACS Binding between CD70 antibody Hab058.21a and 786-O cells;

Figure 8 is the FACS Binding results of CD70 antibodies Hab003.22a, Hab035.31a, Hab055.13, Hab077.12, Hab095.21 and A549 cells;

Figure 9 shows the results of FACS Binding between CD70 antibody Hab058.21a and A549 cells;

Figure 10A shows the results of FACS Binding between CD70 antibodies Hab003.22a, Hab035.31a, Hab055.13, Hab077.12, Hab095.21 and Raji cells;

Figure 10B shows the results of FACS Binding between CD70 antibody Hab058.21a and Raji cells;

Figure 11 shows the FACS Binding results of CD70 antibodies Hab003.22a, Hab035.31a, Hab055.13, Hab077.12, Hab095.21 and cell LCL 8664;

Figure 12 shows the results of blocking experiments on 786-O by CD70 antibodies Hab003.22a, Hab035.31a, Hab055.13, Hab077.12 and Hab095.21;

Figure 13 shows the ADCC killing effect of CD70 fucose knockout antibodies Hab003.22a fut8KO, Hab035.31a fut8KO, Hab055.13 fut8KO, Hab077.12fut8KO and Hab095.21fut8KO on A498 cells;

Figure 14 shows the ADCC killing effect of CD70 fucose knockout antibodies Hab003.22a fut8KO, Hab035.31a fut8KO, Hab055.13 fut8KO, Hab077.12fut8KO, Hab095.21fut8KO on 786-O cells

Figure 15 shows the ADCC killing effect of CD70 fucose knockout antibodies Hab003.22a fut8KO, Hab035.31a fut8KO, Hab055.13 fut8KO, Hab077.12fut8KO and Hab095.21fut8KO on Raji cells;

Figure 16 shows the ADCC killing effect of CD70 fucose knockout antibodies Hab003.22a fut8KO, Hab035.31a fut8KO, Hab055.13 fut8KO, Hab077.12fut8KO, Hab095.21fut8KO on A549 cells;

Figure 17 shows the CDC killing effect of CD70 fucose knockout antibodies Hab003.22a fut8KO, Hab035.31a fut8KO, Hab055.13 fut8KO, Hab077.12 fut8KO, Hab095.21 fut8KO on Raji cells;

Figure 18 shows the ADCP killing effect of CD70 antibodies Hab003.22a, Hab035.31a, Hab055.13, Hab077.12 and Hab095.21 on Raji cells;

Figure 19 shows the inhibitory effect of CD70 antibodies Hab003.22a, Hab035.31a, Hab055.13, Hab077.12, Hab095.21 on IL-8 secretion;

Figure 20 shows the efficacy of CD70 antibody in Raji xenograft tumor model;

Figure 21 shows the survival curve of MOLM-13 model mice.

In all drawings, 41D12 is a positive control, the heavy chain is shown in SEQ ID NO:63, and the light chain is shown in SEQ ID NO:65:

In Figures 13-21, the isotype is hIgG1.

Detailed ways

The present invention will be further described below with reference to specific embodiments, and the advantages and characteristics of the present invention will become clearer with the description. If the specific conditions are not indicated in the examples, the routine conditions are used, such as the Antibody Technology Experiment Manual of Cold Spring Harbor, the Molecular Cloning Manual; or the conditions suggested by the raw material or commodity manufacturers. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

The embodiments of the present invention are only exemplary, and do not constitute any limitation on the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Example 1 Preparation of CD70-related recombinant antigens and purification methods of related antigens or antibodies

1. Antigen design and expression

1. Construction of tagged CD70 antigen with human and rhesus monkey CD70

Using human CD70 protein (UniProt number: P32970) and rhesus monkey CD70 (UniProt number: F7GPA5) as the CD70 template of the present invention, a tagged fusion protein was designed, cloned into the pTT5 vector, and transiently expressed in HEK293 cells to obtain It encodes the antigen and detection protein of the present invention. The specific information of the fusion protein is shown below, in which the double underlines are His and TNC tags, and the italics are the corresponding CD70 proteins.

huCD70.ECD-TNC.his (His, fusion protein of TNC tag and the extracellular domain of human CD70 mature protein), can be used for immunization and screening:

macaca CD70.ECD-TNC.his (His, fusion protein of TNC tag and the extracellular domain of rhesus monkey CD70 mature protein), can be used for immunization and screening:

2. Purification of CD70-related recombinant proteins, as well as purification of anti-human CD70 hybridoma antibodies and recombinant antibodies

1. Purification of hybridoma supernatant/recombinant antibody by Protein A affinity chromatography

Antibody-expressing cell culture supernatants were first collected by high-speed centrifugation (specifically, fucose knockout antibodies were expressed in fut8KO cells). The ProteinA affinity column was washed with 6M guanidine hydrochloride for 3-5 column volumes, and then washed with pure water for 3-5 column volumes. The column is equilibrated for 3-5 column volumes using a buffer system such as 1×PBS (pH 7.4) as the equilibration buffer. The cell supernatant is loaded and combined with low flow rate, and the flow rate is controlled so that the retention time is about 1min or longer. After the combination is completed, the column is washed with 1×PBS (pH7.4) for 3-5 times the column volume until the UV absorption falls back to the baseline. . Use 0.1M acetic acid/sodium acetate (pH3.0-3.5) buffer for sample elution, collect elution peaks according to UV detection, and use 1M Tris-HCl (pH8.0) to quickly adjust the pH of the eluted product to 5-6 temporarily live. For the eluted product, solution replacement can be carried out by methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube and solution replacement to the desired buffer system, or molecular exclusion such as G-25 desalting to replace the desired buffer system. Buffer system, or use a high-resolution size exclusion column such as Superdex 200 to remove the polymer component in the eluted product to improve the purity of the sample.

2. Nickel column purification of CD70-related recombinant proteins such as huCD70.ECD-TNC.his

After constructing and expressing CD70-related recombinant antigens according to the above steps "1. Design and expression of antigens", purify them as follows:

The cell expression supernatant samples were centrifuged at high speed to remove impurities, the buffer was replaced with PBS, and imidazole was added to a final concentration of 5 mM. Equilibrate the nickel column with 5 mM imidazole in PBS and rinse 2-5 column volumes. Combine the replaced supernatant sample on the column, and the medium can choose nickel columns from different companies. The column was rinsed with 5 mM imidazole in PBS until the A280 reading dropped to baseline. Then, the column was washed with PBS+10mM imidazole to remove non-specifically bound impurity proteins, and the effluent was collected. The target protein was eluted with a PBS solution containing 300 mM imidazole, and the elution peaks were collected.

The collected eluted product can be further purified by gel chromatography Superdex200 (GE) after concentration. The mobile phase is PBS to remove aggregates and impurity protein peaks, and collect the eluted peaks of the target product. The obtained protein was identified as correct by electrophoresis, peptide map and LC-MS.

Example 2 Preparation of anti-human CD70 mouse monoclonal antibody

1. Immunization

Anti-human CD70 monoclonal antibodies were produced by immunizing mice. SJL white mice were used in the experiment, female, 6-8 weeks old (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001). Breeding environment: SPF grade. After the mice were purchased, they were reared in a laboratory environment for 1 week, regulated by a 12/12 hour light/dark cycle, the temperature was 20-25°C, and the humidity was 40-60%. The acclimated mice were immunized according to the following protocol. The immunizing antigen was CHO cells overexpressing CD70 (purchased from Kangyuan Bochuang, KC-1267).

Immunization scheme: immunize mice with human CD70-overexpressing CHO cell line, 1×10 ⁷ cells/mouse/time, intraperitoneally. After the cells were collected, they were diluted with PBS to 1×10 ⁸ /ml, injected intraperitoneally (IP) with 100 μl/cell on day 0, and boosted every 7 days thereafter. Blood was collected on the 21st, 35th, 49th and 63rd days, and the antibody titers in the mouse serum were determined by ELISA. After 7-9 immunization, mice with high antibody titers in serum and titers approaching a plateau were selected for lymphocyte and splenocyte fusion. Three days before the fusion of splenocytes, the huCD70.ECD-TNC.his antigen solution was injected intraperitoneally (IP).

The fusion of spleen cells

Splenic lymphocytes were combined with myeloma cells Sp2/0 (

CRL-8287 ^™ ) was fused to obtain hybridoma cells. The fused hybridoma cells were cultured at a density of 0.5-1×10 ⁵ /ml in complete medium (containing 20% FBS, 1× HAT, 1× bovine insulin, 1× non-essential amino acids, 1× double antibody, 1× IL -6 in DMEM medium), resuspended in 200 μl/well in a 96-well plate, incubate at 37° C., 5% CO ₂ for 7-11 days until the formation of pinpoint colonies. Remove the supernatant, add 200 μl/well of HT complete medium (DMEM medium containing 10% FBS, 1×HT and 1× bovine insulin, 1× non-essential amino acids, 1× double antibody), 37°C, 5% CO _2. ELISA or FACS detection was performed after culturing for 1 day.

3. Screening of hybridoma cells

According to the growth density of hybridoma cells, the hybridoma culture supernatant was detected by human CD70 binding ELISA method (see Example 5 "1. ELISA experiment of CD70 antibody binding to human CD70 protein"). The cell supernatants of the positive wells detected by binding ELISA were subjected to human CD70 CHO-K1, 786-O cell binding experiments (see Example 5) and CD70-CD27 binding inhibition experiments (see Example 6). The well cells that were positive in the human CD70 CHO-K1, 786-O cell binding assay and CD70-CD27 binding inhibition assay were promptly expanded, cryopreserved and subcloned until a single-cell clone was obtained.

Subcloned cells also need to be subjected to human CD70 binding ELISA, human CD70 CHO-K1, 786-O cell binding assay and CD70-CD27 binding inhibition assay. The hybridoma clones were obtained through the above experimental screening, the antibodies were further prepared by serum-free cell culture method, and the antibodies were purified by the purification method described in Example 1 for subsequent use.

4. Sequence determination of hybridoma positive clones

The procedure for cloning sequences from positive hybridomas is as follows. Hybridoma cells in logarithmic growth phase were collected, RNA was extracted with Trizol (Invitrogen, Cat No. 15596-018) according to the kit instructions, and reverse transcribed with PrimeScript ^™ Reverse Transcriptase kit (Takara, Cat No. 2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326Rev.B 0503) and then sent to a sequencing company for sequencing. The murine anti-human CD70 antibodies Mab003, Mab035, Mab055, Mab058, Mab077 and Mab095 were obtained by sequencing, and their heavy chain variable region (HCVR) and light chain variable region (LCVR) amino acid sequences are shown below.

Mab003 HCVR:

Mab003 LCVR:

Mab035 HCVR:

Mab035 LCVR:

Mab055 HCVR:

Mab055 LCVR:

Mab058 HCVR:

Mab058 LCVR:

Mab077 HCVR:

Mab077 LCVR:

Mab095 HCVR:

Mab095 LCVR:

The CDR regions of the above CD70 monoclonal antibodies were analyzed by bioinformatics methods, wherein the CDR regions were identified and annotated using the Kabat numbering system, the Chothia numbering system and the IMGT numbering system (http://www.abysis.org/ abysis/sequence_input/key_annotation/key_annotation.cgi; http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results), the specific results are shown in Table 1.

