SEMG2 ANTIBODY AND USE THEREOF
20230080534 · 2023-03-16
Inventors
Cpc classification
C07K16/2875
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
C07K2317/24
CHEMISTRY; METALLURGY
C07K2317/73
CHEMISTRY; METALLURGY
A61K39/3955
HUMAN NECESSITIES
C07K2317/76
CHEMISTRY; METALLURGY
G01N2333/70578
PHYSICS
C07K2317/34
CHEMISTRY; METALLURGY
A61K47/6843
HUMAN NECESSITIES
C07K14/70578
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
G01N2500/02
PHYSICS
A61P35/00
HUMAN NECESSITIES
International classification
A61K39/395
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K47/68
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
The present invention provides a compound agonizing or antagonizing the interaction between SEMG2 and CD27, comprising a small molecule inhibitor, a polypeptide, an antibody, or an antigen-binding fragment. The present invention further discloses methods of preparing antibodies for blocking the binding between SEMG2 and CD27 using the polypeptides as an immunogen with high efficiency. The present invention discloses methods of promoting anti- tumor immunity by blocking the contact of SEMG2 expressed by tumor cells with CD27 expressed by immune cells, also discloses a screening method for screening a therapeutic drug by blocking the binding between SEMG2 and CD27.
Claims
1. A compound agonizing or antagonizing an interaction between SEMG2 and CD27, wherein the interaction between SEMG2 and CD27 is located on the amino acid site at positions 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, and 508 of SEMG2, and the amino acid sequence of the SEMG2 protein is shown in SEQ ID NO:1.
2. (canceled)
3. The compound of claim 1, wherein the compound is a small molecule inhibitor, polypeptide, antibody, or antigen binding fragment; wherein the polypeptide comprises an amino acid sequence of SEQ ID NO:2 (QIEKLVEGKS), SEQ ID NO:86 (QIEKLVEGKS(x)I(x)), SEQ ID NO:87 (QIEKLVEGKS(x)I), or SEQ ID NO:88 (QIEKLVEGKS(x)); or the polypeptide comprises an amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 (QIEKLVEGKSQIQ), SEQ ID NO:4 (QIEKLVEGKSQ), or SEQ ID NO:5 (QIEKLVEGKSQI), or an amino acid sequence at least 90% identity to an amino acid sequence as provided in SEQ ID NO: 2-5, wherein the x is selected from any amino acid; wherein the antibody specifically binds to native or mutant SEMG2 protein, the antibody binds to an antigenic epitope peptide derived from SEMG2 protein, the antigenic epitope peptide comprises an amino acid sequence of SEQ ID NO:2 (QIEKLVEGKS), SEQ ID NO:3 (QIEKLVEGKSQIQ), SEQ ID NO:4 (QIEKLVEGKSQ), or SEQ ID NO:5 (QIEKLVEGKSQI); and/or wherein the antibody specifically binds to native or mutant SEMG2 protein, the antibody recognizes at least one amino acid residue at positions 497, 498, 499, 500, 501, 502, 503, 504, 505, 506 and 508 of the native SEMG2 protein, or recognizes an amino acid residue in the corresponding position of the mutant SEMG2 protein, the amino acid sequence of the native SEMG2 protein is shown in SEQ ID NO:1.
4. (canceled)
5. (canceled)
6. (canceled)
7. The compound of claim 3, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 defined by IMGT; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 defined by IMGT, the HCDR1 consists of or comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:6-11, SEQ ID NOs:60-61 and SEQ ID NO:76; the HCDR2 consists of or comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:12-16 and SEQ ID NOs:62-64; the HCDR3 consists of or comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:17-20, SEQ ID NOs:65-67 and SEQ ID NOs:77- 81; the LCDR1 consists of or comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:21-25, SEQ ID NOs:68-70 and SEQ ID NO:82; the LCDR2 consists of or comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:26-29, SEQ ID NOs:71-72, SEQ ID NOs:83-84 and SEQ ID NO:28; the LCDR3 consists of or comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:30-34, SEQ ID NOs:73-75, SEQ ID NO:85 and SEQ ID NO:95.
