Disclosure of Invention
The invention can achieve better therapeutic effect by constructing the bi/tri-specific antibody and simultaneously generating inhibition or killing effects on tumor cells from different directions.
The present invention provides bispecific antibodies comprising CCR8 antigen binding domains. The bi/tri-specific antibody molecules can bind to CCR8 and simultaneously block VEGF or/and PDL1 pathways. Also provided are methods of treating diseases, such as cancer, using the antibodies and antibody conjugates of the invention, pharmaceutical compositions and articles of manufacture thereof.
In a first aspect of the invention there is provided an anti-CCR 8 antibody or antigen binding fragment thereof, said antibody comprising the following three heavy chain variable region CDRs:
HCDR1, having an amino acid sequence as set forth in SEQ ID NO 1,4,7,10,15,18,20,24,28 or 30;
HCDR2 having an amino acid sequence as set forth in SEQ ID NO 2,5,8,11,13,16,21,23,25 or 31, and
HCDR3 having an amino acid sequence as shown in SEQ ID No. 3,6,9,12,14,17,19,22,26,27,29 or 32;
And, three light chain variable region CDRs:
LCDR1, having an amino acid sequence as set forth in SEQ ID NO 33,36,39,50 or 55;
LCDR2 having an amino acid sequence as set forth in SEQ ID NO 34,37,40,44,48,51,53,56 or 58, and
LCDR3, having an amino acid sequence as set forth in SEQ ID NO:35,38,42,45,49,52,54 or 57.
In another preferred embodiment, the anti-CCR 8 antibody or antigen binding fragment thereof comprises three heavy chain variable region CDRs (HCDR), and three light chain variable region CDRs (LCDR), selected from the group consisting of:
In another preferred embodiment, the anti-CCR 8 antibody or antigen binding fragment thereof comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs 59 to 72 and/or a light chain variable region having at least 80% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs 73 to 86.
In another preferred embodiment, the anti-CCR 8 antibody or antigen binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID No. 59 and a light chain variable region as set forth in SEQ ID No. 73.
In another preferred embodiment, the anti-CCR 8 antibody or antigen binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID No. 60 and a light chain variable region as set forth in SEQ ID No. 74.
In another preferred embodiment, the anti-CCR 8 antibody or antigen binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID No. 61 and a light chain variable region as set forth in SEQ ID No. 75.
In another preferred embodiment, the anti-CCR 8 antibody or antigen binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID No. 62 and a light chain variable region as set forth in SEQ ID No. 76.
In another preferred embodiment, the anti-CCR 8 antibody or antigen binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID No. 63 and a light chain variable region as set forth in SEQ ID No. 77.
In another preferred embodiment, the anti-CCR 8 antibody or antigen binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID No. 64 and a light chain variable region as set forth in SEQ ID No. 78.
In another preferred embodiment, the anti-CCR 8 antibody or antigen binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID No. 65 and a light chain variable region as set forth in SEQ ID No. 79.
In another preferred embodiment, the anti-CCR 8 antibody or antigen binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID No. 66 and a light chain variable region as set forth in SEQ ID No. 80.
In another preferred embodiment, the anti-CCR 8 antibody or antigen binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID No. 67 and a light chain variable region as set forth in SEQ ID No. 81.
In another preferred embodiment, the anti-CCR 8 antibody or antigen binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID No. 68 and a light chain variable region as set forth in SEQ ID No. 82.
In another preferred embodiment, the anti-CCR 8 antibody or antigen binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID No. 69 and a light chain variable region as set forth in SEQ ID No. 83.
In another preferred embodiment, the anti-CCR 8 antibody or antigen binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID No. 70 and a light chain variable region as set forth in SEQ ID No. 84.
In another preferred embodiment, the anti-CCR 8 antibody or antigen binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID No. 71 and a light chain variable region as set forth in SEQ ID No. 85.
In another preferred embodiment, the anti-CCR 8 antibody or antigen binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID No. 72 and a light chain variable region as set forth in SEQ ID No. 86.
In another preferred embodiment, the antibody or antigen binding fragment thereof is of human, murine, humanized or chimeric origin.
In another preferred embodiment, the antibody or antigen binding fragment thereof is a human antibody.
In a second aspect of the invention there is provided a multispecific antibody comprising an antibody, or antigen-binding fragment thereof, which is anti-CCR 8 according to the first invention.
In another preferred embodiment, the multispecific antibody comprises:
A first targeting domain comprising one or more CCR8 antigen binding domains;
A second targeting domain that binds VEGF or PD-L1;
Optionally, a third targeting domain is included that binds VEGF or PD-L1;
and, the second targeting domain and the third targeting domain each bind a different protein.
In another preferred embodiment, the targeting domain is in the form of a single domain antibody (sdAb), a fragment variable (Fv) heterodimer, a single chain Fv (scFv), a Fab fragment, triFab, or a combination thereof.
In another preferred embodiment, the CCR8 antigen binding domain comprises an antibody or antigen binding fragment thereof against CCR8 according to the first aspect of the present invention.
In another preferred embodiment, the CCR8 antigen binding domain comprises the following three heavy chain variable region CDRs:
HCDR1, which has an amino acid sequence as set forth in SEQ ID NO. 1;
HCDR2 having an amino acid sequence as set forth in SEQ ID NO. 2, and
HCDR3, which has the amino acid sequence shown in SEQ ID NO. 3;
And, three light chain variable region CDRs:
LCDR1, having the amino acid sequence set forth in SEQ ID NO. 33;
LCDR2 having the amino acid sequence shown in SEQ ID NO 34, and
LCDR3, having the amino acid sequence shown in SEQ ID NO. 35.
In another preferred embodiment, the CCR8 antigen binding domain comprises the following three heavy chain variable region CDRs:
HCDR1, which has the amino acid sequence shown as SEQ ID NO. 18;
HCDR2 having an amino acid sequence as set forth in SEQ ID NO. 5, and
HCDR3, which has the amino acid sequence shown in SEQ ID NO. 19;
And, three light chain variable region CDRs:
LCDR1, having the amino acid sequence set forth in SEQ ID NO. 46;
LCDR2 having the amino acid sequence shown in SEQ ID NO 34, and
LCDR3, having the amino acid sequence set forth in SEQ ID NO: 38.
In another preferred embodiment, the anti-CCR 8 antibody or antigen binding fragment thereof comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID No. 59 and/or a light chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID No. 73.
In another preferred embodiment, the CCR8 antigen binding domain comprises a heavy chain variable region as set forth in SEQ ID NO. 59 and a light chain variable region as set forth in SEQ ID NO. 73.
In another preferred embodiment, the anti-CCR 8 antibody or antigen binding fragment thereof comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID No. 65 and/or a light chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID No. 79.
In another preferred embodiment, the CCR8 antigen binding domain comprises a heavy chain variable region as set forth in SEQ ID NO. 65 and a light chain variable region as set forth in SEQ ID NO. 79.
In another preferred embodiment, the CCR8 antigen binding domain is selected from the group consisting of scFv, fab, or a combination thereof.
In another preferred embodiment, the CCR8 antigen binding domain is an scFv.
In another preferred embodiment, the CCR8 antigen binding domain is a Fab which comprises the heavy chain variable region as shown in SEQ ID NO. 59 and the light chain variable region as shown in SEQ ID NO. 73, or the heavy chain variable region as shown in SEQ ID NO. 65 and the light chain variable region as shown in SEQ ID NO. 76, and
A heavy chain constant region CH1 as shown in SEQ ID NO. 111 or 119, and a light chain constant region CL as shown in SEQ ID NO. 121 or 122, or a heavy chain constant region CH1 as shown in SEQ ID NO. 112, and a light chain constant region CL as shown in SEQ ID NO. 123.
In another preferred embodiment, the multispecific antibody further comprises an Fc fragment.
In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4.
In another preferred embodiment, the Fc fragment is an Fc fragment derived from IgG1, which has the amino acid sequence shown in SEQ ID NO. 113 or 114.
In another preferred embodiment, the Fc fragment comprises a mutation for forming a knob-in-hole structure, and/or a mutation for enhancing ADCC.
In another preferred embodiment, the Fc fragment derived from IgG1 has a mutation selected from the group consisting of:
Y349C/K370E/K409D/K439E,
S354C/D356K/E357K/D399K;
S354C/T366W,
Y349C/T366S/L368A/Y407V。
In another preferred embodiment, the Fc fragment has an amino acid sequence as set forth in any one of SEQ ID NOS.115-118.
In another preferred embodiment, the IgG4 Fc fragment has a mutation selected from the group consisting of:
Y349C/K370E/R409D/K439E,
S354C/E356K/E357K/D399K, or
S354C/T366W,
Y349C/T366S/L368A/Y407V。
In another preferred embodiment, the Fc fragment is an Fc fragment derived from IgG4, which has the amino acid sequence shown in SEQ ID NO. 120.
In another preferred embodiment, the multispecific antibody is a bispecific/trispecific antibody.
In another preferred embodiment, the multispecific antibody is a bispecific antibody.
In another preferred embodiment, the bispecific antibody comprises:
A first targeting domain comprising one or more CCR8 antigen binding domains, and a second targeting domain which is a VEGF antigen binding domain.
In another preferred embodiment, the bispecific antibody comprises:
A first targeting domain comprising one or more CCR8 antigen binding domains, and a second targeting domain which is a PD-L1 antigen binding domain.
In another preferred embodiment, the multispecific antibody is a trispecific antibody.
In another preferred embodiment, the trispecific antibody comprises:
A first targeting domain comprising one or more CCR8 antigen binding domains;
A second targeting domain, the second targeting domain being a VEGF antigen binding domain;
a third targeting domain that is a PD-L1 antigen binding domain.
In another preferred embodiment, the VEGF antigen binding domain comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 87 or 88, and a light chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 93 or 94.
In another preferred embodiment, the VEGF antigen binding domain comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 89, and a light chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 95.
In another preferred embodiment, the VEGF antigen binding domain comprises a mutation capable of reducing the hydrophobicity of an antibody.
In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs in a non-CDR 3 region of an anti-VEGR antigen binding domain having a heavy chain variable region as set forth in SEQ ID NO. 89 and a light chain variable region as set forth in SEQ ID NO. 95.
In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs at an amino acid position of the heavy chain variable region shown as SEQ ID NO. 89 selected from the group consisting of positions 28, 30, 31, 32, 33, 35, or a combination thereof.
In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs at an amino acid position of the light chain variable region shown as SEQ ID NO. 95 selected from the group consisting of positions 24, 49, 50, 51, 52, 53, 56, or a combination thereof.
In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs in the region from position 46 to 57 of the light chain variable region as shown in SEQ ID NO. 95.
In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody is a mutation of the above amino acid site to a hydrophilic amino acid, such as aspartic acid (D), glutamic acid (E), lysine (K) or arginine (R).
In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs at serine (S) at position 30 of the heavy chain variable region as shown in SEQ ID NO:89, preferably serine (S) at position 30 is mutated to aspartic acid (D), glutamic acid (E), lysine (K) or arginine (R).
In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs at serine (S) at position 50 and/or serine (S) at position 52 of the light chain variable region as shown in SEQ ID NO:95, preferably serine (S) at position 50 is mutated to aspartic acid (D), glutamic acid (E), lysine (K) or arginine (R), and/or serine (S) at position 52 is mutated to aspartic acid (D), glutamic acid (E), lysine (K) or arginine (R).
In another preferred embodiment, the VEGF antigen binding domain comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs 90-92, 149-150.
In another preferred embodiment, the VEGF antigen binding domain comprises a light chain variable region having at least 80% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs 96-102.
In another preferred embodiment, the VEGF antigen binding domain is selected from the group consisting of scFv, fab, or a combination thereof.
In another preferred embodiment, the PD-L1 antigen-binding domain comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 103 or 104 and a light chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 107 or 108.
