ANTI-CD73-ANTI-PD-1 BISPECIFIC ANTIBODY AND USE THEREOF
20230151111 · 2023-05-18
Inventors
- Peng ZHANG (Zhongshan, Guangdong, CN)
- Baiyong LI (Zhongshan, Guangdong, CN)
- Yu XIA (Zhongshan, Guangdong, CN)
- Zhongmin WANG (Zhongshan, Guangdong, CN)
Cpc classification
C07K2317/24
CHEMISTRY; METALLURGY
G01N2333/70596
PHYSICS
C07K2317/64
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
G01N33/57492
PHYSICS
A61K47/6849
HUMAN NECESSITIES
C07K16/2896
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/71
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C07K16/28
CHEMISTRY; METALLURGY
A61K47/68
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
Provided are an anti-CD73-anti-PD-1 bispecific antibody, a pharmaceutical composition thereof and a use thereof.
Claims
1. An anti-CD73/anti-PD-1 bispecific antibody comprising: a first protein functional region targeting PD-1, and a second protein functional region targeting CD73, wherein the first protein functional region comprises: HCDR1, HCDR2 and HCDR3 contained in a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 44, wherein preferably the amino acid sequences of HCDR1, HCDR2 and HCDR3 are sequences set forth in SEQ ID NOs: 45-47, respectively, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequences set forth in SEQ ID NOs: 45-47, or amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the sequences set forth in SEQ ID NOs: 45-47; and LCDR1, LCDR2 and LCDR3 contained in a light chain variable region having an amino acid sequence set forth in SEQ ID NO: 49, wherein preferably the amino acid sequences of LCDR1, LCDR2 and LCDR3 are sequences set forth in SEQ ID NOs: 50-52, respectively, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequences set forth in SEQ ID NOs: 50-52, or amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the sequences set forth in SEQ ID NOs: 50-52; or the second protein functional region comprises: HCDR1, HCDR2 and HCDR3 contained in a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 2, wherein preferably the amino acid sequences of HCDR1, HCDR2 and HCDR3 are sequences set forth in SEQ ID NOs: 3-5, respectively, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequences set forth in SEQ ID NOs: 3-5, or amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the sequences set forth in SEQ ID NOs: 3-5; and LCDR1, LCDR2 and LCDR3 contained in a light chain variable region having an amino acid sequence set forth in SEQ ID NO: 7, wherein preferably the amino acid sequences of LCDR1, LCDR2 and LCDR3 are sequences set forth in SEQ ID NOs: 8-10, respectively, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequences set forth in SEQ ID NOs: 8-10, or amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the sequences set forth in SEQ ID NOs: 8-10.
2. The anti-CD73/anti-PD-1 bispecific antibody according to claim 1, wherein: the first protein functional region comprises: a sequence having an amino acid sequence set forth in SEQ ID NO: 44 or SEQ ID NO: 62, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 44 or 62, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the sequences set forth in SEQ ID NO: 44 or 62; and a sequence having an amino acid sequence correspondingly set forth in SEQ ID NO: 49 or SEQ ID NO: 64, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 49 or 64, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the sequences set forth in SEQ ID NO: 49 or 64; and/or, the second protein functional region comprises a sequence having an amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 20, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 2 or 20, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the sequences set forth in SEQ ID NO: 2 or 20; and a sequence having an amino acid sequence correspondingly set forth in SEQ ID NO: 7 or SEQ ID NO: 22, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 7 or 22, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the sequences set forth in SEQ ID NO: 7 or 22; or the second protein functional region comprises a sequence having an amino acid sequence set forth in SEQ ID NO: 20, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 20, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the sequences set forth in SEQ ID NO: 20; and a sequence having an amino acid sequence set forth in SEQ ID NO: 24, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 24, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the sequences set forth in SEQ ID NO: 24.
3. The anti-CD73/anti-PD-1 bispecific antibody according to claim 1, wherein: the numbers of the first protein functional region and the second protein functional region are independently 1, 2 or more.
4. The anti-CD73/anti-PD-1 bispecific antibody according to claim 1, wherein: the first protein functional region and the second protein functional region are linked directly or via a linker; preferably, the linker is (GGGGS)n, and n is a positive integer, e.g., 1, 2, 3, 4, 5 or 6.
5. The anti-CD73/anti-PD-1 bispecific antibody according to claim 1, wherein: the first protein functional region and the second protein functional region are independently an immunoglobulin or an antigen-binding fragment, such as a half-antibody, Fab, F(ab′).sub.2 or a single chain fragment variable, preferably, the first protein functional region is an immunoglobulin and the second protein functional region is an antigen-binding fragment; or the first protein functional region is an antigen-binding fragment and the second protein functional region is an immunoglobulin.
6. The anti-CD73/anti-PD-1 bispecific antibody according to claim 1, wherein: the N terminus of the heavy chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the C terminus of CH1 of the immunoglobulin, and the N terminus of the light chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the C terminus of the light chain constant region CL of the immunoglobulin; or the N terminus of the heavy chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the C terminus of the light chain constant region CL of the immunoglobulin, and the N terminus of the light chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the C terminus of the heavy chain constant region CH1 of the immunoglobulin, or the C terminus of the heavy chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the N terminus of the heavy chain of the immunoglobulin, and the C terminus of the light chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the N terminus of the light chain of the immunoglobulin; or the C terminus of the heavy chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the N terminus of the light chain of the immunoglobulin, and the C terminus of the light chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the N terminus of the heavy chain of the immunoglobulin.
7. The anti-CD73/anti-PD-1 bispecific antibody according to claim 1, wherein: the antigen-binding fragment is a single chain fragment variable; preferably, the first protein functional region is an immunoglobulin and the second protein functional region is a single chain fragment variable; or the first protein functional region is a single chain fragment variable and the second protein functional region is an immunoglobulin.
8. The anti-CD73/anti-PD-1 bispecific antibody according to claim 7, wherein: the single chain fragment variable is a molecule formed by connecting an antibody heavy chain variable region (V.sub.H) and an antibody light chain variable region (V.sub.L) via a linker; preferably, the single chain fragment variable has the following structure: NH.sub.2-V.sub.L-linker-V.sub.H-COOH or NH.sub.2-V.sub.H-linker-V.sub.L-COOH.
9. The anti-CD73/anti-PD-1 bispecific antibody according to claim 7, wherein: when the single chain fragment variable is linked to the C terminus of the heavy chain of the immunoglobulin (C.sub.H) (or the N terminus of the heavy chain, the C terminus of CH1 of the heavy chain constant region) via a linker, the antibody heavy chain variable region (V.sub.H) of the single chain fragment variable is firstly linked, or the antibody light chain variable region (V.sub.L) of the single chain fragment variable is firstly linked; preferably, the single chain fragment variable may have the following structure: linker-V.sub.H-linker-V.sub.L-COOH, or, linker-V.sub.L-linker-V.sub.H-COOH, preferably, the heavy chain variable region of the immunoglobulin comprises CDRs having amino acid sequences set forth in SEQ ID NOs: 3-5, and the light chain variable region of the immunoglobulin comprises CDRs having amino acid sequences set forth in SEQ ID NOs: 8-10; the heavy chain variable region of the single chain fragment variable comprises CDRs having amino acid sequences set forth in SEQ ID NOs: 45-47, and the light chain variable region of the single chain fragment variable comprises CDRs having amino acid sequences set forth in SEQ ID NOs: 50-52, preferably, when the single chain fragment variable (such as NH.sub.2-V.sub.L-linker-V.sub.H-COOH or NH.sub.2-V.sub.H-linker-V.sub.L-COOH) is linked to the C terminus of the heavy chain of the immunoglobulin via a linker, the antibody heavy chain variable region (V.sub.H) of the single chain fragment variable comprising CDRs having amino acid sequences set forth in SEQ ID NOs: 45-47 may be firstly linked, or the antibody light chain variable region (V.sub.L) of the single chain fragment variable comprising CDRs having amino acid sequences set forth in SEQ ID NOs: 50-52 may be firstly linked, or preferably, the heavy chain variable region of the immunoglobulin comprises CDRs having amino acid sequences set forth in SEQ ID NOs: 45-47, and the light chain variable region of the immunoglobulin comprises CDRs having amino acid sequences set forth in SEQ ID NOs: 50-52; and/or, the heavy chain variable region of the single chain fragment variable comprises CDRs having amino acid sequences set forth in SEQ ID NOs: 3-5, and the light chain variable region of the single chain fragment variable comprises CDRs having amino acid sequences set forth in SEQ ID NOs: 8-10, wherein, when the single chain fragment variable (such as NH.sub.2-V.sub.L-linker-V.sub.H-COOH or NH.sub.2-V.sub.H-linker-V.sub.L-COOH) is linked to the C terminus of the heavy chain of the immunoglobulin via a linker, the antibody heavy chain variable region (V.sub.H) of the single chain fragment variable comprising CDRs having amino acid sequences set forth in SEQ ID NOs: 3-5 may be firstly linked, or the antibody light chain variable region (V.sub.L) of the single chain fragment variable comprising CDRs having amino acid sequences set forth in SEQ ID NOs: 8-10 may be firstly linked, preferably, one immunoglobulin molecule is linked to two single chain fragment variable molecules, and more preferably, the two single chain fragment variable molecules are identical.
10. The anti-CD73/anti-PD-1 bispecific antibody according to claim 1, wherein: the immunoglobulin is IgG, IgA, IgD, IgE or IgM, preferably IgG, e.g., IgG1, IgG2, IgG3 or IgG4.
11. The anti-CD73/anti-PD-1 bispecific antibody according to claim 1, wherein: the single chain fragment variable is linked to the C terminus of the heavy chain of the immunoglobulin, preferably, one immunoglobulin molecule is linked to two single chain fragment variable molecules, and more preferably, the two single chain fragment variable molecules are identical.
12. The anti-CD73/anti-PD-1 bispecific antibody according to claim 1, wherein: the heavy chain variable region of the immunoglobulin comprises CDRs having amino acid sequences set forth in SEQ ID NOs: 45-47, and the light chain variable region of the immunoglobulin comprises CDRs having amino acid sequences set forth in SEQ ID NOs: 50-52; and/or, the heavy chain variable region of the single chain fragment variable comprises CDRs having amino acid sequences set forth in SEQ ID NOs: 3-5, and the light chain variable region of the single chain fragment variable comprises CDRs having amino acid sequences set forth in SEQ ID NOs: 8-10, preferably, when the single chain fragment variable is linked to the C terminus of the heavy chain of the immunoglobulin via a linker, the antibody heavy chain variable region (V.sub.H) of the single chain fragment variable comprising CDRs having amino acid sequences set forth in SEQ ID NOs: 3-5 may be firstly linked, or the antibody light chain variable region (V.sub.L) of the single chain fragment variable comprising CDRs having amino acid sequences set forth in SEQ ID NOs: 8-10 may be firstly linked.
13. The anti-CD73/anti-PD-1 bispecific antibody according to claim 1, wherein: the heavy chain variable region of the immunoglobulin comprises CDRs having amino acid sequences set forth in SEQ ID NOs: 3-5, and the light chain variable region of the immunoglobulin comprises CDRs having amino acid sequences set forth in SEQ ID NOs: 8-10; and/or, the heavy chain variable region of the single chain fragment variable comprises CDRs having amino acid sequences set forth in SEQ ID NOs: 45-47, and the light chain variable region of the single chain fragment variable comprises CDRs having amino acid sequences set forth in SEQ ID NOs: 50-52, wherein, when the single chain fragment variable is linked to the C terminus of the heavy chain of the immunoglobulin via a linker, the antibody heavy chain variable region (V.sub.H) of the single chain fragment variable comprising CDRs having amino acid sequences set forth in SEQ ID NOs: 45-47 may be firstly linked, or the antibody light chain variable region (V.sub.L) of the single chain fragment variable comprising CDRs having amino acid sequences set forth in SEQ ID NOs: 50-52 may be firstly linked.
14. The anti-CD73/anti-PD-1 bispecific antibody according to claim 1, wherein: the heavy chain variable region of the immunoglobulin has an amino acid sequence selected from SEQ ID NO: 44 and SEQ ID NO: 62, and the light chain variable region of the immunoglobulin has an amino acid sequence correspondingly selected from SEQ ID NO: 49 and SEQ ID NO: 64; and/or, the heavy chain variable region of the single chain fragment variable has an amino acid sequence selected from SEQ ID NO: 2 and SEQ ID NO: 20, and the light chain variable region of the single chain fragment variable has an amino acid sequence correspondingly selected from SEQ ID NO: 7 and SEQ ID NO: 22; or the heavy chain variable region of the single chain fragment variable has an amino acid sequence set forth in SEQ ID NO: 20, and the light chain variable region of the single chain fragment variable has an amino acid sequence set forth in SEQ ID NO: 24; wherein, when the single chain fragment variable is linked to the C terminus of the heavy chain of the immunoglobulin via a linker, the antibody heavy chain variable region (V.sub.H) of the single chain fragment variable may be firstly linked, or the antibody light chain variable region (V.sub.L) of the single chain fragment variable may be firstly linked.
15. The anti-CD73/anti-PD-1 bispecific antibody according to claim 1, wherein: the heavy chain variable region of the immunoglobulin has an amino acid sequence selected from SEQ ID NO: 2 and SEQ ID NO: 20, and the light chain variable region of the immunoglobulin has an amino acid sequence selected from SEQ ID NO: 7 and SEQ ID NO: 22; or the heavy chain variable region of the single chain fragment variable has an amino acid sequence set forth in SEQ ID NO. 20, and the light chain variable region of the single chain fragment variable has an amino acid sequence set forth in SEQ ID NO. 24; and/or, the heavy chain variable region of the single chain fragment variable has an amino acid sequence selected from SEQ ID NO: 44 and SEQ ID NO: 62, and the light chain variable region of the single chain fragment variable has an amino acid sequence selected from SEQ ID NO: 49 and SEQ ID NO: 64, wherein, when the single chain fragment variable is linked to the C terminus of the heavy chain via a linker, the antibody heavy chain variable region (V.sub.H) of the single chain fragment variable may be firstly linked, or, the antibody light chain variable region (V.sub.L) may be linked firstly.
16. The anti-CD73/anti-PD-1 bispecific antibody according to claim 5, wherein: the immunoglobulin comprises a non-CDR region, and the non-CDR region is derived from a non-murine species, such as from a human antibody; more preferably, the constant region of the immunoglobulin is humanized; for example, the heavy chain constant region is an Ig gamma-1 chain C region, ACCESSION: P01857; and the light chain constant region is an Ig kappa chain C region, ACCESSION: P01834, or the heavy chain constant region of the immunoglobulin mutates at any 2 or 3 of positions 234, 235 and 237 based on Ig gamma-1 chain C region, ACCESSION: P01857, and after the mutation, the bispecific antibody has a reduced affinity constant to FcγRIa, FcγRIIIa and/or C1q compared with that before the mutation; more preferably, according to the EU numbering system, the heavy chain constant region has the following mutations at positions 234, 235 and/or 237 based on Ig gamma-1 chain C region, ACCESSION: P01857: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A and G237A, even more preferably, the heavy chain constant region of the immunoglobulin also has one or more mutations selected from the following mutations: N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A and K320A, preferably, the anti-CD73/anti-PD-1 bispecific antibody has a structure shown as heavy chain-light chain-linker 1-scFv, and the scFv is selected from 14C12H1V-linker 2-14C12L1V, 14C12H1V-linker 1-14C12L1V, 14C12H1V-linker 2-14C12L1V and 14C12H1V-linker 1-14C12L1V, particularly selected from the group consisting of: (1) NTPDV1, of which the heavy chain has an amino acid sequence set forth in SEQ ID NO: 85, the light chain has an amino acid sequence set forth in SEQ ID NO: 28, the linker 1 has an amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V has an amino acid sequence set forth in SEQ ID NO: 66, the linker 2 has an amino acid sequence set forth in SEQ ID NO: 81, and 14C12L1V has an amino acid sequence set forth in SEQ ID NO: 68; (2) NTPDV2, of which the heavy chain has an amino acid sequence set forth in SEQ ID NO: 85, the light chain has an amino acid sequence set forth in SEQ ID NO: 28, the linker 1 has an amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V has an amino acid sequence set forth in SEQ ID NO: 66, the linker 1 has an amino acid sequence set forth in SEQ ID NO: 79, and 14C12L1V has an amino acid sequence set forth in SEQ ID NO: 68; (3) NTPDV3, of which the heavy chain has an amino acid sequence set forth in SEQ ID NO: 85, the light chain has an amino acid sequence set forth in SEQ ID NO: 96, the linker 1 has an amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V has an amino acid sequence set forth in SEQ ID NO: 66, the linker 2 has an amino acid sequence set forth in SEQ ID NO: 81, and 14C12L1V has an amino acid sequence set forth in SEQ ID NO: 68; and (4) NTPDV4, of which the heavy chain has an amino acid sequence set forth in SEQ ID NO: 85, the light chain has an amino acid sequence set forth in SEQ ID NO: 96, the linker 1 has an amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V has an amino acid sequence set forth in SEQ ID NO: 66, the linker 1 has an amino acid sequence set forth in SEQ ID NO: 79, and 14C12L1V has an amino acid sequence set forth in SEQ ID NO: 68.
17-21. (canceled)
22. The anti-CD73/anti-PD-1 bispecific antibody according to claim 1, which is encoded by a nucleic acid, or which is contained in a conjugate, a kit, or a pharmaceutical composition, wherein the conjugate further comprises a conjugated moiety, wherein the conjugated moiety is a detectable label; specifically, the conjugated moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme; wherein, preferably, the kit further comprises a secondary antibody that specifically recognizes the bispecific antibody; optionally, the secondary antibody further comprises a detectable label, e.g., a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme; or wherein the pharmaceutical composition optionally comprises a pharmaceutically acceptable carrier and/or excipient.
23-25. (canceled)
26. A method selected from the group consisting of the followings: (1) a method for preventing and/or treating a tumor or anemia, or in diagnosing a tumor or anemia; (2) a method for detecting the level of CD73 in a sample; (3) a method for inhibiting the enzyme activity reaction of CD73; (4) a method for blocking the binding of PD-1 to PD-L1; (5) a method for down-regulating the activity or level of PD-1; (6) a method for relieving the immunosuppression of PD-1 in an organism; (7) a method for elevating IL-2 expression in T lymphocytes; and (8) a method for elevating IFN-γ expression in T lymphocytes, wherein the method comprises administering the anti-CD73/anti-PD-1 bispecific antibody according to claim 1.
27-28. (canceled)
29. An in vivo or in vitro method comprising: administering to a cell or administering to a subject in need thereof an effective amount of the anti-CD73/anti-PD-1 bispecific antibody according to claim 1.
30. A hybridoma cell line, selected from: hybridoma cell line LT014 (also called CD73-19F3) deposited at China Center for Type Culture Collection (CCTCC) on Jun. 19, 2018 with an accession number of CCTCC NO: C2018137; or hybridoma cell line LT003 (also called PD-1-14C12) deposited at China Center for Type Culture Collection (CCTCC) on Jun. 16, 2015 with an accession number of CCTCC NO: C2015105.
