JPWO2021213475A5 - - Google Patents

Description

In a specific embodiment, said anti-CD73/anti-PD-1 bispecific antibody has the structure shown as heavy chain-light chain-linker1-scFv, and said scFv is 14C12H1V-linker2- selected from 14C12L1V, 14C12H1V-linker1-14C12L1V, 14C12H1V-linker2-14C12L1V and 14C12H1V-linker1-14C12L1V, specifically:
(1) NTPDV1, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO:85; the light chain of which has the amino acid sequence set forth in SEQ ID NO:28, and the linker 1, which has the amino acid sequence set forth in SEQ ID NO:79 14C12H1V has the amino acid sequence set forth in SEQ ID NO: 62 , Linker 2 has the amino acid sequence set forth in SEQ ID NO: 81, and 14C12L1V has the amino acid sequence set forth in SEQ ID NO: 64 ;
(2) NTPDV2, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO : 83 , the light chain of which has the amino acid sequence set forth in SEQ ID NO: 28, and the linker 1 of which the amino acid sequence is set forth in SEQ ID NO: 79 14C12H1V has the amino acid sequence set forth in SEQ ID NO: 62 , Linker 1 has the amino acid sequence set forth in SEQ ID NO: 79, and 14C12L1V has the amino acid sequence set forth in SEQ ID NO: 64 ;
(3) NTPDV3, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO:85, the light chain of which has the amino acid sequence set forth in SEQ ID NO:96, and the linker 1 of which the amino acid sequence is set forth in SEQ ID NO:79 14C12H1V has the amino acid sequence set forth in SEQ ID NO: 62 , Linker 2 has the amino acid sequence set forth in SEQ ID NO: 81, and 14C12L1V has the amino acid sequence set forth in SEQ ID NO: 64 ; and (4) NTPDV4, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO : 83 , the light chain of which has the amino acid sequence set forth in SEQ ID NO: 96, and the linker 1 of which the amino acid set in SEQ ID NO: 79 14C12H1V has the amino acid sequence set forth in SEQ ID NO: 62 , Linker 1 has the amino acid sequence set forth in SEQ ID NO: 79, and 14C12L1V has the amino acid sequence set forth in SEQ ID NO: 64 ,
selected from the group consisting of

(1) NTPDV1 has the amino acid sequence shown in SEQ ID NO: 85 in its heavy chain, the amino acid sequence shown in SEQ ID NO: 28 in its light chain, and the amino acid sequence shown in SEQ ID NO: 79 in linker 1. , 14C12H1V has the amino acid sequence set forth in SEQ ID NO: 62 , linker 2 has the amino acid sequence set forth in SEQ ID NO: 81, and 14C12L1V has the amino acid sequence set forth in SEQ ID NO: 64 ;
(2) NTPDV2 has a heavy chain having the amino acid sequence set forth in SEQ ID NO: 83 , a light chain having the amino acid sequence set forth in SEQ ID NO: 28, and a linker 1 having the amino acid sequence set forth in SEQ ID NO: 79. and 14C12H1V has the amino acid sequence set forth in SEQ ID NO: 62 , Linker 1 has the amino acid sequence set forth in SEQ ID NO: 79, and 14C12L1V has the amino acid sequence set forth in SEQ ID NO : 64 ;
(3) NTPDV3 has a heavy chain having an amino acid sequence set forth in SEQ ID NO: 85, a light chain having an amino acid sequence set forth in SEQ ID NO: 96, and a linker 1 having an amino acid sequence set forth in SEQ ID NO: 79. and 14C12H1V has the amino acid sequence set forth in SEQ ID NO: 62 , Linker 2 has the amino acid sequence set forth in SEQ ID NO: 81, and 14C12L1V has the amino acid sequence set forth in SEQ ID NO: 64 ; and (4) NTPDV4 has a heavy chain having an amino acid sequence set forth in SEQ ID NO: 83 , a light chain having an amino acid sequence set forth in SEQ ID NO: 96, a linker 1 having an amino acid sequence set forth in SEQ ID NO: 79, and 14C12H1V has the amino acid sequence set forth in SEQ ID NO: 62 , linker 1 has the amino acid sequence set forth in SEQ ID NO:79, and 14C12L1V has the amino acid sequence set forth in SEQ ID NO: 64 .