Example 3 Preparation of human IgG1 chimeric antibody

According to the sequencing results of the hybridoma antibodies Mab003, Mab035, Mab055, Mab058, Mab077 and Mab095 and their variable region coding genes obtained in Example 2, primers were designed, and the sequencing genes were used as templates to construct the VH/VK gene fragments of each antibody through PCR . The obtained VH/VK gene fragment was then subjected to homologous recombination with the expression vector pTT5 (with a signal peptide and hIgG1/hkappa constant region gene (CH1-Fc/CL) fragment) to construct a recombinant chimeric antibody full-length expression plasmid VH-CH1-Fc -pTT5/VL-CL-pTT5, forming six chimeric antibodies ChAb003, ChAb035, ChAb055, ChAb058, ChAb077, ChAb095. The amino acid sequences of the heavy chain constant region and the light chain constant region are shown in SEQ ID NOs: 48 and 49 (Example 4).

Example 4 Humanization of murine anti-human CD70 antibody

1. Construction of humanized variable region of anti-human CD70 antibody

By comparing the IMGT (http://imgt.cines.fr) human antibody heavy and light chain variable region germline gene database and the MOE (Molecular Operating Environment, molecular operating environment) software, respectively select the high homology with the mouse antibody The heavy chain and light chain variable region germline genes are used as templates, and the CDRs of the murine antibody are respectively grafted into the corresponding human templates to form variable sequences in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 region sequence. Back-mutations and/or hot-spot mutations were performed as needed. The amino acid residues of the antibodies of this example are numbered according to the Kabat numbering system, and the CDR regions are determined by the Kabat numbering system .

1. Humanization of Mab003

1.1, Mab003 architecture selection

The humanized light chain templates of the murine antibody Mab003 are IGKV3-11*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-46*01 and IGHJ6*01, and the CDRs of the murine antibody Mab003 were transplanted into them respectively. In the human template, namely the humanized antibody Hab003 obtained from Mab003, the sequence of its variable region is shown below, in which italics indicate the FR sequence, and the underline indicates the CDR sequence.

Hab003 HCVR (also known as VH-CDR graft):

Hab003 LCVR (also known as VL-CDR graft):

1.2. Back mutation design of humanized antibody of Mab003

As needed, the key amino acids in the FR region sequence of the Mab003 humanized antibody were back-mutated to the amino acids corresponding to the mouse antibody to ensure the original affinity. The specific back-mutation design is shown in Table 2.

Table 2 Humanized antibody back mutation design of Mab003

Note: Grafted represents the insertion of mouse antibody CDRs into the human germline FR region sequence; Y49S represents the mutation of Y at position 49 of Grafted back to S, and so on.

1.3. Mab003 humanized antibody

The back-mutation designs of the humanized antibodies of Mab003 in Table 2 were combined to finally obtain a variety of humanized antibodies of Mab 003 (see Table 3 for details).

Table 3 The corresponding amino acid sequence of the variable region of Mab003 humanized antibody

	Hab003.VH1Hab003.VH1	Hab003.VH2Hab003.VH2
Hab003.VL1Hab003.VL1	Hab003.11Hab003.11	Hab003.12Hab003.12
Hab003.VL2Hab003.VL2	Hab003.21Hab003.21	Hab003.22Hab003.22

Note: Hab003.12 indicates that the Mab003 humanized antibody Hab003.12 has a light chain variable region as described in Hab003.VL1 and a heavy chain variable region as described in Hab003.VH2, and so on.

The amino acid sequences of Hab003.VH1, Hab003.VH2, Hab003.VL1 and Hab003.VL2 are shown below, italics indicate FR sequences, underlines indicate CDR sequences, and bold indicate back mutations.

Hab003.VH1 (same as Hab003 HCVR):

Hab003.VH2:

Hab003.VL1 (same as Hab003 LCVR):

Hab003.VL2:

1.4. Hot spot mutation of Mab003 humanized antibody

Based on the existence of a high-risk easy-to-modify site NG in HCDR2 in the heavy chain CDR2 region of 003, the amino acid mutation of NG based on the antibody structure was carried out by computer simulation to eliminate the risk of molecular modification. In one of the preferred embodiments, amino acid mutation is performed on the NG of HCDR2 of Hab003.VH2 to obtain Hab003.VH2a, the amino acid sequence of which is as follows, wherein the border with characters represents the hot spot mutation position:

Table 4 The variable region corresponding to the hot spot mutant antibody of Mab003 humanized antibody

	Hab003.VL2Hab003.VL2
Hab003.VH2aHab003.VH2a	Hab003.22aHab003.22a

Note: Hab003.22a indicates that the Mab003 humanized antibody Hab003.22a has a light chain variable region as described in Hab003.VL2 and a heavy chain variable region as described in Hab003.VH2a.

2. Humanization of Mab035

2.1, Mab035 architecture selection

The humanized light chain templates of the murine antibody Mab035 are IGKV2-29*02 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ6*01, and the CDRs of the murine antibody Mab035 were transplanted into them respectively. In the humanized template, the humanized antibody Hab035 of Mab035 is obtained. The Hab035 variable region sequence is shown below, with italics indicating framework regions and underlining indicating CDR regions.

Hab035 HCVR (also known as VH-CDR graft):

Hab035 LCVR (also known as VL-CDR graft):

2.2. Back mutation design of humanized antibody of Mab035

As needed, the key amino acids in the FR region sequence of the humanized antibody of Mab035 were back-mutated to the amino acids corresponding to the murine antibody to ensure the original affinity. The specific back-mutation design is shown in Table 5.

Table 5 Humanized antibody back mutation design of Mab 035

Note: Grafted represents the insertion of murine antibody CDRs into the human germline FR region sequence; F71Y represents the mutation of the 71st F of Grafted back to Y, and so on.

2.3. Mab035 humanized antibody

The reverse mutation design of the Mab035 humanized antibody in the above Table 5 was combined, and finally a variety of Mab 035 humanized antibodies were obtained. The specific combination is shown in Table 6.

Table 6 The corresponding amino acid sequence of the variable region of Mab035 humanized antibody

	Hab035.VH1Hab035.VH1	Hab035.VH2Hab035.VH2
Hab035.VL1Hab035.VL1	Hab035.11Hab035.11	Hab035.12Hab035.12
Hab035.VL2Hab035.VL2	Hab035.21Hab035.21	Hab035.22Hab035.22
Hab035.VL3Hab035.VL3	Hab035.31Hab035.31	Hab035.32Hab035.32

Note: Hab035.12 indicates that the Mab035 humanized antibody Hab035.12 has a light chain variable region as described in Hab035.VL1 and a heavy chain variable region as described in Hab035.VH2, and so on.

The specific sequences of Hab035.VH1, Hab035.VH2, Hab035.VL1, Hab035.VL2 and Hab035.VL3 are shown below, in which italics indicate framework regions, underlines indicate CDRs, and bold indicates back mutations.

Hab035.VH1:

Hab035.VH2:

Hab035.VL1 (same as Hab035 LCVR):

Hab035.VL2:

Hab035.VL3:

2.4. Hot spot mutation of Mab035 humanized antibody

Based on the existence of a high-risk easy-to-modify site NG in HCDR2 in the heavy chain CDR2 region of the humanized antibody of Mab035, the amino acid mutation of NG based on the antibody structure was carried out by computer simulation to eliminate the risk of molecular modification. In one of the preferred embodiments, amino acid mutation is performed on the NG of HCDR2 of Hab035.VH1, and the sequence after mutation of Hab035.VH1 is as follows, wherein the border with characters indicates the hot spot mutation position.

The amino acid sequence of Hab035.VH1a is shown below:

Table 7 The variable region corresponding to the hot spot mutant antibody of Mab035 humanized antibody

	Hab035.VL3Hab035.VL3
Hab035.VH1aHab035.VH1a	Hab035.31aHab035.31a

Note: Hab035.31a indicates that the Mab035 humanized antibody Hab035.31a has a light chain variable region as described in Hab035.VL3 and a heavy chain variable region as described in Hab035.VH1a, and so on.

3. Humanization of Mab055

3.1 Mab055 Architecture Selection

The humanized light chain templates of the murine antibody Mab055 are IGKV4-1*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV2-26*01 and IGHJ1*01, and the CDRs of the murine antibody Mab055 were transplanted into them respectively. In the human template, that is, the humanized antibody Hab055 from which Mab055 is obtained, the variable region sequence thereof is shown below, in which italics indicate FR sequences and underlines indicate CDR sequences.

Hab055 HCVR (also known as VH-CDR graft):

Hab055 LCVR (also known as VL-CDR graft):

3.2. Back mutation design of humanized antibody of Mab055

As required, the key amino acids in the FR region sequence of the humanized antibody of Mab 055 were back-mutated to the amino acids corresponding to the mouse-derived antibody to ensure the original affinity. The specific back-mutation design is shown in Table 8.

Table 8 Humanized antibody back mutation design of Mab 055

Note: Grafted represents the insertion of murine antibody CDRs into the human germline FR region sequence; V3Q represents the mutation of the 3rd V of Grafted back to Q, and so on.

3.3. Mab055 humanized antibody:

The reverse mutation design of the humanized antibody of Mab055 in the above table 8 was combined, and finally a variety of humanized antibodies of Mab055 were obtained. The amino acid sequence of the variable region of each antibody is as follows, italics indicates the framework region, underline indicates CDR, bold Indicates a back mutation.

Table 9 Mab055 Humanized Antibody Variable Region Corresponding Amino Acid Sequences

	Hab055.VH1Hab055.VH1	Hab055.VH2Hab055.VH2	Hab055.VH3Hab055.VH3
Hab055.VL1Hab055.VL1	Hab055.11Hab055.11	Hab055.12Hab055.12	Hab055.13Hab055.13
Hab055.VL2Hab055.VL2	Hab055.21Hab055.21	Hab055.22Hab055.22	Hab055.23Hab055.23

Note: Hab055.12 indicates that the Mab055 humanized antibody Hab055.12 has a light chain variable region as described in Hab055.VL1 and a heavy chain variable region as described in Hab055.VH2, and so on.

Hab055.VH1 (same as Hab055 HCVR):

Hab055.VH2:

Hab055.VH3:

Hab055.VL1 (same as Hab055 LCVR):

Hab055.VL2:

4. Humanization of Mab058

4.1, Mab058 architecture selection

The humanized light chain templates of the murine antibody Mab058 are IGKV1-33*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV7-4-1*04 and IGHJ6*01. The CDRs of the murine antibody Mab058 were transplanted respectively To its humanized template, the humanized antibody Hab058 of Mab058 is obtained, and its variable region sequence is as follows, italics indicate FR sequences, and underlines indicate CDR sequences.

Hab058 HCVR (also known as VH-CDR graft):

Hab058 LCVR (also known as VL-CDR graft):

4.2. Back mutation design of humanized antibody of Mab058:

As needed, the key amino acids in the FR region sequence of the humanized antibody of Mab058 were back-mutated to the amino acids corresponding to the murine antibody to ensure the original affinity. The specific back-mutation design is shown in Table 10.

Table 10 Humanized antibody back mutation design of Mab058

Note: Grafted represents the insertion of murine antibody CDRs into the human germline FR region sequence; A43G represents the mutation of A back to G at position 43 of Grafted, and so on.

4.3. Mab058 humanized antibody:

The Mab058 humanized antibody back mutation design in Table 10 was combined to finally obtain a variety of Mab058 humanized antibodies (see Table 11 for details).

Table 11 The corresponding amino acid sequence of the variable region of Mab058 humanized antibody

	Hab058.VH1Hab058.VH1	Hab058.VH2Hab058.VH2
Hab058.VL1Hab058.VL1	Hab058.11Hab058.11	Hab058.12Hab058.12
Hab058.VL2Hab058.VL2	Hab058.21Hab058.21	Hab058.22Hab058.22

Note: Hab058.12 indicates that the Mab058 humanized antibody Hab058.12 has a light chain variable region as described in Hab058.VL1 and a heavy chain variable region as described in Hab058.VH2, and so on.