In another preferred embodiment, the PD-L1 antigen-binding domain comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 105 or 106 and a light chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 109 or 110.
In another preferred embodiment, the PD-L1 antigen binding domain is selected from the group consisting of scFv, fab, or a combination thereof.
In another preferred embodiment, the multispecific antibody has a structure according to formula I (e.g., a in fig. 7A):
Fab1-Fc1
║
Fab2-Fc2(I)
Wherein "-" are each independently a peptide bond or a connecting peptide "-" ║ "is a connecting bond between peptide chains;
Fab1 is the first targeting domain, said Fab1 being an anti-CCR 8 Fab;
fab2 is a second targeting domain, said Fab2 being an anti-VEGF Fab or an anti-PD-L1 Fab;
Fc1 and Fc2 are each independently an Fc fragment.
In another preferred embodiment, the Fab2 is an anti-VEGF Fab.
In another preferred embodiment, the anti-CCR 8 Fab comprises the heavy chain variable region as shown in SEQ ID NO. 59 and the light chain variable region as shown in SEQ ID NO. 73, or
The anti-CCR 8 Fab comprises a heavy chain variable region as shown in SEQ ID NO. 65 and a light chain variable region as shown in SEQ ID NO. 79.
In another preferred embodiment, the anti-VEGF Fab comprises a heavy chain variable region as shown in SEQ ID NO. 87 or 88 and a light chain variable region as shown in SEQ ID NO. 93 or 94, or
The anti-VEGF Fab comprises the heavy chain variable region as set forth in any one of SEQ ID NOS: 89-92, 149-150, and the light chain variable region as set forth in any one of SEQ ID NOS: 95-102.
In another preferred embodiment, the anti-PD-L1 Fab comprises the heavy chain variable region as shown in SEQ ID NO. 103 or and the light chain variable region as shown in SEQ ID NO. 107 or 108, or
The anti-PD-L1 Fab comprises a heavy chain variable region as shown in SEQ ID NO. 105 or 106 and a light chain variable region as shown in SEQ ID NO. 109 or 110.
In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4
In another preferred embodiment, the Fc fragment is derived from IgG1.
In another preferred embodiment, the Fc fragment has an amino acid sequence as set forth in any one of SEQ ID NOs 113 to 118.
In another preferred embodiment, the Fc1 has the amino acid sequence shown as SEQ ID NO. 115 and the Fc2 has the amino acid sequence shown as SEQ ID NO. 117.
In another preferred example, the amino acid sequence of "HC1 (heavy chain) -Fc1" in "Fab1-Fc1" is shown as SEQ ID NO. 125, and the amino acid sequence of "LC1 (light chain)" is shown as SEQ ID NO. 124.
In another preferred example, the amino acid sequence of "HC2 (heavy chain) -Fc2" in "Fab2-Fc2" is shown as SEQ ID NO. 126, and the amino acid sequence of "LC2 (light chain)" is shown as SEQ ID NO. 127.
In another preferred embodiment, the multispecific antibody has a structure according to formula II (e.g., b in fig. 7A):
Fab1-Fc1-scFv2
║
Fab1-Fc1-scFv2(II)
Wherein "-" are each independently a peptide bond or a connecting peptide "-" ║ "is a connecting bond between peptide chains;
Fab1 is the first targeting domain, said Fab1 being an anti-CCR 8 Fab;
scFv2 is a second targeting domain, the scFv2 being an anti-VEGF scFv or an anti-PD-L1 scFv;
Fc1 is an Fc fragment.
In another preferred embodiment, the "║" is a disulfide bond.
In another preferred embodiment, the scFv2 is an anti-VEGF scFv.
In another preferred embodiment, the anti-CCR 8 Fab comprises the heavy chain variable region as shown in SEQ ID NO. 59 and the light chain variable region as shown in SEQ ID NO. 73, or
The anti-CCR 8 Fab comprises a heavy chain variable region as shown in SEQ ID NO. 65 and a light chain variable region as shown in SEQ ID NO. 79.
In another preferred embodiment, the anti-VEGF scFv comprises a heavy chain variable region as set forth in SEQ ID NO. 87 or 88 and a light chain variable region as set forth in SEQ ID NO. 93 or 94, or
The anti-VEGF scFv comprises a heavy chain variable region as set forth in any one of SEQ ID NOs 89-92, 149-150, and a light chain variable region as set forth in any one of SEQ ID NOs 95-102.
In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as set forth in SEQ ID NO. 103 or, and a light chain variable region as set forth in SEQ ID NO. 107 or 108, or
The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO. 105 or 106, and a light chain variable region as shown in SEQ ID NO. 109 or 110.
In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4
In another preferred embodiment, the Fc fragment is derived from IgG1.
In another preferred embodiment, the Fc fragment has an amino acid sequence as set forth in any one of SEQ ID NOs 113 to 118.
In another preferred embodiment, the Fc1 has the amino acid sequence shown as SEQ ID NO. 113.
In another preferred embodiment, the amino acid sequence of "HC1 (heavy chain) -Fc1-scFv2" in the "Fab1-Fc1-scFv2" is shown as SEQ ID NO. 128, and the amino acid sequence of "LC1 (light chain)" is shown as SEQ ID NO. 124.
In another preferred embodiment, the multispecific antibody has a structure according to formula III (e.g., c in fig. 7A) as follows:
Fab1-Fc1-scFv2
║
Fab1-Fc2(III)
Wherein "-" are each independently a peptide bond or a connecting peptide "-" ║ "is a connecting bond between peptide chains;
Fab1 is the first targeting domain, said Fab1 being an anti-CCR 8 Fab;
scFv2 is a second targeting domain, the scFv2 being an anti-VEGF scFv or an anti-PD-L1 scFv;
Fc1 and Fc2 are each independently an Fc fragment.
In another preferred embodiment, the scFv2 is an anti-VEGF scFv.
In another preferred embodiment, the anti-CCR 8 Fab comprises the heavy chain variable region as shown in SEQ ID NO. 59 and the light chain variable region as shown in SEQ ID NO. 73, or
The anti-CCR 8 Fab comprises a heavy chain variable region as shown in SEQ ID NO. 65 and a light chain variable region as shown in SEQ ID NO. 79.
In another preferred embodiment, the anti-VEGF scFv comprises a heavy chain variable region as set forth in SEQ ID NO. 87 or 88 and a light chain variable region as set forth in SEQ ID NO. 93 or 94, or
The anti-VEGF scFv comprises a heavy chain variable region as set forth in any one of SEQ ID NOs 89-92, 149-150, and a light chain variable region as set forth in any one of SEQ ID NOs 95-102.
In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as set forth in SEQ ID NO. 103 or, and a light chain variable region as set forth in SEQ ID NO. 107 or 108, or
The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO. 105 or 106, and a light chain variable region as shown in SEQ ID NO. 109 or 110.
In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4
In another preferred embodiment, the Fc fragment is derived from IgG1.
In another preferred embodiment, the Fc fragment has an amino acid sequence as set forth in any one of SEQ ID NOs 113 to 118.
In another preferred embodiment, the Fc1 has the amino acid sequence shown as SEQ ID NO. 115 and the Fc2 has the amino acid sequence shown as SEQ ID NO. 118.
In another preferred embodiment, the amino acid sequence of "HC1 (heavy chain) -Fc1-scFv2" in the "Fab1-Fc1-scFv2" is shown as SEQ ID NO. 128, and the amino acid sequence of "LC1 (light chain)" is shown as SEQ ID NO. 124.
In another preferred example, the amino acid sequence of "HC1 (heavy chain) -Fc2" in "Fab1-Fc2" is shown as SEQ ID NO. 125, and the amino acid sequence of "LC1 (light chain)" is shown as SEQ ID NO. 124.
In another preferred embodiment, the multispecific antibody has a structure according to formula IV (e.g., d in fig. 7A) as follows:
scFv2-Fc1
║
Fab1-Fc2(IV)
Wherein "-" are each independently a peptide bond or a connecting peptide "-" ║ "is a connecting bond between peptide chains;
Fab1 is the first targeting domain, said Fab1 being an anti-CCR 8 Fab;
scFv2 is a second targeting domain, the scFv2 being an anti-VEGF scFv or an anti-PD-L1 scFv;
Fc1 and Fc2 are each independently an Fc fragment.
In another preferred embodiment, the scFv2 is an anti-VEGF scFv.
In another preferred embodiment, the anti-CCR 8 Fab comprises the heavy chain variable region as shown in SEQ ID NO. 59 and the light chain variable region as shown in SEQ ID NO. 73, or
The anti-CCR 8 Fab comprises a heavy chain variable region as shown in SEQ ID NO. 65 and a light chain variable region as shown in SEQ ID NO. 79.
In another preferred embodiment, the anti-VEGF scFv comprises a heavy chain variable region as set forth in SEQ ID NO. 87 or 88 and a light chain variable region as set forth in SEQ ID NO. 93 or 94, or
The anti-VEGF scFv comprises a heavy chain variable region as set forth in any one of SEQ ID NOs 89-92, 149-150, and a light chain variable region as set forth in any one of SEQ ID NOs 95-102.
In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as set forth in SEQ ID NO. 103 or, and a light chain variable region as set forth in SEQ ID NO. 107 or 108, or
The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO. 105 or 106, and a light chain variable region as shown in SEQ ID NO. 109 or 110.
In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4
In another preferred embodiment, the Fc fragment is derived from IgG1.
In another preferred embodiment, the Fc fragment has an amino acid sequence as set forth in any one of SEQ ID NOs 113 to 118.
In another preferred embodiment, the Fc1 has the amino acid sequence shown as SEQ ID NO. 115 and the Fc2 has the amino acid sequence shown as SEQ ID NO. 118.
In another preferred embodiment, the amino acid sequence of said "scFv2-Fc1" is shown as SEQ ID NO. 129.
In another preferred example, the amino acid sequence of "HC1 (heavy chain) -Fc2" in "Fab1-Fc2" is shown as SEQ ID NO. 125, and the amino acid sequence of "LC1 (light chain)" is shown as SEQ ID NO. 124.
In another preferred embodiment, the multispecific antibody has a structure according to formula V (e.g., e in fig. 7A):
scFv1-scFv2-Fc1
║
Fab1-Fc2(V)
Wherein "-" are each independently a peptide bond or a connecting peptide "-" ║ "is a connecting bond between peptide chains;
scFv1 and Fab1 are the first targeting domain, the scFv1 is an anti-CCR 8scFv and the Fab1 is an anti-CCR 8Fab;
scFv2 is a second targeting domain, the scFv2 being an anti-VEGF scFv or an anti-PD-L1 scFv;
Fc1 and Fc2 are each independently an Fc fragment.
In another preferred embodiment, the scFv2 is an anti-VEGF scFv.
In another preferred embodiment, the anti-CCR 8 Fab comprises the heavy chain variable region as shown in SEQ ID NO. 59 and the light chain variable region as shown in SEQ ID NO. 73, or
The anti-CCR 8 Fab comprises a heavy chain variable region as shown in SEQ ID NO. 65 and a light chain variable region as shown in SEQ ID NO. 79.
In another preferred embodiment, the anti-CCR 8 scFv comprises a heavy chain variable region as set forth in SEQ ID NO:59 and a light chain variable region as set forth in SEQ ID NO:73, or
The anti-CCR 8 scFv comprises a heavy chain variable region as shown in SEQ ID NO. 65 and a light chain variable region as shown in SEQ ID NO. 79.
In another preferred embodiment, the anti-VEGF scFv comprises a heavy chain variable region as set forth in SEQ ID NO. 87 or 88 and a light chain variable region as set forth in SEQ ID NO. 93 or 94, or
The anti-VEGF scFv comprises a heavy chain variable region as set forth in any one of SEQ ID NOs 89-92, 149-150, and a light chain variable region as set forth in any one of SEQ ID NOs 95-102.