31. An anti-CD73 monoclonal antibody, wherein the antibody is 19F3H2L3(hG1TM), and has a heavy chain amino acid sequence set forth in SEQ ID NO: 30 and a light chain amino acid sequence set forth in SEQ ID NO: 28.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0168]
[0169]
[0170]
[0171]
[0172]
[0173]
[0174]
[0175]
[0176]
[0177]
[0178]
[0179]
[0180]
[0181]
[0182]
[0183]
[0184]
[0185]
[0186]
[0187]
[0188]
[0189]
[0190]
[0191]
[0192]
[0193]
[0194]
[0195]
[0196]
[0197]
[0198]
[0199]
[0200]
[0201]
[0202]
Collection Information of Biological Materials:
[0203] The hybridoma cell line LT003 (also called PD-1-14C12), which was deposited at China Center for Type Culture Collection (CCTCC) on Jun. 16, 2015 with a collection number of CCTCC NO: C2015105 and a collection address of Wuhan University, Wuhan, China, postal code: 430072.
[0204] The hybridoma cell line LT014 (also called CD73-19F3), which was deposited at China Center for Type Culture Collection (CCTCC) on Jun. 21, 2018 with a collection number of CCTCC NO: C2018137 and a collection address of Wuhan University, Wuhan, China, postal code: 430072.
DETAILED DESCRIPTION
[0205] The embodiments of the present invention will be described in detail below with reference to the examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention, and should not be construed as limitations on the scope of the present invention. In the cases where the techniques or conditions are not specified, the examples were implemented according to the techniques or conditions described in the literature in the art (e.g., see, Molecular Cloning: A Laboratory Manual, authored by J. Sambrook et al., and translated by Peitang Huang et al., 3.sup.rd Edition, Science Press) or according to the product manual. Reagents or instruments used are commercially available conventional products if the manufacturers thereof are not specified. For example, MDA-MB-231 cells and U87-MG cells can be purchased from ATCC.
[0206] In the following examples of the present invention, BALB/c mice used were purchased from Guangdong Medical Laboratory Animal Center.
[0207] In the following examples of the present invention, the positive control antibody MED19447 (generic name: Oleclumab) used was produced by Zhongshan Akesobio Co. Ltd., the sequence of which is identical to the antibody SEQ ID NOs: 21-24 described in the Medmmune Limited's Patent Publication No. US20160129108A1.
[0208] In the following examples of the present invention, the marketed antibody nivolumab (trade name: Opdivo) for the same target was used, which was purchased from Bristol-Myers Squibb.
[0209] In the following examples of the present invention, the cell line 293T-PD1 used was constructed by Zhongshan Akesobio Co. Ltd. The cell line 293T-PD1 was prepared by viral infection of HEK293T cells using 3rd Generation Lentiviral Systems (see, e.g., A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel R J, Nguyen M, Trono D, and Naldini L., J Virol., 1998. 72(11):8463-8471), wherein the lentivirus expression vector used was pCDH-CMV-PD-1FL-Puro (PD1, Genebank ID: NM_005018; vector pCDH-CMV-Puro, purchased from Youbio, Cat. No. VT1480).
[0210] In the following examples of the present invention, the cell line Raji-PDL1 used was constructed by Zhongshan Akesobio Co. Ltd. The cell line Raji-PDL1 was prepared by viral infection of Raji cells using 3rd Generation Lentiviral Systems (see, e.g., A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel R J, Nguyen M, Trono D, and Naldini L., J Virol., 1998. 72(11):8463-8471), wherein the lentivirus expression vector used was plenti6.3-PDL1 (PDL1, Genebank ID: NP_054862.1; vector plenti6.3, purchased from Invitrogen, Cat. No. K5315-20).
[0211] In the following examples of the present invention, the cell line CHO-K1-PD1 used was constructed by Zhongshan Akesobio Co. Ltd. The cell line CHO-K1-PD1 was prepared by viral infection of CHO-K1 cells using 3rd Generation Lentiviral Systems (see, e.g., A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel R J, Nguyen M, Trono D, and Naldini L., J Virol., 1998. 72(11):8463-8471), wherein the lentivirus expression vector used was pCDH-CMV-PD-1FL-Puro (PD1, Genebank ID: NM_005018; vector pCDH-CMV-Puro, purchased from Youbio, Cat. No. VT1480).
[0212] Nivolumab (trade name: Opdivo), an IgG4 subtype anti-PD-1 antibody carrying S228P mutation, was used as a control antibody in the examples and was purchased from Bristol-Myers Squibb.
[0213] In the following examples of the present invention, the isotype control antibody used, i.e., hIgG1, was an antibody targeting human anti-hen egg lysozyme (HEL), and the variable region sequence of the antibody is from the study reported by Acierno et al., entitled “Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies” (Acierno et al., J Mol biol., 2007; 374(1): 130-46). In the constant region fragment of hIgG1, the heavy chain constant region is Ig gamma-1 chain C region, ACCESSION: P01857, and the light chain constant region is Ig kappa chain C region, ACCESSION: P01834; the hIgG1 was prepared in the laboratory of Zhongshan Akesobio Co. Ltd.
Example 1: Preparation of Anti-CD73 Antibody 19F3
1. Preparation of Hybridoma Cell Line LT014
[0214] The antigen used to prepare the anti-CD73 antibody was human NTSE-his (for NT5E, Genbank ID: NP_002517.1, position: 1-552). Spleen cells of immunized mice were fused with myeloma cells of the mice to prepare hybridoma cells. With human NTSE-Biotin (for NTSE, Genbank ID: NP_002517.1, position: 1-552) taken as an antigen, the hybridoma cells were screened by indirect ELISA to obtain hybridoma cells capable of secreting antibodies that can specifically bind to CD73. The hybridoma cells obtained by ELISA screening were subjected to limiting dilution to obtain a stable hybridoma cell line. The above hybridoma cell line was named as hybridoma cell line LT014, and the monoclonal antibody secreted therefrom was named 19F3.
[0215] The hybridoma cell line LT014 (also called CD73-19F3) was deposited at China Center for Type Culture Collection (CCTCC) on Jun. 21, 2018 with a collection number of CCTCC NO: C2018137 and a collection address of Wuhan University, Wuhan, China, postal code: 430072.
2. Preparation of Anti-CD73 Antibody 19F3
[0216] The cell line LT1014 prepared above was cultured with a chemical defined medium (CD medium, containing 1% Penicillin-Streptomycin) in a 5% CO.sub.2, 37° C. incubator. After 7 days, the supernatants were collected and purified by high-speed centrifugation, vacuum filtration through a microfiltration membrane and a HiTrap protein A HP column to obtain an antibody 19F3.
Example 2: Sequence Analysis of Anti-CD73 Antibody 19F3
[0217] mRNA was extracted from the cell line LT014 prepared in Example 1 according to the method described in the manual of RNAprep pure Cell/Bacteria Kit (Tiangen, Cat. No. DP430). cDNA was synthesized according to the manual of Invitrogen SuperScript® III First-Strand Synthesis System for RT-PCR and amplified by PCR.
[0218] The PCR-amplified products were directly subjected to TA cloning according to the manual of the pEASY-T1 Cloning Kit (Transgen CT101).
[0219] The TA-cloned products were directly sequenced, and the sequencing results are as follows:
[0220] The nucleotide sequence (363 bp) of 19F3 heavy chain variable region is set forth in SEQ ID NO: 1, and the encoded amino acid sequence (121 aa) is set forth in SEQ ID NO: 2.
[0221] According to the IMGT numbering system, the heavy chain CDR1 has a sequence set forth in SEQ ID NO: 3, the heavy chain CDR2 has a sequence set forth in SEQ ID NO: 4, and the heavy chain CDR3 has a sequence set forth in SEQ ID NO: 5.
[0222] The nucleotide sequence (339 bp) of 19F3 light chain variable region is set forth in SEQ ID NO: 6, and the encoded amino acid sequence (113 aa) is set forth in SEQ ID NO: 7.
[0223] According to the IMGT numbering system, the light chain CDR1 has a sequence set forth in SEQ ID NO: 8, the light chain CDR2 has a sequence set forth in SEQ ID NO: 9, and the light chain CDR3 has a sequence set forth in SEQ ID NO: 10.
[0224] The amino acid sequences of 4 framework regions (FR-H1 to FR-H4) of the 19F3 heavy chain are set forth in SEQ ID NO: 11 to SEQ ID NO: 14, respectively; the amino acid sequences of 4 framework regions (FR-H1 to FR-H4) of the 19F3 light chain are set forth in SEQ ID NO: 15 to SEQ ID NO: 18, respectively.
Example 3: Design, Preparation and Detection of Humanized Anti-Human CD73 Antibodies
1. Design of Light and Heavy Chain Sequences of Humanized Antibodies 19F3H2L3 and 19F3H2L2
[0225] Based on the three-dimensional crystal structure of human CD73 protein (Hage T, Reinemer P, Sebald W., Crystals of a 1:1 complex between human interleukin-4 and the extracellular domain of its receptor alpha chain, Eur J Biochem., 1998; 258(2):831-6) and the sequence of murine antibody 19F3 obtained in Example 2, the variable region sequences of antibodies 19F3H1L1, 19F3H2L3 and 19F3H2L3 were obtained by computer modeling and mutation design. The corresponding heavy chain variable region sequences were 19F3H1 and 19F3H2, respectively (with amino acid sequences set forth in SEQ ID NO: 93 and SEQ ID NO: 97, respectively), and the light chain variable region sequences were 19F3L1, 19F3L2 and 19F3L3, respectively (with amino acid sequences set forth in SEQ ID NO: 95, SEQ ID NO: 98 and SEQ ID NO: 99, respectively). The antibody constant region sequences are from NCBI database: the heavy chain constant region is Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region is Ig kappa chain C region, ACCESSION: P01834, 19F3H2L3 is also known as 19F3H2L3(hGIWT) in the Chinese Patent Application No. 202110270671.X, wherein the light and heavy chain variable regions of 19F3H1L1, 19F3H2L2 and 19F3H2L3 can further be noted as 19F3H1V (or 19F3H1v), 19F3H2V (or 19F3H2v), 19F3L1V (or 19F3L1v), 19F3L2V (or 19F3L2v) and 19F3L3V (or 19F3L3v).
[0226] (1) Heavy chain variable region and light chain variable region sequences of humanized monoclonal antibody 19F3H1L1 are as follows:
[0227] The nucleotide sequence (363 bp) of the heavy chain variable region is set forth in SEQ ID NO: 92, and the encoded amino acid sequence (121 aa) is set forth in SEQ ID NO: 93.
[0228] The nucleotide sequence (339 bp) of the light chain variable region is set forth in SEQ ID NO: 94, and the encoded amino acid sequence (113 aa) is set forth in SEQ ID NO: 95.
[0229] (2) Heavy chain variable region and light chain variable region sequences of humanized monoclonal antibody 19F3H2L2 are as follows:
[0230] The nucleotide sequence (363 bp) of the heavy chain variable region 19F3H2 is set forth in SEQ ID NO: 19, and the encoded amino acid sequence (121 aa) is set forth in SEQ ID NO: 20.
[0231] The nucleotide sequence (339 bp) of the light chain variable region 19F3L3 is set forth in SEQ ID NO: 21, and the encoded amino acid sequence (113 aa) is set forth in SEQ ID NO: 22.
[0232] (3) Heavy chain variable region and light chain variable region sequences of humanized monoclonal antibody 19F3H2L3 are as follows:
[0233] The nucleotide sequence (363 bp) of the heavy chain variable region 19F3H2 is set forth in SEQ ID NO: 19, and the encoded amino acid sequence (121 aa) is set forth in SEQ ID NO: 20.
[0234] The nucleotide sequence (339 bp) of the light chain variable region 19F3L3 is set forth in SEQ ID NO: 23, and the encoded amino acid sequence (113 aa) is set forth in SEQ ID NO: 24.
2. Preparation of Humanized Antibodies 19F3H1L1, 19F3H2L2 and 19F3H2L3
[0235] Heavy chain constant regions used Ig gamma-1 chain C region. ACCESSION: P01857; the light chain constant regions used Ig kappa chain C region, ACCESSION: P01834.
[0236] The heavy chain cDNA and light chain cDNA of 19F3H1L1, 19F3H2L2 and 19F3H2L3 were separately cloned into pUC57simple (provided by GenScript) vectors to obtain pUC57simple-19F3H1, pUC57simple-19F3L1, pUC57simple-19F3H2, pUC57simple-19F3L2 and pUC57simple-19F3L3. Referring to the standard techniques described in Molecular Cloning: A Laboratory Manual (Second Edition), the heavy and light chain full-length genes synthesized by EcoRI&HindIII digestion were subcloned into an expression vector pcDNA3.1 through digestion by an restriction enzyme (EcoRI&HindIII) to obtain expression plasmids pcDNA3.1-19F3H1, pcDNA3.1-19F3L1, pcDNA3.1-19F3H2, pcDNA3.1-19F3L2, and pcDNA3.1-19F3L3, and the heavy/light chain genes of the recombinant expression plasmids were further subjected to sequencing analysis. Then the designed gene combinations comprising the corresponding light and heavy chain recombinant plasmids (pcDNA3.1-19F3H1/pcDNA3.1-19F3L1, pcDNA3.1-19F3H2/pcDNA3.1-19F3L2, and pcDNA3.1-19F3H2/pcDNA3.1-19F3L3) were separately co-transfected into 293F cells, and the culture solutions were collected and purified. After the sequences were verified, endotoxin-free expression plasmids were prepared, and were transiently transfected into HEK293 cells for antibody expression. The culture solutions were collected after 7 days, and subjected to affinity purification on a Protein A column to obtain humanized antibodies.
3. Design of Light and Heavy Chain Sequences of Humanized Antibodies 19F3112L3(hG1M) and 19F3H2L3(hG1TM)
[0237] On the basis of 19F3H2L3 obtained in of Example 3 1, 19F3H2L3(hG1M) was obtained by introducing a leucine-to-alanine point mutation at position 234 (L234A) and a leucine-to-alanine point mutation at position 235 (L235A) of the heavy chain. The nucleotide and amino acid sequences of the heavy chain of 19F3H12L3(hG1M) are set forth in SEQ ID NO: 25 and SEQ ID NO: 26, respectively; the light chain constant region of 19F3H2L3(hG1M) is an Ig kappa chain C region, ACCESSION: P01834, and the nucleotide and amino acid sequences of the light chain of 19F3H2L3(hG1M) are set forth in SEQ ID NO: 27 and SEQ ID NO: 28, respectively.
[0238] On the basis of 19F3H2L3 obtained in step 1 of Example 3, 19F3H2L3(hG1TM) was obtained by introducing a leucine-to-alanine point mutation at position 234 (L234A), a leucine-to-alanine point mutation at position 235 (L235A), and a glycine-to-alanine point mutation at position 237 (G237A) in the heavy chain. The nucleotide and amino acid sequences of the heavy chain are set forth in SEQ ID NO: 29 and SEQ ID NO: 30, respectively; and the light chain is identical to 19F3H2L3(hG1M).
[0239] The amino acid sequences of 4 framework regions (FR-H1 to FR-H4) of 19F3H2 are set forth in SEQ ID NO: 31 to SEQ ID NO: 34, respectively;
[0240] the amino acid sequences of 4 framework regions (FR-H1 to FR-H4) of the 19F3L2 light chain are set forth in SEQ ID NO: 35 to SEQ ID NO: 38, respectively; and
[0241] the amino acid sequences of 4 framework regions (FR-H1 to FR-H4) of the 19F3L3 light chain are set forth in SEQ ID NO: 39 to SEQ ID NO: 42, respectively.
4. Preparation of Humanized Antibody 19F3H2L3(hG1M)
[0242] The heavy chain cDNA and light chain cDNA of 19F3H2L3(hG1M) were separately cloned into vector pUC57simple (provided by Genscript) to obtain pUC57simple-19F3H2(hG1M) and pUC57simple-19F3L3, respectively. Referring to the standard techniques described in Molecular Cloning: A Laboratory Manual (Second Edition), the heavy and light chain full-length genes synthesized by EcoRI&HindIII digestion were subcloned into an expression vector pcDNA3.1 through digestion by an restriction enzyme (EcoRI&HindIII) to obtain expression plasmids pcDNA3.1-19F3H2(hG1M) and pcDNA3.1-19F3L3, and the heavy/light chain genes of the recombinant expression plasmids were further subjected to sequencing analysis. Then the designed gene combination comprising the corresponding light and heavy chain recombinant plasmids pcDNA3.1-19F3H2(hG1M)/pcDNA3.1-19F3L3 was co-transfected into 293F cells, and the culture solutions were collected and purified. After the sequences were verified, endotoxin-free expression plasmids were prepared, and were transiently transfected into HEK293 cells for antibody expression. The culture solutions were collected after 7 days, and subjected to affinity purification on a Protein A column to obtain a humanized antibody 19F3H2L3(hG1M).
Example 4: Preparation of Anti-PD-1 Antibody 14C12
1. Preparation of the Hybridoma Cell Line LT003
[0243] Using PD-1-mFc fusion protein (PD-1 GenBank: NM-005018, mFc SEQ ID NO: 89) as an antigen, spleen cells of an immunized BALB/c mice (purchased from Guangdong Medical Laboratory Animal Center) and mouse myeloma cells were fused into hybridoma cells, and the established methods (e.g., Stewart, S. J., “Monoclonal Antibody Production”, in Basic Methods in Antibody Production and Characterization, Eds. G. C. Howard and D. R. Bethell, Boca Raton: CRC Press, 2000) were referred to.
[0244] The plate was coated with PD-1-hFc (PD-1, Genbank ID: NM 005018, hFc is a human IgG Fc purification tag, specifically Ig gamma-1 chain C region, Genbank ID: P01857, positions 114-330) for indirect ELISA. By screening, hybridoma cells secreting new antibodies specifically binding to PD-1 were obtained.
[0245] Hybridoma cell lines capable of secreting a monoclonal antibody that competes with the ligand PD-L1-hFc (PD-L1 Genbank ID: NP 054862.1) for binding to PD-1 were screened by competitive ELISA, and a stable hybridoma cell line was obtained by limiting dilution. The LT003 stable cell line (PD-1-14C12) was obtained by limiting dilution, and the secreted monoclonal antibody was named as 14C12.
[0246] The hybridoma cell line LT003 (also called PD-1-14C12) was deposited at China Center for Type Culture Collection (CCTCC) on Jun. 16, 2015 with a collection number of CCTCC NO: C2015105 and a collection address of Wuhan University, Wuhan, China, postal code: 430072.
2. Preparation of Anti-PD-1 Antibody 14C12
[0247] The LT003 cells prepared above were cultured in an IMDM medium containing 10% low IgG fetal bovine serum (IMDM medium containing 1% Penicillin-Streptomycin, cultured in a 5% CO.sub.2, 37° C. cell incubator). After 7 days, the cell culture supernatant was collected and purified to obtain antibody 14C12.
Example 5: Sequence Analysis of Anti-PD-1 Antibody 14C12
[0248] mRNA was extracted from the hybridoma cell line LT003 prepared in Example 1 according to the method described in the manual of RNAprep pure Cell/Bacteria Kit (Tiangen, Cat. No. DP430).
[0249] cDNA was synthesized according to the manual of Invitrogen SuperScript® III First-Strand Synthesis System for RT-PCR and amplified by PCR.
[0250] The PCR-amplified products were directly subjected to TA cloning according to the manual of the pEASY-T1 Cloning Kit (Transgen CT101).
[0251] The TA-cloned products were directly sequenced, and the sequencing results are as follows:
[0252] The nucleotide sequence (354 bp) of the heavy chain variable region is set forth in SEQ ID NO: 43, and the encoded amino acid sequence (118 aa) is set forth in SEQ ID NO: 44.