The specific sequences of Hab058.VH1, Hab058.VH2, Hab058.VL1 and Hab058.VL2 are shown below, wherein italics indicate framework regions, underlines indicate CDRs, and bold indicates back mutations.

Hab058.VH1:

Hab058.VH2:

Hab058.VL1:

Hab058.VL2:

4.4. Hot spot mutation of Mab058 humanized antibody

Based on the existence of a high-risk easy-to-modify site DD in HCDR2 in the heavy chain CDR2 region of the Mab058 humanized antibody, we used computer simulation to perform amino acid mutations on DD based on the antibody structure to eliminate the risk of molecular modification. In one of the preferred embodiments, we carried out amino acid mutation on the DD of HCDR2 of Hab058.VH1. The sequence after mutation of Hab058.VH1 is as follows, in which the border with characters represents the hot spot mutation.

The amino acid sequence of Hab058.VH1a is shown below:

Table 12. Variable regions corresponding to hot spot mutant antibodies of Mab058 humanized antibody

	Hab058.VL2Hab058.VL2
Hab058.VH1aHab058.VH1a	Hab058.21aHab058.21a

Note: Hab058.21a indicates that the Mab058 humanized antibody Hab058.21a has a light chain variable region as described in Hab058.VL2 and a heavy chain variable region as described in Hab058.VH1a, and so on.

5. Humanization of Mab077

5.1, Mab077 architecture selection

The humanized light chain templates of the murine antibody Mab077 are IGKV4-1*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV2-70*04 and IGHJ6*01, and the CDRs of the murine antibody Mab077 were transplanted into them respectively. In the human template, namely the humanized antibody Hab077 obtained from Mab077, the sequence of its variable region is as follows, the italics indicate the FR sequence, and the underline indicates the CDR sequence.

Hab077 HCVR (also known as VH-CDR graft):

Hab077 LCVR (also known as VL-CDR graft):

5.2. Back mutation design of humanized antibody of Mab077:

As needed, the key amino acids in the FR region sequence of the humanized antibody of Mab077 were back-mutated to the amino acids corresponding to the mouse-derived antibody to ensure the original affinity. The specific back-mutation design is shown in Table 13.

Table 13 Humanized antibody back mutation design of Mab 077

Note: Grafted represents the insertion of mouse antibody CDRs into the human germline FR region sequence; M4L represents the mutation of the 4th M of Grafted back to L, and so on.

5.3. Mab077 humanized antibody:

The reverse mutation design of the humanized antibody of Mab077 in Table 13 above was combined to finally obtain a variety of humanized antibodies of Mab077, see Table 14 for details.

Table 14 The corresponding amino acid sequence of the variable region of Mab077 humanized antibody

	Hab077.VH1Hab077.VH1	Hab077.VH2Hab077.VH2
Hab077.VL1Hab077.VL1	Hab077.11Hab077.11	Hab077.12Hab077.12
Hab077.VL2Hab077.VL2	Hab077.21Hab077.21	Hab077.22Hab077.22

Note: Hab077.12 indicates that the Mab077 humanized antibody Hab077.12 has a light chain variable region as described in Hab077.VL1 and a heavy chain variable region as described in Hab077.VH2, and so on.

The amino acid sequences of Hab077.VH1, Hab077.VH2, Hab077.VL1, and Hab077.VL2 are shown below. Italics indicate FR sequences, underlines indicate CDR sequences, and bold indicates back mutations.

Hab077.VH1 (also known as VH-CDR graft):

Hab077.VH2:

Hab077.VL1 (also known as VL-CDR graft):

Hab077.VL2:

6. Humanization of Mab095

6.1, Mab095 architecture selection

The humanized light chain templates of the murine antibody Mab095 are IGKV4-1*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV3-7*01 and IGHJ1*01, and the CDRs of the murine antibody Mab095 were transplanted into them respectively. In the human template, namely the humanized antibody Hab095 obtained from Mab095, the sequence of its variable region is as follows, the italics indicate the FR sequence, and the underline indicates the CDR sequence.

Hab095 HCVR (also known as VH-CDR graft):

Hab095 LCVR (also known as VL-CDR graft):

6.2. Back mutation design of the humanized antibody of Mab095:

As required, the key amino acids in the FR region sequence of the humanized antibody of Mab 095 were back-mutated to the amino acids corresponding to the mouse-derived antibody to ensure the original affinity. The specific back-mutation design is shown in Table 15,

Table 15 Humanized antibody back mutation design of Mab 095

Note: Grafted represents the insertion of mouse antibody CDRs into the human germline FR region sequence; G44R represents the mutation of the 44th G of Grafted back to R, and so on.

6.3. Mab095 humanized antibody:

The reverse mutation design of the humanized antibody of Mab095 in the above Table 15 was combined, and finally a variety of Mab095 humanized antibodies were obtained. The amino acid sequence of the variable region of each antibody is as follows, italics indicate FR sequences, underlines indicate CDR sequences, and bold indicates back mutation.

Table 16 The corresponding amino acid sequence of the variable region of Mab095 humanized antibody

	Hab095.VH1Hab095.VH1	Hab095.VH2Hab095.VH2
Hab095.VL1Hab095.VL1	Hab095.11Hab095.11	Hab095.12Hab095.12
Hab095.VL2Hab095.VL2	Hab095.21Hab095.21	Hab095.22Hab095.22

Note: Hab095.21 indicates that the Mab095 humanized antibody Hab095.21 has a light chain variable region as described in Hab095.VL2 and a heavy chain variable region as described in Hab095.VH1, and so on.

Hab095.VH1:

Hab095.VH2:

Hab095.VL1 (same as Hab095 LCVR):

Hab095.VL2:

2. Construction and expression of anti-CD70 humanized full-length antibody

Design primers PCR to build each humanized antibody VH/VK gene fragment, and then carry out homologous recombination with the expression vector pTT5 (with signal peptide and constant region gene (CH1-FC/CL) fragment) to construct the full-length antibody expression vector VH- CH1-FC-pHr/VK-CL-pHr, wherein the positive control 41D12 heavy chain and light chain variable region sequences were obtained from patent WO2012123586A1.

Wherein, the heavy chain constant region amino acid sequence is as follows:

The amino acid sequence of the light chain constant region is shown below:

The amino acid sequence of the heavy chain of the Hab003.22a antibody is shown below:

The amino acid sequence of the light chain of the Hab003.22a antibody is shown below:

The amino acid sequence of the heavy chain of the Hab035.31a antibody is shown below:

The amino acid sequence of the light chain of the Hab035.31a antibody is shown below:

The amino acid sequence of the heavy chain of the Hab055.13 antibody is shown below:

The amino acid sequence of the light chain of the Hab055.13 antibody is shown below:

The amino acid sequence of the heavy chain of the Hab058.21a antibody is shown below:

The amino acid sequence of the light chain of the Hab058.21a antibody is shown below:

The amino acid sequence of the heavy chain of the Hab077.12 antibody is shown below:

The amino acid sequence of the light chain of the Hab077.12 antibody is shown below:

The amino acid sequence of the heavy chain of the Hab095.21 antibody is shown below:

The amino acid sequence of the light chain of the Hab095.21 antibody is shown below:

The amino acid sequence of the variable region of the heavy chain of the 41D12 antibody is shown below:

The amino acid sequence of the heavy chain of the 41D12 antibody is shown below:

The amino acid sequence of the light chain variable region of the 41D12 antibody is shown below:

The amino acid sequence of the light chain of the 41D12 antibody is shown below:

Example 5 Detection of CD70 antibody binding activity

1. ELISA experiment of CD70 antibody binding to human CD70 protein

The binding capacity of anti-human CD70 antibody was detected by ELISA experiments of antibody and human CD70 protein. The fusion protein huCD70.ECD-TNC.his was immobilized in a 96-well microtiter plate, and the intensity of the signal after the addition of the antibody was used to judge the binding activity of the antibody and CD70. The specific experimental methods are as follows:

huCD70.ECD-TNC.his was diluted to 2 μg/ml with PBS (HyClone, Cat No. SH30256.01) buffer at pH 7.4 and added to a 96-well microtiter plate (Corning, Cat No. 50 μl/well). 9018) in a refrigerator at 4°C overnight (16-18 hours). Discard the liquid and wash the plate 3 times with PBST buffer (pH7.4PBS containing 0.05%tweeen-20), then add 2%BSA (Sangon Bioengineering, Cat No.A500023-0100) blocking solution diluted with PBS 250μl/ The wells were incubated at 37°C in an incubator (Shanghai Yiheng, Cat No. BPC250) for 2 hours or placed at 4°C overnight (16-18 hours) for blocking. After blocking, discard the blocking solution and wash the plate three times with PBST buffer (pH 7.4 PBS containing 0.05% tweeen-20), then add 50 μl/well of different concentrations diluted with sample diluent (2% BSA-PBS). The test antibody (hybridoma purified antibody or humanized antibody) was incubated at room temperature for 2 hours. After the incubation, the plate was washed three times with PBST, and 50 μl/well of HRP-labeled goat anti-mouse secondary antibody (Jackson Immuno Research, Cat No. 115-035-003) diluted with sample diluent (2% BSA-PBS) or Goat anti-human secondary antibody (Merck, Cat No. AP113P) was incubated at room temperature for 1 hour. After washing the plate 5 times with PBST, add 50 μl/well of TMB chromogenic substrate (KPL, Cat No. 52-00-03), incubate at room temperature for 5-10 min, add 50 μl/well of 1M HCl to stop the reaction, and use a microplate reader (PE, EnSight) read the absorbance at a wavelength of 450 nm, analyze the data with GraphPad Prism 8, and calculate the binding EC50 value of CD70 antibody to human CD70 protein.

The experimental results are shown in Figure 1-2 and Table 17-18. The experimental results show that the murine hybridoma antibodies of the present invention Mab003, Mab035, Mab055, Mab058, Mab077 and Mab095 and their humanized antibodies Hab003.22a, Hab035.31a, Hab055.13, Hab077.12, Hab095.21, Hab058. 21a had good binding effect with human CD70 protein, among which, the EC50 value of humanized antibody binding to human CD70 protein was lower than or basically the same as that of positive control antibody 41D12.