Claims (31)

the following:
An anti-CD73/anti-PD-1 bispecific antibody comprising a first protein functional domain that targets PD-1; and a second protein functional domain that targets CD73,
Said first protein functional region comprises HCDR1, HCDR2 and HCDR3 contained in a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 44, wherein preferably said amino acid sequences of HCDR1, HCDR2 and HCDR3 are , SEQ ID NOS: 45-47, respectively, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86% relative to said sequences set forth in SEQ ID NOS: 45-47 , 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or have one or more (preferably 1, 2 or 3) conservative amino acid variations (preferably substitutions, insertions or deletions) compared to said sequences set forth in SEQ ID NOS: 45-47 and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region region having the amino acid sequence set forth in SEQ ID NO:49, wherein preferably said amino acid sequence of LCDR1, LCDR2 and LCDR3 is SEQ ID NO:50 52, respectively, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% relative to said sequences set forth in SEQ ID NOs:50-52 %, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or Is the amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid variations (preferably substitutions, insertions or deletions) compared to said sequence according to numbers 50-52;
or said second protein functional region is HCDR1, HCDR2 and HCDR3 contained in a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 2, wherein preferably said amino acid sequences of HCDR1, HCDR2 and HCDR3 are is an amino acid sequence set forth in SEQ ID NOS: 3-5, respectively, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86% of said sequences set forth in SEQ ID NOS: 3-5 %, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity , or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid variations (preferably substitutions, insertions or deletions) compared to said sequences set forth in SEQ ID NOS: 3-5 and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region having the amino acid sequence set forth in SEQ ID NO:7, wherein preferably said amino acid sequences of LCDR1, LCDR2 and LCDR3 are set forth in SEQ ID NOS:8-10 at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or sequences having 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or sequences set forth in SEQ ID NOs:8-10 an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid variations (preferably substitutions, insertions or deletions) compared to the sequence;
An anti-CD73/anti-PD-1 bispecific antibody. The anti-CD73/anti-PD-1 bispecific antibody of claim 1,
The first protein functional domain comprises
a sequence having the amino acid sequence set forth in SEQ ID NO: 44 or SEQ ID NO: 62, or at least 80%, 81%, 82%, 83%, 84%, 85% of said sequence set forth in SEQ ID NO: 44 or 62, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity sequence or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid variations (preferably substitutions, insertions or deletions) compared to said sequence set forth in SEQ ID NO: 44 or 62 and at least 80%, 81%, 82%, 83%, 84% relative to a sequence having the amino acid sequence set forth in SEQ ID NO: 49 or SEQ ID NO: 64, respectively, or said sequence set forth in SEQ ID NO: 49 or 64, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or have one or more (preferably 1, 2 or 3) conservative amino acid variations (preferably substitutions, insertions or deletions) compared to said sequence set forth in SEQ ID NO: 49 or 64 comprising an amino acid sequence;
and/or
The second protein functional region is a sequence having the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:20 or at least 80%, 81%, 82% of said sequence set forth in SEQ ID NO:2 or SEQ ID NO:20 , 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% % or 99% sequence identity or one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, substitutions, insertion or deletion); %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, A sequence having 97%, 98% or 99% sequence identity or one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably 1, 2 or 3) compared to said sequence set forth in SEQ ID NO: 7 or 22 (preferably substitutions, insertions or deletions);
or
The second protein functional region is a sequence having the amino acid sequence set forth in SEQ ID NO:20 or at least 80%, 81%, 82%, 83%, 84%, 85% relative to said sequence set forth in SEQ ID NO:20 %, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or having one or more (preferably 1, 2 or 3) conservative amino acid variations (preferably substitutions, insertions or deletions) compared to said sequence set forth in SEQ ID NO: 20 and a sequence having the amino acid sequence set forth in SEQ ID NO:24 or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% relative to said sequence set forth in SEQ ID NO:24 %, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or comprising an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid variations (preferably substitutions, insertions or deletions) compared to said sequence set forth in SEQ ID NO: 24 , or
The second protein functional region is a sequence having the amino acid sequence set forth in SEQ ID NO:93 or at least 80%, 81%, 82%, 83%, 84%, 85% relative to said sequence set forth in SEQ ID NO:93 %, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or having one or more (preferably 1, 2 or 3) conservative amino acid variations (preferably substitutions, insertions or deletions) compared to said sequence set forth in SEQ ID NO:93 ;as well as
a sequence having the amino acid sequence set forth in SEQ ID NO:95 or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% relative to said sequence set forth in SEQ ID NO:95, a sequence with 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or a SEQ ID NO: Amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid variations (preferably substitutions, insertions or deletions) compared to said sequence according to 95.