[0253] According to the IMGT numbering system, the heavy chain CDR1 has a sequence set forth in SEQ ID NO: 45, the heavy chain CDR2 has a sequence set forth in SEQ ID NO: 46, and the heavy chain CDR3 has a sequence set forth in SEQ ID NO: 47.
[0254] The nucleotide sequence (321 bp) of the light chain variable region is set forth in SEQ ID NO: 48, and the encoded amino acid sequence (107 aa) is set forth in SEQ ID NO: 49.
[0255] According to the IMGT numbering system, the light chain CDR1 has a sequence set forth in SEQ ID NO: 50, the light chain CDR2 has a sequence set forth in SEQ ID NO: 51, and the light chain CDR3 has a sequence set forth in SEQ ID NO: 52.
[0256] The amino acid sequences of 4 framework regions (FR-H1 to FR-H4) of the 14C12 heavy chain are set forth in SEQ ID NO: 53 to SEQ ID NO: 56, respectively; the amino acid sequences of 4 framework regions (FR-H1 to FR-H4) of the 14C12 light chain are set forth in SEQ ID NO: 57 to SEQ ID NO: 60, respectively.
Example 6: Design and Preparation of Humanized Anti-PD-1 Antibodies 14C12H1L1 and 14C12H111(hG1TM)
1. Design of Humanized Anti-PD-1 Antibody 14C12H1L1
[0257] The light and heavy chain sequences of the humanized antibody 14C12H1L1 were designed according to the three-dimensional crystal structure of PD-1 protein (Shinohara T, et al., Structure and chromosomal localization of the human PD-1 gene (PDCD1). Genomics 1995, 23 (3): 704-6) and the sequence of antibody 14C12 obtained in Example 5 by computer simulation of antibody model and designing mutations according to the model to obtain the variable region sequences of antibody 14C12H1L1.
[0258] The designed variable region sequences are as follows.
[0259] The nucleotide sequence (354 bp) of the heavy chain variable region 14C12H11 of the humanized monoclonal antibody 14C12H1L1 is set forth in SEQ ID NO: 61, and the encoded amino acid sequence (118 aa) is set forth in SEQ ID NO: 62.
[0260] The nucleotide sequence (321 bp) of the light chain variable region 14C12L1 of the humanized monoclonal antibody 14C12H1L1 is set forth in SEQ ID NO: 63, and the encoded amino acid sequence (107 aa) is set forth in SEQ ID NO: 64.
[0261] The constant region of the antibody 14C12H1L1 is from NCBI database (the heavy chain constant region is Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region is Ig kappa chain C region, ACCESSION: P01834). The nucleotide sequence and amino acid sequence of the 14C12H1L1 heavy chain are set forth in SEQ ID NOs: 65 and 66, respectively, and the nucleotide sequence and amino acid sequence of the 14C12H1L1 light chain are set forth in SEQ ID NOs: 67 and 68, respectively, 14C12H1L1 is also known as 14C12H1L1 (hG1WT) herein and in Chinese Patent Application No. 202110270671.X, wherein the heavy chain variable region and the light chain variable region of 14C12H1L1 are also known as 14C12H1V (or 14C12H1v) and 14C12L1v (or 14C12L1v) herein and in Chinese Patent Application No. 202110270671.X.
2. Design of Light and Heavy Chain Sequences of Humanized Antibody 14C12H1L1(hG1TM)
[0262] On the basis of 14C2H1L1 obtained in step 1 of Example 6, 14C12H1L1 (hG1TM) was obtained by introducing a leucine-to-alanine point mutation at position 234 (L234A), a leucine-to-alanine point mutation at position 235 (L235A), and a glycine-to-alanine point mutation at position 237 (G237A) in the heavy chain. The nucleotide and amino acid sequences of the heavy chain of 14C12H1L1(hG1TM) are set forth in SEQ ID NO: 69 and SEQ ID NO: 70, respectively; and the light chain is identical to 14C12H1L1.
[0263] The amino acid sequences of 4 framework regions (FR-H1 to FR-H4) of 14C12H1 are set forth in SEQ ID NO: 71 to SEQ ID NO: 74, respectively;
the amino acid sequences of 4 framework regions (FR-H1 to FR-H4) of the 14C12L1 light chain are set forth in SEQ ID NO: 75 to SEQ ID NO: 78, respectively.
3. Preparation of Humanized Antibodies 14C12H1L1 and 14C12H1L1(hG1TM)
[0264] The heavy chain cDNA and light chain cDNA of 14C12H1L1(hG1TM) and 14C12H1L1 were separately cloned into pUC57simple (provided by GenScript) vectors to obtain pUC57simple-14C12H1, pUC57simple-14C12L1 and pUC57simple-14C12H1(hG1TM). Referring to the standard techniques described in Molecular Cloning: A Laboratory Manual (Second Edition), the heavy and light chain full-length genes synthesized by EcoRI&HindIII digestion were subcloned into an expression vector pcDNA3.1 through digestion by an restriction enzyme (EcoRI&HindIII) to obtain expression plasmids pcDNA3.1-14C12H1, pcDNA3.1-14C12L1 and pcDNA3.1-14C12H1(hG1TM), and the heavy/light chain genes of the recombinant expression plasmids were further subjected to sequencing analysis. Then the designed gene combinations comprising the corresponding light and heavy chain recombinant plasmids (pcDNA3.1-14C12H1(hG1TM)/pcDNA3.1-14C12L1, and pcDNA3.1-14C12H1/pcDNA3.1-14C12L1) were separately co-transfected into 293E cells, and the culture solutions were collected and purified. After the sequences were verified, endotoxin-free expression plasmids were prepared, and were transiently transfected into HEK293 cells for antibody expression. The culture solutions were collected after 7 days, and subjected to affinity purification on a Protein A column to obtain humanized antibodies.
Example 7: Sequence Design and Expression of Anti-PD-1/CD73 Bifunctional Antibodies
1. Sequence Design
[0265] The structure of the bifunctional antibody described herein is in the Morrison form (IgG-scFv), i.e., C-termini of two heavy chains of an IgG antibody are each linked to a scFv fragment of another antibody, and the main composition design of the heavy and light chains is as shown in Table 1 below.
TABLE-US-00001 TABLE 1 Table 1. Composition design of heavy and light chains of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, PID7V07 and P1D7V08. Bispecific Heavy chain antibody IgG Light No. moiety Linker scFv moiety chain P1D7V01 14C12H1 Linker1 19F3H2v-Linker1- 14C12L1 19F3L3v P1D7V02R 14C12H1 Linker1 19F3L3v-Linker1- 14C12L1 19F3H2v P1D7V03 14C12H1 Linker2 19F3H2v-Linker1- 14C12L1 19F3L3v P1D7V04R 14C12H1 Linker2 19F3L3v-Linker1- 14C12L1 19F3H2v P1D7V07 19F3H2 Linker1 14C12H1 v-Linker1- 19F3L3 14C12L1v P1D7V08 19F3H2 Linker2 14C12H1 v-Linker1- 19F3L3 14C12L1v
[0266] In the Table 1 above:
[0267] (1) Those with “V” label at lower right corner refer to the variable region of corresponding heavy chain or the variable region of corresponding light chain. For those without “V” label, the corresponding heavy or light chain is the full length comprising the constant region. The corresponding sequences described in the above examples are referred to for the amino acid sequences of these variable regions or the full length and the nucleotide sequences encoding them.
[0268] (2) The amino acid sequence of linker 1 is (GGGGS).sub.4 (the nucleotide sequence is SEQ ID NO: 80, and the amino acid sequence is SEQ ID NO: 79), and the amino acid sequence of linker 2 is (GGGGS).sub.3 (the nucleotide sequence is SEQ ID NO: 82, and the amino acid sequence is SEQ ID NO: 81).
2. Expression and Purification of Antibodies
[0269] The heavy chain cDNA sequence and the light chain cDNA sequence of P1D7V01 were each cloned into vector pUC57simple (provided by Genscript) to obtain plasmids pUC57simple-VP101H and pUC57simple-VP101L, respectively.
[0270] Plasmids pUC57simple-VP101H and pUC57simple-VP101L were enzyme-digested (HindIII&EcoRI), and heavy and light chains isolated by electrophoresis were subcloned into vector pcDNA3.1, and recombinant plasmids were extracted to co-transfect 293F cells. After 7 days of cell culture, the culture medium was separated by centrifugation at high speed, and the supernatant was concentrated and loaded onto a HiTrap MabSelect SuRe column. The protein was eluted in one step with an elution buffer. The target sample was isolated and the buffer was exchanged into PBS.
[0271] The purified antibodies P1D7V02R, P1D7V03, P1D7V04R, P1D7V07 and P1D7V08 were obtained according to the above expression and purification methods for P1D7V01.
Example 8: Assay for Binding Activity of Anti-CD73/Anti-PD-1 Bispecific Antibodies to Antigens by ELISA
1. Binding Activity of P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R to Antigen PD-1-mFc Determined by ELISA the Method is Specifically as Follows.
[0272] A microplate was coated with PD-1-mFc at 0.5 μg/mL and incubated at 4° C. overnight. Then the microplate coated with antigens was washed once with PBST, and then blocked with a PBS solution containing 1% BSA as blocking solution at 37° C. for 2 h. After blocking, the microplate was washed 3 times with PBST. The antibodies serially diluted with PBST solution (the dilution gradients for the antibody are shown in Table 2) were added. The microplate containing the test antibodies was incubated at 37° C. for 30 min, and then washed 3 times with PBST. After washing, HRP-labeled goat anti-human IgG (H+L) (Jackson, Cat. No. 109-035-088) secondary antibody working solution diluted in a ratio of 1:5000 was added, and then the microplate was incubated at 37° C. for 30 min. After incubation, the plate was washed 4 times with PBST, TMB (Neogen, 308177) was added in the dark for chromogenesis for 5 min, and then a stop solution was added to terminate chromogenic reaction. The microplate was put into a microplate reader immediately, and the OD value of each well in the microplate was read at 450 nm. The data were analyzed and processed by SoftMax Pro 6.2.1,
[0273] The results are shown in Table 2 and
[0274] The above experimental results showed that in the same experimental condition, the binding activities of P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R to PD-1-mFc are comparable to that of the reference drugs 14C12H1L1 and nivolumab for the same target, suggesting that P1D7V01, P1D7V02R, P1D7V03, P1D7V04R have the activity of effectively binding to PD-1-mFc.
TABLE-US-00002 TABLE 2 Binding of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1 and nivolumab to PD-1-mFc determined by ELISA Antibody dilution concentration Antigen coating PD-1-mFc 0.5 μg/mL (μg/mL) P1D7V01 P1D7V02R P1D7V03 P1D7V04R 14C12H1L1 Nivolumab 1 2.345 2.300 2.169 2.323 2.406 2.292 2.225 2.393 2.441 2.609 2.386 2.473 1:3 2.461 2.295 2.114 2.361 2.288 2.303 2.199 2.302 2.414 2.548 2.441 2.600 1:9 2.279 2.085 2.063 2.094 2.135 2.178 2.075 2.043 2.461 2.525 2.519 2.572 1:27 1.838 1.765 1.701 1.696 1.777 1.764 1.659 1.659 2.236 2.295 2.196 2.219 1:81 1.153 1.097 1.089 1.120 1.149 1.154 1.062 1.131 1.858 1.954 1.711 1.712 1:243 0.629 0.684 0.570 0.582 0.675 0.686 0.584 0.592 1.251 1.318 0.980 0.965 1:729 0.361 0.364 0.334 0.349 0.360 0.370 0.340 0.341 0.683 0.709 0.521 0.498 0 0.201 0.198 0.187 0.210 0.197 0.197 0.213 0.257 0.208 0.199 0.196 0.190 Secondary antibody Goat anti-human IgG (H + L), HRP (1:5000) EC50(nM) 0.078 0.078 0.075 0.089 0.033 0.051
2. Binding Activity of P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R to Antigen Human NT5E-Biotin Determined by ELISA
[0275] A microplate was coated with streptavidin at 2 μg/mL and then incubated at 4° C. overnight. After incubation, the microplate coated with streptavidin was washed once with PBST, and blocked with a PBS solution containing 1% BSA as a microplate blocking solution at 37° C. for 2 h. After blocking, the microplate was washed 3 times with PBST. Then, 0.5 μg/mL antigen human NTSE-Biotin was added and incubated at 37° C. for 30 min. Then, the plate was washed 3 times with PBST. The antibodies serially diluted with PBST solution (the dilution gradients for the antibody are shown in Table 3) were added to wells of the microplate. The microplate containing the test antibodies was incubated at 37° C. for 30 min, and then washed 3 times with PBST. After washing, HRP-labeled goat anti-human IgG (H+L) (Jackson, Cat. No. 109-035-088) secondary antibody working solution diluted in a ratio of 1:5000 was added, and the microplate was incubated at 37° C. for 30 min. After incubation, the plate was washed 4 times with PBST, TMB (Neogen, 308177) was added in the dark for chromogenesis for 5 min, and then a stop solution was added to terminate chromogenic reaction. The microplate was put into a microplate reader immediately, and the OD value of each well in the microplate was read at 450 nm. The data were analyzed and processed by SoftMax Pro 6.2.1.
[0276] The results are shown in Table 3 and
[0277] The above experimental results showed that in the same experimental condition, the binding activities of bispecific antibodies P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R to human NTSE-biotin are comparable to that of the reference drugs 19F3H2L3 and MED19447 for the same target, suggesting that P1D7V01, P1D7V02R, P1D7V03 and P1 D7V44R have the activity of effectively binding to human NTSE-biotin.
TABLE-US-00003 TABLE 3 Binding of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 19F3H2L3 and MEDI9447 to human NT5E-biotin determined by ELISA Antibody dilution Antigen coating: SA (2 μg/mL) concentration Human NT5E-biotin (0.5 μg/mL) (μg/mL) P1D7V01 P1D7V02R P1D7V03 P1D7V04R 19F3 H2L3 MEDI9447 0.333 2.465 2.254 2.125 2.024 2.245 2.311 1.992 1.864 2.345 2.413 2.460 2.570 1:3 2.310 2.207 1.718 1.680 2.202 2.194 1.385 1.397 2.366 2.376 2.434 2.528 1:9 1.899 1.856 1.090 1.086 1.834 1.782 0.886 0.898 2.164 2.120 2.219 2.345 1:27 1.276 1.239 0.556 0.554 1.199 1.132 0.464 0.465 1.681 1.631 1.729 1.810 1:81 0.704 0.654 0.289 0.279 0.610 0.601 0.235 0.230 1.048 0.978 1.091 1.166 1:243 0.363 0.333 0.183 0.178 0.308 0.302 0.155 0.156 0.548 0.504 0.547 0.587 1:729 0.199 0.197 0.138 0.141 0.183 0.184 0.130 0.122 0.283 0.278 0.288 0.305 0 0.135 0.119 0.115 0.117 0.116 0.119 0.115 0.111 0.120 0.126 0.121 0.121 Secondary antibody Goat anti-human IgG (H + L), HRP (1:5000) EC50(nM) 0.063 0.230 0.068 0.439 0.045 0.042
Example 9: Activity of Anti-CD73/Anti-PD-1 Bispecific Antibodies Completing with Human PD-L1-mFc for Binding to Human PD-1-mFc-Biotin Determined by Competitive ELISA
[0278] A microplate was coated with human PD-L1-mFc (PD-L1 Genbank ID: NP_054862.1, mFc SEQ ID NO: 143) at 2 μg/mL and incubated at 4° C. overnight. After incubation, the microplate was blocked with a PBS solution containing 1% BSA at 37° C. for 2 h. After blocking, the plate was washed three times and dried. The antibody was serially diluted to 7 concentrations in a gradient ratio of 1:3 on a dilution plate with 10 μg/mL as the starting concentration, and a blank control was set. Then an equal volume of 0.3 μg/mL human PD-1-mFc-biotin solution was added, and the system was mixed well and incubated at room temperature for 20 min. Then the mixture after reaction was added to the coated microplate, and the microplate was incubated for at 37° C. for 30 min. After incubation, the plate was washed three times with PBST and dried. SA-HRP (KPL, 14-30-00) working solution was added, and the plate was incubated at 37° C. for 30 min. After incubation, the plate was washed four times and patted dry. Then TMB (Neogen, 308177) was added in the dark for chromogenesis for 5 min, and a stop solution was added to terminate chromogenic reaction. Then the microplate was put into a microplate reader immediately, and the OD value of each well in the microplate was read at 450 nm. The data were analyzed and processed by SoftMax Pro 6.2.1.
[0279] The results are shown in
[0280] The results showed that P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1 and nivolumab (as a control) can effectively block the binding of the antigen human PD-1-mFc-biotin to its receptor human PD-L1-mFc in a dose-dependent manner. The EC.sub.50 values of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1 and nivolumab for blocking the binding of human PD-1-mFc-biotin to its ligand human PD-L1-mFc were 1.115 nM, 1.329 nM, 1.154 nM, 1.339 nM, 1.459 nM and 1.698 nM, respectively.
TABLE-US-00004 TABLE 4 Activity of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1 and nivolumab competing with human PD-L1-mFc for binding to human PD-1-mFc-biotin Antibody dilution Antigen coating: human PD-L1-mFc 2 μg/mL (μg/mL) P1D7V01 P1D7V02R P1D7V03 P1D7V04R 14C12 H1L1 Nivolumab 5 0.049 0.047 0.047 0.048 0.048 0.048 0.048 0.051 0.046 0.047 0.057 0.057 1:3 0.050 0.050 0.051 0.051 0.052 0.050 0.052 0.054 0.048 0.050 0.075 0.076 1:9 0.070 0.065 0.097 0.104 0.067 0.068 0.105 0.107 0.053 0.055 0.107 0.100 1:27 1.056 1.109 1.253 1.216 1.109 1.121 1.187 1.158 0.817 0.849 1.014 0.875 1:81 1.460 1.505 1.414 1.372 1.453 1.491 1.424 1.337 1.263 1.299 1.235 1.152 1:243 1.474 1.551 1.503 1.505 1.449 1.447 1.442 1.382 1.316 1.383 1.387 1.207 1:729 1.499 1.604 1.501 1.493 1.569 1.452 1.436 1.424 1.336 1.403 1.347 1.284 0 1.399 1.283 1.467 1.456 1.288 1.252 1.343 1.342 1.302 1.391 1.265 1.156 Human PD-1-mFc-biotin: 0.3 μg/mL .sup. Secondary antibody SA-HRP(1:4000) EC50(nM) 1.115 1.329 1.154 1.339 1.459 1.698
Example 10: Kinetic Parameters for Binding of Anti-CD73/Anti-PD-1 Bispecific Antibodies to Antigen Human PD-1-mFc Determined by Fortebio System
[0281] The sample dilution buffer was PBST, 0.1% BSA, pH 7.4. The antibody was immobilized on an AHC sensor at a concentration of 5 μg/mL with an immobilization height of about 0.4 nm. The sensor was equilibrated in a buffer for 60 s, and the binding of the immobilized antibody on the sensor to the antigen PD-1-mFc at concentrations of 0.6-50 nM (three-fold dilution) was determined in 120 s. The protein was dissociated in the buffer for 180 s. The detection temperature was 37° C., the detection frequency was 0.3 Hz, and the sample plate shaking rate was 1000 rpm. The data were analyzed by 1:1 model fitting to obtain affinity constants.