8. The compound of claim 7, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 defined by IMGT; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 defined by IMGT, the CDR sequence of the antibody is selected from any one of the combinations in (a)-(k): (a) the HCDR1 comprises the amino acid sequence of SEQ ID NO:6; the HCDR2 comprises the amino acid sequence of SEQ ID NO:12; the HCDR3 comprises the amino acid sequence of SEQ ID NO:17; the LCDR1 comprises the amino acid sequence of SEQ ID NO:21; the LCDR2 comprises the amino acid sequence of SEQ ID NO:26; the LCDR3 comprises the amino acid sequence of SEQ ID NO:30; (b) the HCDR1 comprises the amino acid sequence of SEQ ID NO:7; the HCDR2 comprises the amino acid sequence of SEQ ID NO:13; the HCDR3 comprises the amino acid sequence of SEQ ID NO:18; the LCDR1 comprises the amino acid sequence of SEQ ID NO:22; the LCDR2 comprises the amino acid sequence of SEQ ID NO:27; the LCDR3 comprises the amino acid sequence of SEQ ID NO:31 or SEQ ID NO:95; (c) the HCDR1 comprises the amino acid sequence of SEQ ID NO:6; the HCDR2 comprises the amino acid sequence of SEQ ID NO:16; the HCDR3 comprises the amino acid sequence of SEQ ID NO:17; the LCDR1 comprises the amino acid sequence of SEQ ID NO:21; the LCDR2 comprises the amino acid sequence of SEQ ID NO:26; the LCDR3 comprises the amino acid sequence of SEQ ID NO:30; (d) the HCDR1 comprises the amino acid sequence of SEQ ID NO:8; the HCDR2 comprises the amino acid sequence of SEQ ID NO:13; the HCDR3 comprises the amino acid sequence of SEQ ID NO:18; the LCDR1 comprises the amino acid sequence of SEQ ID NO:23; the LCDR2 comprises the amino acid sequence of SEQ ID NO:27; the LCDR3 comprises the amino acid sequence of SEQ ID NO:32; (e) the HCDR1 comprises the amino acid sequence of SEQ ID NO:9; the HCDR2 comprises the amino acid sequence of SEQ ID NO:14; the HCDR3 comprises the amino acid sequence of SEQ ID NO:19; the LCDR1 comprises the amino acid sequence of SEQ ID NO:24; the LCDR2 comprises the amino acid sequence of SEQ ID NO:28; the LCDR3 comprises the amino acid sequence of SEQ ID NO:33; (f) the HCDR1 comprises the amino acid sequence of SEQ ID NO:10; the HCDR2 comprises the amino acid sequence of SEQ ID NO:15; the HCDR3 comprises the amino acid sequence of SEQ ID NO:20; the LCDR1 comprises the amino acid sequence of SEQ ID NO:25; the LCDR2 comprises the amino acid sequence of SEQ ID NO:29; the LCDR3 comprises the amino acid sequence of SEQ ID NO:34; (g) the HCDR1 comprises the amino acid sequence of SEQ ID NO:11; the HCDR2 comprises the amino acid sequence of SEQ ID NO:15; the HCDR3 comprises the amino acid sequence of SEQ ID NO:20; the LCDR1 comprises the amino acid sequence of SEQ ID NO:25; the LCDR2 comprises the amino acid sequence of SEQ ID NO:29; the LCDR3 comprises the amino acid sequence of SEQ ID NO:34; (h) the HCDR1 comprises the amino acid sequence of SEQ ID NO:60; the HCDR2 comprises the amino acid sequence of SEQ ID NO:62; the HCDR3 comprises the amino acid sequence of SEQ ID NO:65; the LCDR1 comprises the amino acid sequence of SEQ ID NO:68; the LCDR2 comprises the amino acid sequence of SEQ ID NO:71; the LCDR3 comprises the amino acid sequence of SEQ ID NO:73; (i) the HCDR1 comprises the amino acid sequence of SEQ ID NO:61; the HCDR2 comprises the amino acid sequence of SEQ ID NO:63; the HCDR3 comprises the amino acid sequence of SEQ ID NO:66; the LCDR1 comprises the amino acid sequence of SEQ ID NO:69; the LCDR2 comprises the amino acid sequence of SEQ ID NO:72; the LCDR3 comprises the amino acid sequence of SEQ ID NO:74; (j) the HCDR1 comprises the amino acid sequence of SEQ ID NO:60; the HCDR2 comprises the amino acid sequence of SEQ ID NO:64; the HCDR3 comprises the amino acid sequence of SEQ ID NO:67; the LCDR1 comprises the amino acid sequence of SEQ ID NO:70; the LCDR2 comprises the amino acid sequence of SEQ ID NO:28; the LCDR3 comprises the amino acid sequence of SEQ ID NO:75; (k) the HCDR1 comprises the amino acid sequence of SEQ ID NO:60 or 76; the HCDR2 comprises the amino acid sequence of SEQ ID NO:64 or 62; the HCDR3 comprises the amino acid sequence of SEQ ID NO:77, 78 or 79; and/or the LCDR1 comprises the amino acid sequence of SEQ ID NO:70 or 82; the LCDR2 comprises the amino acid sequence of SEQ ID NO:28, 83 or 84; the LCDR3 comprises the amino acid sequence of SEQ ID NO:75 or 85.