An anti-CD73/anti-PD-1 bispecific antibody. 3. The anti-CD73/anti-PD-1 bispecific antibody of claim 1 or 2, wherein the number of said first protein functional domain and said second protein functional domain is independently 1, 2 or more An anti-CD73/anti-PD-1 bispecific antibody, which is the above. The anti-CD73/anti-PD-1 bispecific antibody according to any one of claims 1 to 3, wherein the first protein functional region and the second protein functional region are directly or via a linker preferably said linker is (GGGGS)n and n is a positive integer such as 1, 2, 3, 4, 5 or 6;
Anti-CD73/anti-PD-1 bispecific antibody 5. The anti-CD73/anti-PD-1 bispecific antibody of any one of claims 1-4, wherein said first protein functional domain and said second protein functional domain independently comprise an immunoglobulin or an antigen binding fragment, such as a half antibody, Fab, F(ab')2 or single chain variable region fragment, preferably said first protein functional domain is an immunoglobulin and said second the protein functional domain is an antigen-binding fragment; or the first protein functional domain is an antigen-binding fragment and the second protein functional domain is an immunoglobulin,
An anti-CD73/anti-PD-1 bispecific antibody. 6. The anti-CD73/anti-PD-1 bispecific antibody of any one of claims 1-5, wherein the N-terminus of the heavy chain variable region of said antigen-binding fragment comprises directly (or via a linker) to the C-terminus of CH1, and the N-terminus of the light chain variable region of the antigen-binding fragment is directly (or via a linker) the C-terminus of the immunoglobulin light chain constant region CL; or the N-terminus of the heavy chain variable region of said antigen-binding fragment is linked directly (or via a linker) to the C-terminus of said immunoglobulin light chain variable region CL, and said The N-terminus of the light chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the C-terminus of the heavy chain constant region of the immunoglobulin, or
The C-terminus of the heavy chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the N-terminus of the heavy chain of the immunoglobulin, and the C-terminus of the light chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the N-terminus of the light chain of said immunoglobulin; or the C-terminus of the heavy chain variable region of said antigen-binding fragment is linked to the N-terminus of said immunoglobulin light chain and the C-terminus of the light chain variable region of said antigen-binding fragment is linked directly (or via a linker) to the N-terminus of said immunoglobulin heavy chain;
An anti-CD73/anti-PD-1 bispecific antibody. The anti-CD73/anti-PD-1 bispecific antibody of any one of claims 1-6, wherein said antigen-binding fragment is a single chain variable region fragment; wherein the protein functional region is an immunoglobulin and said second protein functional region is a single chain variable region fragment; or said first protein functional region is a single chain variable region fragment; the second protein functional domain is an immunoglobulin,
An anti-CD73/anti-PD-1 bispecific antibody. 8. The anti-CD73/anti-PD-1 bispecific antibody of claim 7, wherein said single chain variable region fragment comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL). , is a molecule formed by joining via a linker; preferably said single chain variable region fragment has the structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH an anti-CD73/anti-PD-1 bispecific antibody having 9. The anti-CD73/anti-PD-1 bispecific antibody of claim 7 or 8, wherein said single chain variable region fragment is connected via a linker to the C-terminus (CH) of said immunoglobulin heavy chain (or said N-terminus of the heavy chain, C-terminus of CH1 of said heavy chain constant region), the antibody heavy chain variable region (VH) of said single chain variable region fragment is first linked, or said single The antibody light chain variable region (VL) of the chain variable region fragment is first ligated; preferably said single chain variable region fragment has the following structure: linker-VH-linker-VL-COOH or linker- may have VL-linker-VH-COOH,