[0282] The determination results of the affinity constants of the humanized antibodies P1D7V01, 14C12H1L1 and nivolumab (as a control antibody) for human PD-1-mFc are shown in Table 5, and the detection results are shown in
TABLE-US-00005 TABLE 5 Determination of affinity constants of P1D7V01, 14C12H1L1 and nivolumab for PD-1-mFc Test antibodies KD (M) Kon (1/Ms) SE (kon) Kdis (1/s) S E (kdis) Rmax (nm) P1D7V01 1.76E−10 4.19E+05 1.52E+04 7.37E−05 3.12E−05 0.13-0.17 14C12H1L1 1.64E−10 4.55E+05 1.61E+04 7.47E−05 2.98E−05 0.24-0.28 Nivolumab 2.32E−10 5.85E+05 2.03E+04 1.36E−04 3.47E−05 0.02-0.14 K.sub.D is the affinity constant; K.sub.D = kdis/kon
Example 11: Kinetic Parameters for Binding of Anti-CD73/Anti-PD-1 Bispecific Antibodies to Antigen Human NT5E(1-552)-his Determined by Fortebio System
[0283] The sample dilution buffer was PBST, pH 7.4. The antibody was immobilized on a Protein A sensor at a concentration of 5 μg/mL with the immobilization time of about 15 s. The sensor was equilibrated in a buffer for 120 s, and the binding of the immobilized antibody on the sensor to the antigen human NT5E(1-552)-his at concentrations of 3.125-200 nM (two-fold dilution) was determined for 120 s. The protein was dissociated in the buffer for 600 s. The sensor was refreshed with 10 mM Gly solution, pH 1.5. The detection temperature was 37° C., the detection frequency was 0.6 Hz, and the sample plate shaking rate was 1000 rpm. The data were analyzed by 1:1 model fitting to obtain affinity constants.
[0284] The determination results of the affinity constants of the humanized antibodies P1D7V01 and MED19447 (as a control antibody) for human NT5E(1-552)-his are shown in Table 6, and the detection results are shown in
[0285] The above experimental results showed that the binding ability of P1D7V01 is comparable to that of MEDI9447, suggesting that the humanized antibody P1D7V01 has stronger binding ability to human NT5E(1-552)-his.
TABLE-US-00006 TABLE 6 Affinity constants of P1D7V01 and MEDI9447 for NTSE(1-552)-his Test antibodies KD (M) Kon (1/Ms) S E (kon) Kdis (1/s) S E (kdis) Rmax (nm) P1D7V01 2.29E−10 1.81E+05 2.30E+03 4.13E−05 4.54E−06 0.47-0.57 MEDI 9447 1.04E−10 2.34E+05 3.20E+03 2.44E−05 5.02E−06 0.59-0.82 K.sub.D is the affinity constant; K.sub.D = kdis/kon
Example 12: Binding Activity of Anti-CD73/Anti-PD-1 Bispecific Antibodies Determined by FACS
1. Binding Activity of Anti-CD73/Anti-PD-1 Bispecific Antibody to PD-1 on 293T-PD1 Membrane Surface Determined by FACS
[0286] 293T-PD1 cells in logarithmic growth phase were collected and transferred to a 1.5 mL centrifuge tube at 3×10.sup.5 cells/tube. 500 μL of PBSA was added, and the mixture was centrifuged at 5600 rpm for 5 min to remove the supernatant. 100 μL of antibodies diluted by PBSA (at the final concentrations of 100 nM, 33.33 nM, 11.11 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.14 nM and 0.05 nM, respectively) were added, respectively. The system was mixed gently and uniformly, and then was incubated on ice for 1 h. Then 500 μL of PBSA was added, and the mixture was centrifuged at 5600 rpm for 5 min to remove the supernatant. The 500-fold diluted FITC labeled goat anti-human IgG secondary antibody (Jackson, Cat. No. 109-095-098) was added to resuspend and mix well, and the mixture was incubated on ice in the dark for 0.5 h. 500 μL of PBSA was added, and the mixture was centrifuged at 5600 rpm for 5 min to remove the supernatant. At last, 200 μL of PBSA was added to resuspend cell precipitates, and the mixture was transferred to a flow tube for FACSCalibur detection.
[0287] The experimental results are shown in Table 7 and
[0288] Under the same experimental conditions, EC.sub.50 values of P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R binding to 293T-PD1 were 1.000 nM, 1.075 nM, 1.377 nM and 1.57 nM, respectively, and the EC.sub.50 value of 14C12H1L1 binding to 293T-PD1 was 2.111 nM.
[0289] The above experimental results showed that in the same experimental condition, P1D7V01, P1 D7V02R, P1 D7V03, P1D7V04R and 293T-PD1 all have binding activity superior to that of the PD1 single-target control antibody 14C12H1L1, suggesting that P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R have the activity of effectively binding to PD-1 on the 293T-PD1 membrane surface.
TABLE-US-00007 TABLE 7 Binding activity of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R and 14C12H1L1 to PD-1 on 293T-PD1 cell surface determined by FACS. Concentration (nM) 0.05 0.14 0.41 1.23 3.70 11.11 33.33 100.00 EC50 14C12H1L1 35.69 57.70 129.42 437.62 974.97 1384.91 1174.75 1500.20 2.111 P1D7V01 26.65 58.65 150.57 307.19 530.77 577.76 479.34 531.15 1.000 P1D7V02R 23.44 42.98 108.44 217.56 404.33 426.06 405.29 301.40 1.075 P1D7V03 24.05 49.21 118.03 289.08 384.62 459.40 808.38 343.48 1.377 P1D7V04R 24.79 52.43 112.56 288.79 466.72 1284.73 400.33 360.79 1.57.0
2. Binding Activity of Anti-CD73/Anti-PD-1 Bispecific Antibody to CD73 on MDA-MB-231 Membrane Surface Determined by FACS
[0290] MDA-MB-231 cells in logarithmic phase were digested with conventional trypsin and transferred to a 1.5 mL centrifuge tube at 3×10.sup.5 cells/tube. 500 μL of PBSA was added, and the mixture was centrifuged at 5600 rpm for 5 min to remove the supernatant. 100 μL of antibodies diluted by PBSA (at the final concentrations of 100 nM, 33.33 nM, 11.11 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.14 nM and 0.05 nM, respectively) were added, respectively. The system was mixed gently and uniformly, and then was incubated on ice for 1 h. Then 500 μL of PBSA was added, and the mixture was centrifuged at 5600 rpm for 5 min to remove the supernatant. The 500-fold diluted FITC labeled goat anti-human IgG secondary antibody (Jackson, Cat. No. 109-095-098) was added to resuspend and mix well, and the mixture was incubated on ice in the dark for 0.5 h. 500 μL of PBSA was added, and the mixture was centrifuged at 5600 rpm for 5 min to remove the supernatant. At last, 200 μL of PBSA was added to resuspend cell precipitates, and the mixture was transferred to a flow tube for FACSCalibur detection.
[0291] The experimental results are as shown in Table 8 and
[0292] The above experimental results showed that P1D7V01, P1D7V03, 19F3H2L3 and the reference drug MED19447 for the same target can specifically bind to CD73 on the MDA-MB-231 membrane surface in a dose-dependent manner. The binding activities of P1D7V01 and P1D7V03 were superior to that of 19F3H2L3, wherein P1D7V01 was superior to the reference drug MEDI9447 for the same target. It was suggested that P1D7V01 and P1D7V03 have the activity of effectively binding to CD73 on the MDA-MB-231 membrane surface.
TABLE-US-00008 TABLE 8 Binding activity of P1D7V01, P1D7V03, MEDI9447 and 19F3H2L3 to CD73 on the MDA-MB-231 cell surface determined by FACS Concentration (nM) 0.05 0.14 0.41 1.23 3.70 11.11 33.33 100.00 EC50 MEDI9447 32.59 64.38 157.75 388.51 697.80 829.29 930.26 878.04 1.589 19F3H2L3 25.95 45.13 102.56 189.07 466.87 658.72 843.09 639.57 2.773 P1D7V01 23.37 38.02 76.84 271.72 571.56 560.81 525.91 705.50 1.384 P1D7V03 16.96 44.05 106.39 233.32 457.61 525.37 576.19 677.66 2.009
Example 13: Detection of Inhibition of Anti-CD73/Anti-PD-1 Bispecific Antibody on Enzyme Activity of CD73 on Cell Membrane Surface
1. Detection of Inhibition of Anti-CD73/Anti-PD-1 Bispecific Antibody on Enzyme Activity of CD73 on MDA-MB-231 Membrane Surface
[0293] The experimental procedures were as follows. MDA-MB-231 cells in logarithmic phase in good condition were taken, resuspended in a serum-free RPMI-1640 culture solution, and then counted. The MDA-MB-231 cells were seeded into a 96-well plate at 2×10.sup.4 cells/100 μL/well. The antibody was diluted with the serum-free RPMI-1640 culture solution (serial 2.5-fold dilution). The antibody was added to the 96-well plate at 50 μL/well, and the plate was incubated at 37° C. for 1 h. After 1 h, 50 μL of 1200 μM RPMI-1640-diluted AMP (TCL, Cat. No. A0157) was added to each well. After 3 h, 25 μL of cell culture supernatant was taken and transferred to a new 96-well plate, and 25 μL of 100 μM ATP (TCL, Cat. No. A0158) was added to each well. 50 μL of CTG (CellTiterGlo, promega, Cat. No. G8641) chromogenic solution was added to each well for chromogenesis, and relative fluorescence intensity RLU was read by a multi-label microplate tester (PerkinElmer 2140-0020).
[0294] The experimental results are as shown in
[0295] The above experimental results showed that the added AMP can be converted into adenosine A without antibodies by the enzyme activity of CD73 on the MDA-MB-231 cell surface, and that after the addition of the antibody, the enzyme-catalyzed activity was decreased due to the binding of antibodies P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R to CD73, so that the AMP was not converted into adenosine A. It was suggested that the antibodies effectively inhibit the enzyme activity reaction thereof in a non-substrate competition mode and reduce the production of adenosine.
2. Detection of Inhibition of Anti-CD73/Anti-PD-1 Bispecific Antibody on Enzyme Activity of CD73 on U87-MG Membrane Surface
[0296] U87-MG cells in logarithmic phase in good condition were taken, resuspended in a serum-free RPMI-1640 culture solution, and then counted. The U87-MG cells were seeded into a 96-well plate at 2×10.sup.4 cells/100 μL/well. The antibody was diluted with the serum-free RPMI-1640 culture solution (serial 2.5-fold dilution). The antibody was added to the 96-well plate at 50 μL/well, and the plate was incubated at 37° C. for 1 h. After 1 h, 50 μL of 1200 μM RPMI-1640-diluted AMP was added to each well. After 3 h, 25 μL of cell culture supernatant was taken and transferred to a new 96-well plate, and 25 μL of 100 μM ATP was added to each well. 50 μL of CTG (CellTiterGlo) chromogenic solution was added to each well for chromogenesis, and relative fluorescence intensity RLU were read by a multi-label microplate tester (PerkinElmer 2140-0020).
[0297] The experimental results are as shown in
[0298] The above experimental results showed that the added AMP can be converted into adenosine A without antibodies by the enzyme activity of CD73 on the U87-MG cell surface, and that after the addition of the antibody, the enzyme-catalyzed function was decreased due to the binding of antibodies P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R to CD73, so that the AMP was not converted into adenosine A. It was suggested that the antibodies effectively inhibit the enzyme activity reaction thereof in a non-substrate competition mode and reduce the production of adenosine.
Example 14: Biological Activity of Anti-CD73/Anti-PD-1 Bispecific Antibodies for Promoting IFN-γ and IL-2 Secretion Determined by Mixed Lymphocyte Reaction (MLR)
1. Biological Activity of Anti-CD73/Anti-PD-1 Bispecific Antibodies for Promoting IFN-γ Secretion in Raji-PDL1 Mixed Lymphocyte Reaction System
[0299] Raji-PDL1 cells were normally subcultured. PBMCs were thawed, incubated with 10 mL of a 1640 complete medium, and stimulated with 0.5 μg/mL SEB (Dianotech, Cat. No. S010201) for two days. Raji-PDL1 cells were treated with 25 μg/mL MMC (Sigma, Cat. No. M4287) and placed in a 37° C. incubator for 1 h. PBMCs (peripheral blood mononuclear cells) stimulated with SEB for 2 days and Raji-PDL1 cells treated with MMC for 1 h were collected, washed twice with PBS, resuspended in the complete medium, and counted. The cells were separately added to a U-shaped 96-well plate at 100,000 cells/well. The antibodies were added according to the study design and cultured in an incubator for 3 days. After 3 days, the cell culture supernatant was collected and determined for IFN-γ by ELISA.
[0300] As shown in
2. Biological Activity of Anti-CD73/Anti-PD-1 Bispecific Antibodies for Promoting IL-2 Secretion in Raji-PDL1 Mixed Lymphocyte Reaction System
[0301] Raji-PDL1 cells were normally subcultured. PBMCs were thawed, incubated with 10 mL of a 1640 complete medium, and stimulated with SEB (0.5 μg/mL) for two days. Raji-PDL1 cells were treated with 25 μg/mL MMC and placed in a 37° C. incubator for 1 h. PBMCs (peripheral blood mononuclear cells) stimulated with SEB for 2 days and Raji-PDL1 cells treated with MMC for 1 h were collected, washed twice with PBS, resuspended in the complete medium, and counted. The cells were separately added to a U-shaped 96-well plate at 100,000 cells/well. The antibodies were added according to the study design and cultured for 3 days. The cell culture supernatant was collected and determined for IL-2 by ELISA.
[0302] As shown in
3. Bioactivity Detection of Anti-CD73/Anti-PD-1 Bispecific Antibody Promoting DC Mixed Lymph Reaction System to Secrete IFN-γ
[0303] PBMCs in normal human peripheral blood were isolated, resuspended in a complete medium, and seed into a culture dish. The culture dish was placed in an incubator overnight for culture. The suspended PBMCs were collected and removed. The adherent cells at the bottom of the dish were washed with PBS buffer and then subjected to DC maturation induction. 10 mL of RPMI1640 complete medium containing GM-CSF and IL-4 was added to each dish with GM-CSF and IL-4 each at a concentration of 1000 U/mL. The dishes were cultured in a 37° C., 5% carbon dioxide incubator for three days. Then half of the medium was exchanged, 1000 U/ml GM-CSF and IL-4 each was added, and the dishes were put into a 37° C., 5% carbon dioxide incubator for continuous culture for three days. After three days, half amount of the medium was changed again, 1000 U/mL GM-CSF and IL-4 each, and 100 U/mL TNF-α were added, and the dishes were cultured for another two days. PBMCs from other donors were freshly isolated and seeded into a 96-well plate at 100,000 cells/well after cell counting. DCs induced to mature were collected and washed once with the complete medium. The cells were counted and seed into the 96-well plate containing PBMC at 10,000 cells/well. The antibodies were added according to the study design. The system was mixed well and cultured in a 37° C., 5% carbon dioxide incubator for 5 days for co-culture. After 5 days, the cell culture supernatant was collected and quantitatively determined for IFN-γ by ELISA.
[0304] The results are as shown in
Biological Activity of Anti-CD73/Anti-PD-1 Bispecific Antibodies for Promoting IL-2 Secretion in DC Mixed Lymphocyte Reaction System
[0305] PBMCs in normal human peripheral blood were isolated, resuspended in a complete medium, and seed into a culture dish. The culture dish was placed in an incubator overnight for culture. The suspended PBMCs were collected and removed. The adherent cells at the bottom of the dish were washed with PBS buffer and then subjected to DC maturation induction. 10 mL of RPMI1640 complete medium containing GM-CSF and IL-4 was added to each dish with GM-CSF and IL-4 each at a concentration of 2000 U/mL. The dishes were cultured in a 37° C., 5% carbon dioxide incubator for three days. Then half amount of the medium was changed, 50 ng/mL IFN-γ and 100 ng/mL LPS were added, and the dishes were cultured for another two days. PBMCs from other donors were thawed and seeded into a 96-well plate at 100,000 cells/well after cell counting. DCs induced to mature were collected and washed once with the complete medium. The cells were counted and seed into the 96-well plate containing PBMCs at 10,000 cells/well. The antibodies were added according to the study design. The system was mixed well and cultured in a 37° C., 5% carbon dioxide incubator for 5 days for co-culture. After 5 days, the cell culture supernatant was collected and quantitatively determined for IL-2 by ELISA. The results are as shown in
Example 15: Preparation of Anti-PD-1/CD73 Bispecific Antibodies NTPDV1, NTPDV2, NTPDV3 and NTPDV4
[0306] The structural patterns of the bispecific antibodies NTPDV1, NTPDV2, NTPDV3 and NTPDV4 are in the Morrison format (IgG-scFv), i.e., C termini of two heavy chains of one IgG antibody are separately linked to the scFv fragment of another antibody via linkers. The components for the heavy and light chain design are shown in Table 9 below.
[0307] NTPDV1, NTPDV2, NTPDV3 and NTPDV4 are known as NTPDV1(hG1TM), NTPDV2(hG1TM), NTPDV3(hG1TM) and NTPDV4(hG1TM) herein and in Chinese Patent Application No. 202110270671.X, because the constant regions of the immunoglobulin moieties thereof have introduced amino acid mutations to eliminate their binding activity to FcγR.
TABLE-US-00009 TABLE 9 Sequence design of NTPDV1, NTPDV2, NTPDV3 and NTPDV4 Heavy chain Immuno- globulin Light moiety Linker scFv moiety chain NTPDV1 19F3H2 Linker1 14C12H1v-Linker 2- 19F3L3 (hGITM) 14C2L1v NTPDV2 14C12H1v-Linker 1- 14C2L1v NTPDV3 14C12H1v-Linker 2- 19F3L2 14C2L1v NTPDV4 14C12H1v-Linker1- 14C12L1v
[0308] In the Table 9 above:
[0309] Those with “V” label at lower right corner refer to the variable region of corresponding heavy chain or the variable region of corresponding light chain. For those without “V” label, the corresponding heavy or light chain is the full length comprising the constant region. The corresponding sequences described in the above examples are referred to for the amino acid sequences of these variable regions or the full length and the nucleotide sequences encoding them.
[0310] The amino acid sequence of linker1 consists of 4 repeats of (GGGGS), i.e., (GGGGS).sub.4 or (G.sub.4S).sub.4 (nucleotide sequence SEQ ID NO: 80, and amino acid sequence SEQ ID NO: 79).
[0311] The amino acid sequence of linker2 consists of 3 repeats of (GGGGS), i.e., (GGGGS).sub.4 or (G.sub.4S).sub.3 (nucleotide sequence SEQ ID NO: 82, and amino acid sequence SEQ ID NO: 81).
[0312] The 3 CDR sequences of the light chains 19F3L2 and 19F3L3 of the immunoglobulin moiety in NTPDV1, NTPDV2, NTPDV3 and NTPDV4 are identical to the light chain CDR sequences of 19F3.
[0313] The 3 CDR sequences of 19F3H2(hG1TM) of the immunoglobulin moiety in NTPDV1, NTPDV2, NTPDV3 and NTPDV4 are identical to the heavy chain CDR sequences of 19F3.