9. The compound of claim 3, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:35-41, 48-51, 54-56 and 96-100, or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to any sequence of SEQ ID NOs:35-41, 48-51, 54-56 and 96-100; the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:42-47, 52-53, 57-69 and 101-103, or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to any sequences of SEQ ID NOs:42-47, 52-53, 57-69 and 101-103.
10. The compound of claim 9, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region are selected from any one of the combinations in (a)-(o): (a) the heavy chain variable region comprises SEQ ID NO:35 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:35; the light chain variable region comprises SEQ ID NO:42 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:42; (b) the heavy chain variable region comprises SEQ ID NO:36 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:36; the light chain variable region comprises SEQ ID NO:43 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:43; (c) the heavy chain variable region comprises SEQ ID NO:37 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:37; the light chain variable region comprises SEQ ID NO:44 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:44; (d) the heavy chain variable region comprises SEQ ID NO:38 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:38; the light chain variable region comprises SEQ ID NO:45 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:45; (e) the heavy chain variable region comprises SEQ ID NO:39 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:39; the light chain variable region comprises SEQ ID NO:46 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:46; (f) the heavy chain variable region comprises SEQ ID NO:40 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:40; the light chain variable region comprises SEQ ID NO:47 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:47; (g) the heavy chain variable region comprises SEQ ID NO:41 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:41; the light chain variable region comprises SEQ ID NO:47 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:47; (h) the heavy chain variable region comprises SEQ ID NO:48, 49, 50, 51 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:48, 49, 50 or 51; the light chain variable region comprises SEQ ID NO:52 or 53 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% to SEQ ID NO:52 or 53; (i) the heavy chain variable region comprises SEQ ID NO:54 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:54; the light chain variable region comprises SEQ ID NO:57 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:57; (j) the heavy chain variable region comprises SEQ ID NO:55 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:55; the light chain variable region comprises SEQ ID NO:58 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:58; (k) the heavy chain variable region comprises SEQ ID NO:56 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:56; the light chain variable region comprises SEQ ID NO:59 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:59; (l) the heavy chain variable region comprises SEQ ID NO:96 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:96; the light chain variable region comprises SEQ ID NO:59, 101, 102, 103 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:59, 101, 102 or 103; (m) the heavy chain variable region comprises SEQ ID NO:97 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:97; the light chain variable region comprises SEQ ID NO:59 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:59; (n) the heavy chain variable region comprises SEQ ID NO:98 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:98; the light chain variable region comprises SEQ ID NO:103 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:103; (o) the heavy chain variable region comprises SEQ ID NO:99 or 100 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:99 or 100; the light chain variable region comprises SEQ ID NO:57 or an amino acid sequence at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO:57.
11. The compound of any of the claim 3, wherein the antibody further comprises a coupling moiety linked to the polypeptide, the coupling moiety is selected from the group consisting one or more of radionuclides, drugs, toxins, cytokines, enzymes, fluorescein, carrier proteins, lipids, and biotin, wherein the polypeptide or antibody is selectively linked to the coupling moiety by a linker, preferably the linker is a peptide or polypeptide.
12. The compound of any of the claim 3, wherein the antibody is selected from monoclonal antibodies, polyclonal antibodies, antisera, chimeric antibodies, humanized antibodies, and human antibodies; wherein the antibody is selected from multispecific antibodies, single chain Fv (scFv), single chain antibodies, anti-idiotype (anti-Id) antibodies, diabodies, minibodies, nanobodies, single domain antibodies, Fab fragments, F(ab′) Fragments, disulfide-linked bispecific Fvs (sdFv) and intracellular antibodies.
13. (canceled)
14. (canceled)
15. A protein, wherein the protein comprises the antigenic epitope peptide of claim 11 and an optional tag sequence which can selectively be linked at the N-terminus or C-terminus; preferably wherein the protein comprises an amino acid sequence of SEQ ID NO:2 (QIEKLVEGKS), SEQ ID NO:86 (QIEKLVEGKS(x)I(x)), SEQ ID NO:87 (QIEKLVEGKS(x))I), or SEQ ID NO:88 (QIEKLVEGKS(x)), preferably the polypeptide comprises an amino acid sequence of SEQ ID NO:3 (QIEKLVEGKSQIQ), SEQ ID NO:4 (QIEKLVEGKSQ), or SEQ ID NO:5 (QIEKLVEGKSQI) or an amino acid sequence at least 90% identity to any one of SEQ ID NOs:2-5, more preferably SEQ ID NOs:89-94 and SEQ ID NO:3.
16. (canceled)
17. A method for preparing an antibody, including using a protein of claim 15 as immunogen to immunize mammals or obtained by screening in natural antibody library.