Preferably,
The immunoglobulin heavy chain variable region comprises CDRs having the amino acid sequences set forth in SEQ ID NOs:3-5, and the immunoglobulin light chain variable region has the CDRs having the amino acid sequences set forth in SEQ ID NOs:8-10. includes;
The heavy chain variable region of said single chain variable region fragment comprises CDRs having the amino acid sequences set forth in SEQ ID NOs:45-47, and the light chain variable region of said single chain variable region fragment is set forth in SEQ ID NOs:50-52. comprising a CDR having an amino acid sequence of
Preferably, the single-chain variable region fragment (such as NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH) is linked via a linker to the C-terminus of the heavy chain of the immunoglobulin. 45-47, the antibody heavy chain variable regions (VH) of said single chain variable region fragments comprising CDRs having the amino acid sequences set forth in SEQ ID NOS: 45-47 may first be ligated, or the sequence The antibody light chain variable regions (VL) of single chain variable region fragments comprising CDRs having the amino acid sequences set forth in numbers 50-52 may be first ligated,
or preferably
The immunoglobulin heavy chain variable region comprises CDRs having the amino acid sequences set forth in SEQ ID NOs:45-47, and the immunoglobulin light chain variable region has the CDRs having the amino acid sequences set forth in SEQ ID NOs:50-52. and/or the heavy chain variable region of said single chain variable region fragment comprises CDRs having the amino acid sequences set forth in SEQ ID NOS:3-5, and the light chain variable region of said single chain variable region fragment comprises the sequence comprising CDRs having the amino acid sequences set forth in numbers 8-10;
Here, the single-chain variable region fragment (NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH, etc.) is linked via a linker to the C-terminus of the heavy chain of the immunoglobulin. In some cases, the antibody heavy chain variable regions (VH) of single chain variable region fragments containing CDRs having the amino acid sequences set forth in SEQ ID NOs: 3-5 may be first ligated, or the amino acids set forth in SEQ ID NOs: 8-10. The antibody light chain variable regions (VL) of single chain variable region fragments containing CDRs having the sequences may first be ligated,
Preferably,
An anti-CD73/anti-PD-1 bispecific antibody wherein one immunoglobulin molecule is bound to two single chain variable region fragment molecules, and more preferably said two single chain variable region fragment molecules are identical. Anti-CD73/anti-PD-1 bispecific antibody according to claim 1 or 2, wherein said immunoglobulin is IgG, IgA, IgD, IgE or IgM, preferably IgG, such as IgG1, IgG2, IgG3 or IgG4. An anti-CD73/anti-PD-1 bispecific antibody. 3. The anti-CD73/anti-PD-1 bispecific antibody of claim 1 or 2, wherein said single chain variable region fragment is attached to the C-terminus of said immunoglobulin heavy chain, preferably one immune An anti-CD73/anti-PD-1 bispecific antibody, wherein the globulin molecule is attached to two single chain variable region fragment molecules, and more preferably said two single chain variable region fragment molecules are identical. 3. The anti-CD73/anti-PD-1 bispecific antibody of claim 1 or 2, wherein the immunoglobulin heavy chain variable region comprises CDRs having the amino acid sequences set forth in SEQ ID NOS: 45-47, and said immunoglobulin light chain variable region comprises CDRs having the amino acid sequences set forth in SEQ ID NOS:50-52;