[0314] The CDR sequences of 14C12H1V-Linker2-14C12L1V and 14C12H1V-Linker1-14C12L1V of the scFv moiety in NTPDV1, NTPDV2, NTPDV3 and NTPDV4 are identical to the heavy chain CDRs and light chain CDRs of 14C12.
[0315] The amino acid sequences of the heavy chains of NTPDV2 and NTPDV4 are identical and are marked as NTPDH2/4 (SEQ ID NO: 83), and the nucleotide sequence of the heavy chains of NTPDV2 or NTPDV4 is SEQ ID NO: 84.
[0316] The amino acid sequences of the heavy chains of NTPDV1 and NTPDV3 are identical and are marked as NTPDH1/3 (SEQ ID NO: 85), and the nucleotide sequence of the heavy chains of NTPDV1 or NTPDV3 is SEQ ID NO: 86.
[0317] The 3 CDR sequences of 19F3L3 and 19F3L2 of the immunoglobulin moiety in NTPDV1, NTPDV2, NTPDV3 and NTPDV4 are identical to the three CDRs of the light chain of antibody 19F3.
[0318] The amino acid sequence of the light chain 19F3L3 of the immunoglobulin moiety in NTPDV1 or NTPDV2 is identical to the light chain sequence (SEQ ID NO: 28) of antibody 19F3H2L3(G1M), and the nucleotide sequence of the light chain 19F3L3 of the immunoglobulin moiety in NTPDV1 or NTPDV2 is SEQ ID NO: 27. The amino acid sequence of the light chain 19F3L2 of the immunoglobulin moiety in NTPDV3 or NTPDV4 is SEQ ID NO: 96, and the nucleotide sequence of the light chain 19F3L2 of the immunoglobulin moiety in NTPDV3 or NTPDV4 is SEQ ID NO: 100.
[0319] (1) NTPDV1, of which the heavy chain has an amino acid sequence set forth in SEQ ID NO: 85, the light chain has an amino acid sequence set forth in SEQ ID NO: 28, the linker 1 has an amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V has an amino acid sequence set forth in SEQ ID NO: 66, the linker 2 has an amino acid sequence set forth in SEQ ID NO: 81, and 14C12L1V has an amino acid sequence set forth in SEQ ID NO: 68;
[0320] (2) NTPDV2, of which the heavy chain has an amino acid sequence set forth in SEQ ID NO: 85, the light chain has an amino acid sequence set forth in SEQ ID NO: 28, the linker 1 has an amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V has an amino acid sequence set forth in SEQ ID NO: 66, the linker 1 has an amino acid sequence set forth in SEQ ID NO: 79, and 14C12L1V has an amino acid sequence set forth in SEQ ID NO: 68;
[0321] (3) NTPDV3, of which the heavy chain has an amino acid sequence set forth in SEQ ID NO: 85, the light chain has an amino acid sequence set forth in SEQ ID NO: 96, the linker 1 has an amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V has an amino acid sequence set forth in SEQ ID NO: 66, the linker 2 has an amino acid sequence set forth in SEQ ID NO: 81, and 14C12L1V has an amino acid sequence set forth in SEQ ID NO: 68; and
[0322] (4) NTPDV4, of which the heavy chain has an amino acid sequence set forth in SEQ ID NO: 85, the light chain has an amino acid sequence set forth in SEQ ID NO: 96, the linker 1 has an amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V has an amino acid sequence set forth in SEQ ID NO: 66, the linker 1 has an amino acid sequence set forth in SEQ ID NO: 79, and 14C12L1V has an amino acid sequence set forth in SEQ ID NO: 68.
Expression and Purification of Antibodies
[0323] The heavy chain cDNA sequences of NTPDV1 and NTPDV3, the heavy chain cDNA sequences of NTPDV2 and NTPDV4, and the light chain cDNA sequences thereof were separately cloned into vector pUC57simple (provided by Genscript) to obtain plasmids pUC57simple-NTPDH2/4, pUC57simple-NTPDH1/3, pUC57simple-19F3L3 and pUC57simple-19F3L2, respectively.
[0324] Plasmid pUC57simple-NTPDH2/4 and plasmid pUC57simple-19F3L3, plasmid pUC57simple-NTPDH2/4 and plasmid pUC57simple-19F3L2, plasmid pUC57simple-NTPDH1/3 and plasmid pUC57simple-19F3L3, and plasmid pUC57simple-NTPDH1/3 and plasmid pUC57simple-19F3L2 were digested (HindIII&EcoRI). The recovered heavy and light chains were separately subcloned into vector pcDNA3.1 to obtain plasmids pcDNA3.1-NTPDH2/4 and pcDNA3.1-19F3L3, plasmids pcDNA3.1-NTPDH2/4 and pcDNA3.1-19F3L2, plasmids pcDNA3.1-NTPDH1/3 and pcDNA3.1-19F3L3, and plasmids pcDNA3.1-NTPDH1/3 and pcDNA3.1-19F3L2. The recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture medium was separated by centrifugation at high speed, and the supernatant was concentrated and loaded onto a HiTrap MabSelect SuRe column. The protein was eluted in one step with an elution buffer. The target sample was isolated and the buffer was exchanged into PBS.
[0325] The purified antibodies NTPDV1, NTPDV2, NTPDV3 and NTPDV4 were obtained according to the expression and purification methods mentioned in the above preparation examples.
Example 16: Assay for Binding Activity of Anti-CD73/Anti-PD-1 Bispecific Antibodies to Antigens by ELISA
1. Binding Activity of NTPDV1, NTPDV2, NTPDV3 and NTPDV4 to Antigen PD-1-mFc Determined by ELISA
[0326] A microplate was coated with PD-1-mFc (0.5 μg/mL) and incubated at 4° C. overnight. Then the microplate coated with antigens was washed once with PBST, and then blocked with a PBS solution containing 1% BSA as blocking solution at 37° C. for 2 h. After blocking, the microplate was washed 3 times with PBST. The antibodies serially diluted with PBST solution (the dilution gradients for the antibody are shown in Table 2) were added. The microplate containing the test antibodies was incubated at 37° C. for 30 min, and then washed 3 times with PBST. After washing, HRP-labeled goat anti-human IgG Fc (Jackson, Cat. No. 109-035-098) secondary antibody working solution diluted in a ratio of 1:5000 was added, and then the microplate was incubated at 37° C. for 30 min. After incubation, the plate was washed 4 times with PBST, TMB (Neogen, 308177) was added in the dark for chromogenesis for 8 min, and then a stop solution was added to terminate chromogenic reaction. The microplate was put into a microplate reader immediately, and the OD value of each well in the microplate was read at 450 nm. The data were analyzed and processed by SoftMax Pro 6.2.1.
[0327] The results are shown in Table 10. It can be seen that NTPDV1, NTPDV2, NTPDV3 and NTPDV4 can effectively bind to the antigen PD-1-mFc in a dose-dependent manner. The absorbance intensity of each dose is shown in Table 10. By quantitative analysis of the absorbance of the bound antibodies, the binding efficiency EC.sub.50 values of the antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4 and 14C12H1L1(hG1TM) (as a control) obtained by curve fitting were 0.101 nM, 0.119 nM, 0.110 nM, 0.123 nM and 0.031 nM, respectively.
[0328] The above experimental results showed that in the same experimental condition, the binding activities of NTPDV1, NTPDV2, NTPDV3 and NTPDV4 to PD-1-mFc are comparable to that of the reference drugs 14C12H1L1(hG1TM) for the same target, suggesting that NTPDV1, NTPDV2, NTPDV3 and NTPDV4 have the activity of effectively binding to PD-1-mFc.
TABLE-US-00010 TABLE 10 Binding of NTPDV1, NTPDV2, NTPDV3, NTPDV4 and 14C12H1L1(hG1TM) to PD-1-mFc determined by ELISA Antibody dilution concentration Antigen coating PD-1-mFc 0.5 μg/mL (nM) NTPDV1 NTPDV2 NTPDV3 NTPDV4 14C12H1L1 (hG1TM) 5 2.222 2.231 2.249 2.249 2.204 2.171 2.244 2.289 2.399 2.524 1:3 2.206 2.195 2.160 2.223 2.211 2.167 2.222 2.209 2.361 2.441 1:9 2.039 2.027 1.922 2.013 2.010 1.971 1.920 1.944 2.273 2.401 1:27 1.514 1.530 1.425 1.446 1.489 1.414 1.417 1.464 2.071 2.091 1:81 0.880 0.844 0.804 0.802 0.829 0.807 0.794 0.804 1.644 1.691 1:243 0.400 0.396 0.363 0.369 0.386 0.381 0.373 0.379 1.006 1.059 1:729 0.183 0.184 0.176 0.174 0.175 0.174 0.170 0.178 0.503 0.531 0 0.053 0.056 0.055 0.056 0.056 0.056 0.054 0.059 0.057 0.059 Secondary antibody HRP-labeled goat anti-human IgG Fc (1:5000) EC50 (nM) 0.101 0.119 0.110 0.123 0.031
2. Binding Activity of NTPDV1, NTPDV2, NTPDV3 and NTPDV4 to Antigen Human NT5E-Biotin Determined ELISA
[0329] A microplate was coated with streptavidin SA (2 μg/mL) and then incubated at 4° C. overnight. After incubation, the microplate coated with streptavidin was washed once with PBST, and blocked with a PBS solution containing 1% BSA as a microplate blocking solution at 37° C. for 2 h. After blocking, the microplate was washed 3 times with PBST. Then, 0.5 μg/mL antigen human NT5E-Biotin was added and incubated at 37° C. for 30 min. Then, the plate was washed 3 times with PBST. The antibodies serially diluted with PBST solution (the dilution gradients for the antibody are shown in Table 11) were added to wells of the microplate. The microplate containing the test antibodies was incubated at 37° C. for 30 min, and then washed 3 times with PBST. After washing, HRP-labeled goat anti-human IgG Fc (Jackson, Cat. No. 109-035-098) secondary antibody working solution diluted in a ratio of 1:5000 was added, and the microplate was incubated at 37° C. for 30 min. After incubation, the plate was washed 4 times with PBST, TMB (Neogen, 308177) was added in the dark for chromogenesis for 7 min, and then a stop solution was added to terminate chromogenic reaction. The microplate was put into a microplate reader immediately, and the OD value of each well in the microplate was read at 450 nm. The data were analyzed and processed by SoftMax Pro 6.2.1.
[0330] The results are shown in Table 11. It can be seen that NTPDV1, NTPDV2, NTPDV3, NTPDV4 and 19F3H2L3(hG1M) can effectively bind to the antigen human NTSE-biotin in a dose-dependent manner (the absorbance intensity of each dose is shown in Table 11). By quantitative analysis of the absorbance of the bound antibodies, the binding efficiency EC.sub.50 values of the antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4 and 19F3H2L3(hG1M) (as a control antibody) obtained by curve fitting were 0.079 nM, 0.082 nM, 0.084 nM, 0.077 nM and 0.029 nM, respectively.
[0331] The above experimental results showed that in the same experimental condition, the binding activities of bispecific antibodies NTPDV1, NTPDV2, NTPDV3 and NTPDV4 to human NT5E-biotin are comparable to that of the reference drug 19F3H2L3(hG1M) for the same target, suggesting that NTPDV1, NTPDV2, NTPDV3 and NTPDV4 have the activity of effectively binding to human NTSE-biotin.
TABLE-US-00011 TABLE 11 Binding of NTPDV1, NTPDV2, NTPDV3, NTPDV4 and 19F3H2L3(hG1M) to human NT5E-biotin determined by ELISA Antibody dilution Antigen coating SA: 2 μg/mL concentration Human NT5E-biotin (0.5 μg/mL) (nM) NTPDV1 NTPDV2 NTPDV3 NTPDV4 19F3H2L3 (hG1M) 5 2.286 2.300 2.322 2.333 2.364 2.286 2.298 2.291 2.371 2.383 1:3 2.412 2.347 2.450 2.409 2.381 2.412 2.427 2.421 2.420 2.506 1:9 2.227 2.177 2.226 2.238 2.230 2.081 2.186 2.222 2.322 2.345 1:27 1.795 1.729 1.744 1.746 1.768 1.698 1.734 1.759 2.125 2.162 1:81 1.071 1.070 1.103 1.058 1.068 1.025 1.095 1.125 1.649 1.752 1:243 0.528 0.527 0.540 0.551 0.552 0.534 0.552 0.611 1.067 1.143 1:729 0.273 0.274 0.271 0.275 0.279 0.267 0.275 0.309 0.542 0.601 0 0.092 0.092 0.090 0.087 0.092 0.090 0.092 0.095 0.112 0.206 Secondary antibody HRP-labeled goat anti-human IgG Fc (1:5000) EC50 (nM) 0.079 0.082 0.084 0.077 0.029
Example 17: Activity of Anti-CD73/Anti-PD-1 Bispecific Antibodies Completing with Human PD-L1-mFc for Binding to Human PD-1-mFc-Biotin Determined by Competitive ELISA
[0332] A microplate was coated with human PD-L1-mFc (PD-L1 Genbank ID: NP 054862.1, mFc SEQ ID NO: 89) at 2 μg/mL and incubated at 4° C. overnight. After incubation, the microplate was blocked with a PBS solution containing 1% BSA at 37° C. for 2 h. After blocking, the plate was washed once and dried. The antibody was serially diluted to 7 concentrations in a gradient ratio of 1:3 on a dilution plate with 10 μg/mL as the starting concentration, and a blank control was set. Then an equal volume of 0.3 μg/mL human PD-1-mFc-biotin solution was added, and the system was mixed well and incubated at room temperature for 10 min. Then the mixture after reaction was added to the coated microplate, and the microplate was incubated for at 37° C. for 30 min. After incubation, the plate was washed three times with PBST and dried. SA-HRP (KPL, 14-30-00) working solution was added, and the plate was incubated at 37° C. for 30 min. After incubation, the plate was washed four times and patted dry. Then TMB (Neogen, 308177) was added in the dark for chromogenesis for 5 min, and a stop solution was added to terminate chromogenic reaction. Then the microplate was put into a microplate reader immediately, and the OD value of each well in the microplate was read at 450 nm. The data were analyzed and processed by SoftMax Pro 6.2.1.
[0333] The OD values for all the dosages are shown in Table 12. By quantitative analysis of the absorbance intensity of the bound antibody, the curve simulation was performed to give the binding efficiency EC.sub.50 of the antibody (Table 12).
[0334] The results showed that NTPDV1, NTPDV2, NTPDV3, NTPDV4 and 14C12H1L1(hG1TM) (as a control) can effectively block the binding of the antigen human PD-1-mFc-biotin to its receptor human PD-L1-mFc in a dose-dependent manner. The EC.sub.50 values of NTPDV1, NTPDV2, NTPDV3, NTPDV4 and 14C12H1L1(hG1TM) for blocking the binding of human PD-1-mFc-biotin to its ligand human PD-L1-mFc were 2.249 nM, 2.253 nM, 2.332 nM, 2.398 nM, 2.216 nM and 2.231 nM, respectively.
TABLE-US-00012 TABLE 12 Activity of NTPDV1, NTPDV2, NTPDV3, NTPDV4 and 14C12H1L1(hG1TM) competing with human PD-L1-mFc for binding to human PD-1-mFc-biotin Antibody Antigen coating: human PD-L1-mFc 2 μg/mL dilution (nM) NTPDV1 NTPDV2 NTPDV3 NTPDV4 14C12H1L1 (hG1TM) 50 0.086 0.082 0.084 0.081 0.077 0.087 0.076 0.076 0.081 0.077 1:3 0.083 0.083 0.083 0.084 0.083 0.085 0.084 0.090 0.087 0.082 1:9 0.202 0.195 0.204 0.204 0.195 0.183 0.189 0.214 0.120 0.119 1:27 0.604 0.616 0.564 0.593 0.628 0.598 0.521 0.584 0.576 0.634 1:81 0.837 0.821 0.790 0.809 0.816 0.884 0.782 0.768 0.797 0.871 1:243 0.946 0.938 0.822 0.929 0.895 0.851 0.926 0.891 0.880 0.934 1:729 0.956 0.938 0.884 0.878 0.928 0.923 0.854 0.902 0.901 0.964 0 1.012 0.952 0.891 0.966 0.918 0.983 0.919 0.966 0.904 0.954 Human PD-1-mFc-biotin: 0.3 μg/mL .sup. Secondary antibody SA-HRP (1:4000) .sup. EC50 (nM) 2.253 2.332 2.398 2.216 2.231
Example 18: Kinetic Parameters for Binding of Anti-CD73/Anti-PD-1 Bispecific Antibodies to Antigen Human PD-1-mFc Determined by Fortebio System
[0335] The sample dilution buffer was PBS (0.02% Tween-20, 0.1% BSA, pH 7.4). PD1-mFc was immobilized on the AMC sensor at a concentration of 5 μg/mL with an immobilization height of about 0.1 nM (time 60 s). The sensor was equilibrated in a buffer for 60 s, and the binding of the immobilized PD1-mFc on the sensor to the antibodies at concentrations of 0.62-50 nM (three-fold dilution) was determined for 120 s. The protein was dissociated in the buffer for 300 s. The detection temperature was 30° C., the detection frequency was 0.3 Hz, and the sample plate shaking rate was 1000 rpm. The data were analyzed by 1:1 model fitting to obtain affinity constants.
[0336] The determination results of the affinity constants of humanized antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4 and nivolumab (as control antibody) for human PD-1-mFc are shown in Table 13, and the detection results are shown in
TABLE-US-00013 TABLE 13 Affinity constants of 14C12H1L1(hG1TM), NTPDV1, NTPDV2, NTPDV3, NTPDV4 and nivolumab for PD-1-mFc Test antibodies KD (M) Kon (1/Ms) S E (kon) Kdis (1/s) S E (kdis) Rmax (nm) 14C12H1L1(hG1TM) 4.45E−11 1.18E+06 5.74E+04 5.23E−05 4.47E−05 0.38-0.57 Nivolumab 2.26E−10 1.52E+06 7.79E+04 3.43E−04 4.66E−05 0.25-0.37 NTPDV1 1.40E−10 6.09E+05 2.01E+04 8.55E−05 3.18E−05 0.38-0.52 NTPDV2 7.39E−11 6.83E+05 2.47E+04 5.05E−05 3.50E−05 0.40-0.55 NTPDV3 1.25E−10 6.68E+05 2.52E+04 8.35E−05 3.44E−05 0.38-0.50 NTPDV4 1.13E−11 5.35E+05 1.59E+04 6.03E−06 2.77E−05 0.35-0.47 K.sub.D is the affinity constant; K.sub.D = kdis/kon
Example 19: Kinetic Parameters for Binding of Anti-CD73/Anti-PD-1 Bispecific Antibodies to Antigen Human NTSE (1-552)-his Determined by Fortebio System
[0337] The sample dilution buffer was PBS (0.02% Tween-20, 0.1% BSA, pH 7.4). HNT5E(1-552)-his was immobilized on the HIS1K sensor at a concentration of 5 μg/mL with an immobilization height of about 0.4 nM (time 50 s). The sensor was equilibrated in a buffer for 60 s, and the binding of the immobilized HNT5E(1-552)-his on the sensor to the antibodies at concentrations of 0.31-25 nM (three-fold dilution) was determined for 100 s. The protein was dissociated in the buffer for 180 s. The detection temperature was 30° C., the detection frequency was 0.3 Hz, and the sample plate shaking rate was 1000 rpm. The data were analyzed by 1:1 model fitting to obtain affinity constants.