18. An isolated polynucleotide encoding the compound of claim 3.
19. A recombinant cloning vector or an expression vector comprising the polynucleotide of claim 18; wherein the regulatory sequence is selected from a leading sequence, a polyadenylation sequence, a leader-peptide sequence, a promoter, a signal sequence, a transcription terminator, or any combination thereof.
20. (canceled)
21. A host cell comprising the recombinant vector of claim 18; wherein, the host cell is a prokaryotic cell or a eukaryotic cell.
22. (canceled)
23. A pharmaceutical composition comprising the compound of; wherein, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or adjuvant.
24. (canceled)
25. A method for agonizing or antagonizing the interaction between SEMG2 and CD27, comprising administrating the compound of claim 1.
26. (canceled)
27. (canceled)
28. The method of claim 25, wherein; the tumor is selected from one or more of colorectal cancer, lung cancer, melanoma, lymphoma, liver cancer, head and neck cancer, stomach cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, endometrial cancer, breast cancer and ovarian cancer.
29. (canceled)
30. (canceled)
31. (canceled)
32. The method of claim 37, wherein the subject has received or is receiving or will receive additional anti-cancer therapy; wherein the additional anti-cancer therapy comprises surgery, radiotherapy, chemotherapy, immunotherapy, or hormone therapy.
33. (canceled)
34. (canceled)
35. (canceled)
36. The compound for use of claim, wherein the method comprises the following steps: A) analyzing the expression of SEMG2 in tumor cells; B) contacting the tumor cells with an antibody recognizing SEMG2, the binding of the antibody to SEMG2 is KD<2×10.sup.−8; C) contacting T lymphocytes with the antibody and tumor cells
37. A method for preventing or treating tumors, comprising administrating the compound of claim 1 to the subject, wherein SEMG2 is expressed in tumor cells and CD27 is expressed in immune cells.
38. A method for modulating an immune response elicited against tumors, or detecting the presence or absence of SEMG2 in a biological sample in vitro, comprising contacting immune cells such as lymphocytes and/or tumor cells of the subject with an effective dose of the compound of claim 1; optionally, the expression of SEMG2 in tumor cells is detected before contacting immune cells such as lymphocytes and/or tumor cells of the subject with an effective dose of the compound.
Description
DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0101] The following makes further explanations to the invention by detailed description.
[0102] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.
[0103] In this application, the singular forms “a”, “an” and “the” include plural reference, unless the context clearly dictates otherwise.
[0104] As used herein, the term “subject” includes any human or nonhuman animals. The term “nonhuman primate” includes all vertebrates, such as mammals or nonmammals, for example nonhuman primates, sheep, canines, felines, equines, bovines, chickens, rats, mice, amphibians, reptiles, and the like. Unless otherwise specified, the terms “patient” and “subject” can be used interchangeably. In the present invention, a subject is preferably human.
[0105] As used herein, the term “SEMG2” is human semenogelin 2, one of the major components in human semen, secreted by seminal gland, and forms colloidal material to coat sperm cells and restrict their movement. The proteolytic enzymes and fibrinolytic enzymes secreted by the prostate gland in semen can break down the semenogelin and promote semen liquefaction, allowing sperm to move more freely. See Yoshida K, Karzai Z T, Krishna Z, Yoshida M, Kawano N, Yoshida M, et al., Cell Motil Cytoskeleton. 2009; 66(2):99-108. The “SgII A” polypeptide isolated from SEMG2 protein has antibacterial activity, and the sequence is H-KQEGRDHDKSKGHFHMIVIHHKGGQAHHG-OH. It should be noted that different from the key amino acid sequence for binding between SEMG2 and CD27 described in the present invention, the antimicrobial peptide sequence is located in a completely different region of SEMG2. See Edström A M, Maim J, Frohm B, Martellini J A, Giwercman A, Mörgelin M, et al., J Immunol. 2008; 181(5):3413-21. In addition, SEMG2 has also been reported to bind to zinc ions and affect the activity of prostatic proteolytic enzyme PSA. See Jonsson M, Linse S, Frohm B, Lundwall A, Malm J. Biochem J. 2005; 387(Pt 2):447-53.
[0106] As used herein, the term “antibody” includes intact antibody and any antigen-binding fragment (i.e., “antigen-binding part”) or the single chain thereof. “Antibody” refers to a protein containing at least two heavy (H) chains and two light (L) chains connected by disulfide bond, or its antigen-binding part. Each heavy chain consists of a heavy chain variable region (short for VH herein) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (short for VL herein) and a light chain constant region. The light chain constant region consists of a CL domain. The VH and VL regions can be further subdivided into high-variable regions, known as complementary decision area (CDR), scattered over more conservative regions known as framework region (FR). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of heavy and light chains contain binding domains for antigen interaction.