and/or
The heavy chain variable region of said single chain variable region fragment comprises CDRs having the amino acid sequences set forth in SEQ ID NOs:3-5, and the light chain variable region of said single chain variable region fragment is set forth in SEQ ID NOs:8-10. comprising a CDR having an amino acid sequence of
Preferably, when the single-chain variable region fragment is ligated via a linker to the C-terminus of the heavy chain of the immunoglobulin, the single-chain variable region fragment comprises CDRs having the amino acid sequences set forth in SEQ ID NOS:3-5. may be ligated first, or the antibody light chain variable regions (VL) of single chain variable region fragments comprising CDRs having the amino acid sequences set forth in SEQ ID NOs: 8-10 may be first ligated may be concatenated with
An anti-CD73/anti-PD-1 bispecific antibody. The anti-CD73/anti-PD-1 bispecific antibody of claim 1 or 2,
The immunoglobulin heavy chain variable region comprises CDRs having the amino acid sequences set forth in SEQ ID NOs:3-5, and the immunoglobulin light chain variable region has the CDRs having the amino acid sequences set forth in SEQ ID NOs:8-10. and/or
The heavy chain variable region of said single chain variable region fragment comprises CDRs having the amino acid sequences set forth in SEQ ID NOs:45-47, and the light chain variable region of said single chain variable region fragment is set forth in SEQ ID NOs:50-52. comprising a CDR having an amino acid sequence of
Here, when the single-chain variable region fragment is linked to the C-terminus of the heavy chain of the immunoglobulin via a linker, the single-chain variable region fragment comprising CDRs having the amino acid sequences set forth in SEQ ID NOS: 45-47 may be ligated first, and the antibody light chain variable regions (VL) of single chain variable region fragments comprising CDRs having the amino acid sequences set forth in SEQ ID NOs: 50-52 may be first ligated may be concatenated,
An anti-CD73/anti-PD-1 bispecific antibody. The anti-CD73/anti-PD-1 bispecific antibody of claim 1 or 2,
The immunoglobulin heavy chain variable region has an amino acid sequence selected from SEQ ID NO:44 and SEQ ID NO:62, and the immunoglobulin light chain variable region is selected from SEQ ID NO:49 and SEQ ID NO:64, respectively. having an amino acid sequence;
and/or the heavy chain variable region of said single chain variable region fragment has an amino acid sequence selected from SEQ ID NO: 2 and SEQ ID NO: 20, and the light chain variable region of said single chain variable region fragment has SEQ ID NO: 7 and or the heavy chain variable region of said single chain variable region fragment has the amino acid sequence set forth in SEQ ID NO:20, and the light chain variable region of said single chain variable region fragment has an amino acid sequence each selected from SEQ ID NO:22; has the amino acid sequence set forth in SEQ ID NO: 24; or the heavy chain variable region of said single chain variable region fragment has the amino acid sequence set forth in SEQ ID NO: 93 and the light chain variable region of said single chain variable region fragment has the amino acid sequence set forth in SEQ ID NO:95,