[0338] The determination results of the affinity constants of the humanized antibodies 19F3H2L3(hG1M) (as a control antibody), NTPDV1, NTPDV2, NTPDV3 and NTPDV4 for human NTSE(1-552)-his are shown in Table 14, and the detection results are shown in FIGS. 24-27. The affinity constants of the humanized antibodies NTPDV1, NTPDV2, NTPDV3 and NTPDV4 for human NTSE(1-552)-his were 3.29E-11 M, 2.88E-11 M, 7.92E-11 M and 5.77E-11 M, respectively.
[0339] The above experimental results showed that the binding ability of 19F3H2L3(hG1M), NTPDV1, NTPDV2, NTPDV3 and NTPDV4 is comparable, suggesting that the humanized antibodies NTPDV1, NTPDV2. NTPDV3 and NTPDV4 has stronger binding ability to human NTSE(1-552)-his.
TABLE-US-00014 TABLE 14 Affinity constants of 19F3H2L3(hG1M), NTPDV1, NTPDV2, NTPDV3 and NTPDV4 for human NTSE(1-552)-his Test antibodies KD (M) kon (1/Ms) S E (kon) kdis (1/s) S E (kdis) Rmax (nm) 19F3H2L3 (hG1M) 4.05E−11 1.16E+06 6.50E+04 4.68E−05 8.96E−05 0.36-0.77 NTPDV1 3.29E−11 1.16E+06 3.27E+04 3.80E−05 4.63E−05 0.49-0.61 NTPDV2 2.88E−11 1.27E+06 3.84E+04 3.66E−05 4.88E−05 0.63-0.71 NTPDV3 7.92E−11 1.28E+06 4.08E+04 1.02E−04 5.29E−05 0.55-0.65 NTPDV4 5.77E−11 1.23E+06 3.82E+04 7.09E−05 4.95E−05 0.52-0.63 K.sub.D is the affinity constant; K.sub.D = kdis/kon
Example 20: Detection of Inhibition of Anti-CD73/Anti-PD-1 Bispecific Antibody on Enzyme Activity of CD73 on U87-MG Cell Membrane Surface
[0340] U87-MG cells in logarithmic phase in good condition were taken, resuspended in a serum-free RPMI-1640 culture solution, and then counted. The U87-MG cells were seeded into a 96-well plate at 2.5×10.sup.4 cells/60 μL/well. The antibody was diluted according to the study design with the serum-free RPMI-1640 culture solution. The antibody was added to the 96-well plate at 60 μL/well, and the plate was incubated at 37° C. for 1 h. After 1 h, 60 μL of 600 μM RPMI-1640-diluted AMP was added to each well. After 3 h, 100 μL of cell culture supernatant was taken and transferred to a new 96-well plate, 40 μL of CTG (CellTiterGlo) chromogenic solution was added to each well, and the plate was placed in the dark at room temperature for 5 min. After 5 min, 10 μL of 300 μM ATP was added to each well for chromogenesis. Relative fluorescence intensity RLU were read by a multi-label microplate tester (PerkinElmer 2140-0020).
[0341] The experimental results are as shown in Table 15 and
[0342] The above experimental results showed that the added AMP can be converted into adenosine A without antibodies by the enzyme activity of CD73 on the U87-MG cell surface, and that after the addition of the antibody, the enzyme-catalyzed function was decreased due to the binding of antibody NTPDV2 to CD73, so that the AMP was not converted into adenosine A. It was suggested that the antibodies effectively inhibit the enzyme activity reaction thereof in a non-substrate competition mode and reduce the production of adenosine.
TABLE-US-00015 TABLE 15 Detection of inhibition of anti-CD73/anti-PD-1 bispecific antibody on enzyme activity of CD73 on U87-MG cell membrane surface Name of antibody MEDI9447 19E3H2L3(hG1M) NTPDV2 IC50 (nM) 0.1189 0.3647 0.5551
Example 21: Biological Activity of Anti-CD73/Anti-PD-1 Bispecific Antibodies for Promoting IFN-γ and IL-2 Secretion Determined by Mixed Lymphocyte Reaction (MLR)
1. Biological Activity of Anti-CD73/Anti-PD-1 Bispecific Antibodies for Promoting IFN-γ Secretion in Raji-PDL1 Mixed Lymphocyte Reaction System
[0343] Raji-PDL1 cells were normally subcultured. PBMCs were thawed, incubated with 10 mL of a 1640 complete medium, and stimulated with 0.5 μg/mL SEB (Dianotech, Cat. No. S010201) for two days. Raji-PDL1 cells were treated with 25 μg/mL MMC (mitomycin C, Stressmarq, catalog: SIH-246, Cat. No. SM286474) and placed in a 37° C. incubator for 1 h. PBMCs (peripheral blood mononuclear cells) stimulated with SEB for 2 days and Raji-PDL1 cells treated with MMC for 1 h were collected, washed twice with PBS, resuspended in the complete medium, and counted. The cells were separately added to a U-shaped 96-well plate at 1×10.sup.5 cells/well. The antibodies were added according to the study design and cultured in an incubator for 3 days. After 3 days, the cell culture supernatant was collected and determined for IFN-γ by ELISA.
[0344] As shown in
2. Biological Activity of Anti-CD73/Anti-PD-1 Bispecific Antibodies for Promoting IL-2 Secretion in Raji-PDL1 Mixed Lymphocyte Reaction System
[0345] Raji-PDL cells were normally subcultured. PBMCs were thawed, incubated with 10 mL of a 1640 complete medium, and stimulated with SEB (0.5 μg/mL) for two days. Raji-PDL1 cells were treated with 25 μg/mL MMC and placed in a 37° C. incubator for 1 h. PBMCs (peripheral blood mononuclear cells) stimulated with SEB for 2 days and Raji-PDL1 cells treated with MMC for 1 h were collected, washed twice with PBS, resuspended in the complete medium, and counted. The cells were separately added to a U-shaped 96-well plate at 1×10.sup.5 cells/well. The antibodies were added according to the study design and cultured for 3 days. The cell culture supernatant was collected and determined for IL-2 by ELISA.
[0346] As shown in
Example 22: Experiment of Inhibition of Tumor Growth in Vivo by Anti-CD73/Anti-PD-1 Bispecific Antibody
[0347] In order to determine the anti-tumor activity in vivo of the anti-CD73/anti-PD-1 bispecific antibody, firstly, MC38-hPDL1/hCD73 cells (from GemPharmatech Co., Ltd.) were subcutaneously inoculated into female C57BL6-hPD1hPDL1hCD73 triple-transgenic mice aged 5-7 weeks (from GemPharmatech Co., Ltd.). The modeling and specific administration methods are shown in Table 16. Ater the administration, the length and width of each group of tumors were measured, and the tumor volume was calculated.
TABLE-US-00016 TABLE 16 Study design Grouping n Tumor xenograft Condition of administration Isotype 8 MC38-hPDL1/hCD73, Isotype control antibody hIgG, control 1 million cells/mouse, 10 mg/kg s.c. Twice weekly, for three weeks 19F3H2L3 8 19F3H2L3(hG1M)m 10 mg/kg (hG1M) Twice weekly, for three weeks NTPDV2 8 NTPDV2, 13.3 mg/kg Twice weekly, for three weeks NTPDV2 8 NTPDV2, 1.33 mg/kg Twice weekly, for three weeks
The experimental results are as shown in
Experimental Example 23: Effective Elimination of Bispecific Immune Checkpoint Inhibitor PD-1/CD73 Bispecific Antibody-Mediated IL-8 and IL-6 Secretions in Human Macrophages by the Amino Acid Mutations of Fc Segments
[0348] HPMMs were prepared by induction of PBMCs. PBMCs used in this study were isolated and prepared in Zhongshan Akesobio Co. Ltd., with informed consent of the donor.
[0349] Ficoll-Paque PLUS lymphocyte isolation solution (GE, Cat. No. 17-1440-03); RPMI 1640 (Gibco, Cat. No. 22400-105); CHO-K1-PD1 cells (constructed by Zhongshan Akesobio Co. Ltd.); U87-MG cells (cells from ATCC, purchased from Beijing Zhongyuan Ltd.); FBS (Fetal Bovine Serum, Excell bio, Cat. No, FSP 500); human IFN-γ protein (Sinobio, Cat. No. 11725-HNAS-100); LPS (lipopolysaccharide) (Sigma, Cat. No. L4391); a 96-well cell culture plate (Corning).
[0350] Healthy human PBMCs were isolated according to the instructions of insolation solution Ficoll-Paque™ Plus reagent and resuspended in a 1640 medium containing 2% FBS. The plate was incubated in a 5% CO.sub.2 cell incubator at 37° C. After 2 h, the supernatant was removed, and the adherent cells were washed twice with PBS and 1640 complete medium (containing 10% FBS) and 100 ng/mL human M-CSF were added for 7 days of induction. On day 3 and day 5, the medium was exchanged, and M-CSF was added to induce HPMM. On day 7, after induction of HPMM, the cells were collected, adjusted to the concentration of 0.1 million cells/mL with the complete medium, and filled into a 96-well plate. The recombinant human IFN-γ (50 ng/mL) was added, and incubated in an incubator for 24 h. After 24 h, CHO-K1-PD1 cells expressing human PD-1 or U87-MG cells constitutively expressing human CD73 in logarithmic phase were collected. The cells were resuspended and then adjusted to the concentration of 0.3 million cells/mL with the complete medium. The antibody was diluted with the complete medium to working concentrations of 25 nM, 2.5 nM and 0.25 nM. An isotype control antibody and a blank control were designed simultaneously. The supernatant in the 96-well plate was removed, the CHO-K1-PD1 or U87-MG cell suspension and antibodies were added (with a final volume of 200 μL). The system was mixed well and incubated in an incubator for 24 h. The mixture was centrifuged at 500×g for 5 min, the supernatant was collected, and the secretion amounts of IL-8 and IL-6 were determined with a Dakewe kit. LPS was used as a positive control and was adjusted to a concentration of 100 ng/mL with the complete medium in the experiment.
[0351] In this example, the co-culture of CHO-K1-PD1 and U87-MG cells as target cells with HPMM induced the activation of HPMM, and after the activated HPMM was linked to the target cells by antibody Fab, the Fc fragment of the antibody interacted with FcγR on HPMM, causing the secretion of cytokine by HPMM.
3. Experimental Results
[0352] The results are shown in
[0353] The results showed that compared with the wild-type IgG1 subtype PD-1 antibody or CD73 antibody, the anti-PD-1/CD73 bispecific antibody carrying the Fc fragment mutation is able to effectively eliminate the secretion of IL-6 and/or IL-8 in immune cells.
[0354] The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited to the embodiments. Those skilled in the art can make various equivalent modifications or replacements without violating the spirit of the present invention. These equivalent modifications or replacements are included in the scope defined by the claims of the present application.
TABLE-US-00017 SEQUENCE LISTING Nucleotide sequence of 19F3 heavy chain variable region: (SEQ ID NO: 1, with CDR sequences underlined) GAGGTGCAGCTGCAGCAGTCCGGACCAGAGCTGGTGAAGCCTGGCGCCTCCATGCGGATGTCTT GTAAGGCCTCTGGCTACAGCTTCACCGGCTATACAATGAACTGGGTGAAGCAGTCTCACGGCA AGAATCTGGAGTGGATCGGCCTGATCAACCCTTACAATGCCGGCACCAGCTATAACCAGAAGT TTAAGGGCAAGGCCACCCTGACAGTGGACAAGAGCTCCTCTACCGCCTACATGGAGCTGCTGTCC CTGACATCTGAGGATAGCGCCGTGTACTATTGCGCCCGGTCCGAGTACAGATATGGCGGCGAC TACTTTGATTATTGGGGCCAGGGCACCACACTGACAGTGAGCTCC Amino acid sequence of 19F3 heavy chain variable region: (SEQ ID NO: 2, with CDR sequences underlined) EVQLQQSGPELVKPGASMRMSCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNAGTSYNQKFK GKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSEYRYGGDYFDYWGQGTTLTVSS HCDR1 of 19F3: (SEQ ID NO: 3) GYSFTGYT HCDR2 of 19F3: (SEQ ID NO: 4) INPYNAGT HCDR3 of 19F3: (SEQ ID NO: 5) ARSEYRYGGDYFDY Nucleotide sequence of 19F3 light chain variable region: (SEQ ID NO: 6, with CDR sequences underlined) GACATCGTGATGACCCAGTCTCCAAGCTCCCTGGCAATGAGCGTGGGACAGAAGGTGACAATGT CTTGTAAGTCTAGCCAGAGCCTGCTGAACTCCTCTAATCAGAAGAACTACCTGGCCTGGTATC AGCAGAAGCCAGGCCAGTCTCCCAAGCTGCTGGTGTACTTTGCCAGCACCAGGGAGTCCGGAGT GCCTGACAGATTCATCGGCTCCGGCTCTGGCACAGACTTCACCCTGACAATCAGCTCCGTGCAGG CAGAGGACCTGGCAGATTATTTCTGCCAGCAGCACTACGACACCCCTTATACATTTGGCGGCG GCACCAAGCTGGAGATCAAG Amino acid sequence of 19F3 light chain variable region: (SEQ ID NO: 7, with CDR sequences underlined) DIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPD RFIGSGSGTDFTLTISSVQAEDLADYFCQQHYDTPYTFGGGTKLEIK LCDR1 amino acid sequence of 19F3: (SEQ ID NO: 8) QSLLNSSNQKNY LCDR2 amino acid sequence of 19F3: (SEQ ID NO: 9) FAS LCDR3 amino acid sequence of 19F3: (SEQ ID NO: 10) QQHYDTPYT Amino add sequences of 19F3 heavy chain framework regions: FR-H1: (SEQ ID NO: 11) EVQLQQSGPELVKPGASMRMSCKAS FR-H2: (SEQ ID NO: 12) MNWVKQSHGKNLEWIGL FR-H3: (SEQ ID NO: 13) SYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYC FR-H4: (SEQ ID NO: 14) WGQGTTLTVSS Amino acid sequences of 19F3 light chain framework regions: FR-L1: (SEQ ID NO: 15) DIVMTQSPSSLAMSVGQKVTMSCKSS FR-L2: (SEQ ID NO: 16) LAWYQQKPGQSPKLLVY FR-L3: (SEQ ID NO: 17) TRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFC FR-L4: (SEQ ID NO: 18) FGGGTKLEIK Nucleotide sequence of 19F3H2: (SEQ ID NO: 19, with CDR sequences underlined) CAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGGTGAAGCCAGGAGCCTCTGTGAAGGTGAGCT GTAAGGCCAGCGGCTACTCCTTCACCGGCTATACAATGAACTGGGTGAGGCAGGCACCAGGAC AGAATCTGGAGTGGATCGGCCTGATCAACCCTTACAATGCCGGCACCTCTTATAACCAGAAGTT TCAGGGCAAGGTGACCCTGACAGTGGACAAGTCCACCTCTACAGCCTACATGGAGCTGAGCTCCC TGCGGAGCGAGGATACAGCCGTGTACTATTGCGCCCGGTCCGAGTACAGATATGGCGGCGAC TACTTTGATTATTGGGGCCAGGGCACCACACTGACCGTGTCTAGC Amino acid sequence of 19F3H2: (SEQ ID NO: 20, with CDR sequences underlined) QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAPGQNLEWIGLINPYNAGTSYNQKFQ GKVTLTVDKSTSTAYMELSSLRSEDTAVYYCARSEYRYGGDYFDYWGQGTTLTVSS Nucleotide sequence of 19F3L2: (SEQ ID NO: 21, with CDR sequences underlined) GACATCGTGATGACCCAGTCCCCAAGCTCCCTGGCCGTGTCTGTGGGAGAGCGGGTGACAATCTC CTGTAAGTCTAGCCAGTCTCTGCTGAACTCCTCTAATCAGAAGAACTACCTGGCCTGGTATCA GCAGAAGCCCGGCCAGGCCCCTAAGCTGCTGATCTACTTCGCCTCTACCAGGGAGAGCGGAGTG CCAGACAGATTCTCTGGCAGCGGCTCCGGCACAGACTTCACCCTGACAATCAGCTCCGTGCAGGC AGAGGACGTGGCAGATTACTATTGCCAGCAGCACTACGATACCCCCTATACATTTGGCGGCGG CACCAAGCTGGAGATCAAG Amino acid sequence of 19F3L2: (SEQ ID NO: 22, with CDR sequences underlined) DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQKPGQAPKLLIYFASTRESGVPDRF SGSGSGTDFTLTISSVQAEDVADYYCQQHYDTPYTFGGGTKLEIK Nucleotide sequence of 19F3L3: (SEQ ID NO: 23, with CDR sequences underlined) GACATCGTGATGACCCAGTCCCCAAGCTCCCTGGCCGTGTCTGTGGGAGAGCGGGTGACAATCTC CTGTAAGTCTAGCCAGTCTCTGCTGAACTCCTCTAATCAGAAGAACTACCTGGCCTGGTATCA GCAGAAGCCCGGCCAGGCCCCTAAGCTGCTGATCTACTTCGCCTCTACCAGGGAGAGCGGAGTG