In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as set forth in SEQ ID NO. 103 or, and a light chain variable region as set forth in SEQ ID NO. 107 or 108, or
The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO. 105 or 106, and a light chain variable region as shown in SEQ ID NO. 109 or 110.
In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4
In another preferred embodiment, the Fc fragment is derived from IgG1.
In another preferred embodiment, the Fc fragment has an amino acid sequence as set forth in any one of SEQ ID NOs 113 to 118.
In another preferred embodiment, the Fc1 has the amino acid sequence shown as SEQ ID NO. 115 and the Fc2 has the amino acid sequence shown as SEQ ID NO. 118.
In another preferred embodiment, the amino acid sequence of said "scFv1-scFv2-Fc1" is shown in SEQ ID NO. 130.
In another preferred example, the amino acid sequence of "HC1 (heavy chain) -Fc2" in "Fab1-Fc2" is shown as SEQ ID NO. 125, and the amino acid sequence of "LC1 (light chain)" is shown as SEQ ID NO. 124.
In another preferred embodiment, the multispecific antibody has a structure according to formula VI below (e.g., f in fig. 7A):
Fab2-Fab1-Fc1
║
Fab1-Fc2(VI)
Wherein "-" are each independently a peptide bond or a connecting peptide "-" ║ "is a connecting bond between peptide chains;
Fab1 is the first targeting domain, said Fab1 being an anti-CCR 8 Fab;
fab2 is a second targeting domain, said Fab2 being an anti-VEGF Fab or an anti-PD-L1 Fab;
Fc1 and Fc2 are each independently an Fc fragment.
In another preferred embodiment, the Fab2 is an anti-VEGF Fab.
In another preferred embodiment, the anti-CCR 8 Fab comprises the heavy chain variable region as shown in SEQ ID NO. 59 and the light chain variable region as shown in SEQ ID NO. 73, or
The anti-CCR 8 Fab comprises a heavy chain variable region as shown in SEQ ID NO. 65 and a light chain variable region as shown in SEQ ID NO. 79.
In another preferred embodiment, the anti-VEGF Fab comprises a heavy chain variable region as shown in SEQ ID NO. 87 or 88 and a light chain variable region as shown in SEQ ID NO. 93 or 94, or
The anti-VEGF Fab comprises the heavy chain variable region as set forth in any one of SEQ ID NOS: 89-92, 149-150, and the light chain variable region as set forth in any one of SEQ ID NOS: 95-102.
In another preferred embodiment, the anti-PD-L1 Fab comprises the heavy chain variable region as shown in SEQ ID NO. 103 or and the light chain variable region as shown in SEQ ID NO. 107 or 108, or
The anti-PD-L1 Fab comprises a heavy chain variable region as shown in SEQ ID NO. 105 or 106 and a light chain variable region as shown in SEQ ID NO. 109 or 110.
In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4
In another preferred embodiment, the Fc fragment is derived from IgG1.
In another preferred embodiment, the Fc fragment has an amino acid sequence as set forth in any one of SEQ ID NOs 113 to 118.
In another preferred embodiment, the Fc1 has the amino acid sequence shown as SEQ ID NO. 116 and the Fc2 has the amino acid sequence shown as SEQ ID NO. 117.
In another preferred example, the amino acid sequence of "HC2 (heavy chain) -HC1 (heavy chain) -Fc1" in the "Fab2-Fab1-Fc1" is shown as SEQ ID NO. 132, the amino acid sequence of "LC1 (light chain)" is shown as SEQ ID NO. 131, and the amino acid sequence of "LC2 (light chain)" is shown as SEQ ID NO. 134.
In another preferred example, the amino acid sequence of "HC1 (heavy chain) -Fc2" in the "Fab1-Fc2" is shown as SEQ ID NO. 133, and the amino acid sequence of "LC1 (light chain)" is shown as SEQ ID NO. 131.
In another preferred embodiment, the multispecific antibody has a structure according to formula VII below (e.g., g in fig. 7A):
Fab2-Fab1-Fc1
║
scFv1-Fc2(VII)
Wherein "-" are each independently a peptide bond or a connecting peptide "-" ║ "is a connecting bond between peptide chains;
Fab1 and scFv1 are the first targeting domain, said Fab1 is an anti-CCR 8Fab, and said scFv1 is an anti-CCR 8scFv;
fab2 is a second targeting domain, said Fab2 being an anti-VEGF Fab or an anti-PD-L1 Fab;
Fc1 and Fc2 are each independently an Fc fragment.
In another preferred embodiment, the Fab2 is an anti-VEGF Fab.
In another preferred embodiment, the anti-CCR 8 Fab comprises the heavy chain variable region as shown in SEQ ID NO. 59 and the light chain variable region as shown in SEQ ID NO. 73, or
The anti-CCR 8 Fab comprises a heavy chain variable region as shown in SEQ ID NO. 65 and a light chain variable region as shown in SEQ ID NO. 79.
In another preferred embodiment, the anti-CCR 8 scFv comprises a heavy chain variable region as set forth in SEQ ID NO:59 and a light chain variable region as set forth in SEQ ID NO:73, or
The anti-CCR 8 scFv comprises a heavy chain variable region as shown in SEQ ID NO. 65 and a light chain variable region as shown in SEQ ID NO. 79.
In another preferred embodiment, the anti-VEGF scFv comprises a heavy chain variable region as set forth in SEQ ID NO. 87 or 88 and a light chain variable region as set forth in SEQ ID NO. 93 or 94, or
The anti-VEGF scFv comprises a heavy chain variable region as set forth in any one of SEQ ID NOs 89-92, 149-150, and a light chain variable region as set forth in any one of SEQ ID NOs 95-102.
In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as set forth in SEQ ID NO. 103 or, and a light chain variable region as set forth in SEQ ID NO. 107 or 108, or
The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO. 105 or 106, and a light chain variable region as shown in SEQ ID NO. 109 or 110.
In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4
In another preferred embodiment, the Fc fragment is derived from IgG1.
In another preferred embodiment, the Fc fragment has an amino acid sequence as set forth in any one of SEQ ID NOs 113 to 118.
In another preferred embodiment, the Fc1 has the amino acid sequence shown as SEQ ID NO. 116 and the Fc2 has the amino acid sequence shown as SEQ ID NO. 118.
In another preferred example, the amino acid sequence of "HC2 (heavy chain) -HC1 (heavy chain) -Fc1" in the "Fab2-Fab1-Fc1" is shown as SEQ ID NO. 132, the amino acid sequence of "LC1 (light chain)" is shown as SEQ ID NO. 131, and the amino acid sequence of "LC2 (light chain)" is shown as SEQ ID NO. 134.
In another preferred embodiment, the amino acid sequence of said "scFv1-Fc2" is shown in SEQ ID NO. 135.
In another preferred embodiment, the multispecific antibody has a structure according to formula VIII (e.g., h in fig. 7A):
scFv2-Fab1-Fc1
║
scFv2-Fab1-Fc1(VIII)
Wherein "-" are each independently a peptide bond or a connecting peptide "-" ║ "is a connecting bond between peptide chains;
Fab1 is the first targeting domain, said Fab1 being an anti-CCR 8 Fab;
scFv2 is a third targeting domain, the scFv2 being an anti-PD-L1 scFv or an anti-VEGF scFv;
Fc1 is an Fc fragment.
In another preferred embodiment, the "║" is a disulfide bond.
In another preferred embodiment, the anti-CCR 8 Fab comprises the heavy chain variable region as shown in SEQ ID NO. 59 and the light chain variable region as shown in SEQ ID NO. 73, or
The anti-CCR 8 Fab comprises a heavy chain variable region as shown in SEQ ID NO. 65 and a light chain variable region as shown in SEQ ID NO. 79.
In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as set forth in SEQ ID NO. 103 or, and a light chain variable region as set forth in SEQ ID NO. 107 or 108, or
The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO. 105 or 106, and a light chain variable region as shown in SEQ ID NO. 109 or 110.
In another preferred embodiment, the anti-VEGF scFv comprises a heavy chain variable region as set forth in SEQ ID NO. 87 or 88 and a light chain variable region as set forth in SEQ ID NO. 93 or 94, or
The anti-VEGF scFv comprises a heavy chain variable region as set forth in any one of SEQ ID NOs 89-92, 149-150, and a light chain variable region as set forth in any one of SEQ ID NOs 95-102.
In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4
In another preferred embodiment, the Fc fragment is derived from IgG1.
In another preferred embodiment, the Fc fragment has an amino acid sequence as set forth in any one of SEQ ID NOs 113 to 118.
In another preferred embodiment, the Fc1 has the amino acid sequence shown as SEQ ID NO. 113.
In another preferred embodiment, the amino acid sequence of "scFv2-HC1 (heavy chain) -Fc1" in the "scFv2-Fab1-Fc1" is shown as SEQ ID NO. 136, and the amino acid sequence of "LC1 (light chain)" is shown as SEQ ID NO. 124.
In another preferred embodiment, the multispecific antibody has a structure according to formula IX (e.g., i in fig. 7A):
Fab2-Fab1-Fc1
║
Fab2-Fab1-Fc1(IX)
Wherein "-" are each independently a peptide bond or a connecting peptide "-" ║ "is a connecting bond between peptide chains;
Fab1 is the first targeting domain, said Fab1 being an anti-CCR 8 Fab;
Fab2 is a second targeting domain, said Fab2 being an anti-PDL 1 Fab or an anti-VEGF Fab;
Fc1 is an Fc fragment.
In another preferred embodiment, the "║" is a disulfide bond.
In another preferred embodiment, the anti-CCR 8 Fab comprises the heavy chain variable region as shown in SEQ ID NO. 59 and the light chain variable region as shown in SEQ ID NO. 73, or
The anti-CCR 8 Fab comprises a heavy chain variable region as shown in SEQ ID NO. 65 and a light chain variable region as shown in SEQ ID NO. 79.
In another preferred embodiment, the anti-PD-L1 Fab comprises the heavy chain variable region as shown in SEQ ID NO. 103 or and the light chain variable region as shown in SEQ ID NO. 107 or 108, or
The anti-PD-L1 Fab comprises a heavy chain variable region as shown in SEQ ID NO. 105 or 106 and a light chain variable region as shown in SEQ ID NO. 109 or 110.
In another preferred embodiment, the anti-VEGF Fab comprises a heavy chain variable region as shown in SEQ ID NO. 87 or 88 and a light chain variable region as shown in SEQ ID NO. 93 or 94, or
The anti-VEGF Fab comprises the heavy chain variable region as set forth in any one of SEQ ID NOS: 89-92, 149-150, and the light chain variable region as set forth in any one of SEQ ID NOS: 95-102.
In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4
In another preferred embodiment, the Fc fragment is derived from IgG1.
In another preferred embodiment, the Fc fragment has an amino acid sequence as set forth in any one of SEQ ID NOs 113 to 118.
In another preferred embodiment, the Fc1 has the amino acid sequence shown as SEQ ID NO. 114.
In another preferred example, the amino acid sequence of "HC2 (heavy chain) -HC1 (heavy chain) -Fc1" in the "Fab2-Fab1-Fc1" is shown as SEQ ID NO:138, the amino acid sequence of "LC1 (light chain)" is shown as SEQ ID NO:137, and the amino acid sequence of "LC2 (light chain)" is shown as SEQ ID NO: 139.
In another preferred example, the amino acid sequence of "HC2 (heavy chain) -HC1 (heavy chain) -Fc1" in the "Fab2-Fab1-Fc1" is shown as SEQ ID NO:140, the amino acid sequence of "LC1 (light chain)" is shown as SEQ ID NO:137, and the amino acid sequence of "LC2 (light chain)" is shown as SEQ ID NO: 134.