Table 17 ELISA test results of CD70 hybridoma antibody binding to human CD70 protein

受试样品Test sample	与人CD70蛋白结合ELISA实验EC50(μg/mL)Binding to human CD70 protein ELISA experiment EC50 (μg/mL)
Mab003Mab003	0.0058810.005881
Mab035Mab035	0.0054060.005406
Mab055Mab055	0.0018290.001829
Mab058Mab058	0.0082490.008249
Mab077Mab077	0.0055990.005599
Mab095Mab095	0.0069240.006924

Table 18 ELISA test results of CD70 humanized antibody binding to human CD70 protein

受试样品Test sample	与人CD70蛋白结合ELISA实验EC50(μg/mL)Binding to human CD70 protein ELISA experiment EC50 (μg/mL)
Hab003.22aHab003.22a	0.008170.00817
Hab035.31aHab035.31a	0.0062820.006282
Hab055.13Hab055.13	0.0076550.007655
Hab077.12Hab077.12	0.0081990.008199
Hab095.21Hab095.21	0.0075860.007586
41D1241D12	0.010060.01006
受试样品Test sample	与人CD70蛋白结合ELISA实验EC50(μg/mL)Binding to human CD70 protein ELISA experiment EC50 (μg/mL)
Hab058.21aHab058.21a	0.0012530.001253
41D1241D12	0.0022730.002273

2. ELISA experiment of CD70 antibody binding to rhesus monkey CD70 protein

The monkey cross-binding capacity of the anti-human CD70 antibody was tested by ELISA experiments of the antibody with the rhesus CD70 protein. The macaca CD70.ECD-TNC.his fusion protein was directly coated on a 96-well microtiter plate, and the strength of the signal after the addition of the antibody was used to judge the binding activity of the antibody to rhesus monkey CD70. The specific experimental methods are as follows:

macaca CD70.ECD-TNC.his was diluted to a concentration of 2 μg/ml with PBS (HyClone, Cat No. SH30256.01) buffer at pH 7.4 and added to a 96-well microtiter plate (Corning, Cat. No. 9018), placed in a refrigerator at 4°C overnight (16-18 hours). Discard the liquid and wash the plate 3 times with PBST buffer (pH7.4PBS containing 0.05% tweeen-20), then add 2% BSA diluted with PBS (Sangon Bioengineering, Cat No.A500023-0100) blocking solution 250μl/ The wells were incubated at 37°C in an incubator (Shanghai Yiheng, Cat No. BPC250) for 2 hours or placed at 4°C overnight (16-18 hours) for blocking. After blocking, discard the blocking solution and wash the plate three times with PBST buffer (pH 7.4 PBS containing 0.05% tweeen-20), then add 50 μl/well of different concentrations diluted with sample diluent (2% BSA-PBS). The test antibody (hybridoma purified antibody or humanized antibody) was incubated at room temperature for 2 hours. After the incubation, the plate was washed three times with PBST, and 50 μl/well of HRP-labeled goat anti-mouse secondary antibody (Jackson Immuno Research, Cat No. 115-035-003) diluted with sample diluent (2% BSA-PBS) or Goat anti-human secondary antibody (Merck, Cat No. AP113P) was incubated at room temperature for 1 hour. After washing the plate 5 times with PBST, add 50 μl/well of TMB chromogenic substrate (KPL, Cat No. 52-00-03), incubate at room temperature for 5-10 min, add 50 μl/well of 1M HCl to stop the reaction, and use a microplate reader (PE, EnSight) read the absorbance at a wavelength of 450 nm, analyze the data with GraphPad Prism 8, and calculate the binding EC50 value of CD70 antibody to monkey CD70 protein.

The experimental results are shown in Figure 3-4 and Table 19-20. The experimental results show that the CD70 murine hybridoma antibodies Mab003, Mab035, Mab055, Mab058, Mab077 and Mab095 of the present invention and their humanized antibodies Hab003.22a, Hab035.31a, Hab055.13, Hab077.12, Hab095.21, Hab058.21a has a good binding effect with monkey CD70 protein. The humanized antibody showed substantially equivalent or better binding ability to the positive control 41D12.

Table 19 ELISA test results of CD70 hybridoma antibody binding to rhesus monkey CD70 protein

受试样品Test sample	与猴CD70蛋白结合ELISA实验EC50(μg/mL)Binding to monkey CD70 protein ELISA experiment EC50 (μg/mL)
Mab003Mab003	0.076680.07668
Mab035Mab035	0.0071670.007167
Mab055Mab055	0.0016760.001676
Mab058Mab058	0.0069280.006928
Mab077Mab077	0.0096160.009616
Mab095Mab095	0.0050080.005008

Table 20 ELISA test results of CD70 antibody binding to rhesus monkey CD70 protein

受试样品Test sample	与猴CD70蛋白结合ELISA实验EC50(μg/mL)Binding to monkey CD70 protein ELISA experiment EC50 (μg/mL)
Hab003.22aHab003.22a	0.0081980.008198
Hab035.31aHab035.31a	0.003290.00329
Hab055.13Hab055.13	0.001540.00154
Hab077.12Hab077.12	0.0026590.002659
Hab095.21Hab095.21	0.0041180.004118
41D1241D12	0.0033940.003394
受试样品Test sample	与猴CD70蛋白结合ELISA实验EC50(μg/mL)Binding to monkey CD70 protein ELISA experiment EC50 (μg/mL)
Hab058.21aHab058.21a	0.0043180.004318
41D1241D12	0.0023040.002304

3. Binding experiment of CD70 antibody and human CD70 CHO-K1 cells

The binding capacity of the anti-human CD70 antibody was detected by ELISA experiments of the antibody with the human CD70 CHO-K1 cell line that highly expresses human CD70. Human CD70 CHO-K1 cells were directly coated into a 96-well culture plate, and the intensity of the signal after the addition of the antibody was used to judge the binding activity of the antibody to human CD70 CHO-K1 cells. The specific experimental methods are as follows:

huCD70-CHO-K1 cells were adjusted to a density of ⁵ x 105 cells/ml with Ham's F-12K (Kaighn's) Medium complete medium (Gibco, Cat No. 21127030) and added to a 96-well cell culture plate at a volume of 100 μl/well (Corning, Cat No. 3599) overnight (16-18 hours) in a 37°C cell incubator (ESCO). The medium was discarded, and 50 μl/well of immunostaining fixative (Beyotime, Cat No. P0098) was added for 40 min at room temperature. Discard the fixative and wash the plate 3 times with PBST buffer (pH7.4PBS containing 0.05% tweeen-20), then use PBS diluted 5% milk (Sangon Bioengineering, Cat No.A600669-0250) blocking solution 250μl/ Wells were blocked by incubating at 37°C for 2 hours or overnight at 4°C (16-18 hours). After blocking, discard the blocking solution and wash the plate three times with PBST buffer (pH 7.4 PBS containing 0.05% tweeen-20), then add 50 μl/well of different concentrations diluted with sample diluent (2% BSA-PBS). Test antibody (hybridoma-purified antibody or humanized antibody), and incubate at room temperature for 2 hours. After the incubation, the plate was washed three times with PBST, and 50 μl/well of HRP-labeled goat anti-mouse secondary antibody (Jackson Immuno Research, Cat No. 115-035-003) diluted with sample diluent (2% BSA-PBS) or Goat anti-human secondary antibody (Merck, Cat No. AP113P) was incubated for 1 hour at room temperature. After washing the plate with PBST for 5 times, add 50 μl/well of TMB chromogenic substrate (KPL, Cat No. 52-00-03), incubate at room temperature for 5-10 min, add 50 μl/well of 1M HCl to stop the reaction, and use a microplate reader (PE, EnSight) read the absorbance at a wavelength of 450 nm, analyze the data with GraphPad Prism 8, and calculate the binding EC50 value of CD70 antibody to human CD70.

The experimental results are shown in Figure 5 and Table 21. The experimental results show that the CD70 antibodies Hab003.22a, Hab035.31a, Hab055.13, Hab058.21a, Hab077.12 and Hab095.21 of the present invention all have a good binding effect with human CD70 CHO-K1 cells, which is better than positive The control 41D12 was basically equivalent to the positive control.

Table 21 Results of the binding experiment between CD70 antibody and human CD70 CHO-K1 cells

受试样品Test sample	与human CD70 CHO-K1细胞结合ELISA实验EC50(μg/mL)Binding to human CD70 CHO-K1 cells ELISA experiment EC50 (μg/mL)
Hab003.22aHab003.22a	0.0052520.005252
Hab035.31aHab035.31a	0.0069430.006943
Hab055.13Hab055.13	0.0078290.007829
Hab058.21aHab058.21a	0.016030.01603
Hab077.12Hab077.12	0.012470.01247
Hab095.21Hab095.21	0.0082350.008235
41D1241D12	0.0082670.008267

4. Binding experiment of CD70 antibody and 786-O cells

The binding capacity of the anti-human CD70 antibody was tested by FACS experiments of the antibody with the CD70-expressing tumor cell line 786-O (ATCC, CRL-1932). The 786-O cells were collected into a 96-well cell plate, and the intensity of the signal after the addition of the antibody was used to judge the binding activity of the antibody to human 786-O. The specific experimental methods are as follows:

786-O cells were collected, adjusted to a cell density of 5×10 ⁵ cells/ml, added to a 96-well culture plate (Corning, Cat No. 3799) at a volume of 100 μl/well, centrifuged at 4° C., 1500 rpm, and 5 min. Discard the supernatant, add 250 μl/well PBS (HyClone, Cat No. SH30256.01), and centrifuge at 4° C., 1500 rpm, 5 min. After discarding the supernatant, add 100 μl/well of different concentrations of the antibody to be tested (hybridoma purified antibody or humanized antibody) diluted with sample diluent (2% BSA-PBS), and incubate at 4°C for 1 hour. After the end, centrifuge at 4°C, 1500rpm, 5min. The supernatant was discarded, 250 μl/well of PBS (HyClone, Cat No. SH30256.01) was added, and centrifugation (Thermo) at 4° C., 1500 rpm, 5 min. After discarding the supernatant, add 100 μl/well of Alexa Fluor-647-labeled goat anti-mouse secondary antibody (Jackson Immuno Research, Cat No. 115-605-003) or sheep Anti-human secondary antibody (Jackson Immuno Research, Cat No. 109-605-088) was incubated at 4°C for 1 hour. After incubation, the cells were washed 3 times by centrifugation (Thermo) at 4°C, 1500 rpm, 5 min. Finally, the supernatant was discarded, and 80 μl/well of PBS was added to resuspend the cells. Flow cytometry (BD, Canto II) was used to detect the intensity of the fluorescent signal, and FlowJo and GraphPad Prism 8 were used to analyze the data to calculate the effect of CD70 antibody on human 786-O cells. Combined EC50 values.

The experimental results are shown in Figures 6-7 and Table 22. The experimental results show that the CD70 antibodies Hab003.22a, Hab035.31a, Hab055.13, Hab077.12, Hab095.21 and Hab058.21a of the present invention all have a good binding effect with the tumor cell line 786-O, and the Emax values are all higher than the positive control antibody 41D12.

Table 22 The experimental results of CD70 antibody binding to 786-O cells

5. Binding experiment of CD70 antibody and A549 cells

The binding capacity of the anti-human CD70 antibody was tested by FACS experiments of the antibody with the tumor cell line A549 (ATCC, CCL-185), which expresses low human CD70. The A549 cells were collected into a 96-well cell plate, and the intensity of the signal after the addition of the antibody was used to judge the binding activity of the antibody to human A549. The specific experimental methods are as follows:

A549 cells were collected, adjusted to a cell density of 5×10 ⁵ cells/ml, added to a 96-well culture plate (Corning, Cat No. 3799) at a volume of 100 μl/well, centrifuged at 4° C., 1500 rpm, and 5 min. Discard the supernatant, add 250 μl/well PBS (HyClone, Cat No. SH30256.01), and centrifuge at 4° C., 1500 rpm, 5 min. After discarding the supernatant, add 100 μl/well of different concentrations of the antibody to be tested (humanized antibody) diluted with sample diluent (2% BSA-PBS), and incubate in a refrigerator at 4°C for 1 hour. , 1500rpm, 5min centrifugation. Discard the supernatant, add 250 μl/well PBS (HyClone, Cat No. SH30256.01), and centrifuge at 4° C., 1500 rpm, 5 min. After discarding the supernatant, add 100 μl/well of Alexa Fluor-647-labeled goat anti-human secondary antibody (Jackson Immuno Research, Cat No. 109-605-088) diluted with sample diluent (2% BSA-PBS), Incubate at 4°C for 1 hour. After incubation, the cells were washed 3 times by centrifugation (Thermo) at 4°C, 1500 rpm, 5 min. Finally, the supernatant was discarded, and 80 μl/well of PBS was added to resuspend the cells. The fluorescence signal intensity was detected by flow cytometer (BD, Canto II), and the data were analyzed by FlowJo and GraphPad Prism 8, and the binding EC50 of CD70 antibody to human A549 cells was calculated. value.