CCAGACAGATTCTCTGGCAGCGGCTCCGGCACAGACTTCACCCTGACAATCAGCTCCCTGCAGGC AGAGGACGTGGCCGTGTACTATTGCCAGCAGCACTACGATACCCCCTATACATTTGGCGGCGG CACCAAGCTGGAGATCAAG Amino acid sequence of 19F3L3: (SEQ ID NO: 24, with CDR sequences underlined) DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQKPGQAPKLLIYFASTRESGVPDRF SGSGSGTDFTLTISSLQAEDVAVYYCQQHYDTPYTFGGGTKLEIK Nucleotide sequence of 19F3H2L3(G1M) heavy chain (SEQ ID NO: 25, with non-variable region sequences underlined) CAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGGTGAAGCCAGGAGCCTCTGTGAAGGTGAGCT GTAAGGCCAGCGGCTACTCCTTCACCGGCTATACAATGAACTGGGTGAGGCAGGCACCAGGACA GAATCTGGAGTGGATCGGCCTGATCAACCCTTACAATGCCGGCACCTCTTATAACCAGAAGTTTC AGGGCAAGGTGACCCTGACAGTGGACAAGTCCACCTCTACAGCCTACATGGAGCTGAGCTCCCT GCGGAGCGAGGATACAGCCGTGTACTATTGCGCCCGGTCCGAGTACAGATATGGCGGCGACTAC TTTGATTATTGGGGCCAGGGCACCACACTGACCGTGTCTAGCGCCTCCACAAAG ggG ccC agc gtg ttt cct ctc gcc ccc tcc tcc aaa a gcaccagcggaggaaccgctgctctcggatgtctggtgaaggactacttccctgaacccgtcaccgtgagctgg aatagcggcgctctgacaagcggagtccatacattccctgctgtgctgcaaagcagcggactctattccctgtc cagcgtcgtcacagtgcccagcagcagcctgggcacccagacctacatctgtaacgtcaaccacaagccctcca acaccaaggtggacaagaaagtggagcccaaatcctgcgacaagacacacacctgtcccccctgtcctgctccc gaaGCTGCTggaggccctagcgtcttcctctttcctcccaaacccaaggacaccctcatgatcagcagaaccc ctgaagtcacctgtgtcgtcgtggatgtcagccatgaggaccccgaggtgaaattcaactggtatgtcgatggc gtcgaggtgcacaacgccaaaaccaagcccagggaggaacagtacaactccacctacagggtggtgtccgtgct gacagtcctccaccaggactggctgaacggcaaggagtacaagtgcaaggtgtccaacaaggctctccctgccc ccattgagaagaccatcagcaaggccaaaggccaacccagggagccccaggtctatacactgcctccctccagg gacgaactcaccaagaaccaggtgtccctgacctgcctggtcaagggcttttatcccagcgacatcgccgtcga gtgggagtccaacggacagcccgagaataactacaagaccacccctcctgtcctcgactccgacggctccttct tcctgtacagcaagctgaccgtggacaaaagcaggtggcagcagggaaacgtgttctcctgcagcgtgatgcac gaagccctccacaaccactacacccagaaaagcctgtccctgagccccggcaaatga Amino acid sequence of 19F3H2L3(G1M) heavy chain (SEQ ID NO: 26, with non-variable region sequences underlined) QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAPGQNLEWIGLINPYNAGTSYNQKFQGKVTLTVDK STSTAYMELSSLRSEDTAVYYCARSEYRYGGDYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK Nucleotide sequence of 19F3H2L3(G1M) light chain. (SEQ ID NO: 27, with non-variable region sequences underlined) GACATCGTGATGACCCAGTCCCCAAGCTCCCTGGCCGTGTCTGTGGGAGAGCGGGTGACAATCTC CTGTAAGTCTAGCCAGTCTCTGCTGAACTCCTCTAATCAGAAGAACTACCTGGCCTGGTATCAGC AGAAGCCCGGCCAGGCCCCTAAGCTGCTGATCTACTTCGCCTCTACCAGGGAGAGCGGAGTGCC AGACAGATTCTCTGGCAGCGGCTCCGGCACAGACTTCACCCTGACAATCAGCTCCCTGCAGGCAG AGGACGTGGCCGTGTACTATTGCCAGCAGCACTACGATACCCCCTATACATTTGGCGGCGGCACC AAGCTGGAGATCAAGCGTACGGTGGCAGCCCCATCTGTCTTCATTTTTCCCCCTAGTGACGAGCAGCTGAA ATCCGGAACAGCCTCTGTGGTCTGTCTGCTGAACAATTTCTACCCTCGCGAAGCCAAGGTGCAGTGGAAAGTCG ATAACGCTCTGCAGAGTGGCAATTCACAGGAGAGCGTGACTGAACAGGACTCCAAGGATTCTACCTATAGTCTG AGCTCCACTCTGACCCTGTCCAAAGCAGATTACGAAAAGCACAAAGTGTATGCCTGTGAGGTCACCCACCAGGG GCTGAGTTCTCCAGTCACCAAATCCTTCAACAGAGGCGAATGT Amino acid sequence of 19F3H2L3(G1M) light chain (SEQ ID NO: 28, with non-variable region sequences underlined) DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQKPGQAPKLLIYFASTRESGVPDRFSGSGSG TDFTLTISSLQAEDVAVYYCQQHYDTPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Nucleotide sequence of 19F3H2L3(hG1TM) heavy chain (SEQ ID NO: 29, with non-variable region sequences underlined) CAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGGTGAAGCCAGGAGCCTCTGTGAAGGTGAGCT GTAAGGCCAGCGGCTACTCCTTCACCGGCTATACAATGAACTGGGTGAGGCAGGCACCAGGACA GAATCTGGAGTGGATCGGCCTGATCAACCCTTACAATGCCGGCACCTCTTATAACCAGAAGTTTC AGGGCAAGGTGACCCTGACAGTGGACAAGTCCACCTCTACAGCCTACATGGAGCTGAGCTCCCT GCGGAGCGAGGATACAGCCGTGTACTATTGCGCCCGGTCCGAGTACAGATATGGCGGCGACTAC TTTGATTATTGGGGCCAGGGCACCACACTGACCGTGTCTAGCGCCTCCACAAAG ggG ccC agc gtg ttt cct ctc gcc ccc tcc tcc aaa a gcaccagcggaggaaccgctgctctcggatgtctggtgaaggactacttccctgaacccgtcaccgtgagctgg aatagcggcgctctgacaagcggagtccatacattccctgctgtgctgcaaagcagcggactctattccctgtc cagcgtcgtcacagtgcccagcagcagcctgggcacccagacctacatctgtaacgtcaaccacaagccctcca acaccaaggtggacaagaaagtggagcccaaatcctgcgacaagacacacacctgtcccccctgtcctgctccc gaaGCTGCTggaGCCcctagcgtcttcctctttcctcccaaacccaaggacaccctcatgatcagcagaaccc ctgaagtcacctgtgtcgtcgtggatgtcagccatgaggaccccgaggtgaaattcaactggtatgtcgatggc gtcgaggtgcacaacgccaaaaccaagcccagggaggaacagtacaactccacctacagggtggtgtccgtgct gacagtcctccaccaggactggctgaacggcaaggagtacaagtgcaaggtgtccaacaaggctctccctgccc ccattgagaagaccatcagcaaggccaaaggccaacccagggagccccaggtctatacactgcctccctccagg gacgaactcaccaagaaccaggtgtccctgacctgcctggtcaagggcttttatcccagcgacatcgccgtcga gtgggagtccaacggacagcccgagaataactacaagaccacccctcctgtcctcgactccgacggctccttct tcctgtacagcaagctgaccgtggacaaaagcaggtggcagcagggaaacgtgttctcctgcagcgtgatgcac gaagccctccacaaccactacacccagaaaagcctgtccctgagccccggcaaatga Amino acid sequence of 19F3H2L3(hG1TM) heavy chain variable region: (SEQ ID NO: 30) QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAPGQNLEWIGLINPYNAGTSYNQKFQ GKVTLTVDKSTSTAYMELSSLRSEDTAVYYCARSEYRYGGDYFDYWGQGTTLTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSEFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Amino acid sequences of 19F3H2 framework regions: FR-H1: (SEQ ID NO: 31) QVQLVQSGAEVVKPGASVKVSCKAS FR-H2: (SEQ ID NO: 32) MNWVRQAPGQNLEWIGL FR-H3: (SEQ ID NO: 33) SYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYC FR-H4: (SEQ ID NO: 34) QVQLVQSGAEVVKPGASVKVSCKAS Amino acid sequences of 19F3L2 framework regions: FR-L1: (SEQ ID NO: 35) DIVMTQSPSSLAVSVGERVTISCKSS FR-L2: (SEQ ID NO: 36) LAWYQQKPGQAPKLLIY FR-L3: (SEQ ID NO: 37) TRESGVPDRFSGSGSGTDFTLTISSVQAEDVADYYC FR-L4: (SEQ ID NO: 38) FGGGTKLEIK Amino acid sequences of 19F3L3 framework regions: FR-L1: (SEQ ID NO: 39) DIVMTQSPSSLAVSVGERVTISCKSS FR-L2: (SEQ ID NO: 40) LAWYQQKPGQAPKLLIY FR-L3: (SEQ ID NO: 41) TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC FR-L4: (SEQ ID NO: 42) FGGGTKLEIK Nucleotide sequence of 14C12 heavy chain variable region: (SEQ ID NO: 43) GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAAGCCCGGCGGGTCACTGAAACTGAGCT GCGCCGCTTCCGGCTTCGCCTTTAGCTCCTACGACATGTCATGGGTGAGGCAGACCCCTGAGAAG CGCCTGGAATGGGTCGCTACTATCAGCGGAGGCGGGCGATACACCTACTATCCTGACTCTGTCAA AGGGAGATTCACAATTAGTCGGGATAACGCCAGAAATACTCTGTATCTGCAGATGTCTAGTCTGC GGTCCGAGGATACAGCTCTGTACTATTGTGCAAACCGGTACGGCGAAGCATGGTTTGCCTATTGG GGACAGGGCACCCTGGTGACAGTCTCTGCC Amino acid sequence of 14C12 heavy chain variable region: (SEQ ID NO: 44) EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVATISGGGRYTYYPDSVKG RFTISRDNARNTLYLQMSSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA HCDR1 amino acid sequence of 14C12: (SEQ ID NO: 45) GFAFSSYD HCDR2 amino acid sequence of 14C12: (SEQ ID NO: 46) ISGGGRYT HCDR3 amino acid sequence of 14C12: (SEQ ID NO: 47) ANRYGEAWFAY Nucleotide sequence of 14C12 light chain variable region: (SEQ ID NO: 48) GACATTAAGATGACACAGTCCCCTTCCTCAATGTACGCTAGCCTGGGCGAGCGAGTGACCTTCAC ATGCAAAGCATCCCAGGACATCAACACATACCTGTCTTGGTTTCAGCAGAAGCCAGGCAAAAGC CCCAAGACCCTGATCTACCGGGCCAATAGACTGGTGGACGGGGTCCCCAGCAGATTCTCCGGATC TGGCAGTGGGCAGGATTACTCCCTGACCATCAGCTCCCTGGAGTATGAAGACATGGGCATCTACT ATTGCCTGCAGTATGATGAGTTCCCTCTGACCTTTGGAGCAGGCACAAAACTGGAACTGAAG Amino acid sequence of 14C12 light chain variable region: (SEQ ID NO: 49) DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSG QDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELK LCDR1 amino acid sequence of 14C12: (SEQ ID NO: 50) QDINTY LCDR2 amino acid sequence of 14C12: (SEQ ID NO: 51) RAN LCDR3 amino acid sequence of 14C12: (SEQ ID NO: 52) LQYDEFPLT Amino acid sequences of 14C12 heavy chain framework regions: FR-H1: (SEQ ID NO: 53) EVKLVESGGGLVKPGGSLKLSCAAS FR-H2: (SEQ ID NO: 54) MSWVRQTPEKRLEWVAT FR-H3: (SEQ ID NO: 55) YYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYC FR-H4: (SEQ ID NO: 56) WGQGTLVTVSA Amino acid sequences of 14C12 light chain framework regions: HR-L1: (SEQ ID NO: 57) DIKMTQSPSSMYASLGERVTFTCKAS HR-L2: (SEQ ID NO: 58) LSWFQQKPGKSPKTLIY HR-L3: (SEQ ID ND: 59) RLVDGVPSRFSGSGSGQDYSLLISSLEYEDMGIYYC HR-L4: (SEQ ID NO: 60) FGAGTKLELK Nucleotide sequence of 14C12H1: (SEQ ID NO: 61) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCT GCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAG GGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAA GGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTG AGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTG GGGGCAGGGAACCCTGGTGACAGTCTCTAGT Amino acid sequence of 14C12H1: (SEQ ID NO: 62) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKG Nucleotide sequence of 14C12L1: (SEQ ID NO: 63) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCAC ATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGC CCCAAGACACTGATCTACCGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTC AGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTAC TATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAG Amino acid sequence of 14C12L1: (SEQ ID NO: 64) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSG QDYTLTTSSLQPEDMATYYCLQYDEFPLTFGAGTKLELK Nucleotide sequence of 14C12H1L1 heavy chain (SEQ ID NO: 65) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCT GCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAG GGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAA GGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTG AGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTG GGGGCAGGGAACCCTGGTGACAGTCTCTAGTGCCAGCACCAAAGGACCTAGCGTGTTTCCTCTCG CCCCCTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTACTTC CCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTGACAAGCGGAGTCCATACATTCCCTGC TGTGCTGCAAAGCAGCGGACTCTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGG GCACCCAGACCTACATCTGTAACGTCAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAAGT GGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCGAACTCCTCGGAG GCCCTAGCGTCTTCCTCTTTCCTCCCAAACCCAAGGACACCCTCATGATCAGCAGAACCCCTGAA GTCACCTGTGTCGTCGTGGATGTCAGCCATGAGGACCCCGAGGTGAAATTCAACTGGTATGTCGA TGGCGTCGAGGTGCACAACGCCAAAACCAAGCCCAGGGAGGAACAGTACAACTCCACCTACAGG GTGGTGTCCGTGCTGACAGTCCTCCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGG TGTCCAACAAGGCTCTCCCTGCCCCCATTGAGAAGACCATCAGCAAGGCCAAAGGCCAACCCAG GGAGCCCCAGGTCTATACACTGCCTCCCTCCAGGGACGAACTCACCAAGAACCAGGTGTCCCTGA CCTGCCTGGTCAAGGGCTTTTATCCCAGCGACATCGCCGTCGAGTGGGAGTCCAACGGACAGCCC GAGAATAACTACAAGACCACCCCTCCTGTCCTCGACTCCGACGGCTCCTTCTTCCTGTACAGCAA GCTGACCGTGGACAAAAGCAGGTGGCAGCAGGGAAACGTGTTCTCCTGCAGCGTGATGCACGAA GCCCTCCACAACCACTACACCCAGAAAAGCCTGTCCCTGAGCCCCGGCAAA Amino add sequence of 14C12H1L1 heavy chain variable region: (SEQ ID NO: 66) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKG RFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Nucleotide sequence of 14C12H1L1 light chain (SEQ ID NO: 67) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCAC ATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGC CCCAAGACACTGATCTACCGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTC AGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTAC TATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAGCG AACTGTGGCCGCTCCCTCCGTCTTCATTTTTCCCCCTTCTGACGAACAGCTGAAATCAGGCACAGC CAGCGTGGTCTGTCTGCTGAACAATTTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATA ACGCCCTGCAGTCCGGCAACAGCCAGGAGAGTGTGACTGAACAGGACTCAAAAGATAGCACCTA TTCCCTGTCTAGTACACTGACTCTGTCCAAGGCTGATTACGAGAAGCACAAAGTGTATGCATGCG AAGTGACACATCAGGGACTGTCAAGCCCCGTGACTAAGTCTTTTAACCGGGGCGAATGT Amino acid sequence of 14C12H1L1 light chain: (SEQ ID NO: 68) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSG QDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC Nucleotide sequence of 14C12H1L1(G1TM) heavy chain (SEQ ID NO: 69) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCT GCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAG GGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAA GGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTG AGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTG GGGGCAGGGAACCCTGGTGACAGTCTCTAGTGCCAGCACCAAAGGGCCCAGCGTGTTTCCTCTCG CCCCCTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTACTTC CCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTGACAAGCGGAGTCCATACATTCCCTGC TGTGCTGCAAAGCAGCGGACTCTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGG GCACCCAGACCTACATCTGTAACGTCAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAAGT GGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCGAAGCTGCTGGAG CCCCTAGCGTCTTCCTCTTTCCTCCCAAACCCAAGGACACCCTCATGATCAGCAGAACCCCTGAA GTCACCTGTGTCGTCGTGGATGTCAGCCATGAGGACCCCGAGGTGAAATTCAACTGGTATGTCGA TGGCGTCGAGGTGCACAACGCCAAAACCAAGCCCAGGGAGGAACAGTACAACTCCACCTACAGG GTGGTGTCCGTGCTGACAGTCCTCCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGG TGTCCAACAAGGCTCTCCCTGCCCCCATTGAGAAGACCATCAGCAAGGCCAAAGGCCAACCCAG GGAGCCCCAGGTCTATACACTGCCTCCCTCCAGGGACGAACTCACCAAGAACCAGGTGTCCCTGA CCTGCCTGGTCAAGGGCTTTTATCCCAGCGACATCGCCGTCGAGTGGGAGTCCAACGGACAGCCC GAGAATAACTACAAGACCACCCCTCCTGTCCTCGACTCCGACGGCTCCTTCTTCCTGTACAGCAA GCTGACCGTGGACAAAAGCAGGTGGCAGCAGGGAAACGTGTTCTCCTGCAGCGTGATGCACGAA GCCCTCCACAACCACTACACCCAGAAAAGCCTGTCCCTGAGCCCCGGCAAA Amino acid sequence of 14C12H1L1(G1TM) heavy chain variable region: (SEQ ID NO: 70) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKG RFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Amino acid sequences of 14C12H1 heavy chain framework regions: FR-H1: (SEQ ID NO: 71) EVQLVESGGGLVQPGGSLRLSCAAS FR-H2: (SEQ ID NO: 72) MSWVRQAPGKGLDWVAT FR-H3: (SEQ ID NO: 73) YYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYC FR-H4: (SEQ ID NO: 74) WGQGTLVTVSS Amino acid sequences of 14C12L1 light chain framework regions: FR-L1: (SEQ ID NO: 75) DIQMTQSPSSMSASVGDRVTFTCRAS FR-L2: (SEQ ID NO: 76) LSWFQQKPGKSPKTIIY FR-L3: (SEQ ID NO: 77) RLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYC FR-L4: (SEQ ID NO: 78) FGAGTKLELK Amino acid sequence of Linker1: (SEQ ID NO: 79) GGGGSGGGGSGGGGSGGGGS Nucleotide sequence of Linker: (SEQ ID NO: 80) GGCGGCGGCGGCAGCGGCGGCGGCGGCTCCGGAGGAGGCGGCTCTGGCGGCGGCGGCAGC Amino acid sequence of Linker2: (SEQ ID NO: 81) GGGGSGGGGSGGGGS Nucleotide sequence of Linker2: (SEQ ID NO: 82) GGCGGCGGCGGCTCCGGAGGAGGCGGCTCTGGCGGCGGCGGCAGC The amino acid sequence of the heavy chain of NTPDV2 and NTPDV4 (SEQ ID NO: 83): wherein the CDR regions of 19F3H2(hG1TM) in the immunoglobulin moiety are marked in bold underlines, the CDR regions of 14C12H1V- Linker1-14C12L1V in the scFv part are marked in bold underlines, the mutated amino acids in the heavy chain region are marked in bold italics, and the linker regions are marked in bold: QVQLVQSGAEVVKPGASVKVSCKAS MNWVRQAPGQNLEWIGL