In another preferred embodiment, the multispecific antibody has a structure according to formula X (e.g., a in fig. 8A):
Fab1-Fc1-scFv2
║
Fab1-Fc2-scFv3(X)
Wherein "-" are each independently a peptide bond or a connecting peptide "-" ║ "is a connecting bond between peptide chains;
Fab1 is the first targeting domain, said Fab1 being an anti-CCR 8 Fab;
scFv2 is a second targeting domain, said scFv2 being an anti-VEGF scFv;
scFv3 is a third targeting domain, the scFv3 being an anti-PD-L1 scFv;
Fc1 and Fc2 are each independently an Fc fragment.
In another preferred embodiment, the anti-CCR 8 Fab comprises the heavy chain variable region as shown in SEQ ID NO. 59 and the light chain variable region as shown in SEQ ID NO. 73, or
The anti-CCR 8 Fab comprises a heavy chain variable region as shown in SEQ ID NO. 65 and a light chain variable region as shown in SEQ ID NO. 79.
In another preferred embodiment, the anti-VEGF scFv comprises a heavy chain variable region as set forth in SEQ ID NO. 87 or 88 and a light chain variable region as set forth in SEQ ID NO. 93 or 94, or
The anti-VEGF scFv comprises a heavy chain variable region as set forth in any one of SEQ ID NOs 89-92, 149-150, and a light chain variable region as set forth in any one of SEQ ID NOs 95-102.
In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as set forth in SEQ ID NO. 103 or, and a light chain variable region as set forth in SEQ ID NO. 107 or 108, or
The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO. 105 or 106, and a light chain variable region as shown in SEQ ID NO. 109 or 110.
In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4
In another preferred embodiment, the Fc fragment is derived from IgG1.
In another preferred embodiment, the Fc fragment has an amino acid sequence as set forth in any one of SEQ ID NOs 113 to 118.
In another preferred embodiment, the Fc1 fragment has an amino acid sequence as shown in SEQ ID NO. 115 and the Fc2 fragment has an amino acid sequence as shown in SEQ ID NO. 118.
In another preferred embodiment, the amino acid sequence of "HC1 (heavy chain) -Fc1-scFv2" in the "Fab1-Fc1-scFv2" is shown as SEQ ID NO. 141, and the amino acid sequence of "LC1 (light chain)" is shown as SEQ ID NO. 124.
In another preferred embodiment, the amino acid sequence of "HC1 (heavy chain) -Fc2-scFv3" in the "Fab1-Fc2-scFv3" is shown as SEQ ID NO:142, and the amino acid sequence of "LC1 (light chain)" is shown as SEQ ID NO: 124.
In another preferred embodiment, the multispecific antibody has the structure shown in formula XI below (b in FIG. 8A):
Fab1-Fc1-scFv3
║
Fab2-Fc2-scFv3(XI)
Wherein "-" are each independently a peptide bond or a connecting peptide "-" ║ "is a connecting bond between peptide chains;
Fab1 is the first targeting domain, said Fab1 being an anti-CCR 8 Fab;
Fab2 is the second targeting domain, and said Fab1 is an anti-VEGF Fab;
scFv3 is a third targeting domain, the scFv3 being an anti-PD-L1 scFv;
Fc1 and Fc2 are each independently an Fc fragment.
In another preferred embodiment, the anti-CCR 8 Fab comprises the heavy chain variable region as shown in SEQ ID NO. 59 and the light chain variable region as shown in SEQ ID NO. 73, or
The anti-CCR 8 Fab comprises a heavy chain variable region as shown in SEQ ID NO. 65 and a light chain variable region as shown in SEQ ID NO. 79.
In another preferred embodiment, the anti-VEGF Fab comprises a heavy chain variable region as shown in SEQ ID NO. 87 or 88 and a light chain variable region as shown in SEQ ID NO. 93 or 94, or
The anti-VEGF Fab comprises the heavy chain variable region as set forth in any one of SEQ ID NOS: 89-92, 149-150, and the light chain variable region as set forth in any one of SEQ ID NOS: 95-102.
In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as set forth in SEQ ID NO. 103 or, and a light chain variable region as set forth in SEQ ID NO. 107 or 108, or
The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO. 105 or 106, and a light chain variable region as shown in SEQ ID NO. 109 or 110.
In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4
In another preferred embodiment, the Fc fragment is derived from IgG1.
In another preferred embodiment, the Fc fragment has an amino acid sequence as set forth in any one of SEQ ID NOs 113 to 118.
In another preferred embodiment, the Fc1 fragment has the amino acid sequence shown as SEQ ID NO. 116 and the Fc2 fragment has the amino acid sequence shown as SEQ ID NO. 118.
In another preferred example, the amino acid sequence of "HC1 (heavy chain) -Fc1-scFv3" in the "Fab1-Fc1-scFv3" is shown as SEQ ID NO:144, and the amino acid sequence of "LC1 (light chain)" is shown as SEQ ID NO: 143.
In another preferred embodiment, the amino acid sequence of "HC1 (heavy chain) -Fc2-scFv3" in the "Fab2-Fc2-scFv3" is shown as SEQ ID NO. 145, and the amino acid sequence of "LC2 (light chain)" is shown as SEQ ID NO. 134.
In another preferred embodiment, the multispecific antibody has a structure according to formula XII (e.g., c in FIG. 8A):
Fab1-Fc1-scFv3
║
scFv2-Fc2-scFv3(XII)
Wherein "-" are each independently a peptide bond or a connecting peptide "-" ║ "is a connecting bond between peptide chains;
Fab1 is the first targeting domain, said Fab1 being an anti-CCR 8 Fab;
scFv2 is a second targeting domain, said scFv2 being an anti-VEGF scFv;
scFv3 is a third targeting domain, the scFv3 being an anti-PD-L1 scFv;
Fc1 and Fc2 are each independently an Fc fragment.
In another preferred embodiment, the anti-CCR 8 Fab comprises the heavy chain variable region as shown in SEQ ID NO. 59 and the light chain variable region as shown in SEQ ID NO. 73, or
The anti-CCR 8 Fab comprises a heavy chain variable region as shown in SEQ ID NO. 65 and a light chain variable region as shown in SEQ ID NO. 79.
In another preferred embodiment, the anti-VEGF scFv comprises a heavy chain variable region as set forth in SEQ ID NO. 87 or 88 and a light chain variable region as set forth in SEQ ID NO. 93 or 94, or
The anti-VEGF scFv comprises a heavy chain variable region as set forth in any one of SEQ ID NOs 89-92, 149-150, and a light chain variable region as set forth in any one of SEQ ID NOs 95-102.
In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as set forth in SEQ ID NO. 103 or, and a light chain variable region as set forth in SEQ ID NO. 107 or 108, or
The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO. 105 or 106, and a light chain variable region as shown in SEQ ID NO. 109 or 110.
In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4
In another preferred embodiment, the Fc fragment is derived from IgG1.
In another preferred embodiment, the Fc fragment has an amino acid sequence as set forth in any one of SEQ ID NOs 113 to 118.
In another preferred embodiment, the Fc1 fragment has an amino acid sequence as shown in SEQ ID NO. 115 and the Fc2 fragment has an amino acid sequence as shown in SEQ ID NO. 118.
In another preferred embodiment, the amino acid sequence of "HC1 (heavy chain) -Fc1-scFv3" in the "Fab1-Fc1-scFv3" is shown as SEQ ID NO:142, and the amino acid sequence of "LC1 (light chain)" is shown as SEQ ID NO: 124.
In another preferred embodiment, the amino acid sequence of said "scFv2-Fc2-scFv3" is shown in SEQ ID NO. 146.
In another preferred embodiment, the multispecific antibody has a structure according to formula XIII (e.g., d in FIG. 8A):
scFv2-Fab1-Fc1-scFv3
║
scFv2-Fab1-Fc1-scFv3(XIII)
Wherein "-" are each independently a peptide bond or a connecting peptide "-" ║ "is a connecting bond between peptide chains;
Fab1 is the first targeting domain, said Fab1 being an anti-CCR 8 Fab;
scFv2 is a second targeting domain, said scFv2 being an anti-VEGF scFv;
scFv3 is a second targeting domain, the scFv3 being an anti-PD-L1 scFv;
Fc1 is an Fc fragment.
In another preferred embodiment, the "║" is a disulfide bond.
In another preferred embodiment, the anti-CCR 8 Fab comprises the heavy chain variable region as shown in SEQ ID NO. 59 and the light chain variable region as shown in SEQ ID NO. 73, or
The anti-CCR 8 Fab comprises a heavy chain variable region as shown in SEQ ID NO. 65 and a light chain variable region as shown in SEQ ID NO. 79.
In another preferred embodiment, the anti-VEGF scFv comprises a heavy chain variable region as set forth in SEQ ID NO. 87 or 88 and a light chain variable region as set forth in SEQ ID NO. 93 or 94, or
The anti-VEGF scFv comprises a heavy chain variable region as set forth in any one of SEQ ID NOs 89-92, 149-150, and a light chain variable region as set forth in any one of SEQ ID NOs 95-102.
In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as set forth in SEQ ID NO. 103 or, and a light chain variable region as set forth in SEQ ID NO. 107 or 108, or
The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO. 105 or 106, and a light chain variable region as shown in SEQ ID NO. 109 or 110.
In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4
In another preferred embodiment, the Fc fragment is derived from IgG1.
In another preferred embodiment, the Fc fragment has an amino acid sequence as set forth in any one of SEQ ID NOs 113 to 118.
In another preferred embodiment, the Fc1 fragment has the amino acid sequence shown as SEQ ID NO. 113.
In another preferred embodiment, the amino acid sequence of "scFv2-HC1 (heavy chain) -Fc1-scFv3" in the "scFv2-Fab1-Fc1-scFv3" is shown as SEQ ID NO:147, and the amino acid sequence of "LC1 (light chain)" is shown as SEQ ID NO: 124.
In a third aspect of the invention there is provided a polynucleotide encoding an anti-CCR 8 antibody or antigen binding fragment thereof according to the first aspect of the invention, or a multispecific antibody according to the second aspect of the invention.
In a fourth aspect of the invention there is provided an expression vector comprising a polynucleotide according to the third aspect of the invention.
In another preferred embodiment, the expression vector comprises a prokaryotic expression vector and a eukaryotic expression vector.
In a fifth aspect of the invention there is provided a host cell comprising an expression vector according to the fourth aspect of the invention, or having integrated into its genome a polynucleotide according to the third aspect of the invention.
In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.
In another preferred embodiment, the host cell is selected from the group consisting of E.coli, yeast cells, HEK 293T cells, CHO cells.
In a sixth aspect of the invention there is provided the use of an anti-CCR 8 antibody or antigen binding fragment thereof as described in the first aspect of the invention, or a multispecific antibody as described in the second aspect of the invention, for the manufacture of a medicament for the treatment of cancer/tumour.
In another preferred embodiment, the cancer/tumor is a CCR 8-highly expressed cancer/tumor.
In another preferred embodiment, the cancer/tumor comprises a solid tumor and a hematological tumor.
In another preferred embodiment, the cancer/tumor is a solid tumor.
In another preferred embodiment, the cancer/tumor is selected from the group consisting of rectal cancer, non-small cell lung cancer, glioblastoma, renal cell carcinoma, cervical cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, or a combination thereof.
In a seventh aspect of the invention, there is provided an immunoconjugate comprising:
(i) An anti-CCR 8 antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a multispecific antibody according to the second aspect of the invention, and
(Ii) A conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
In another preferred embodiment, the conjugate is selected from the group consisting of a fluorescent or luminescent label, a radiolabel, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biotoxin, a cytokine (e.g., IL-2, etc.), an antibody, an Fc fragment of an antibody, an scFv fragment of an antibody, a gold nanoparticle/nanorod, a viral particle, a liposome, a nanomagnetic particle, a prodrug-activating enzyme (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), a chemotherapeutic agent (e.g., cisplatin), or any form of nanoparticle, etc.