The experimental results are shown in Figures 8-9 and Table 23. The experimental results show that the binding Emax of the CD70 antibodies Hab003.22a, Hab035.31a, Hab055.13, Hab077.12, Hab095.21 and Hab058.21a of the present invention to the tumor cell line A549 is better than that of the control antibody 41D12.

Table 23 Experimental results of binding of CD70 antibody to A549 cells

6. Binding experiment of CD70 antibody and Raji cells

The binding capacity of the anti-human CD70 antibody was detected by FACS experiments between the antibody and human Raji cells expressing CD70 (ATCC, CCL-86). Raji cells were collected into 96-well cell plates, and the intensity of the signal after the addition of the antibody was used to judge the binding activity of the antibody to human Raji. The specific experimental methods are as follows:

Raji cells were collected, adjusted to a cell density of 5×10 ⁵ cells/ml, added to a 96-well culture plate (Corning, Cat No. 3799) at a volume of 100 μl/well, centrifuged at 4° C., 1500 rpm, and 5 min. Discard the supernatant, add 50 μl/well of 4% FBS-PBS buffer, and incubate at 4°C for 1 hour. After the incubation, centrifuge at 4°C, 1500 rpm, and 5 min. After discarding the supernatant, add 100 μl/well of different concentrations of the antibody to be tested diluted with sample diluent (2% FBS-PBS), and incubate at 4°C for 1 hour. After the incubation, centrifuge at 4°C, 1500rpm, 5min. The supernatant was discarded, 250 μl/well of PBS (HyClone, Cat No. SH30256.01) was added, and centrifugation (Thermo) at 4° C., 1500 rpm, 5 min. After discarding the supernatant, add 100 μl/well of Alexa Fluor-647-labeled goat anti-human secondary antibody (Jackson Immuno Research, Cat No. 109-605-088) diluted with sample diluent (2% FBS-PBS), Incubate at 4°C for 1 hour. After incubation, the cells were washed 3 times by centrifugation (Thermo) at 4°C, 1500 rpm, 5 min. Finally, the supernatant was discarded, and 80 μl/well of PBS was added to resuspend the cells. Flow cytometry (BD, Canto II) was used to detect the intensity of the fluorescent signal, and FlowJo and GraphPad Prism 8 were used to analyze the data to calculate the binding EC50 of CD70 antibody to human Raji cells. value. The specific results are shown in Figures 10A-10B and Table 24. According to the results shown in Figure 10, the humanized antibodies Hab003.22a, Hab035.31a, Hab055.13, Hab077.12, Hab095.21, Hab058.21a of the present invention bind better to human Raji cells than the positive control 41D12 , or basically equivalent to 41D12.

Table 24 Experimental results of binding of CD70 antibody to Raji cells

7. FACS experiment of CD70 antibody binding to LCL8664

The binding capacity of the anti-CD70 antibody to monkey CD70 was detected by FACS experiments of the antibody with the monkey LCL 8664 (ATCC, CRL-1805) tumor cell line expressing monkey CD70. LCL 8664 cells were collected into a 96-well cell plate, and the intensity of the signal after the addition of the antibody was used to judge the binding activity of the antibody to monkey CD70. The specific experimental methods are as follows:

LCL 8664 cells were collected, the cell density was adjusted to 5×10 ⁵ cells/ml, added to a 96-well culture plate (Corning, Cat No. 3799) at a volume of 200 μl/well, centrifuged at 4° C., 1500 rpm, 5 min (Thermo). The supernatant was discarded, 250 μl/well of PBS (HyClone, Cat No. SH30256.01) was added, and centrifugation (Thermo) at 4° C., 1500 rpm, 5 min. After discarding the supernatant, add 100 μl/well of different concentrations of the antibody to be tested (humanized antibody) diluted with sample diluent (2% FBS-PBS), and incubate in a refrigerator at 4°C for 1 hour. , 1500rpm, 5min centrifugation (Thermo). The supernatant was discarded, 250 μl/well of PBS (HyClone, Cat No. SH30256.01) was added, and centrifugation (Thermo) at 4° C., 1500 rpm, 5 min. After discarding the supernatant, add 100 μl/well of Alexa Fluor-647-labeled goat anti-human secondary antibody (Jackson Immuno Research, Cat No. 109-605-088) diluted with sample diluent (2% FBS-PBS), Incubate at 4°C for 1 hour. After incubation, the cells were washed 3 times by centrifugation (Thermo) at 4°C, 1500 rpm, 5 min. Finally, the supernatant was discarded, and 80 μl/well of PBS was added to resuspend the cells. Flow cytometry (BD, Canto II) was used to detect the intensity of the fluorescent signal, and FlowJo and GraphPad Prism 8 were used to analyze the data to calculate the binding EC50 value of CD70 antibody to monkey CD70. . The specific results are shown in Figure 11 and Table 25. The experimental results show that the CD70 antibodies Hab003.22a, Hab035.31a, Hab055.13, Hab077.12 and Hab095.21 of the present invention are well combined with the tumor cell line LCL 8664, and its EC50 value is better than that of the positive control 41D12, or its substantially equivalent.

Table 25 Experimental results of binding of CD70 antibody to LCL 8664 cells

受试样品Test sample	与LCL 8664结合FACS实验EC50(μg/mL)Combined with LCL 8664 FACS experiment EC50 (μg/mL)
Hab003.22aHab003.22a	0.2440.244
Hab035.31aHab035.31a	0.063050.06305
Hab055.13Hab055.13	0.060450.06045
Hab077.12Hab077.12	0.0670.067
Hab095.21Hab095.21	0.13560.1356
41D1241D12	0.15690.1569

8. Biacore determination of CD70 antibody

In this experiment, the Biacore 8K (GE) instrument was used to measure the affinity of the CD70 antibody to be tested with human CD70 (huCD70.ECD-TNC hiss) and monkey CD70 (macaca CD70.ECD-TNC hiss) by multi-cycle kinetics.

The experimental running buffer was 1×HBS-EP+ buffer solution (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20) (Cat.#BR-1006-69, GE), the temperature of the flow-through cell was set to 25°C, and the sample The chamber temperature was set to 16°C. Both were pretreated with running buffer. A protein A biosensor chip (Cat.#29127556, GE) was used to affinity capture a certain amount of the antibody to be tested, and then a certain concentration of human and monkey CD70 antigens flowed on the surface of the chip, and the reaction was detected in real time using a Biacore 8K instrument (GE). signal to obtain binding and dissociation curves. After each cycle of dissociation, the antigen-antibody complex was washed and regenerated with a pH 1.5 glycine-hydrochloric acid regeneration solution (Cat. #BR-1003-54, GE). The binding process was detected by injecting different concentrations of human CD70 and monkey CD70 antigen 240s in the solution at a flow rate of 30 μL/min, starting from 50 nM (see detailed results for the actual concentration tested), diluted 1:1, and a series of concentrations were set Gradient; dissociation time up to 900s, and finally washing with 10mM glycine-hydrochloric acid solution (pH 1.5) at a flow rate of 30μL/min for 30s to complete the regeneration of the chip surface.

The data obtained in the experiment was fitted with the (1:1) Langmuir model by GE Biacore 8K Evaluation version 2.0 software, and the association rate (Ka), dissociation rate (Kd) and affinity value (KD) were obtained, as shown in Table 26- 27 shown. The experimental results show that the CD70 antibodies Hab003.22a, Hab035.31a, Hab055.13, Hab058.21a, Hab077.12 and Hab095.21 of the present invention can bind to human CD70 and monkey CD70 with high affinity, which is better than the positive control 41D12 or substantially equivalent.

Table 26 Reaction affinity of CD70 antibody with huCD70 protein

抗体Antibody	Ka(1/Ms)Ka(1/Ms)	Kd(1/s)Kd(1/s)	KD(M)KD(M)
Hab003.22aHab003.22a	4.66E+054.66E+05	7.62E-057.62E-05	1.64E-101.64E-10
Hab035.11Hab035.11	6.21E+056.21E+05	1.05E-041.05E-04	1.70E-101.70E-10
Hab035.12Hab035.12	5.63E+055.63E+05	9.61E-059.61E-05	1.71E-101.71E-10
Hab035.21Hab035.21	6.56E+056.56E+05	9.44E-059.44E-05	1.44E-101.44E-10
Hab035.22Hab035.22	6.26E+056.26E+05	7.60E-057.60E-05	1.21E-101.21E-10
Hab035.31Hab035.31	6.89E+056.89E+05	8.86E-058.86E-05	1.29E-101.29E-10
Hab035.32Hab035.32	6.11E+056.11E+05	9.48E-059.48E-05	1.55E-101.55E-10
Hab035.31aHab035.31a	6.06E+056.06E+05	9.81E-059.81E-05	1.62E-101.62E-10
Hab055.13Hab055.13	1.57E+061.57E+06	1.21E-041.21E-04	7.69E-117.69E-11
Hab058.21aHab058.21a	6.52E+056.52E+05	8.87E-058.87E-05	1.36E-101.36E-10
Hab077.11Hab077.11	1.09E+061.09E+06	1.12E-041.12E-04	1.03E-101.03E-10
Hab077.12Hab077.12	1.32E+061.32E+06	1.08E-041.08E-04	8.15E-118.15E-11
Hab077.21Hab077.21	1.06E+061.06E+06	1.12E-041.12E-04	1.06E-101.06E-10
Hab077.22Hab077.22	9.41E+059.41E+05	9.49E-059.49E-05	1.01E-101.01E-10
Hab095.11Hab095.11	3.44E+053.44E+05	7.19E-057.19E-05	2.09E-102.09E-10
Hab095.12Hab095.12	4.17E+054.17E+05	7.02E-057.02E-05	1.68E-101.68E-10
Hab095.21Hab095.21	4.61E+054.61E+05	8.83E-058.83E-05	1.91E-101.91E-10
Hab095.22Hab095.22	4.66E+054.66E+05	7.22E-057.22E-05	1.55E-101.55E-10
41D1241D12	4.36E+054.36E+05	7.69E-057.69E-05	1.76E-101.76E-10

Table 27 Reaction affinity of CD70 antibody with monkey CD70 protein

Example 6 Blocking experiment of CD70 antibody

The blocking rate of anti-human CD70 antibody was detected by FACS experiment in which the antibody competed with CD27 for binding to 786-O. The 786-O cells were collected into a 96-well cell plate, the antibody was mixed with CD27 and added to the cell plate, and the intensity of the detected signal was used to judge the ability of the antibody to block the interaction between CD70 and CD27. The CD27 used for competition in this method is huCD27.ECD.hFc or huCD27.ECD.mFc (referred to as huCD27.ECD.hFc/mFc); wherein huCD27.ECD.hFc was purchased from Acro, and the product number is CD7-5254. It was used when synthesizing antibodies; huCD27.ECD.mFc was purchased from Acro, the catalog number is CD7-5257, and it was used when detecting hybridoma purified antibodies. The specific experimental methods are as follows:

Collect 786-O cells, adjust the cell density to 5×10 ⁵ cells/ml, add 100 μl/well to a 96-well culture plate (Corning, Cat No. 3799), 4°C, 1500 rpm, 5 min centrifugation (Thermo) . The supernatant was discarded, 250 μl/well of PBS (HyClone, Cat No. SH30256.01) was added, and centrifugation (Thermo) at 4° C., 1500 rpm, 5 min. After discarding the supernatant, add 100 μl/well of different concentrations of the antibody to be tested (hybridoma purified antibody or humanized antibody) diluted with sample diluent (2% BSA-PBS) and 20 μg/ml huCD27.ECD.hFc/mFc The mixture was placed in a refrigerator at 4°C and incubated for 1 hour. After the incubation, centrifuge (Thermo) at 4°C, 1500 rpm, and 5 min. The supernatant was discarded, 250 μl/well of PBS (HyClone, Cat No. SH30256.01) was added, and centrifugation (Thermo) at 4° C., 1500 rpm, 5 min. After discarding the supernatant, add 100 μl/well APC-labeled mouse anti-human secondary antibody (Biolegend, Cat No. 409306) or goat anti-mouse secondary antibody (Invitrogen, Cat No. 2) diluted with sample diluent (2% BSA-PBS). .31630) and incubate at 4°C for 1 hour. After the incubation, the cells were washed 3 times by centrifugation (Thermo) at 4°C, 1500 rpm, 5 min. Finally, the supernatant was discarded, and 80 μl/well of PBS was added to resuspend the cells. Flow cytometry (BD, Canto II) was used to detect the intensity of the fluorescent signal, and FlowJo and GraphPad Prism 8 were used to analyze the data to calculate the effect of CD70 antibody on the interaction between CD70 and CD27. Blocking IC50 values. The specific results are shown in Figure 12 and Table 28. The experimental results show that the CD70 antibodies Hab003.22a, Hab035.31a, Hab055.13, Hab077.12 and Hab095.21 of the present disclosure can significantly inhibit the binding activity of CD70 and CD27 proteins expressed on the tumor cell line 786-0, IC50 The value was comparable or better than that of the positive control antibody 41D12 (see Table 28), indicating that the antibody of the present invention has a good ability to inhibit the binding of CD70 and CD27, and compared with the positive control 41D12, the effect is better or basically equivalent.