[0107] The term “antibody” refers to immunoglobulins or their fragments or derivatives thereof, and includes any polypeptides that contain antigen binding sites, whether they are produced in vitro or in vivo. The term includes, but is not limited to, multi-clone, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybridized, mutation, and graft antibodies. The term “antibody” also includes antibody fragments such as Fab, F(ab′) 2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen binding function, i.e., can specifically bind to PD-1. Generally, such fragments will contain antigen binding fragments.
[0108] The terms “antigen-binding fragment”, “antigen-binding domain” and “binding fragment” refer to an antibody molecule, which contains amino acids responsible for the binding between specific antibodies and antigens. For example, where the antigen is large and the antigen-binding fragment binds only a portion of the antigen. That is, the part of the antigen molecule responsible for the specific interaction with the antigen binding fragment is called “epitope” or “antigenic determinant”.
[0109] An antigen-binding fragment typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), however, it does not necessarily have to comprise both. For example, a so-called Fd antibody fragment consists only of a VH domain, but still retains some of the antigen binding functions of the intact antibody.
[0110] The term “epitope” is defined as an antigenic determinant which specifically binds/recognizes a binding fragment. Binding fragments can specifically bind/react with a conformation that is unique to the target structure or a contiguous epitope, the conformation or discontinuous epitope is characterized by that the polypeptide antigen being two or more separated discrete amino acid residues in the primary sequence, but the polypeptides are aggregated together on the surface of the molecule when they are folded into native proteins/antigens. Two or more discrete amino acid residues of an epitope exist in separate parts of one or more polypeptide chains. When the polypeptide chain folds into a three-dimensional structure, these residues gather on the surface of the molecule to form an epitope. In contrast, contiguous or linear epitopes, consisting of two or more discrete amino acid residues, are present in a single linear segment of a polypeptide chain.
[0111] The terms “treating” or “treatment” refer to both therapeutic treatment and prophylactic/preventing measures. Those in need of treatment include individuals who already have a particular medical condition, as well as those who may eventually acquire the condition.
[0112] The term “vector” as used herein refers to a molecular tool for the transport, transduction, and expression in a target cell of a contained exogenous gene of interest (for example, a polynucleotide according to the present invention). The tool provides a suitable nucleotide sequence that initiates transcription, i.e., the promoter.
[0113] The terms “tag protein” and “protein tag” in the present invention are interchangeable, and refer to a polypeptide or protein fused and expressed with the target protein by using DNA in vitro recombination technology, to facilitate protein expression, detection, tracking and purification. Tag proteins include, but not limited to, His6, Flag, GST, MBP, HA, GFP and Myc.
EXAMPLES
[0114] Unless otherwise specifically explained, the implementation methods in the following examples are all conventional method. The invention will be further understood with reference to the following non-limiting experimental examples.
Example 1: Detection of Binding Between SEMG2 and CD27
[0115] Human HEK293 cells were co-transfected in a 10 cm diameter culture dish. 48 hours after co-transfection with complex including pcDNA3-Flag-SEMG2 plasmid and pcDNA3-HA-CD2 plasmid, cells were collected and lysed, CD27 in lysate was enriched by standard immunoprecipitation procedure. The antibody used for immunoprecipitation was Flag antibody, and IgG nonspecific antibody was used for the control group. Immunoblotting (western blot) experiment was carried out later using HA antibody to detect the amount of co-immunoprecipitated CD27 and using Flag antibody to detect the amount of immunoprecipitated SEMG2. In immunoblotting assay, cells were lysed by Roche Complete protease inhibitor in 1% Triton X-100 (TBS pH7.6) for 30 minutes on ice, and insoluble material was pelleted by centrifugation. Lysate in SDS sample buffer with 50 mM DTT was heated to 100° C. for 10 minutes, separated by SDS-PAGE and transferred to PVDF membrane (Millipore). The cell membrane was blocked in TBS with 5% bovine serum albumin (BSA) and probed with the indicated antibodies. The bands were visualized with West Pico (Thermo Fisher Scientific).
[0116] In co-immunoprecipitation experiment, cells were lysed in IP buffer (Thermo Scientific) and Roche complete protease inhibitor for 10 minutes, followed by the addition of benzonase (sigma) for 25 minutes at room temperature. The lysate was then centrifuged at 15,000 rpm at 4° C. to remove the precipitate. The supernatant was then incubated with primary antibody slowly rotated overnight at 4° C., followed by the addition of protein A or protein G dynabeads and incubated at 4° C. for 2 hours, washed for 4 times in PBST (PBS with 0.01% Tween 20), eluted with 50 mM DTT in SDS sample buffer for 10 minutes at 100° C., separated with SDS, and immunoblotted as previously described.