In an eighth aspect of the invention there is provided a pharmaceutical composition comprising (a) an anti-CCR 8 antibody or antigen-binding fragment thereof as described in the first aspect of the invention, or a multispecific antibody as described in the second aspect of the invention, or an immunoconjugate as described in the seventh aspect of the invention, and (b) a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition is in the form of an injection.
In a ninth aspect of the invention there is provided a method of treating cancer/tumour comprising administering to a subject in need thereof a multispecific antibody according to the first aspect of the invention.
In another preferred embodiment, the subject in need thereof is a human or non-human mammal.
In another preferred embodiment, the cancer/tumor is a CCR 8-highly expressed cancer/tumor.
In another preferred embodiment, the cancer/tumor comprises a solid tumor and a hematological tumor.
In another preferred embodiment, the cancer/tumor is a solid tumor.
In another preferred embodiment, the cancer/tumor is selected from the group consisting of rectal cancer, non-small cell lung cancer, glioblastoma, renal cell carcinoma, cervical cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, or a combination thereof.
In a tenth aspect of the invention there is provided the use of an anti-CCR 8 antibody or antigen binding fragment thereof as described in the first aspect, or an immunoconjugate as described in the seventh aspect of the invention, for the preparation of a detection reagent or kit for detecting CCR8 molecules in a sample.
In another preferred embodiment, the sample comprises an ex vivo sample, such as an ex vivo tissue or cell sample.
In another preferred embodiment, the detection reagent or kit is used as a diagnostic reagent for diagnosing cancer/tumor with high CCR8 expression.
In an eleventh aspect of the invention, there is provided an anti-VEGF antibody mutant comprising a heavy chain variable region having at least 80% sequence identity to the amino acid sequence shown as SEQ ID NO. 89, and a light chain variable region having at least 80% sequence identity to the amino acid sequence shown as SEQ ID NO. 95, and comprising a mutation capable of reducing the hydrophobicity of the antibody.
In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs in a non-CDR 3 region of an anti-VEGR antigen binding domain having a heavy chain variable region as set forth in SEQ ID NO. 89 and a light chain variable region as set forth in SEQ ID NO. 95.
In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs at an amino acid position of the heavy chain variable region shown as SEQ ID NO. 89 selected from the group consisting of positions 28, 30, 31, 32, 33, 35, or a combination thereof.
In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs at an amino acid position of the light chain variable region shown as SEQ ID NO. 95 selected from the group consisting of positions 24, 49, 50, 51, 52, 53, 56, or a combination thereof.
In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs in the region from position 46 to 57 of the light chain variable region as shown in SEQ ID NO. 95.
In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody is a mutation of one or more (e.g., two, three, four) of the above amino acid sites to a hydrophilic amino acid, such as aspartic acid (D), glutamic acid (E), lysine (K), or arginine (R).
In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs at serine (S) at position 30 of the heavy chain variable region as shown in SEQ ID NO:89, preferably serine (S) at position 30 is mutated to aspartic acid (D), glutamic acid (E), lysine (K) or arginine (R).
In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs at serine (S) at position 50 and/or serine (S) at position 52 of the light chain variable region as shown in SEQ ID NO:95, preferably serine (S) at position 50 is mutated to aspartic acid (D), glutamic acid (E), lysine (K) or arginine (R), and/or serine (S) at position 52 is mutated to aspartic acid (D), glutamic acid (E), lysine (K) or arginine (R).
In another preferred embodiment, the anti-VEGF antibody mutant comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs 90-92, 149-150.
In another preferred embodiment, the anti-VEGF antibody mutant comprises a light chain variable region having at least 80% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOS: 96-102.
In another preferred embodiment, the anti-VEGF antibody mutant comprises a heavy chain variable region of the amino acid sequence shown as SEQ ID NO. 91, and a light chain variable region of the amino acid sequence shown as SEQ ID NO. 95.
In another preferred embodiment, the anti-VEGF antibody mutant comprises a heavy chain variable region of the amino acid sequence shown as SEQ ID NO. 89, and a light chain variable region of the amino acid sequence shown as SEQ ID NO. 97.
In another preferred embodiment, the anti-VEGF antibody mutant comprises a heavy chain variable region of the amino acid sequence shown as SEQ ID NO. 89, and a light chain variable region of the amino acid sequence shown as SEQ ID NO. 99.
In another preferred embodiment, the anti-VEGF antibody mutant comprises a heavy chain variable region of the amino acid sequence shown as SEQ ID NO. 89, and a light chain variable region of the amino acid sequence shown as SEQ ID NO. 101.
In another preferred embodiment, the anti-VEGF antibody mutant comprises a heavy chain variable region of the amino acid sequence shown as SEQ ID NO. 91, and a light chain variable region of the amino acid sequence shown as SEQ ID NO. 97.
In another preferred embodiment, the anti-VEGF antibody mutant comprises a heavy chain variable region of the amino acid sequence shown as SEQ ID NO. 91, and a light chain variable region of the amino acid sequence shown as SEQ ID NO. 99.
In another preferred embodiment, the anti-VEGF antibody mutant comprises a heavy chain variable region of the amino acid sequence shown as SEQ ID NO. 91, and a light chain variable region of the amino acid sequence shown as SEQ ID NO. 101.
In another preferred embodiment, the anti-VEGF antibody mutant has a significantly reduced hydrophobicity and a significantly reduced propensity for aggregation of the antibody molecule relative to the original antibody (i.e., an anti-VEGF antibody having the heavy chain variable region amino acid sequence of SEQ ID NO:89 and the light chain variable region sequence of SEQ ID NO: 95).
In another preferred embodiment, the "significantly reduced antibody hydrophobicity" means that the anti-VEGF antibody mutant has a hydrophobicity F1, F1/F0<1, preferably F1/F0≤0.7, more preferably F1/F0≤0.5, as compared to the hydrophobicity F0 of the original antibody.
In another preferred embodiment, the anti-VEGF antibody mutant is used to construct a multispecific antibody that targets VEGF, e.g., a multispecific antibody according to the second aspect of the invention.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Detailed Description
The present inventors have made extensive and intensive studies and have unexpectedly developed a class of multispecific antibodies comprising CCR8 antigen binding domains for the first time. The multispecific antibody comprises a first targeting domain that targets a chemokine (C-C motif) receptor 8 (CCR 8) molecule that is highly expressed on the surface of a tumor-infiltration modulating T cell, the first targeting domain being a CCR8 antibody or antigen binding fragment thereof, and further comprises a second targeting domain and/or a third targeting domain that binds VEGF and/or PD-L1. The multispecific antibody of the invention can combine Treg cells, tumor cells and free VEGF molecules simultaneously, and can be used as an effective therapeutic agent for tumor treatment.
On this basis, the present invention has been completed.
As used herein, the term "chemokine (C-C motif) receptor 8" or "CCR8" refers to a protein encoded by the CCR8 gene in humans. CCR8 is highly expressed on many tumor-infiltrated Treg cells, while it shows low or no expression in thymus, spleen and peripheral blood Treg cells.
VEGF (vascular endothelial growth factor) is a member of the Platelet Derived Growth Factor (PDGF) family. VEGF is a key mediator of angiogenesis in tumors and can mediate the continual formation of new vasculature within and around tumors, whereas structural and functional abnormalities in tumor vessels formed under the action of VEGF can lead to poor bleeding and hypoxia of tumors, thereby further producing more VEGF. The key role of VEGF in tumor angiogenesis makes it a well-known target for anti-tumor.
PD-1/PD-L1 is an important target in tumor Immunity (IO) treatment. Since PD-L1 is highly expressed in most tumors, PD-L1 binding to the surface of T cells will transmit inhibitory signals to T cells. Therefore, blocking PD-1/PD-L1 can effectively activate the killing of T cells to tumor cells.
The term "Fc fragment" or "Fc" refers to a portion of an antibody that does not have antigen binding activity but is initially observed to crystallize readily, and is therefore designated as an Fc fragment (crystalline for the fragment). Such fragments correspond to the paired CH2 and CH3 domains and are the portions of the antibody molecule that interact with effector molecules and cells. The Fc fragments described herein may be derived from IgG1, igG2, and IgG4 antibodies. For particular uses, a particular subclass of IgG may be preferred. For example, igG1 is more effective than IgG2 and IgG4 in mediating ADCC and CDC. Thus, igG2 Fc may be preferred when effector function is undesirable. However, igG2 Fc-containing molecules are generally more difficult to prepare and may not be as stable as IgG1 Fc-containing molecules. Furthermore, effector functions of antibodies may be increased or decreased by introducing one or more mutations into the Fc (see, e.g., strohl, curr. Opin. Biotech.,20:685-691,2009).
As used herein, the term "antibody" or "immunoglobulin" is an iso-tetralin protein of about 150000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H), having identical structural features. Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end, the constant region of the light chain being opposite the first constant region of the heavy chain and the variable region of the light chain being opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.
As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.
Antibodies of the application may include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific, bispecific, human, humanized, primatized, chimeric and single chain antibodies. The antibodies disclosed herein can be from any animal source, including birds and mammals. Preferably, the antibody is a human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibody.
The term "antibody fragment" or "antigen-binding fragment" is used to refer to a portion of an antibody, such as F (ab ') 2,F (ab) 2, fab', fab, fv, single chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies. Regardless of structure, the antibody fragment binds to the same antigen as recognized by the intact antibody. The term "antibody fragment" includes DART and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein comprising immunoglobulin variable regions that act like antibodies by binding to a specific antigen to form a complex. "Single chain fragment variable region" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. In some aspects, the region domain is linked to a short linker peptide of 10 to about 25 amino acids. The linker may be glycine-rich to have flexibility and serine or threonine to have solubility, and may be linked to the N-terminus of VH or the C-terminus of VL, and vice versa. This protein retains the original immunoglobulin specificity despite removal of the constant region and introduction of the linker. With respect to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides having a molecular weight of about 23,000 daltons and two identical heavy chain polypeptides having a molecular weight of 53,000-70,000. The four chains are typically linked by disulfide bonds in a "Y" configuration, with the light chain linked (bracket) to the heavy chain from the mouth of the "Y" and extending through the variable region.
As described above, the variable regions allow the antibodies to selectively recognize and specifically bind to epitopes on antigens. That is, the VL domain and VH domain of an antibody or a subset (subset) of Complementarity Determining Regions (CDRs) of an antibody combine to form a variable region defining a three-dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site present at the end of each arm of each Y configuration. More specifically, the antigen binding site is defined by three CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR 3) on each of the VH and VL chains. In some cases, for example, certain immunoglobulin molecules are derived from or engineered based on camelid species. Alternatively, the immunoglobulin molecule may consist of a heavy chain having no light chain alone or a light chain having no heavy chain alone.
In naturally occurring antibodies, the six CDRs present in each antigen binding domain are short, non-contiguous amino acid sequences, and as the antibody assumes its three-dimensional configuration in an aqueous environment, the CDRs are specifically positioned to form an "antigen binding domain". The remaining amino acids in the antigen binding domain, referred to as the "framework" region, exhibit less intermolecular variability. The framework regions adopt predominantly a β -sheet conformation, and the CDRs form loops that connect and in some cases form part of the β -sheet structure. Thus, the framework regions act to form a scaffold that positions the CDRs in the correct orientation by inter-chain non-covalent interactions. The antigen binding domain formed by the localized CDRs defines a surface complementary to an epitope on the immunoreactive antigen. The complementary surface facilitates non-covalent binding of the antibody to its cognate epitope (cognate epitope). Having been precisely defined, one of ordinary skill in the art can readily identify amino acids comprising CDRs and framework regions, respectively, for any given heavy or light chain variable region.
As used herein, the term "light chain constant region (CL)" includes the amino acid sequence CL (SEQ ID NO:121 or 122) derived from the light chain of an antibody. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain.