Table 28 Experimental results of blocking 786-O cells by CD70 antibody

受试样品Test sample	786-O细胞酶活抑制实验IC50(μg/ml)786-O cell enzyme activity inhibition experiment IC50 (μg/ml)
Hab003.22aHab003.22a	0.1590.159
Hab035.31aHab035.31a	0.093580.09358
Hab055.13Hab055.13	0.057310.05731
Hab077.12Hab077.12	0.069090.06909
Hab095.21Hab095.21	0.11560.1156
41D1241D12	0.092680.09268

Example 7 CD70 antibody ADCC

The cryopreserved human PBMCs were thawed one day in advance, resuspended in 1640 complete medium containing 10% FBS, 50ng/mL IL-2 (R&D, Cat#202-IL-050), and cultured at 37°C, 5% CO ₂ Incubate overnight. The next day, tumor target cells (786-O/Raji/A549/A498) and PBMCs were resuspended in phenol red-free 1640 medium containing 5% heat-inactivated fetal bovine serum, respectively. The ratio of 200,000 PBMCs (50 μL per well) was added to a flat-bottom 96-well plate, and then 50 μL of CD70 antibody or its control molecule was added to each well. Incubate for 4 hours in a 37°C, 5% _CO2 incubator. After 4 hours, the LDH value released by the cells in each well in the supernatant medium was detected by LDH detection kit (DO JINDO, Cat#CK12), and the percentage of target cell killing was calculated.

The experimental results are shown in Figures 13-16. The results show that NK cells in PBMC can be recruited by antibodies to kill CD70-positive tumor cells A498, 786-O, Raji, and A549. The CD70 fucose knockout antibodies Hab003.22a fut8KO, Hab035.31a fut8KO, Hab055.13fut8KO, Hab077.12fut8KO, Hab095.21fut8KO can produce strong ADCC effect, which is stronger than or equivalent to the positive control 41D12fut8KO.

Example 8 CD70 antibody CDC

The tumor cells expressing CD70 Raji were centrifuged and resuspended in medium containing 10% human serum. 20,000 cells per well (50 μl per well) were added to a round-bottom 96-well plate, and then 50 μl of CD70 antibody or 50 μl of CD70 antibody was added to each well. For the control molecule, the antibody was diluted according to a 4-fold concentration gradient (the initial concentration was 10 μg/ml), and was incubated in a 37° C., 5% CO2 incubator for 2 hours. After 2 hours, add 50 μl of PBS-diluted PI staining solution (containing 0.5 μl of 1 mg/ml PI, Invitrogen, Cat#P3566) to each well of a 96-well plate, incubate for 30 minutes, and detect the proportion of PI-positive cells by flow cytometry, namely CDC kill ratio.

The test results are shown in Figure 17. The results show that the antibody can cause CDC effect in the presence of human serum, thereby producing a killing effect on Raji cells. The CD70 fucose knockout antibodies Hab003.22a fut8KO, Hab035.31a fut8KO, Hab055.13fut8KO, Hab077.12fut8KO and Hab095.21fut8KO of the present invention can all induce strong CDC effects.

Example 9 CD70 antibody ADCP

CD14 ⁺ monocytes were isolated and purified from PBMCs prepared from human whole blood using CD14Microbeads (CD14 Microbeads, Miltenyi Biotec, Cat#130-050-201). 25ng/ml recombinant human M-CSF (R&D, Cat#216-MC-025) was added to the isolated and purified CD14 ⁺ monocytes for macrophage differentiation. On the seventh day of differentiation, macrophages were digested and stained with CellTrace ^™ CFSE Cell Proliferation Kit (invitrogen, Cat#C34554), and CD70-expressing tumor targets were detected with CellTrace ^™ Violet Cell Proliferation Kit (invitrogen, Cat#C34557). Cells were stained with Raji. The ratio of 80,000 tumor target cells and 20,000 macrophages per well (50 μl per well) was added to a low-adsorption flat-bottom 96-well plate (Corning, Cat#3474), and then 50 μl of CD70 antibody or its control molecule were added to each well, respectively. The antibody was diluted in a 5-fold concentration gradient (the initial concentration was 10 μg/ml) and incubated in a 37°C, 5% CO2 incubator for 4 hours. After 4 hours, 50 μl of paraformaldehyde fixative was added to each well of a 96-well plate, and the proportion of Cell Trace Violet-positive tumor cells in CFSE-positive macrophages was detected by flow cytometry.

The experimental results are shown in Figure 18. The results show that the CD70 antibody can recruit macrophages and phagocytose CD70-positive Raji cells. The CD70 antibodies Hab003.22a, Hab035.31a, Hab055.13, Hab077.12 and Hab095.21 of the present invention can all produce ADCP killing effect on Raji.

Example 10 Inhibition of IL-8 secretion by CD70 antibody

Tumor cells expressing CD70 in U266 (ATCC, TIB-196) were centrifuged and resuspended in medium containing 10% fetal bovine serum, and 50,000 cells per well (50 μl per well) were added to a round-bottom 96-well plate, and then 50 μl of CD70 antibody or its control molecule was added to each well, and the antibody was diluted according to a 4-fold concentration gradient (the initial concentration was 1 μg/ml), and incubated in a 37°C, 5% CO2 incubator for 1 hour. After 1 hour, 5000 cells of HT1080 (Kangyuan Bochuang, KC-0144), a stably transfected cell line expressing CD27, were added to each well of a round-bottomed 96-well plate, and incubated in a 37°C, 5% CO ₂ incubator. 24 hours. 24 hours later, the supernatant of the medium in the 96-well plate was collected by centrifugation, and the expression level of IL-8 in the supernatant was detected by ELISA (BD, Cat#555244).

After co-incubation of U266 and HT1080-CD27 cells, the CD70-CD27 signaling pathway was activated, resulting in the production of IL-8. As shown in Figure 19, the results show that the CD70 antibody of the present invention with blocking function can block the CD70-CD27 signaling pathway and inhibit the production of IL-8. The CD70 fucose knockout antibodies Hab003.22a fut8KO, Hab035.31a fut8KO, Hab055.13fut8KO, Hab077.12fut8KO and Hab095.21fut8KO of the present invention can significantly inhibit the inhibition of IL-8.

Example 11 Efficacy of CD70 antibody in Raji xenograft tumor model

Human lymphoma Raji-luciferase-eGFP cells (purchased from Beijing Biositu Gene Biotechnology Co., Ltd., 1×10^6 cells) were inoculated into 5-6-week-old female NOD-SCID (purchased from Beijing Weitong) in 200 μl tail vein. Lihua Laboratory Animal Technology Co., Ltd.) mice. On the 5th day after inoculation, the mice were randomly divided into 3 groups according to their body weight: the isotype control antibody group, the control group 41D12fut8KO 0.1 mg/kg, and the test antibody group Hab035.31a fut8KO 0.1 mg/kg (see Table 29 for grouping and dosage). , 6 in each group. All antibodies were injected intraperitoneally, twice a week, for a total of 2 doses. Beginning on day 7 post-inoculation, mice were monitored for body weight and small animal live imaging was performed twice a week, and data were recorded. In vivo imaging operation: the animals were intraperitoneally injected with a dose of 150 mg/kg of luciferase substrate, after isoflurane anesthesia, in vivo imaging was performed 5 minutes after the substrate injection, and the luciferase exposure time was 30 s. The mean ± standard deviation (Mean ± SEM) of the luminescence signal values in each group was expressed and plotted with GraphPad prism, and two-way ANOVA was used for statistical analysis.

Table 29 Groups and doses

The experimental results are shown in Figure 20. The results show that CD70 antibody can recruit immune cells in mice and kill CD70-positive Raji cells. Compared with the negative control group, the CD70 fucose knockout antibody Hab035.31a fut8KO of the present invention can effectively inhibit the growth of Raji tumor, and there is a significant difference with the negative control group.

Example 12 CD70 antibody in

Pharmacodynamics in OLM-13 Xenograft Tumor Models

The anti-tumor activity of CD70 antibody was studied using the MOLM-13 vein tumor model. Twenty-four female CB17-SCID mice (6-7 weeks old, Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were inoculated into the tail vein of 5 × 10^6 human acute myeloid leukemia MOLM-13 cells (CBP60678, Nanjing Kebai Biotechnology Co., Ltd.) Technology Co., Ltd.), and were randomly divided into 3 groups on the 2nd day after inoculation, with 8 animals in each group: the first group was given an isotype control antibody twice a week by intraperitoneal injection, and the second group was given 10 mg/kg 41D12fut8KO antibody twice a week by intraperitoneal injection , the third group was given 10 mg/kg Hab035.31a fut8KO antibody by intraperitoneal injection twice a week. Mice were allowed to eat and drink freely during the whole experiment.

The state of the mice was observed every day, and the mice were paralyzed, lost more than 20% of their body weight for 3 consecutive days, or when other indicators of euthanasia were met, the mice were euthanized. Mice body weights were measured twice a week. Log-rank (Mantel-Cox) test compared the difference of survival trend of mice in each group, when p<0.05, there was a significant difference.

The experimental results are shown in Table 30 (median survival) and Figure 21 (survival). The results show that CD70 antibody can recruit immune cells in mice, and the CD70-positive

M-13 cells produce killing effect. Compared with the negative control group, the CD70 fucose knockout antibody Hab035.31a fut8KO of the present invention can effectively inhibit the growth of Raji tumor, with a statistically significant difference compared with the isotype control antibody group, while the control antibody 41D12fut8KO group There was no statistical difference in the isotype control antibody group.