[0117] The result showed that precipitation with Flag antibody made the precipitated complex contain both SEMG2 and CD27, while the control group did not contain SEMG2 or CD27. See
[0118] Under the above co-transfection experimental conditions, the pre-placed cell slides in a 10 cm dish were fixed, permeabilized, and blocked, and further immunolabeled with antibodies containing HA tag (mouse anti) and Flag tag (rabbit anti) simultaneously for CD27 and SEMG2, and then labeled with secondary antibodies to show red and green colors, respectively. The co-localization of SEMG2 and CD27 in cells was observed under fluorescence confocal microscopy. The results are shown in
Example 2: Binding Between SEMG2(497-509) Fragment and CD27 Protein
[0119] To further confirm which part of SEMG-2 binds to CD27, fragments of SEMG2 protein were designed. The amino acid sequence of full-length SEMG2 protein (SEQ ID NO:1) was divided into 6 segments of sequences, fused with GFP and named as SEMG2-P1, SEMG2-P2, SEMG2-P3, SEMG2-P4, SEMG2-P5 and SEMG2-P6 (See Table 1 for specific sequences, with corresponding abbreviation as P1-P6 respectively). Plasmids expressing these amino acid sequences were co-transfected with CD27 into HEK293 cells, and co-immunoprecipitation experiments were performed to identify the main fragment of SEMG2 that binds to CD27. The co-immunoprecipitation results are shown in
TABLE-US-00001 (human SEMG2): SEQ ID NO: 1 MKSIILFVLSLLLILEKQAAVMGQKGGSKGQLPSGSSQFPHGQKGQHYFG QKDQQHTKSKGSFSIQHTYHVDINDHDWTRKSQQYDLNALHKATKSKQHL GGSQQLLNYKQEGRDHDKSKGHFHMIVIHHKGGQAHHGTQNPSQDQGNSP SGKGLSSQCSNTEKRLWVHGLSKEQASASGAQKGRTQGGSQSSYVLQTEE LVVNKQQRETKNSHQNKGHYQNVVDVREEHSSKLQTSLHPAHQDRLQHGP KDIFTTQDELLVYNKNQHQTKNLSQDQEHGRKAHKISYPSSRTEERQLHH GEKSVQKDVSKGSISIQTEEKIHGKSQNQVTIHSQDQEHGHKENKISYQS SSTEERHLNCGEKGIQKGVSKGSISIQTEEQIHGKSQNQVRIPSQAQEYG HKENKISYQSSSTEERRLNSGEKDVQKGVSKGSISIQTEEKIHGKSQNQV TIPSQDQEHGHKENKMSYQSSSTEERRLNYGGKSTQKDVSQSSISFQIEK LVEGKSQIQTPNPNQDQWSGQNAKGKSGQSADSKQDLLSHEQKGRYKQES SESHNIVITEHEVAQDDHLTQQYNEDRNPIST
TABLE-US-00002 TABLE 1 Corresponding amino acid sequences for construction of SEMG2 expression fragments SEMG2-P1 GSFSIQHTYHVDINDHDWTRKSQQYDLNALHKATKSKQHLGGSQQLLNY SEQ ID NO: 89 KQEGRDHDKSKGHFHMIVIHHKGGQAHHGT SEMG2-P2 QNPSQDQGNSPSGKGLSSQCSNTEKRLWVHGLSKEQASASGAQKGRTQ SEQ ID NO: 90 GGSQSSYVLQTEELVVNKQQRETKNSHQNKGHYQNVVDVREEHSSKLQT SLHPAHQDRLQHGPKDIFTTQDELLVYNKNQHQTKNLSQDQEHGR SEMG2-P3 KAHKISYPSSRTEERQLHHGEKSVQKDVSKGSISIQTEEKIHGKSQNQVTIHS SEQ ID NO: 91 QDQEHGHKENKISYQSSSTEERHLNCGEKGIQKGVSKGSISIQTEEQIHGKS QNQVRIPSQAQ SEMG2-P4 EYGHKENKISYQSSSTEERRLNSGEKDVQKGVSKGSISIQTEEKIHGKSQNQ SEQ ID NO: 92 VTIPSQDQEHGHKENKMSYQSSSTEERRLNY GGKSTQKDVSQSSIS SEMG2-P5 FQIEKLVEGKSQIQTPNPNQDQWSGQNAKGKSGQSADSKQDLLSH SEQ ID NO: 93 SEMG2-P6 EQKGRYKQESSESHNIVITEHEVAQDDHLTQQYNEDRNPIST SEQ ID NO: 94 SEMG2-P7 QIEKLVEGKSQIQ SEQ ID NO: 3
[0120] To further confirm the key amino acids in the binding between SEMG2-P5 sequence and CD27, SEMG2(497-509) fragment was selected and named as SEMG2-P7 (the specific sequence is QIEKLVEGKSQIQ, abbreviated as P7 or SP7). SEMG2-P7(497-509), SEMG2-P5(positive control), SEMG2-P4 (negative control) or SEMG2-P6 (negative control) was co-transfected with CD27 into HEK293 cells respectively, including human CD27 and mouse CD27. The result of co-immunoprecipitation experiment performed later showed that both SEMG2-P7 and SEMG2-P5 bind to CD27, and the results of human CD27 and mouse CD27 were the same. The experimental results are shown in