Here, when the single-chain variable region fragment is ligated to the C-terminus of the immunoglobulin heavy chain via a linker, the antibody heavy chain variable region (VH) of the single-chain variable region fragment is ligated first. the antibody light chain variable region (VL) of said single chain variable region fragment may be ligated first,
An anti-CD73/anti-PD-1 bispecific antibody. The anti-CD73/anti-PD-1 bispecific antibody of claim 1 or 2,
The immunoglobulin heavy chain variable region has an amino acid sequence selected from SEQ ID NO:2 and SEQ ID NO:20, and the immunoglobulin light chain variable region has an amino acid sequence selected from SEQ ID NO:7 and SEQ ID NO:22. or the heavy chain variable region of said single chain variable region fragment has the amino acid sequence set forth in SEQ ID NO:20 and the light chain variable region of said single chain variable region fragment has the sequence set forth in SEQ ID NO:24 or the heavy chain variable region of said single chain variable region fragment has the amino acid sequence set forth in SEQ ID NO:93 and the light chain variable region of said single chain variable region fragment has the amino acid sequence set forth in SEQ ID NO:95 has an amino acid sequence of
and/or the heavy chain variable region of said single chain variable region fragment has an amino acid sequence selected from SEQ ID NO:44 and SEQ ID NO:62, and the light chain variable region of said single chain variable region fragment has SEQ ID NO:49 and having an amino acid sequence selected from SEQ ID NO: 64;
Here, when the single chain variable region fragment is ligated to the C-terminus of the heavy chain via a linker, the antibody heavy chain variable region (VH) of the single chain variable region fragment may be ligated first, Alternatively, the antibody light chain variable region (VL) may be ligated first,
An anti-CD73/anti-PD-1 bispecific antibody. 15. The anti-CD73/anti-PD-1 bispecific antibody of any one of claims 5-14, wherein said immunoglobulin comprises a non-CDR region, and said non-CDR region is human derived from a species other than mouse, such as from an antibody; more preferably, said immunoglobulin constant region is humanized; and said light chain constant region is Ig κ chain C region, Accession No. P01834, or said immunoglobulin heavy chain constant region is Ig γ-1 chain C region, Accession No. P1857, according to position 234, 235 and 237 are mutated at any 2 or 3 positions, and after the mutation the bispecific antibody exhibits a greater degree of FcγRIa, FcγRIIIa and/or C1q compared to the bispecific antibody before the mutation having a reduced affinity constant for
More preferably, according to the EU numbering system, said heavy chain constant region has the following mutations at positions 234, 235 and/or 237, based on Igγ-1 chain C region, accession number P01857:
L234A and L235A;
L234A and G237A;
L235A and G237A;
or having L234A, L235A and G237A,
Even more preferably, said immunoglobulin heavy chain constant region also has the following mutations:
N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P 238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A and K320A,
having one or more mutations selected from
Preferably, said anti-CD73/anti-PD-1 bispecific antibody is:
(i) P1D7V01, the immunoglobulin heavy chain having the amino acid sequence set forth in SEQ ID NO: 66, the immunoglobulin light chain having the amino acid sequence set forth in SEQ ID NO: 68, and Linker 1 having the amino acid sequence set forth in SEQ ID NO: 79, the scFv consists of a heavy chain variable region having the amino acids set forth in SEQ ID NO: 20, Linker 1 has the amino acid sequence set forth in SEQ ID NO: 79, and SEQ ID NO: 24. having a light chain variable region with the indicated amino acids;