Table 30 Median survival time (MST) of MOLM-13 model mice

Claims

An antibody or antigen-binding fragment that specifically binds to CD70, characterized in that the antibody or antigen-binding fragment comprises:

(a) SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 23, 26, 27, 29, 30, 32, 34, 35, 38, 39, 41 , HCDR1, HCDR2 and HCDR3 of the VH of any one of 43, 45 or 46;

and/or, (b) SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 21, 24, 25, 28, 31, 33, 36, 37, 40, 42, 44 or 47 LCDR1, LCDR2 and LCDR3 of any of the indicated VLs;

Preferably, the HCDR1-3 and/or the LCDR1-3 are determined according to the Kabat numbering system, the Chothia numbering system or the IMGT numbering system, more preferably, the HCDR1-3 and/or the LCDR1-3 are selected from the table 1.
The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment comprises: (1) HCDR1-3 of VH shown in SEQ ID NO: 3, 15, 17 or 19, and SEQ ID NO: 3, 15, 17 or 19 ID NO: LCDR1~3 of VL indicated by 4, 16 or 18;

Or, (2) HCDR1-3 of VH shown in SEQ ID NO: 5, 20, 22, 23 or 26, and LCDR1-3 of VL shown in SEQ ID NO: 6, 21, 24, 25;

Or, (3) HCDR1-3 of VH shown in SEQ ID NO: 7, 27, 29 or 30, and LCDR1-3 of VL shown in SEQ ID NO: 8, 28, 31;

Or, (4) HCDR1-3 of VH shown in SEQ ID NO: 9, 32, 34, 35 or 38, and LCDR1-3 of VL shown in SEQ ID NO: 10, 33, 36 or 37;

Or, (5) HCDR1-3 of VH shown in SEQ ID NO: 11, 39 or 41, and LCDR1-3 of VL shown in SEQ ID NO: 12, 40 or 42;

Or, (6) HCDR1-3 of VH shown in SEQ ID NO: 13, 43, 45 or 46, and LCDR1-3 of VL shown in SEQ ID NO: 14, 44 or 47.
The antibody or antigen-binding fragment according to any one of claims 1 to 2, wherein the HCDRs 1 to 3 of the VH shown in SEQ ID NO: 3 are numbered according to the Kabat, Chothia or IMGT numbering system, and have as SEQ ID NO: 66 ~68, the sequence shown in SEQ ID NO: 69 ~ 71 or SEQ ID NO: 72 ~ 74;

HCDRs 1-3 of the VH shown in SEQ ID NO: 15 have the sequence shown in SEQ ID NO: 75-77, SEQ ID NO: 78-80 or SEQ ID NO: 81-83 according to the Kabat, Chothia or IMGT numbering system ;

HCDRs 1-3 of the VH shown in SEQ ID NO: 17 have the sequence shown in SEQ ID NO: 84-86, SEQ ID NO: 87-89 or SEQ ID NO: 90-92 according to the Kabat, Chothia or IMGT numbering system ;

HCDRs 1-3 of the VH shown in SEQ ID NO: 19 have the sequence shown in SEQ ID NO: 93-95, SEQ ID NO: 96-98 or SEQ ID NO: 99-101 according to the Kabat, Chothia or IMGT numbering system ;

HCDRs 1-3 of the VHs shown in SEQ ID NO: 5, 20, 22 or 23 are according to the Kabat, Chothia or IMGT numbering system, with eg SEQ ID NO: 112-114, SEQ ID NO: 115-117 or SEQ ID NO: 118 The sequence shown in ~120;

HCDRs 1-3 of the VH shown in SEQ ID NO: 26 have the sequence shown in SEQ ID NO: 121-123, SEQ ID NO: 124-126 or SEQ ID NO: 127-129 according to the Kabat, Chothia or IMGT numbering system ;

HCDRs 1-3 of the VH shown in SEQ ID NO: 7 have the sequence shown in SEQ ID NO: 140-142, SEQ ID NO: 143-145 or SEQ ID NO: 146-148 according to the Kabat, Chothia or IMGT numbering system ;

HCDRs 1-3 of the VH shown in SEQ ID NO: 27 have the sequence shown in SEQ ID NO: 149-151, SEQ ID NO: 152-154 or SEQ ID NO: 155-157 according to the Kabat, Chothia or IMGT numbering system ;

The HCDRs 1-3 of the VH shown in SEQ ID NO: 29 or 30 are according to the Kabat, Chothia or IMGT numbering system and have as shown in SEQ ID NO: 158-160, SEQ ID NO: 161-163 or SEQ ID NO: 164-166 the sequence of;

HCDRs 1-3 of the VHs shown in SEQ ID NO: 9, 32, 34 or 35 are according to the Kabat, Chothia or IMGT numbering system, with eg SEQ ID NO: 177-179, SEQ ID NO: 180-182 or SEQ ID NO: 183 The sequence shown in ~185;

HCDRs 1-3 of the VH shown in SEQ ID NO: 38 have the sequence shown in SEQ ID NO: 186-188, SEQ ID NO: 189-191 or SEQ ID NO: 192-194 according to the Kabat, Chothia or IMGT numbering system ;

HCDRs 1-3 of the VH shown in SEQ ID NO: 11 have the sequences shown in SEQ ID NO: 205-207, SEQ ID NO: 208-210 or SEQ ID NO: 211-213 according to the Kabat, Chothia or IMGT numbering system ;

HCDRs 1-3 of the VH shown in SEQ ID NO: 39 have the sequence shown in SEQ ID NO: 214-216, SEQ ID NO: 217-219 or SEQ ID NO: 220-223 according to the Kabat, Chothia or IMGT numbering system ;

HCDRs 1-3 of the VH shown in SEQ ID NO: 41 have the sequence shown in SEQ ID NO: 224-226, SEQ ID NO: 227-229 or SEQ ID NO: 230-233 according to the Kabat, Chothia or IMGT numbering system ;

HCDRs 1-3 of the VHs shown in SEQ ID NO: 13, 43, 45 or 46 are according to the Kabat, Chothia or IMGT numbering system, with eg SEQ ID NO: 244-246, SEQ ID NO: 247-249 or SEQ ID NO: 250 The sequence shown in ~252;

LCDRs 1 to 3 of the VL shown in SEQ ID NO: 4, 16 or 18 are according to the Kabat, Chothia or IMGT numbering system, with eg SEQ ID NO: 102 to 104, SEQ ID NO: 105 to 107 or SEQ ID NO: 108 to 111 the sequence shown;

LCDRs 1 to 3 of the VL shown in SEQ ID NO: 6, 21, 24 or 25 are according to the Kabat, Chothia or IMGT numbering system and have as SEQ ID NO: 130 to 132, SEQ ID NO: 133 to 135 or SEQ ID NO: 136 The sequence shown in ~139;

LCDRs 1 to 3 of the VL shown in SEQ ID NO: 8, 28 or 31 are according to the Kabat, Chothia or IMGT numbering system, with eg SEQ ID NO: 167-169, SEQ ID NO: 170-172 or SEQ ID NO: 173-176 the sequence shown;

LCDRs 1 to 3 of the VL shown in SEQ ID NO: 10, 33, 36 or 37 are according to the Kabat, Chothia or IMGT numbering system and have as SEQ ID NO: 195 to 197, SEQ ID NO: 198 to 200 or SEQ ID NO: 201 The sequence shown in ~204;

LCDRs 1 to 3 of the VL shown in SEQ ID NO: 12, 40 or 42 are according to the Kabat, Chothia or IMGT numbering system, with eg SEQ ID NO: 234 to 236, SEQ ID NO: 237 to 239 or SEQ ID NO: 240 to 243 the sequence shown;

LCDRs 1 to 3 of the VL shown in SEQ ID NO: 14, 44 or 47 are according to the Kabat, Chothia or IMGT numbering system, with eg SEQ ID NO: 253-255, SEQ ID NO: 256-258 or SEQ ID NO: 259-262 the sequence shown.
The antibody or antigen-binding fragment according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment is contained in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17 , 19, 20, 22, 23, 26, 27, 29, 30, 32, 34, 35, 38, 39, 41, 43, 45 or 46 of any of the VHs shown in Up to 6 mutated sequences; and/or,

The antibody or antigen-binding fragment is contained in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 21, 24, 25, 28, 31, 33, 36, 37, 40, 42, 44 or a sequence of up to 6 mutations on LCDR1, LCDR2 and/or LCDR3 of the VL shown in any one of 47;

Preferably, the mutation is selected from substitution, deletion or insertion mutation; more preferably, the substitution is a conservative amino acid substitution;

Preferably, the number of mutations may be selected from 1, 2, 3, 4, 5 or 6;

Preferably, the mutation comprises a G55 mutation occurring on HCDR2 of SEQ ID NO: 3, 5, 15, 17, 20, 22 or 23, numbered according to the Kabat numbering system, more preferably, the G55 mutation is a G55A mutation or, according to the Kabat numbering system, the mutation comprises a D61 mutation occurring on HCDR2 of SEQ ID NO: 9, 32, 34 or 35, more preferably, the D61 mutation is a D61Q mutation.
The antibody or antigen-binding fragment according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17 , HCDR1, HCDR2 and/or HCDR3 in the VH of any one of Sequences of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity;

And/or, the antibody or antigen-binding fragment comprises SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 21, 24, 25, 38, 31, 33, 36, 37, 40 , LCDR1, LCDR2 and/or LCDR3 in the VL of any one of 42, 44 or 47 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Sequences of 97%, 98%, 99%, 100% identity.
The antibody or antigen-binding fragment according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment comprises:

(a) a heavy chain variable region comprising said VH CDR1, VH CDR2 and VH CDR3; and/or,

(b) a light chain variable region comprising the VL CDR1, VL CDR2 and VL CDR3.
The antibody or antigen-binding fragment according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment comprises:

(a) SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 23, 26, 27, 29, 30, 32, 34, 35, 38, 39, 41 The sequence shown in any one of , 43, 45 or 46; and/or, (b) SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 21, 24, 25, 28, 31 , 33, 36, 37, 40, 42, 44 or 47 any one of the sequences shown;

Preferably, the antibody or antigen-binding fragment comprises:

(1) the sequence shown in any one of SEQ ID NO: 3, 15, 17 or 19 and the sequence shown in any one of SEQ ID NO: 4, 16 or 18;

(2) the sequence shown in any one of SEQ ID NO:5, 20, 22, 23 or 26 and the sequence shown in any one of SEQ ID NO:6, 21, 24 or 25;

(3) the sequence shown in any one of SEQ ID NO: 7, 27, 29 or 30 and the sequence shown in any one of SEQ ID NO: 8, 28 or 31;

(4) the sequence shown in any one of SEQ ID NO: 9, 32, 34, 35 or 38 and the sequence shown in any one of SEQ ID NO: 10, 33, 36 or 37;

(5) the sequence shown in any one of SEQ ID NO: 11, 39 or 41 and the sequence shown in any one of SEQ ID NO: 12, 40 or 42;

(6) the sequence shown in any one of SEQ ID NO: 13, 43, 45 or 46 and the sequence shown in any one of SEQ ID NO: 14, 44 or 47.
The antibody or antigen-binding fragment of claim 7, wherein the antibody or antigen-binding fragment comprises SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 23, 26, 27, 29, 30, 32, 34, 35, 38, 39, 41, 43, 45 or 46 The framework region of the VH has at least 80%, 85%, 90%, 91% %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical framework region sequences; and/or,

VL shown in any one of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 21, 24, 25, 28, 31, 33, 36, 37, 40, 42, 44, or 47 The framework regions have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical framework region sequences.
The antibody or antigen-binding fragment of claim 7, wherein the antibody or antigen-binding fragment comprises SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, A sequence having up to 15 amino acid mutations compared to the framework regions of the VHs shown in any one of 22, 23, 26, 27, 29, 30, 32, 34, 35, 38, 39, 41, 43, 45 or 46; and/or, comprising any A sequence having up to 15 amino acid mutations compared to the framework regions of the indicated VL;

Preferably, the mutation is selected from substitution, deletion or insertion mutation; more preferably, the substitution is a conservative amino acid substitution;