Example 3: Precise Characterization of the Key Amino Acids of SEMG2 Binding to CD27 Using Glycine Scanning Method
[0121] To characterize the epitope of SEMG2 binding to CD27 with higher resolution, and to demonstrate the contribution of each amino acid of SEMG2(497-509) to binding CD27 protein more accurately, each amino acid of SEMG2(497-509) was replaced one by one with glycine, and the resulting sequences are mutant amino acid sequences numbered 1-13 (see
Example 4: SEMG2 Expressed by Tumor Cells Inhibits Effect of Killing Tumors by Immune Cells
[0122] T cell-mediated killing assay of tumor cells. HCT116 human colorectal cancer cells were stably transfected with SEMG2 expression vector or control empty vector, and the proportion of apoptotic cells after co-culturing with activated PBMCs was determined by caspase3/7 lysis assay (green fluorescence assay). Specifically; HCT116 cells stably expressing SEMG2 were seeded in 96-well plate. Human peripheral blood mononuclear cells (PBMC; 470025, Stem Cell) were activated with 100 ng/mL of CD3 antibody, 100 ng/mL of CD28 antibody, and 10 ng/mL of IL2 (#317303; #302913; #589102, BioLegend) respectively, and co-cultured with the colorectal cancer cells (#4440, Essen Bioscience) at a ratio of 10:1 in presence of fluorescent caspase-3/7 substrate. After 10 hours, cells were observed under a fluorescence microscope. The result is shown in
Example 5: Detection of SEMG2 Expression in Different Tumor Cells
[0123] Different types of human tumor cells, including LOVO colorectal cancer, RKO colorectal cancer, PC3 prostate cancer, A375 malignant melanoma, SW1116 colorectal cancer, DLD1 colorectal cancer, HEK293 human renal epithelial cell line, HepG2 hepatocellular carcinoma, NCM460 human normal colonic epithelial cells, NCI-H1975 human non-small cell lung adenocarcinoma, CaCo2 colonic adenocarcinoma, HT29 colorectal adenocarcinoma, SW1990 human pancreatic adenocarcinoma, AGS human gastric adenocarcinoma, SW480 colorectal cancer, SaOS2 osteosarcoma, GES-1 human gastric mucosal cells, and so like, were incubated with DMEM medium containing 10% calf serum in cell incubator with 5% carbon dioxide at 37° C.
[0124] In immunoblotting assay, cells were lysed by Roche complete protease inhibitor in 1% Triton X-100 (TBS pH7.6) for 30 minutes on ice, and insoluble material was pelleted by centrifugation. Lysate in SDS sample buffer with 50 mM DTT was heated to 100° C. for 10 minutes, separated by SDS-PAGE and transferred to PVDF membrane (Millipore). The cell membrane was blocked in TBS with 5% bovine serum albumin (BSA) and probed with the primary antibodies specifically against SEMG2 and internal control GAPDH, respectively. The primary antibodies were labeled with HRP-conjugated secondary antibodies. The bands were visualized with West Pico (Thermo Fisher Scientific). The results are shown in
Example 6: Detection of SEMG2 Expression in Different Tumor Cells Using Immunohistochemistry (IHC)
[0125] For immunohistochemical staining, we obtained tissue chips of various tumors from Shanghai Xinchao Biotechnology Company. Briefly, tissue specimens were incubated with anti-SEMG2 antibody (HPA042767, purchased from Sigma Aldrich, 1:100 dilution) and a biotin-conjugated secondary antibody, followed by incubation with an anti-biotin-biotin-peroxidase complex, and observed with chromophoric reagent aminoethylcarbazole. As the histological score, staining intensities were divided into four groups: high (3), moderate (2), low (1), and negative (0).