(ii) P1D7V02R, the immunoglobulin heavy chain having the amino acid sequence set forth in SEQ ID NO: 66, the immunoglobulin light chain having the amino acid sequence set forth in SEQ ID NO: 68, and Linker 1 having the amino acid sequence set forth in SEQ ID NO: 79, the scFv consists of a light chain variable region having the amino acids set forth in SEQ ID NO: 24, Linker 1 has the amino acid sequence set forth in SEQ ID NO: 79, and SEQ ID NO: 20. having a heavy chain variable region with the indicated amino acids;
(iii) P1D7V03, the immunoglobulin heavy chain having the amino acid sequence set forth in SEQ ID NO: 66, the immunoglobulin light chain having the amino acid sequence set forth in SEQ ID NO: 68, and Linker 1 having the amino acid sequence set forth in SEQ ID NO: 68 81, the scFv consists of a heavy chain variable region having the amino acids set forth in SEQ ID NO: 20, Linker 1 has the amino acid sequence set forth in SEQ ID NO: 79, and SEQ ID NO: 24. having a light chain variable region with the indicated amino acids;
(iv) P1D7V04R, the immunoglobulin heavy chain having the amino acid sequence set forth in SEQ ID NO: 66, the immunoglobulin light chain having the amino acid sequence set forth in SEQ ID NO: 68, and Linker 1 having the amino acid sequence set forth in SEQ ID NO: 68 81, the scFv consists of a light chain variable region having the amino acids set forth in SEQ ID NO: 24, Linker 1 has the amino acid sequence set forth in SEQ ID NO: 79, and SEQ ID NO: 20. having a heavy chain variable region with the indicated amino acids;
selected from the group consisting of
Preferably, said anti-CD73/anti-PD-1 bispecific antibody has the structure shown as heavy chain-light chain-linker1-scFv, and said scFv is 14C12H1V-linker2-14C12L1V, 14C12H1V- selected from linker 1-14C12L1V, 14C12H1V-linker 2-14C12L1V and 14C12H1V-linker 1-14C12L1V, specifically:
(1) NTPDV1, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO: 85, the light chain of which has the amino acid sequence set forth in SEQ ID NO: 28, and the linker 1 of which the amino acid sequence is set forth in SEQ ID NO: 79 14C12H1V has the amino acid sequence set forth in SEQ ID NO: 62 , Linker 2 has the amino acid sequence set forth in SEQ ID NO: 81, and 14C12L1V has the amino acid sequence set forth in SEQ ID NO: 64 ;
(2) NTPDV2, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO : 83 , the light chain of which has the amino acid sequence set forth in SEQ ID NO: 28, and the linker 1 of which the amino acid sequence is set forth in SEQ ID NO: 79 14C12H1V has the amino acid sequence set forth in SEQ ID NO: 63 , Linker 1 has the amino acid sequence set forth in SEQ ID NO: 79, and 14C12L1V has the amino acid sequence set forth in SEQ ID NO: 64 ;
(3) NTPDV3, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO:85, the light chain of which has the amino acid sequence set forth in SEQ ID NO:96, and the linker 1 of which the amino acid sequence is set forth in SEQ ID NO:79 14C12H1V has the amino acid sequence set forth in SEQ ID NO: 62 , Linker 2 has the amino acid sequence set forth in SEQ ID NO: 81, and 14C12L1V has the amino acid sequence set forth in SEQ ID NO: 64 ; (4) NTPDV4, whose heavy chain has the amino acid sequence set forth in SEQ ID NO: 83 , whose light chain has the amino acid sequence set forth in SEQ ID NO: 96, and whose linker 1 has the amino acid sequence set forth in SEQ ID NO: 79 14C12H1V has the amino acid sequence set forth in SEQ ID NO: 62 , Linker 1 has the amino acid sequence set forth in SEQ ID NO: 79, and 14C12L1V has the amino acid sequence set forth in SEQ ID NO: 64 .