As used herein, the term "heavy chain constant region (CH)" includes amino acid sequences derived from immunoglobulin heavy chains. The polypeptide comprising a heavy chain constant region comprises at least one of a CH1 domain (SEQ ID NO:111 or 119), a hinge region (e.g., upper, middle and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. It will be appreciated that the heavy chain constant regions may be modified such that their amino acid sequences differ from naturally occurring immunoglobulin molecules.
In one embodiment of the invention, the multispecific antibody prepared comprises crossmab structures, i.e., heavy chain CH1 and light chain CL exchange portions of the amino acid sequences to prevent mismatches. Such a structure comprises CH1 as shown in SEQ ID NO. 112, and CL as shown in SEQ ID NO. 113.
As used herein, an antibody, antibody fragment, or "variant" of an antibody domain refers to an antibody, antibody fragment, or antibody domain that (1) has at least 80%,85%,90%,95%,96%,97%,98%, or 99% sequence identity to the original antibody, antibody fragment, or antibody domain, and (2) specifically binds to the same target that specifically binds to the original antibody, antibody fragment, or antibody domain. It is to be understood that where sequence identity is expressed in terms of "at least x% identical" or "at least x% identical," such embodiments include any and all numerical percentages equal to or above the lower limit. Furthermore, it is to be understood that where an amino acid sequence is present in the present application, it is to be construed as additionally disclosing or comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence.
Included within the scope of the multispecific molecules of the invention are various compositions and methods including asymmetric IgG-like antibodies (e.g., trifunctional monoclonal antibodies/tetravalent hybridomas (triomab/quadroma)), buttonhole antibodies (knob-antibodies), cross monoclonal antibodies (Cross mabs), electrostatically matched antibodies, LUZ-Y, chain exchange engineering domain (SEED) bodies, fab exchanged antibodies, symmetric IgG class antibodies, diabodies, cross-linked monoclonal antibodies, MAb2, cov X-bodies, double Variable Domain (DVD) -Ig fusion proteins, igG-like bispecific antibodies, ts2Ab, bsAb, scFv/Fc fusions, bis (scFv) 2-Fabs, F (Ab) 2 fusion proteins, double acting or Bis-Fab, dock-and-Lock (DNL), fab-Fv, monoclonal antibodies and diabody-based antibodies (e.g., bispecific antibodies (BiTEs), diabodies, tandem diabodies, igG-fusion proteins, tandem diabodies, and human TCR fusion proteins, tandem diabodies, igG-diabodies, and human TCR fusion proteins.
As used herein, the phrase "multispecific antibody" refers to a molecule comprising at least two targeting domains with different binding specificities, wherein at least one targeting domain specifically binds to Treg cell surface antigen CCR8. In some embodiments, the multispecific antibody is a polypeptide comprising a scaffold and two or more immunoglobulin antigen binding domains that target different antigens or epitopes. In some embodiments, the multispecific antibody is a bispecific antibody. In other embodiments, the multispecific antibody is a trispecific antibody.
As used herein, the phrase "bispecific" refers to a molecule comprising at least two targeting domains with different binding specificities. Each targeting domain is capable of specifically binding to a target molecule and inhibiting a biological function of the target molecule upon binding to the target molecule. In some embodiments, the bispecific antibody is a polymer molecule having two or more peptides. In some embodiments, the targeting domain comprises an antigen binding domain or CDR of an antibody. In some embodiments, the targeting domain comprises a ligand or fragment thereof that specifically binds to a target protein.
The terms "bispecific antibody", "bispecific molecule" and "diabody" are used interchangeably herein to refer to an antibody that can specifically bind to two different antigens (or epitopes). In some embodiments, the bispecific antibody is a full length antibody that binds one antigen (or epitope) on one of its two binding arms (one pair of HC/LCs) and a different antigen (or epitope) on its second arm (the other pair of HC/LCs). In these embodiments, the bispecific antibody has two different antigen binding arms (both in specificity and CDR sequences), and is monovalent for each antigen to which it binds.
In other embodiments, the bispecific antibody is a full length antibody that can bind two different antigens (or epitopes) in each of its two binding arms (two pairs of HC/LC). In these embodiments, the bispecific antibody has two identical antigen binding arms, has the same specificity and the same CDR sequences, and is bivalent for each antigen to which it binds.
The terms "trispecific antibody", "trispecific molecule" and "trispecific antibody" are used interchangeably herein and relate to a molecule comprising three targeting domains with three different binding specificities. Each targeting domain is capable of specifically binding to a target molecule and inhibiting a biological function of the target molecule upon binding to the target molecule. In some embodiments, the trispecific antagonist is a polymer molecule having two or more peptides. In some embodiments, the targeting domain comprises an antigen binding domain or CDR of an antibody. In some embodiments, the targeting domain comprises a ligand or fragment thereof that specifically binds to a target protein.
In a preferred embodiment of the invention, bispecific antibodies targeting CCR8 and VEGF/PD-L1 are constructed, having the structures shown as a-i in fig. 7A, exemplary molecules of the a-i structures being shown in fig. 7B-K, respectively. The anti-CCR 8 Fab/anti-CCR 8 scFv can be Fab/scFv constructed by using the VH and the VL of any anti-CCR 8 antibody, the anti-VEGF Fab/anti-VEGF scFv can be Fab/scFv constructed by using the VH and the VL of any anti-VEGF antibody, and the anti-PD-L1 Fab/anti-PD-L1 scFv can be Fab/scFv constructed by using the VH and the VL of any anti-PD-L1 antibody.
In another preferred embodiment of the invention, trispecific antibodies targeting CCR8, VEGF and PD-L1 are constructed, having the structure shown as a-d in fig. 8A, exemplary molecules of the a-d structure being shown in fig. 8B-E, respectively. The anti-CCR 8 Fab can be a Fab constructed by using the VH and the VL of any anti-CCR 8 antibody, the anti-VEGF Fab/anti-VEGF scFv can be a Fab/scFv constructed by using the VH and the VL of any anti-VEGF antibody, and the anti-PD-L1 scFv can be a scFv constructed by using the VH and the VL of any anti-PD-L1 antibody.
The invention also provides polynucleotide molecules encoding the antibodies or fragments thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequence of an antibody of the invention or a degenerate variant. As used herein, "degenerate variant" refers to a nucleic acid sequence that encodes a polypeptide having the same amino acid sequence as the polypeptide of the present invention, but differs in the sequence of its coding region.
Polynucleotides encoding the mature polypeptides of the present invention include coding sequences encoding only the mature polypeptide, coding sequences and various additional coding sequences for the mature polypeptide, coding sequences (and optionally additional coding sequences) for the mature polypeptide, and non-coding sequences.
The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences.
The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means (1) hybridization and elution at a relatively low ionic strength and a relatively high temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃, or (2) hybridization with a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42 ℃, etc., or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably 95%.
The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form.
At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell, or a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Representative examples are E.coli, streptomyces, salmonella typhimurium, fungal cells such as yeast, drosophila S2 or Sf9 insect cells, CHO, COS7, 293 cell animal cells, and the like.
Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which are capable of absorbing DNA, can be obtained after an exponential growth phase and treated by the CaCl2 method using procedures well known in the art. Another approach is to use MgCl2. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. may be used.
The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.
The antibodies of the invention may be used alone or in combination or coupling with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents, or enzymes capable of producing a detectable product.
Couplable therapeutic agents include, but are not limited to, insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analogs, albumin, antibody fragments, cytokines, and hormones.
The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to, oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.
The pharmaceutical composition of the invention can be used for treating cancers/tumors, especially solid tumors, especially LCRR high-expression solid tumors.
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the monoclonal antibodies (or conjugates thereof) of the invention as described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 1 microgram per kilogram of body weight to about 10 milligrams per kilogram of body weight per day. In addition, the pharmaceutical compositions of the present invention may also be used with other therapeutic agents.
When a pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 8 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally followed by conventional conditions, such as those described in Sambrook et al, molecular cloning, a laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or by the manufacturer's recommendations. Percentages and parts are weight percentages and parts unless otherwise indicated.
Sequences for preparing the multispecific antibodies of the invention
CCR8 antibody sequences
CCR8 antibody sequences
VEGF antibody sequences
Wherein AVACH and AVACL are mutants of the heavy chain variable region AVAH and the light chain variable region AVAL of the antibody molecule AVA, respectively, and the heavy chain variable region ASH and the light chain variable region ASL of the antibody molecule AS-1 are derived from the mutants of the antibody molecule ASP in US7758859, ASCH, AS2H, AS CH, AS3H, AS CH being VH thereof, ASCL, AS2L, AS CL, AS3L, AS CL, AS4L, AS CL being VL thereof. Wherein the underlined parts are in turn CDR1, CDR2 and CDR3 of the heavy chain variable region or the light chain variable region, and the mutation sites are indicated in bold.
PD-L1 antibody sequences
Wherein PL1CH and PL1CL are mutants of the heavy chain variable region PL1H and the light chain variable region PL1L of the antibody molecule PL1, respectively, and PL2CH and PL2CL are mutants of the heavy chain variable region PL2H and the light chain variable region PL2L of the antibody molecule PL2, respectively.
IgG1 CH sequence
The CH1 sequence is as follows:
the Fc region sequence is shown below:
the Fc region may comprise the following mutations:
In a preferred embodiment of the invention, the Fc region has the following sequence:
in another preferred embodiment of the present invention, lysine (K) at the C-terminal end of the Fc region described above is removed, thereby reducing charge isomer formation of the antibody.
IgG4 CH sequence
The Fc region may comprise the following mutations:
CL sequence
Alternative linker sequences
| G |
| GS |
| SS |
| GSS |
| GSSSG |
| GGGGS |
| GGGGSGGGGS |
| GGGGSGGGGSGGGGS |
| GGGGSGGGGSGGGGSGGGGS |
| GGGGSGGGGSGGGGSGGGGSGGGGS |
| PGGGGSP |
| PGGGGSPGGGGSPGGGGSP |
| GEPGSGE |
| GEPGSGEGEPGSGE |
| GEPGSGEGEPGSGEEGEPGSGE |
| EGEPGSGEEGEPGSGEEGEPGSGEEGEPGSGE |
| GKPGS |
| GKPGSGKPGS |
| GKPGSGKPGSGKPGS |
| GKPGSGKPGSGKPGSGKPGS |
| GKPGSGKPGSGKPGSGKPGSGKPGS |
| SSSSG |
| SSSSGSSSSG |
| SSSSGSSSSGSSSSG |
| SSSSGSSSSGSSSSGSSSSG |
| GRPGSGPGSGRPGSGRPGS |
| GRPGSGPGSGRPGSGRPGSGRGPS |
| GKPGSGRPGSGKGPSGRPGS |
EXAMPLE 1 preparation and validation of anti-CCR 8 monoclonal antibodies
1.1 Screening of anti-CCR 8 monoclonal antibodies
The CCR8 antibodies of the invention are selected by humanized mouse immunohybridoma selection. hCCR8 expression plasmid was constructed, DNA was extracted and endotoxin removed, and heavy and light chain variable region humanized mice (derived from CN114763558 a) were immunized. Spleen of immunized mice was selected, isolated to obtain spleen cells, and fused with Sp2/0-Ag14 multiple myeloma cells. Fusion was performed by electrotransformation according to the hybridoma fusion technique. After fusion, a number of cells were seeded in each well of a 96-well plate and cultured in HAT medium for 10 days. Detecting the culture supernatant of the hybridoma cells by using an HEK293-CCR8 over-expression cell line to obtain positive hybridoma cells. Culturing positive hybridoma cells, taking cell culture supernatant for 5 days, and carrying out small-quantity purification on the antibody by using Protein A magnetic beads to obtain the hybridoma antibody of the human variable region-mouse constant region. And performing binding activity function test on the purified antibody. During the screening process, specific binding molecules were screened against the extracellular domain of CCR8 or against CCR 8-overexpressing cell lines by ELISA and FACS, resulting in 14 monoclonal antibodies (C5, C6, C20, C23, C24, C27, C39, C40, C46, C53, C54, C57, C61 and C62). These 14 antibodies can be classified into 5 classes by sequence analysis. Wherein, C24, C39 and C57 are independently classified into one type, C5 and C27 type, and other antibodies are classified into one type.