Preferably, the number of mutations may be selected from 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: compared with the framework region of the VH shown in SEQ ID NO: 15: numbered according to the Kabat numbering system, T28N, F29I, T30E , V37L, M69I or R71A; more preferably, at least T28N, F29I, T30E, V37L, M69I and R71A mutations;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, Q43K, R71V or T73K compared to the framework region of the VH shown in SEQ ID NO: 20 Mutations, more preferably, at least R71V and T73K mutations or at least Q43K, R71V and T73K mutations;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, G26D, I37V or R94K compared to the framework region of the VH shown in SEQ ID NO: 27 Mutations, more preferably, at least G26D and R94K mutations or at least G26D, I37V and R94K mutations;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, V2I, D72E or V75A compared to the framework region of VH shown in SEQ ID NO: 32 Mutations, more preferably, at least the V2I mutation or at least the D72E and V75A mutations;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, F24V or G26D mutation compared to the framework region of the VH shown in SEQ ID NO: 39, More preferably, at least the F24V and G26D mutations;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, G42E, G44R or N73I compared to the framework region of the VH shown in SEQ ID NO: 43 Mutations, more preferably, at least G42E, G44R and N73I mutations, or at least G44R and N73I mutations;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, Y49S, G57E or I58F compared to the framework region of VL shown in SEQ ID NO: 16 Mutations, more preferably, at least the Y49S, G57E and I58F mutations;

Preferably, the antibody or antigen-binding fragment comprises, compared with the framework region of VL shown in SEQ ID NO: 21, at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, Y36L, P44F, L46G , F71Y or V85D mutations, more preferably, at least F71Y and V85D mutations, or at least Y36L, P44F, L46G and F71Y mutations;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, V3Q, Q38R or D60A compared to the framework region of VL shown in SEQ ID NO: 28 Mutations, more preferably, at least V3Q, Q38R and D60A mutations;

Preferably, the antibody or antigen-binding fragment comprises a framework region sequence having at least one or more mutations selected from the group consisting of: numbering according to the Kabat numbering system, A43G compared to the framework region of VL shown in SEQ ID NO: 33 , Y49H, T69R or F71Y mutation, more preferably, at least A43G and Y49H mutation, or at least A43G, Y49H, T69R and F71Y mutation;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, M4L or N22S mutation, compared to the framework region of VL shown in SEQ ID NO: 40, More preferably, at least M4L and N22S mutations;

Preferably, the antibody or antigen-binding fragment comprises at least a mutated framework region sequence selected from the group consisting of: numbering according to the Kabat numbering system, M4L or N22S mutation, compared to the framework region of VL shown in SEQ ID NO: 44, More preferably, at least the M4L and N22S mutations are present.
The antibody or antigen-binding fragment according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment includes or does not include a heavy chain constant region and/or a light chain constant region;

Preferably, the heavy chain constant region comprises a full-length heavy chain constant region or a heavy chain constant region fragment, which may be selected from a CH1, Fc or CH3 domain;

Preferably, the heavy chain constant region and/or light chain constant region is a human heavy chain constant region and/or a human light chain constant region;

Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1 heavy chain constant region, an IgG2 heavy chain constant region, an IgG3 heavy chain constant region or an IgG4 heavy chain constant region;

Preferably, the heavy chain constant region is a human IgG1 heavy chain constant region, a human IgG2 heavy chain constant region, a human IgG3 heavy chain constant region or a human IgG4 heavy chain constant region;

Preferably, the heavy chain constant region has the amino acid sequence shown in SEQ ID NO:48, and the light chain constant region has the amino acid sequence shown in SEQ ID NO:49;

Preferably, the antibody or antigen-binding fragment has a heavy chain as set forth in any one of SEQ ID NOs: 50, 52, 54, 56, 58 or 60, and/or, the antibody or antigen-binding fragment has a heavy chain as set forth in any of SEQ ID NOs: 50, 52, 54, 56, 58 or 60 ID NO: light chain shown in 51, 53, 55, 57, 59 or 61;

More preferably, the antibody or antigenic fragment has a heavy chain as shown in SEQ ID NO:50 and a light chain as shown in SEQ ID NO:51, or has a heavy chain as shown in SEQ ID NO:52 and SEQ ID The light chain set forth in NO:53, or with the heavy chain set forth in SEQ ID NO:54 and the light chain set forth in SEQ ID NO:55, or with the heavy chain set forth in SEQ ID NO:56 and SEQ ID The light chain set forth in NO: 57, or with the heavy chain set forth in SEQ ID NO: 58 and the light chain set forth in SEQ ID NO: 59, or with the heavy chain set forth in SEQ ID NO: 60 and SEQ ID NO: light chain shown in 61;

Preferably, the antibody lacks fucosylation.
The antibody or antigen-binding fragment according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, Multispecific antibodies (eg bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, Fab, Fab ', Fab'-SH, F(ab') 2 , Fd, Fv, scFv, diabody or single domain antibody.
The antibody or antigen-binding fragment according to any one of claims 1 to 11, wherein the antibody or antigen-binding fragment specifically binds to human CD70 and/or monkey CD70; preferably, the antibody or antigen-binding fragment specifically binds to human CD70 and/or monkey CD70 KD for binding to human CD70 and/or monkey CD70 is less than 1.00-8EM, 1.00E-9M, 1.00E-10M, 2.00E-10M, 3.00E-10M, 4.00E-10M, 5.00E-10M, 6.00 E-10M, 7.00E-10M, 8.00E-10M, 9.00E-10M, 1.00E-11 M, 2.00E-11 M, 3.00E-11 M, 4.00E-11 M, 5.00E-11 M, 6.00 E-11 M, 7.00E-11 M, 8.00E-11 M, 9.00E-11 M or 1.00E-12 M.
The antibody or antigen-binding fragment according to any one of claims 1 to 12, wherein the antibody or antigen-binding fragment inhibits and/or blocks the binding of CD70 to its ligand CD27; preferably, the CD70 is human CD70 and/or monkey CD70.
The antibody or antigen-binding fragment of any one of claims 1 to 13, wherein the antibody or antigen-binding fragment exhibits one or more effector functions selected from the group consisting of: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP).
The antibody or antigen-binding fragment according to any one of claims 1 to 14, wherein the antibody or antigen-binding fragment is further coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioactive Isotopes, chemotherapeutic agents or immunomodulatory agents, the tracers are selected from radiographic contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers.
A multispecific antigen-binding molecule, characterized in that the antigen-binding molecule comprises a first antigen-binding moiety, and the first antigen-binding moiety comprises the antibody or antigen-binding fragment of any one of claims 1 to 15; and a second antigen binding moiety that specifically binds to other antigens than CD70, or binds to a different CD70 epitope from the first antigen binding moiety;

Preferably, the other antigen is selected from CD3, CD3ε, CD16, CD16A, CD28, CD20, CD19, CD47 or CD40L;

Preferably, the multispecific antigen binding molecule is bispecific, trispecific or tetraspecific.
A chimeric antigen receptor (CAR), characterized in that the chimeric antigen receptor comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, the extracellular antigen binding domain The antibody or antigen-binding fragment of any one of claims 1 to 15 is included.
An immune effector cell, characterized in that the immune effector cell expresses the CAR of claim 17 or comprises a nucleic acid fragment encoding the CAR of claim 17; preferably, the immune effector cell is selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), monocytes, macrophages, dendritic cells or mast cells;

Preferably, the immune effector cells are autoimmune effector cells or allogeneic immune effector cells;

Preferably, the T cells are selected from cytotoxic T cells, regulatory T cells or helper T cells.
An isolated nucleic acid fragment, characterized in that the nucleic acid fragment encodes the antibody or antigen-binding fragment of any one of claims 1 to 15, the multispecific antigen-binding molecule of claim 16, or the antigen-binding molecule of claim 17. chimeric antigen receptors.
A vector (vector), characterized in that, the vector comprises the nucleic acid fragment of claim 19 .
A host cell, characterized in that the cell comprises the carrier of claim 20; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), insect cells or mammalian cells Animal cells (CHO cell line or 293T cell line); preferably, the cells lack a fucosyltransferase such as FUT8.
A method for preparing the antibody or antigen-binding fragment of any one of claims 1 to 15, and the multispecific antigen-binding molecule of claim 16, wherein the method comprises culturing the cell of claim 21, and isolating an antibody or antigen-binding fragment expressed by the cell, or isolating a multispecific antigen-binding molecule expressed by the cell.
A method for preparing the immune effector cell of claim 18, wherein the method comprises: introducing a nucleic acid fragment comprising the CAR of claim 17 into the immune effector cell, optionally, the method further Including enabling the immune effector cells to express the CAR of claim 17.
A pharmaceutical composition, characterized in that the composition comprises the antibody or antigen-binding fragment of any one of claims 1 to 15, the multispecific antigen-binding molecule of claim 16, the Chimeric antigen receptor, immune effector cell according to claim 18, nucleic acid fragment according to claim 19, vector according to claim 20 or cell according to claim 21; preferably, the composition further comprises A pharmaceutically acceptable carrier, diluent or adjuvant; preferably, the composition comprises the antibody or antigen-binding fragment in an amount that can be administered to a subject in an amount of 0.1 to 50 mpk, preferably 1 to 20 mpk, and more It is preferably 10 to 20 mpk.
The antibody or antigen-binding fragment of any one of claims 1 to 15, the multispecific antigen-binding molecule of claim 16, the chimeric antibody receptor of claim 17, and the immune effector of claim 18 Use of the cell, the nucleic acid fragment according to claim 19, the vector according to claim 20 or the cell according to claim 21 in the preparation of a medicament for the treatment of cancer or tumor, autoimmune disease or viral infection; preferably, the The cancer or tumor can be selected from renal cell carcinoma, melanoma, myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma, T-cell lymphoma , mantle cell lymphoma or cutaneous T-cell lymphoma; preferably, the medicament comprises the antibody or antigen-binding fragment in an amount capable of being administered to a subject of 0.1-50 mpk, preferably 1-20 mpk, more preferably 10-20 mpk.
A method for treating cancer or tumor, autoimmune disease or viral infection, characterized in that the method comprises administering to a subject an effective amount of the antibody or antigen-binding fragment according to any one of claims 1 to 15, The multispecific antigen-binding molecule of claim 16, the chimeric antibody receptor of claim 17, the immune effector cell of claim 18, the nucleic acid fragment of claim 19, the chimeric antibody of claim 20 The vector or the cell of claim 21; preferably, the cancer or tumor can be selected from renal cell carcinoma, melanoma, myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia ( CLL), non-Hodgkin's lymphoma, T-cell lymphoma, mantle cell lymphoma or cutaneous T-cell lymphoma; preferably, the effective amount of the antibody or antigen-binding fragment is 0.1-50 mpk, preferably 1-20 mpk, more It is preferably 10 to 20 mpk.
The antibody or antigen-binding fragment of any one of claims 1 to 15, the multispecific antigen-binding molecule of claim 16, the chimeric antigen receptor of claim 17, the immune effector cell of claim 18, The nucleic acid fragment of claim 19, the nucleic acid vector of claim 20 or the host cell of claim 21, characterized in that, it is used for the treatment of cancer or tumor, autoimmune disease or viral infection; preferably, The cancer or tumor may be selected from renal cell carcinoma, melanoma, myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma, T-cell lymphoma tumor, mantle cell lymphoma or cutaneous T-cell lymphoma; preferably, the effective amount of the antibody or antigen-binding fragment to a subject is 0.1-50 mpk, preferably 1-20 mpk, more preferably 10-20 mpk.