selected from the group consisting of
An anti-CD73/anti-PD-1 bispecific antibody. 17. The anti-CD73/anti-PD-1 bispecific antibody of any one of claims 1-16, wherein the anti-CD73/anti-PD-1 bispecific antibody is less than about 10 M, such as An anti-CD73/anti-PD-1 bispecific antibody that binds to CD73 protein and/or PD-1 protein with a KD of about 10-6 M, 10-7 M, 10-8 M, 10-9 M, 10-10 M or less. . An isolated nucleic acid molecule comprising a nucleotide sequence capable of encoding a heavy chain variable region of a bispecific antibody,
The heavy chain variable region of said antibody comprises
CDRs having the amino acid sequences of SEQ ID NOS:3-5, CDRs having the amino acid sequences of SEQ ID NOS:45-47, and CDRs having the amino acid sequences of SEQ ID NOS:50-52;
or CDRs having the amino acid sequences of SEQ ID NOs: 45-47, CDRs having the amino acid sequences of SEQ ID NOs: 3-5, and CDRs having the amino acid sequences of SEQ ID NOs: 8-10;
and the heavy chain variable region of said bispecific antibody specifically binds to CD73 and PD-1 antigens as part of said bispecific antibody, and said bispecific antibody further comprises:
CDRs having the amino acid sequences of SEQ ID NOS: 8-10;
or CDRs having the amino acid sequences of SEQ ID NOS:50-52
comprising a light chain variable region comprising
Preferably, the CDRs of said light chain variable region are different from the CDRs of said heavy chain variable region,
An isolated nucleic acid molecule. A vector comprising the isolated nucleic acid molecule of claim 18 . 20. A host cell comprising the isolated nucleic acid molecule of claim 18 or the vector of claim 19. A method for preparing the anti-CD73/anti-PD-1 bispecific antibody of any one of claims 1-17, comprising culturing the host cell of claim 20 under suitable conditions, and isolating said bispecific antibody from said cell culture. 18. A conjugate comprising the anti-CD73/anti-PD-1 bispecific antibody of any one of claims 1-17 and a conjugated moiety, wherein the conjugated moiety comprises a detectable label and specifically the conjugate, wherein the conjugated moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme. A kit comprising an anti-CD73/anti-PD-1 bispecific antibody according to any one of claims 1 to 17 or a conjugate according to claim 22, said kit preferably comprising said bispecific Further comprising a secondary antibody that specifically recognizes the specific antibody, optionally said secondary antibody further comprises a detectable label such as a radioisotope, fluorescent substance, chemiluminescent substance, colored substance or enzyme. Including, kit. Anti-CD73/anti-PD-1 bispecific antibody according to any one of claims 1 to 17 in the preparation of a kit for detecting the presence or level of CD73 and/or PD-1 in a sample use. A pharmaceutical composition comprising the anti-CD73/anti-PD-1 bispecific antibody of any one of claims 1-17 or the conjugate of claim 22, optionally said pharmaceutical composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier and/or excipient. An anti-CD73/anti-PD-1 bispecific antibody according to any one of claims 1 to 17 or a conjugate according to claim 22 in the prevention and/or treatment of tumors or anemia or in the diagnosis of tumors or anemia Use of. Anti-CD73/anti-PD-1 according to any one of claims 1 to 17, in the preparation of a medicament for preventing and/or treating tumors or anemia, or in the preparation of a medicament for diagnosing tumors or anemia Use of a bispecific antibody or a conjugate according to claim 22. The following drugs:
an agent for detecting the level of CD73 in a sample;
an agent for inhibiting the enzymatic activity reaction of CD73;
and or agents for blocking the binding of PD-1 to PDL-1;
agents for down-regulating (eg, down-regulating) the activity or level of PD-1;
agents for alleviating immunosuppression of PD-1 in an organism;
An agent to increase IL-2 expression in T lymphocytes or an agent to increase IFN-γ expression in T lymphocytes.
The use of the anti-CD73/anti-PD-1 bispecific antibody according to any one of claims 1-17 or the conjugate according to claim 22 in the preparation of. A subject in need of or administering to a cell an effective amount of the anti-CD73/anti-PD-1 bispecific antibody of any one of claims 1 to 17 or the conjugate of claim 22 An in vivo or in vitro method comprising administering to the body. the following:
Hybridoma cell line LT014 (also called CD73-19F3) deposited at the China Culture Collection Center (CCTCC) on June 19, 2018 with accession number CCTCCNO: C2018137; or with accession number CCTCCNO: C2015105. , the hybridoma cell line LT003 (also called PD-1-14C12) deposited at the China Culture Collection Center (CCTCC) on June 16, 2015.
A hybridoma cell line selected from An anti-CD73 monoclonal antibody, wherein said antibody is 19F3H2L3 (hG1TM) and has a heavy chain amino acid sequence set forth in SEQ ID NO:30 and a light chain amino acid sequence set forth in SEQ ID NO:28 monoclonal antibody.