1.2 Binding Capacity of anti-CCR 8 monoclonal antibodies to HEK cells expressing CCR8
Further, the CCR8 antibodies of the invention and HEK293 cells expressing CCR8 were tested for binding capacity. HEK293 cells expressing CCR8 are prepared by obtaining CCR8 full-length sequence gene fragments of human, mouse and cynomolgus monkey through total gene synthesis, and then respectively inserting the fragments into the skeleton of a stable-transformation expression vector to obtain the expression plasmid vector. The three plasmids are transfected into HEK293 cells, and monoclonal cell lines (HEK 293-hCCR8 cells, HEK293-mCCR cells and HEK293-cCCR cells) which respectively express human, mouse and cynomolgus monkey CCR8 are screened under the pressure of puromycin, so that the flow detection cell lines used in the embodiment are obtained.
Using HEK 293-hCR 8 cells as described above, 1X105 cells were plated in each well of a 96-well plate and the antibody was diluted to 1. Mu.g/ml starting concentration at 2-fold dilution. Mu.l of diluted antibody was mixed with the cells separately and incubated at 4℃for 45 minutes. The washing liquid was washed 2 times. After decanting the liquid, all cells were resuspended in Goat anti-Mouse IgG PE at a concentration of 50. Mu.l 200ng/ml and incubated at 4℃for 30 min in the absence of light. The washing liquid was washed 3 times. After decanting the liquid, all cells were resuspended in 25 μl of diluent. Detection was performed using a flow cytometer. FACS binding data of the obtained 14 monoclonal antibodies are shown in FIG. 1.
The results show that antibodies C20, C24, C46, C53, C54, C61, C62, C40, C6, C23 have Emax and EC50 superior to BM-1 (the antibody sequence is derived from WO2021194942A 1), C57, C27 have EC50 superior to BM-1, and C5 have Emax superior to BM-1.
The positive hybridoma clones are subjected to antibody sequence sequencing, antibody sequences are cloned to a human IgG1 expression vector, FUT8-/-CHO cells are used for antibody expression, supernatant after cell culture is taken for centrifugation to remove sediment, the supernatant is filtered by using a 0.22 mu m filter membrane, and a protein purifier is used for machine purification, so that the human IgG1 Fc antibody without fucose, namely the recombinant human monoclonal antibody, is obtained.
1.3 Binding affinity of anti-CCR 8 recombinant human monoclonal antibodies to CCR8 proteins
Affinity test was performed on 14 recombinant humanized monoclonal antibodies by the following method:
The antibodies were diluted to 5 μg/ml and the antigen human CCR8 (1-35 aa) -mFc or cynomolgus CCR8-mFc was diluted to 50nM, 25nM, 12.5nM, 6.25nM, 3.12nM, 1.56nM, 0.78nM or 1000nM, 500nM, 250nM, 125nM, 62.5nM, 31.2nM, 15.6nM, respectively, while wells with a concentration of 0 were set as baseline. Antibody affinity assays were performed using a Gator instrument.
As a result, the affinity of the candidate antibody for human CCR8 was in the range of 0.0168 to 1.24nM (Table 1). Multiple candidate antibodies can bind to cynomolgus CCR8 (table 2).
TABLE 1 binding affinity of antibodies to human CCR8
| Antibody name | KD(M) |
| C5 | 1.78E-10 |
| C6 | 1.24E-09 |
| C20 | 3.35E-10 |
| C23 | 7.05E-10 |
| C24 | 1.68E-11 |
| C27 | 4.39E-10 |
| C40 | 8.08E-10 |
| C46 | 1.02E-09 |
| C53 | 5.36E-10 |
| C54 | 3.80E-10 |
| C61 | 4.44E-10 |
| C62 | 3.69E-10 |
| C39 | NA |
| C57 | NA |
TABLE 2 binding of antibodies to cynomolgus CCR8
1.4 ADCC Activity of recombinant human monoclonal antibody against CCR8
The ADCC activity of 14 recombinant humanized monoclonal antibodies against CCR8 was tested as follows:
CHO-K1 overexpressing CCR8 was plated at 2×104 cells per well overnight. The medium was removed and 25. Mu.l of complete medium was added. The antibody dilutions were made in complete medium to a final concentration of 4000ng/ml、1333.3ng/ml、444.4ng/ml、148.1ng/ml、49.38ng/ml、16.5ng/ml、5.48ng/ml、1.82ng/ml、0.61ng/ml、0.20ng/ml、0.06ng/ml、0.02ng/ml,. Mu.l antibody dilutions were added per well. The Jurkat-FcgammaRIIIa-V158 effector cell density was maintained between 0.2-1.0X106 cells/ml, the effector cells were resuspended in medium to a cell density of 6X 106 cells/ml, 25. Mu.l of cell suspension was added to 96 Kong Baiban per well, and the total volume per sample well was 75. Mu.l. The cell culture was incubated in an incubator for 6 hours. The plates were added with luciferase substrate, 50 μl/well, and mixed for 30 seconds. The Lum-TM channel was used for detection using a multifunctional microplate reader.
As a result, as shown in FIG. 2, all candidate antibodies had different degrees of ADCC against the target cells, with C5 having the relatively smallest EC50 and C61 having the relatively highest signal value, all with better ADCC.
1.5 Blocking of CCR8 and ligand CCL1 binding by recombinant human monoclonal antibodies against CCR8
The blocking effect of 14 anti-CCR 8 recombinant humanized monoclonal antibodies on CCR8 and ligand CCL1 binding was tested as follows:
The blocking experiment used a kit of DiscoverX's beta-Arrestin eXpress GPCR ASSAY. Into a 15ml centrifuge tube, 11.5ml CELL PLATING REAGENTE were added. 0.5ml CELL PLATING REAGENT was added to the cryopreservation tube, and PathHunter eXpress. Beta. -ARRESTIN GPCR CELLS was thawed and transferred to the 15ml centrifuge tube, mixed well, added with 100. Mu.l per well, placed in the cell incubator, and incubated for 48 hours. The initial concentration was set at 220000ng/ml (22 Xfinal concentration), three-fold dilution, total 8 gradients. Mu.l of antibody dilution per well in the white plate, complete medium was added to the remaining wells, and the wells were placed in a cell incubator and incubated for 30 minutes. 88nM of CCL1 was prepared (22 times the final concentration of CCL1, 4 nM) and 5 μl was added to each well and placed in a cell incubator and incubated for 90 minutes. 55 mu l Working Detection Solution was added to each well and incubated at room temperature for 60 minutes in the dark and detected using the Lum-TM channel.
As shown in FIG. 3, the test antibodies had a certain blocking effect on CCL1 and CCR8 binding, and antibodies with blocking effect >90% were C5, C20, C23, C57 and C61, the blocking values were 96.83%, 90.08%, 91.39% and 92.46%, respectively, and the IC50 values were 496.8, 377.4, 216.5 and 183.4ng/ml, respectively.
Example 2 preparation and validation of anti-VEGF mutants
AS-1 antibodies (heavy chain variable region SEQ ID NO:89, light chain variable region SEQ ID NO: 95) are inherently more hydrophobic and prone to aggregation. In order to improve the quality of the antibody itself, and also to facilitate the subsequent application of the antibody sequence to a diabody or triabody structure, the inventors designed a series of mutations.
The surface hydrophobic region of the Fab structure of AS-1 (PDB: 2 FJG) is marked with the color_h.py code on the PyMOL Wiki, with the higher hydrophobicity being darker and the grey black. The lower the hydrophobicity the lighter the color, making an off-white color, see fig. 4. In general, protein sequences are highly hydrophobic, which can easily cause aggregation, reducing protein stability. In order to confirm the hydrophobicity of the amino acids related to the antibody surface after conversion to scFv, the present inventors predicted the scFv structure of AS-1 with AlphaFold (scFv sequence was identical to the scFv sequence of AS-1 used in the double and triple antibody molecules of the present invention) and analyzed it with AGGRESCAN server, AS shown in FIG. 5. All amino acids greater than 0 are hydrophobic, thus making the antibody prone to aggregation. The present invention obtains a large number of hydrophobic amino acid sites from the Fab and scFv structures of AS-1 AS potential mutation points.
In addition, in order to analyze both the aggregation propensity and the thermal stability of AS-1 sequences, the present inventors used SolubiS server to analyze regions of AS-1 structure variable region sequences that are prone to aggregation (high TANGO score) and have poor thermal stability (high free energy ΔG). As shown in FIG. 6, 6 peptide fragments were identified. Among them, the light chain L46-S57 region has the highest aggregation tendency and the lowest thermal stability. In addition, amino acid mutations at positions 24, 49, 50, 51, 52, 53, 56 of the light chain and positions 28, 30, 31, 32, 33, 35 of the heavy chain have also been found to be effective in improving antibody aggregation by site-directed mutagenesis (Dudgeon et al,2012, pnas).
For the structural and sequence analysis described above, the present invention mutates part of the amino acids in the main L46-S57 region, as well as in other regions (such as hydrophobic sites on the structure, or reported sites prone to aggregation) to reduce hydrophobicity. In addition, it is known from the structure diagram published on PDB by 2FJG that the VH-CDR3 region plays an important role in VEGF binding and that many amino acids are buried internally, so that the main mutations of the present invention are directed to non-VH-CDR 3 regions and other regions not related to VEGF binding to maintain the binding activity of antibodies to VEGF. In this example 7 mutants of AS-1 were obtained by site-directed mutagenesis.
To confirm whether these mutations changed the hydrophobicity of the antibodies, the retention time of each mutant antibody was differentiated using a Proteomix HIC Butyl-NP5 column. After each mutant of the antibody was loaded onto a column in a solution at 1.5M NaCl,25mM Na3PO4 pH 7.4, the mutants were eluted with a stepwise decreasing salt concentration at pH 7.4 with retention times as shown in table 3. AS-1 can be seen to have the longest retention time, indicating that it is the most hydrophobic. Other mutants have reduced retention times, and some combinations of mutant antibodies, such AS AS-6, have reduced retention times by approximately half, greatly reducing the hydrophobicity of the antibody and its potential aggregation propensity.
TABLE 3 Table 3
To further confirm that the binding of the mutants to VEGF was not affected, each mutant was subjected to an affinity assay. Human VEGF proteins containing his tags were loaded onto anti-his BLI probes. The mutants were dissociated for 300s after 150s binding to VEGF in a solution containing 1X PBS,0.1%BSA,0.02%Tween-20, and 0.05% sodium azide, and KD values were calculated as shown in Table 4 below. From Table 4 it can be seen that only mutants AS-3, AS-6, AS-8 have a significant decrease in affinity for VEGF binding, while the other mutants have a better maintenance of affinity than AS-1.
TABLE 4 Table 4
| Mutant | KD(M) |
| AS-1 | 5.15E-11 |
| AS-2 | 3.29E-11 |
| AS-3 | 3.23E-10 |
| AS-4 | 5.14E-11 |
| AS-5 | 8.53E-11 |
| AS-6 | 1.56E-10 |
| AS-7 | 5.37E-11 |
| AS-8 | 4.61E-10 |
EXAMPLE 3 preparation of multispecific antibody molecules
Based on the 14 different CCR8 antibody sequences obtained in example 1, a variety of multispecific antibody molecules were designed.
The molecular sequence fragment genes of the multispecific antibodies were obtained by total gene synthesis and then the sequences of interest were inserted into expression vectors by conventional gene cloning means (see, e.g., lo.b.k.c methods in Molecular biology.volume 248,2004.Antibody Engineering).