SYNQKFQ GKVTLTVDKSTSTAYMELSSLRSEDTAVYYC
WGQGTTLTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
G
PSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSLKTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGG GGSEVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKG RFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVLVSSGGGGSGGGGSGGGGS GGGGSDIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSG SGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKR Nucleotide sequence of the heavy chain of NTPDV2 and NTPDV4: (SEQ ID NO: 84) CAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGGTGAAGCCAGGAGCCTCTGTGAAGGTGAGCT GTAAGGCCAGCGGCTACTCCTTCACCGGCTATACAATGAACTGGGTGAGGCAGGCACCAGGACA GAATCTGGAGTGGATCGGCCTGATCAACCCTTACAATGCCGGCACCTCTTATAACCAGAAGTTTC AGGGCAAGGTGACCCTGACAGTGGACAAGTCCACCTCTACAGCCTACATGGAGCTGAGCTCCCT GCGGAGCGAGGATACAGCCGTGTACTATTGCGCCCGGTCCGAGTACAGATATGGCGGCGACTAC TTTGATTATTGGGGCCAGGGCACCACACTGACCGTGTCTAGCgcctccacaaaggggcccagcgtgtttcctctcgccc cctcctccaaaagcaccagcggaggaaccgctgctctcggatgtctggtgaaggactacttccctgaacccgtcaccgt gagctggaatagcggcgctctgacaagcggagtccatacattccctgctgtgctgcaaagcagcggactctattccctg tccagcgtcgtcacagtgcccagcagcagcctgggcacccagacctacatctgtaacgtcaaccacaagccctccaaca ccaaggtggacaagaaagtggagcccaaatcctgcgacaagacacacacctgtcccccctgtcctgctcccgaaGCTGC TggagCccctagcgtcttcctctttcctcccaaacccaaggacaccctcatgatcagcagaacccctgaagtcacctgt gtcgtcgtggatgtcagccatgaggaccccgaggtgaaattcaactggtatgtcgatggcgtcgaggtgcacaacgcca aaaccaagcccagggaggaacagtacaactccacctacagggtggtgtccgtgctgacagtcctccaccaggactggct gaacggcaaggagtacaagtgcaaggtgtccaacaaggctctccctgcccccattgagaagaccatcagcaaggccaaa ggccaacccagggagccccaggtctatacactgcctccctccagggacgaactcaccaagaaccaggtgtccctgacct gcctggtcaagggcttttatcccagcgacatcgccgtcgagtgggagtccaacggacagcccgagaataactacaagac cacccctcctgtcctcgactccgacggctccttcttcctgtacagcaaactgaccgtcgataaatctaggtggcagcag ggcaacgtgttctcttgttccgtgatgcatgaagcactgcacaaccattatacccagaagtctctga gcctgtcccccggcaagGGCGGCGGCGGCTCTGGAGGAGGAGGCAGCGGCGGAGGAGGCTCCGGAG GCGGCGGCTCTGAGGTGCAGCTGGTGGAGTCTGGAGGAGGACTGGTGCAGCCTGGAGGCTCCCT GAGGCTGTCTTGCGCAGCAAGCGGATTCGCCTTTAGCTCCTACGACATGAGCTGGGTGCGGCAGG CACCTGGCAAGggtCTGGATTGGGTGGCAACCATCAGCGGAGGCGGCAGATACACATACTATCCC GACTCCGTGAAGGGCAGGTTCACCATCTCCCGCGATAACTCTAAGAACAATCTGTATCTGCAGAT GAACAGCCTGAGGGCCGAGGACACAGCCCTGTACTATTGCGCCAACCGCTACGGCGAGGCCTGG TTTGCCTATTGGGGCCAGGGCACCCTGGTGACAGTGTCTAGCGGCGGCGGCGGCAGCGGCGGCG GCGGCTCCGGAGGAGGCGGCTCTGGCGGCGGCGGCAGCGATATCCAGATGACCCAGTCCCCCTC CTCTATGTCTGCCAGCGTGGGCGACCGGGTGACCTTCACATGTAGAGCCTCCCAGGATATCAACA CCTACCTGTCTTGGTTTCAGCAGAAGCCCGGCAAGAGCCCTAAGACACTGATCTATCGGGCCAAT AGACTGGTGAGCGGAGTGCCTTCCCGGTTCTCCGGCTCTGGCAGCGGACAGGACTATACCCTGAC AATCAGCTCCCTGCAGCCAGAGGATATGGCCACATACTATTGCCTGCAGTATGACGAGTTCCCCC TGACCTTCGGGgctGGCACTAAGCTGGAGCTGAAAAGA The amino acid sequence of the heavy chain of NTPDV1 and NTPDV3 (SEQ ID NO: 85): wherein the CDR regions of 19F3H2(hG1TM) is the immunoglobulin moiety are marked in bold underlines, the CDR regions of 14C12H1V- Linker2-14C12L1V in the scFv part are marked in bold underlines, the mutated amino acids in the heavy chain region are marked in bold italics, and the linker regions are marked in bold: QVQLVQSGAEVVKPGASVKVSCKAS
MNWVRQAPGQNLEWIGL
SYNQKFQ GKVTLTVDKSTSTAYMELSSLRSEDTAVVYC
WGQGTTLTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
G
PSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYACKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSLKTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGG GGSEVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKG RFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVLVSSGGGGSGGGGSGGGGS DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYT LTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKR Nucleotide sequence of the heavy chain of NTPDV1 and NTPDV3: (SEQ ID NO: 86) CAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGGTGAAGCCAGGAGCCTCTGTGAAGGTGAGCT GTAAGGCCAGCGGCTACTCCTTCACCGGCTATACAATGAACTGGGTGAGGCAGGCACCAGGACA GAATCTGGAGTGGATCGGCCTGATCAACCCTTACAATGCCGGCACCTCTTATAACCAGAAGTTTC AGGGCAAGGTGACCCTGACAGTGGACAAGTCCACCTCTACAGCCTACATGGAGCTGAGCTCCCT GCGGAGCGAGGATACAGCCGTGTACTATTGCGCCCGGTCCGAGTACAGATATGGCGGCGACTAC TTTGATTATTGGGGCCAGGGCACCACACTGACCGTGTCTAGCGCCTCCACAAaggggcccagcgtgtttcctct cgccccctcctccaaaagcaccagcggaggaaccgctgctctcggatgtctggtgaaggactacttccctgaacccgtc accgtgagctggaatagcggcgctctgacaagcggagtccatacattccctgctgtgctgcaaagcagcggactctatt ccctgtccagcgtcgtcacagtgcccagcagcagcctgggcacccagacctacatctgtaacgtcaaccacaagccctc caacaccaaggtggacaagaaagtggagcccaaatcctgcgacaagacacacacctgtcccccctgtcctgctcccgaa GCTGCTggagCccctagcgtcttcctctttcctcccaaacccaaggacaccctcatgatcagcagaaccCCTGAAGTCA CCTGTGTCGTCGTGGATGTCAGCCATGAGGACCCCGAGGTGAAATTCAACTGGTATGTCGATGGCGTCGAG GTGCACAACGCCAAAACCAAGCCCAGGGAGGAACAGTACAACTCCACCTACAGGGTGGTGTCCG TGCTGACAGTCCTCCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAA GGCTCTCCCTGCCCCCATTGAGAAGACCATCAGCAAGGCCAAAGGCCAACCCAGGGAGCCCCAG GTCTATACACTGCCTCCCTCCAGGGACGAACTCACCAAGAACCAGGTGTCCCTGACCTGCCTGGT CAAGGGCTTTTATCCCAGCGACATCGCCGTCGAGTGGGAGTCCAACGGACAGCCCGAGAATAAC TACAAGACCACCCCTCCTGTCCTCGACTCCGACGGCTCCTTCTTCCTGTACAGCAAACTGACCGTC GATAAATCTAGGTGGCAGCAGGGCAACGTGTTCTCTTGTTCCGTGATGCATGAAGCACTGCACAA CCATTATACCCAGAAGTCTCTGAGCCTGTCCCCCGGCAAGGGCGGCGGCGGCTCTGGAGGAGGA GGCAGCGGCGGAGGAGGCTCCGGAGGCGGCGGCTCTGAGGTGCAGCTGGTGGAGTCTGGAGGA GGACTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCTTGCGCAGCAAGCGGATTCGCCTTTAGCTC CTACGACATGAGCTGGGTGCGGCAGGCACCTGGCAAGGGTCTGGATTGGGTGGCAACCATCAGC GGAGGCGGCAGATACACATACTATCCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCGCGATA ACTCTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCCGAGGACACAGCCCTGTACTAT TGCGCCAACCGCTACGGCGAGGCCTGGTTTGCCTATTGGGGCCAGGGCACCCTGGTGACAGTGTC TAGCGGCGGCGGCGGCTCCGGAGGAGGCGGCTCTGGCGGCGGCGGCAGCGATATCCAGATGACC CAGTCCCCCTCCTCTATGTCTGCCAGCGTGGGCGACCGGGTGACCTTCACATGTAGAGCCTCCCA GGATATCAACACCTACCTGTCTTGGTTTCAGCAGAAGCCCGGCAAGAGCCCTAAGACACTGATCT ATCGGGCCAATAGACTGGTGAGCGGAGTGCCTTCCCGGTTCTCCGGCTCTGGCAGCGGACAGGAC TATACCCTGACAATCAGCTCCCTGCAGCCAGAGGATATGGCCACATACTATTGCCTGCAGTATGA CGAGTTCCCCCTGACCTTCGGGGCTGGCACTAAGCTGGAGCTGAAAAGATGATAAGAATTC Amino acid sequence of NT5E(1-552)-his (SEQ ID NO: 87) MCPRAARAPATLLLALGAVLWPAAGAWELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFT KVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEFDNGVEGLIEPLL KEAKFPILSANIKAKGPLASQISGLYLPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQP EVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSD DGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQEL GKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTIT WENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLD VLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYP AVEGRIKFSTGSHHHHHH Nucleotide sequence of NT5E(1-552)-his (SEQ ID NO: 88) ATGTGTCCTAGAGCCGCCAGAGCTCCAGCTACACTGCTGCTGGCTCTGGGAGCAGTCCTCTGGCC AGCAGCAGGAGCTTGGGAACTGACCATCCTGCACACCAACGACGTGCACAGCAGGCTGGAACAG ACCAGCGAGGACAGCAGCAAGTGCGTGAACGCCAGTCGCTGTATGGGAGGAGTGGCAAGGCTGT TCACCAAGGTGCAGCAGATCCGGAGAGCCGAACCTAACGTGCTGCTGCTGGACGCCGGAGATCA GTATCAGGGAACCATCTGGTTCACCGTGTACAAGGGCGCCGAAGTGGCCCACTTCATGAACGCTC TGCGCTACGACGCTATGGCCCTGGGCAATCACGAGTTCGATAACGGCGTGGAGGGACTGATCGA GCCTCTGCTGAAGGAGGCCAAGTTCCCCATCCTGAGCGCCAACATCAAGGCCAAGGGACCTCTG GCTAGCCAGATTAGCGGCCTGTACCTGCCTTACAAGGTGCTGCCCGTGGGAGACGAAGTGGTGG GAATCGTGGGCTACACCAGCAAGGAGACCCCTTTCCTGAGCAACCCAGGCACCAACCTGGTGTTC GAGGACGAGATCACCGCTCTGCAGCCAGAGGTGGACAAGCTGAAGACCCTGAACGTGAACAAGA TCATCGCCCTGGGACACAGCGGCTTCGAGATGGACAAGCTGATCGCCCAGAAAGTGCGAGGAGT GGACGTGGTCGTGGGCGGACACAGCAACACCTTCCTGTACACCGGCAACCCTCCTTCTAAGGAAG TGCCAGCCGGCAAGTACCCCTTCATCGTGACCAGCGACGACGGAAGAAAGGTGCCAGTGGTGCA GGCTTACGCCTTCGGCAAGTACCTGGGCTACCTGAAGATCGAGTTCGACGAGCGGGGCAACGTG ATCTCTAGCCACGGCAACCCCATCCTGCTGAACAGCAGCATCCCAGAGGACCCCAGCATCAAGG CCGACATCAACAAGTGGCGGATCAAGCTGGACAACTACAGCACCCAGGAGCTGGGAAAGACCAT CGTGTACCTGGACGGCAGCTCTCAGTCTTGCCGGTTCCGCGAGTGCAACATGGGCAACCTGATTT GCGACGCCATGATCAACAACAACCTGCGGCACACCGACGAGATGTTTTGGAACCACGTCAGCAT GTGCATCCTGAACGGCGGAGGCATCAGAAGCCCTATTGACGAGCGGAACAACGGCACCATCACT TGGGAGAACCTGGCAGCAGTGCTGCCTTTTGGCGGAACATTCGACCTGGTGCAGCTGAAGGGCA GCACACTGAAGAAGGCCTTCGAGCACAGCGTGCACAGATACGGCCAGAGCACAGGCGAGTTCCT GCAGGTCGGAGGAATCCACGTGGTGTACGACCTGAGCAGGAAGCCAGGAGACAGAGTGGTGAA GCTGGACGTGCTCTGCACCAAGTGTCGGGTGCCAAGCTACGACCCCCTGAAGATGGACGAGGTG TACAAGGTCATCCTGCCCAACTTCCTGGCTAACGGAGGAGACGGCTTCCAGATGATCAAGGACG AGCTGCTGAGGCACGACAGCGGAGACCAGGACATCAACGTCGTGTCCACCTACATCAGCAAGAT GAAGGTCATCTACCCCGCCGTGGAAGGCAGGATCAAGTTCAGCACCGGCTCTCACCACCACCATC ACCAC Amino acid sequence of mFc: (SEQ ID NO: 89) PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVH TAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEEKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPP PEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWV ERNSYSCSVVHEGLHNHHTTKSFSRTPGK Amino acid sequence of 19F3H1V-Linker-19F3L2V: (SEQ ID NO: 90) QVQLQQSGAEVVKPGASMKMSCKASGYSFTGYTMNWVKQAHGQNLEWIGLINPYNAGTSYNQKFQ GKATLTVDKSTSTAYMELSSLRSEDTAVYYCARSEYRYGGDYFDYWGQGTTLTVSSGGGGSGGGGS VPDRFSGSGSGTDFTLTISSVQAEDVADYYCQQHYDTPYTFGGGTKLEIK Nucleotide sequence of 19F3H1V-Linker2-19F3L2V: (SEQ ID NO: 91) CAGGTGCAGCTGCAGCAGTCTGGAGCAGAGGTGGTGAAGCCAGGAGCCTCTATGAAGATGAGCT GTAAGGCCAGCGGCTACTCCTTCACCGGCTATACAATGAACTGGGTGAAGCAGGCCCACGGCCA GAATCTGGAGTGGATCGGCCTGATCAACCCTTACAATGCCGGCACCTCTTATAACCAGAAGTTTC AGGGCAAGGCCACCCTGACAGTGGACAAGTCCACCTCTACAGCCTACATGGAGCTGAGCTCCCT GCGGAGCGAGGATACAGCCGTGTACTATTGCGCCCGGTCCGAGTACAGATATGGCGGCGACTAC TTTGATTATTGGGGCCAGGGCACCACACTGACCGTGTCTAGC GGCCTGTACGGCCTGTACGGCCTGTACGGCCTGTACAGCGAGAGGGGCCTGTACGGCCTGTACGG CCTGTACGGCCTGTACAGCGAGAGGGGCCTGTACGGCCTGTACGGCCTGTACGGCCTGTACAGCG AGAGGGACATCGTGATGACCCAGTCCCCAAGCTCCCTGGCCGTGTCTGTGGGAGAGCGGGTGAC AATCTCCTGTAAGTCTAGCCAGTCTCTGCTGAACTCCTCTAATCAGAAGAACTACCTGGCCTGGT ATCAGCAGAAGCCCGGCCAGGCCCCTAAGCTGCTGATCTACTTCGCCTCTACCAGGGAGAGCGG AGTGCCAGACAGATTCTCTGGCAGCGGCTCCGGCACAGACTTCACCCTGACAATCAGCTCCGTGC AGGCAGAGGACGTGGCAGATTACTATTGCCAGCAGCACTACGATACCCCCTATACATTTGGCGGC GGCACCAAGCTGGAGATCAAG Nucleotide sequence of 19F3H1: (SEQ ID NO: 92) CAGGTGCAGCTGCAGCAGTCTGGAGCAGAGGTGGTGAAGCCAGGAGCCTCTATGAAGATGAGCT GTAAGGCCAGCGGCTACTCCTTCACCGGCTATACAATGAACTGGGTGAAGCAGGCCCACGGCCA GAATCTGGAGTGGATCGGCCTGATCAACCCTTACAATGCCGGCACCTCTTATAACCAGAAGTTTC AGGGCAAGGCCACCCTGACAGTGGACAAGTCCACCTCTACAGCCTACATGGAGCTGAGCTCCCT GCGGAGCGAGGATACAGCCGTGTACTATTGCGCCCGGTCCGAGTACAGATATGGCGGCGACTAC TTTGATTATTGGGGCCAGGGCACCACACTGACCGTGTCTAGC Amino acid sequence of 19F3H1: (SEQ ID NO: 93) QVQLQQSGAEVVKPGASMKMSCKASGYSFTGYTMNWVKQAHGQNLEWIGLINPYNAGTSYNQKFQ GKATLTVDKSTSTAYMELSSLRSEDTAVYYCARSEYRYGGDYFDYWGQGTTLTVSS Nucleotide sequence of 19F3L1: (SEQ ID NO: 94) GACATCGTGATGACCCAGTCCCCAAGCTCCCTGGCAATGTCTGTGGGAGAGAGGGTGACAATGTC CTGTAAGTCTAGCCAGTCTCTGCTGAACTCCTCTAATCAGAAGAACTACCTGGCCTGGTATCAGC AGAAGCCCGGCCAGGCCCCTAAGCTGCTGGTGTACTTTGCCTCTACCAGGGAGAGCGGAGTGCC AGACAGATTCTCTGGCAGCGGCTCCGGCACAGACTTCACCCTGACAATCAGCTCCGTGCAGGCAG AGGACCTGGCAGATTATTTCTGCCAGCAGCACTACGATACCCCCTATACATTTGGCGGCGGCACC AAGCTGGAGATCAAG Amino acid sequence of 19F3L1: (SEQ ID NO: 95) DIVMTQSPSSLAMSVGERVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQAPKLLVYFASTRESGVPDR FSGSGSGTDFTLTISSVQAEDLADYFCQQHYDTPVTFGGGTKLEIK Amino acid of 19F3L2 light chain (full length) (SEQ ID NO: 96, with non-variable region sequences underlined) DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQKPGQAPKLLIYFASTRESGVPDRFS GSGSGTDFTLTISSVQAEDVADYYCQQHYDTPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLESGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC Amino acid sequence of 19F3H2 heavy chain variable region with CDR sequences underlined: (SEQ ID NO: 97) QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAPGQNLEWIGLINPYNAGTSYNQKFQ GKVTLTVDKSTSTAYMELSSLRSEDTAVYYCARSEYRYGGDYFDYWGQGTTLTVSS Amino acid sequence of 19F3L2 heavy chain variable region with CDR sequences underlined: (SEQ ID NO: 98) DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQKPGQAPKLLIYFASTRESGVPDRFS GSGSGTDFTLTISSVQAEDVADYYCQQHYDTPYTFGGGTKLEIK Amino acid sequence of 19F3L3 heavy chain variable region with CDR sequences underlined: (SEQ ID NO: 99) DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQKPGQAPKLLIYFASTRESGVPDRFS GSGSGTDFTLTISSLQAEDVAVYYCQQHYDTPYTFGGGTKLEIK Nucleotide sequence of 19F3L2 light chain (full length) (SEQ ID NO: 100) GACATCGTGATGACCCAGTCCCCAAGCTCCCTGGCCGTGTCTGTGGGAGAGCGGGTGACAATCTCCTGTAAGTC TAGCCAGTCTCTGCTGAACTCCTCTAATCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCCC CTAAGCTGCTGATCTACTTCGCCTCTACCAGGGAGAGCGGAGTGCCAGACAGATTCTCTGGCAGCGGCTCCGGC ACAGACTTCACCCTGACAATCAGCTCCGTGCAGGCAGAGGACGTGGCAGATTACTATTGCCAGCAGCACTACGA TACCCCCTATACATTTGGCGGCGGCACCAAGCTGGAGATCAAGCGTACGGTGGCAGCCCCATCTGTCTTCATTT TTCCCCCTAGTGACGAGCAGCTGAAATCCGGAACAGCCTCTGTGGTCTGTCTGCTGAACAATTTCTACCCTCGC GAAGCCAAGGTGCAGTGGAAAGTCGATAACGCTCTGCAGAGTGGCAATTCACAGGAGAGCGTGACTGAACAGGA CTCCAAGGATTCTACCTATAGTCTGAGCTCCACTCTGACCCTGTCCAAAGCAGATTACGAAAAGCACAAAGTGT ATGCCTGTGAGGTCACCCACCAGGGGCTGAGTTCTCCAGTCACCAAATCCTTCAACAGAGGCGAATGT