HEK293E cells were transfected with vectors carrying multispecific antibody molecules. The corresponding molecules were produced in F17 medium (1L F17+10mL 10%PF68+30ml 200mM L-glutamine) after 7 days of incubation at 37℃with 5% CO2. After the expression is finished, the supernatant is harvested and purified to obtain the multi-specific antibody molecule, which can be used for various experimental analysis subsequently.
The anti-VEGF sequence is derived from molecular AVA (SEQ ID NO:87 for the heavy chain variable region and SEQ ID NO:93 for the light chain variable region) and the mutant of VEGF antibody variable region and its CDR regions of patent US7758859, and the anti-PDL 1 sequence is derived from molecular PL1 (SEQ ID NO:103 for the heavy chain variable region and SEQ ID NO:107 for the light chain variable region) or PL2 (SEQ ID NO:105 for the heavy chain variable region and SEQ ID NO:109 for the light chain variable region).
Using the above method, the following multispecific molecules were prepared:
(1) Bispecific antibodies consisting of CCR8 antigen binding domains and VEGF or PD-L1 antigen binding domains of the structure shown as a-i in fig. 7A;
One bispecific antibody of structure a in FIG. 7A was designated 8As-1, the structure of which is shown in FIG. 7B;
a bispecific antibody of structure b designated 8As-2, the structure of which is shown in figure 7C;
one bispecific antibody of structure c was designated 8As-3, the structure of which is shown in FIG. 7D;
a bispecific antibody of structure d, designated 8As-4, has the structure shown in FIG. 7E;
one bispecific antibody of structure e, designated 8As-5, has the structure shown in FIG. 7F;
a bispecific antibody of structure f, designated 8As-6, has the structure shown in FIG. 7G;
a bispecific antibody of structure g, designated 8As-7, was constructed As shown in FIG. 7H;
one bispecific antibody of structure h is designated Pl8-8, the structure of which is shown in FIG. 7I;
one bispecific antibody of structure i was designated Pl8-9, the structure of which is shown in FIG. 7J, another bispecific antibody of structure i was designated 8As-9, the structure of which is shown in FIG. 7K, and it differs from Pl8-9 in that instead of the anti-PD-L1 VH and VL, anti-VEGF VH and VL were used.
(2) A trispecific antibody consisting of CCR8 antigen binding domain and VEGF antigen binding domain and PD-L1 antigen binding domain as shown in a-d in figure 8A.
One bispecific antibody of structure a in FIG. 8A, designated 8AsPl-1, is shown in FIG. 8B;
a bispecific antibody of structure b designated 8AsPl-2, the structure of which is shown in FIG. 8C
A bispecific antibody of structure c designated 8AsPl-3, the structure shown in figure 8D;
A bispecific antibody of structure d was designated 8AsPl-4 or 8AsPl-4v, the structure of which is shown in FIG. 8E.
An exemplary molecule prepared in this example is as follows, wherein the CCR8 antigen binding domain uses VH and VL of C61 and/or C27:
| molecular name | Chain 1 (SEQ ID NO:) | Chain 2 (SEQ ID NO:) | Chain 3 (SEQ ID NO:) | Chain 4 (SEQ ID NO:) |
| 8As-1 | 124 | 125 | 126 | 127 |
| 8As-2 | 124 | 128 | / | / |
| 8As-3 | 124 | 128 | 125 | / |
| 8As-4 | 124 | 129 | 125 | / |
| 8As-5 | 124 | 130 | 125 | / |
| 8As-6 | 131 | 132 | 133 | 134 |
| 8As-7 | 131 | 132 | 135 | 134 |
| Pl8-8 | 124 | 136 | / | / |
| Pl8-9 | 137 | 138 | 139 | / |
| 8As-9 | 137 | 140 | 134 | / |
| 8AsPl-1 | 124 | 141 | 142 | / |
| 8AsPl-2 | 143 | 144 | 145 | 134 |
| 8AsPl-3 | 124 | 142 | 146 | / |
| 8AsPl-4 | 124 | 147 | / | / |
| 8AsPl-4v | 124 | 148 | | |
Wherein the 8AsPl-4v molecule has the same structure as 8AsPl-4 except that the anti-VEGF antigen binding domain comprises a VH as shown in SEQ ID NO:91 and a VL as shown in SEQ ID NO:99, and the K at the C-terminus of the Fc region is removed.
Example 4 Performance detection of CCR8 and VEGF/PD-L1 Multi-specific antibody molecules targeting
4.1 ADCC experiments targeting CCR8 and VEGF/PDL1 multispecific antibody molecules
ADCC effects of the targeted CCR8 and VEGF/PD-L1 multispecific antibody molecules prepared in example 2 (8 As-1, 8As-2, 8As-4, 8As-7, 8As-9, pl8-8, pl8-9, 8AsPl-1, 8AsPl-3, 8 AsPl-4) were assayed, wherein a portion of the antibodies were expressed using Fut8 knockout CHO cells (FKO). The specific method comprises the following steps:
The CHOK 1-hCR 8 cells in logarithmic growth phase (human CCR8 overexpressing cells, obtained by transfecting CHOK1 cells with CCR8 full-length expression plasmid) were collected, prepared in the same manner as HEK 293-hCR 8 cell preparation described in example 1.2, and centrifuged at 300g for 5min. Cells were resuspended in 1ml assay buffer (1640 medium+10% FBS), counted and cell density was adjusted to 1E6/ml with assay buffer. After mixing well, a suspension of CHOK 1-hCR 8 cells was inoculated into 96 Kong Baiban, 25. Mu.l/well. The gradient diluted antibodies were added sequentially to 96 Kong Baiban above with assay buffer, 25. Mu.l/well, and double wells were set. Incubation was performed in a CO2 incubator for 30min. Finally, ADCC Bioassay Effector CELL V VARIANT (HIGH AFFINITY)/NFAT Luciferase Reporter Jurkat Cell Line, which were well grown, were collected into a 50ml centrifuge tube and centrifuged for 300g and 5min. Cells were resuspended in 3ml assay buffer (1640 m+10% FBS), counted and cell density adjusted to 3E6/ml. After mixing, the mixture was added to the above-mentioned 96 Kong Baiban, 50. Mu.l/well. Placed in a CO2 incubator for 5h incubation. 96 Kong Baiban was removed, BRITELITE PLUS reagent was added, and 100. Mu.l/well. Incubation was performed at room temperature for 5min, and the lumi reading was detected with an enzyme-labeled instrument. And plotting with prism software to calculate EC50 values.
The results in FIG. 9 show that the ADCC effect of the antibody expressed with the Fut8 knockdown CHO cells (designation FKO after) was significantly higher than that of the same antibody expressed with wild type CHO cells (e of FIG. 9). The diabodies of various structures showed strong ADCC action (a-d of FIG. 9). Wherein 8As-1,8As-4, pl8-8, pl8-9 are similar to monoclonal antibodies in ADCC effect of CCR8 target, and the double antibody structure does not influence the exertion of ADCC effect. While better ADCC against CCR8 was shown for the tri-antibody molecule 8AsPl-4, ADCC against PD-L1 was very weak (f and g of fig. 9).
4.2 VEGF blocking experiments targeting CCR8 and VEGF/PD-L1 multispecific antibody molecules
The ability of the targeted CCR8 and VEGF/PD-L1 multispecific antibody molecules (8 As-1, 8As-2, 8As-4, 8As-9, 8AsPl-1, 8AsPl-3, 8 AsPl-4) to block VEGF signaling was determined, with AS-1 molecules As control samples. The specific method comprises the following steps:
VEGFR2/NFAT Reporter-HEK293 Recombinat cells in the logarithmic growth phase were collected and centrifuged at 300g for 5min. 1ml assay buffer (MEM/EBSS medium+10% FBS) was used to resuspend cells, counted and the cell density was adjusted to 6E5/ml with assay buffer. After mixing well, a VEGFR2/NFAT Reporter-HEK293 Recombinant Cell Line cell suspension was seeded into 96 Kong Baiban, 50 μl/well. The gradient diluted antibodies were added sequentially to 96 Kong Baiban above with assay buffer, 25. Mu.l/well, and double wells were set. Human VEGF165 his Tag protein was then diluted to 50ng/ml using assay buffer and transferred to 96 well white plates at 25 μl/well. Incubated in a CO2 incubator for 4h. Finally, 96 Kong Baiban was removed, BRITELITE PLUS reagent was added, and 100. Mu.l/well was used. Incubation was performed at room temperature for 5min, and the lumi reading was detected with an enzyme-labeled instrument. And plotting with prism software to calculate IC50 values.
The results are shown in FIG. 10. FIGS. 10a and b show that the blocking effect of each diabody molecule on VEGF differs little, whereas for the triabody molecule (FIG. 10 c), 8AsPl-4 shows a better blocking effect, similar to that of the mab molecule.
4.3 Experiments for blocking the PDL 1 Multispecific antibody molecules targeting CCR8 and VEGF/PD-L1
The ability of a multi-specific antibody molecule targeting CCR8 and VEGF/PD-L1 to block PD-1/PD-L1 binding was determined using the PL1 molecule described above as a control sample. The specific method comprises the following steps:
CHO-hPDL1 cells in log phase were collected and centrifuged at 300g for 5min. Cells were resuspended in 1ml FACS buffer (1XPBS+2% FBS), counted and the cell density was adjusted to 2E6/ml with FACS buffer. After mixing well, the CHO-hPDL1 cell suspension was inoculated into a 96 well V-bottom plate, 25. Mu.l/well. The gradient diluted antibodies (8 AsPl-1, 8AsPl-3, 8AsPl-4, or Pl8-8-FKO, pl 8-9-FKO) were added sequentially to the 96-well V-bottom plate described above using FACS buffer, 50. Mu.l/well, and double wells were set. Human PD-1mFc Tag protein was diluted to 16. Mu.g/ml using FACS buffer and transferred to 96-well V-bottom plates at 25. Mu.l/well. Placed in a 4 ℃ refrigerator and incubated for 30min. Washed twice with FACS buffer, added Goat anti-Mouse IgG Fc Cross-Adsorbed Secondary Antibody PE, placed in a 4 ℃ refrigerator and incubated for an additional 20min. After washing twice with FACS buffer, detection was performed with a flow cytometer. The experimental data were plotted using prism software to calculate IC50 values.
The results are shown in FIG. 11. The results showed that the blocking of PD-L1 by both diabody molecules was substantially identical to that of mab (fig. 11 a). In the mab molecule, the blocking effect of 8AsPl-3 and 8AsPl-4 was better than that of 8AsPl-1 but lower than that of the mab control sample (b of FIG. 11).
Example 5 in vivo efficacy experiments of antibody molecules targeting CCR8
5.1 In vivo efficacy experiment of MC38 mouse model
CCR8 humanized C57BL/6 mice were inoculated with MC38 cells (5X 105 cells/mouse) to construct a model of MC38 mice and the group dosing was started until the average tumor volume grew to about 100mm3. Intravenous injection is carried out, the administration dosage is 10mg/kg, and the administration is carried out twice a week for 5 times continuously.
As a result, the results are shown in FIG. 12, and on day 14 after administration, BM-1, C61, C27 each had an antitumor effect with Tumor Growth Inhibition (TGI) of 30%,34% and 45%, respectively.
5.2 In vivo efficacy experiment of CT26 mouse model
CCR8 humanized Balb/c mice were inoculated with CT26 cells (3X 105 cells/mouse) to construct a CT26 mouse model and group dosing was started after the average tumor volume had grown to about 60mm3. The doses given to each group by intraperitoneal injection were given 4 times continuously twice a week as shown in fig. 13.
As a result, AS shown in FIG. 13 a, on day 14 after administration, tumor Growth Inhibition (TGI) was 70% (AS-1), 4% (C61), 66% (AS-1+C61), 63% (8 As-1), 58% (8 As-2), 74% (8 As-4) in each of the administration groups, respectively. The change in body weight of mice during dosing is shown in fig. 13 b. Therefore, the diabody molecule also shows good anti-tumor effect in mice.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.