[0126] First, the expression of SEMG2 in tissue chips of colorectal cancer tumor was stained, and normal colorectal tissue was used as control; it was found that there was extensively high expression of SEMG2 in colorectal cancer tissue. The result is shown in
[0127] Next, the expression of SEMG2 in different lung cancer tissues was stained, and normal lung tissue was used as control; it was found that there was extensively high expression of SEMG2 in lung cancer tissues. The results are shown in
[0128] Again, the positive expression of SEMG2 in prostate cancer, melanoma, and gastric cancer was stained, and the results are shown in
[0129] Finally, based on the tissue chip staining, the positive rate of SEMG2 expression in the different tumor types indicated was calculated. Positive expression was defined as moderate or strong positive expression in immunohistochemical staining. Statistical results based on tissue chips (each chip comprises more than 50 tissue samples) are shown as a percentage in
Example 7: Demonstration of the Association Between High SEMG2 Expression and Poor Tumor Prognosis
[0130] In immunohistochemical staining, we obtained tissue chips of various tumors from Shanghai Xinchao Biotechnology Co., Ltd., all with follow-up data of survival time information. Immunohistochemical detection was performed by the method described in Example 6. Taking colorectal cancer as an example, the patients were classified according to the expression of SEMG2, and divided into two groups: high SEMG2 (immunohistochemical score of 2, 3) and low SEMG2 (immunohistochemical score of 0, 1). The Kaplan-Meier method was used to compare the overall survival of the two patient groups. The results are shown in
Example 8: Confirmation of the Correlation Between High SEMG2 Expression and Infiltration of Regulatory T cells (Treg) with Immunosuppressive Function
[0131] To analyze the correlation between SEMG2 expression in tumor tissues and infiltration of Regulatory T cells (Treg) with immunosuppressive function, we examined a variety of tumor tissue chips purchased from Shanghai Xinchao Biotechnology Co., Ltd using immunohistochemistry assay. Take lung cancer for example, the infiltration of Treg in tumor tissues (labeled by Foxp3 antibody) was compared according to the expression of SEMG2. It was found that the higher the expression of SEMG2 was, the more Treg infiltrated (there was a statistically significant difference among the tissues, P<0.05), see
Example 9: Preparation of Antibody Using SEMG2 (497-509) Fragment as Immunogen
[0132] Specifically, the following steps are included: (1) antigen preparation, synthesize polypeptide according to SEMG2 (497-509), i.e., the “QIEKLVEGKSQIQ” sequence, and couple to VLP carrier for immunization; use full-length SEMG2 protein (purchased from Cusabio, Cat. No. CSB-YP0211002HU) as immunogen for another group, (2) The first immunization: remove part of the rabbit hair on both hind paws of the rabbit using a pair of scissors, and disinfect the skin with alcohol and iodine. Aspirate 1 mL of antigen solution emulsify by Freund's complete adjuvant (FCA) using a 2 mL syringe, and inject 0.5 mL of which into each sole of the feet subcutaneously. (3) The second immunization: After an interval of 10-14 days, inject the antigen solution into the swollen lymph nodes on bilateral fossa and groin, 0.1 mL for each lymph node and 1 mL for the rest under the skin near the lymph nodes. If the lymph nodes are not swollen or the swelling is not obvious, directly inject it into bilateral fossa and subcutaneous of groin. (4) After an interval of 7-10 days, collect 0.5-1.0 mL of blood from the ear vein, separate the serum, and determine the serum titer using indirect ELISA which coated with 10 μg/mL of antigen. Collect the blood if the titer is 1:64,000 or more. (5) If the titer does not meet the requirements, inject the antigen liquid without adjuvant into the ear vein for immunization. Which is, inject for 3 times within 1 week, 0.1, 0.3 and 0.5 mL for each time, respectively. Repeat the blood test after an interval of 1 week. If the titer meets the requirement, take the blood immediately, and collect all the antiserum.
[0133] The specific experimental steps for polyclonal antibody purification comprise: (1) Preparation of protein A sepharose CL-4B affinity column. To prepare 10 mL of protein A sepharose CL-4B packing, mix equal volume of packing and TBS buffer solution in a vacuum flask, stir and vacuum for 15 minutes to remove air bubbles in the packing. Slowly add Protein A sepharose CL-4B packing into the glass column using the pump to control the filling speed at 1 mL/min-2 mL/min, avoid column dryness, and use 10 times the bed volume of pre-cooled TBS buffer solution to equilibrate the column. (2) Preparation of antiserum. Slowly thaw the antiserum in ice water or in a 4° C. freezer to avoid protein aggregation. Aggregates appeared during protein thawing process can be dissolved by preheating at 37° C. Add solid sodium azide to a concentration of 0.05%, centrifuge at 15,000×g for 5 minutes at 4° C., remove the clarified antiserum and filter through a filter to remove excess lipids. (3) Affinity chromatography. Dilute the antibody with TBS buffer solution at 1:5 and filtered through a filter. Load the antiserum onto the column at a speed of 0.5 mL/min. To ensure the binding of the antiserum to the packing, the column should be loaded continuously for 2 times and the loading effluent should be kept. Wash the column with TBS buffer solution until Aλ280 nm<0.008, add pH 2.7 elution buffer solution, and elute at a speed of 0.5 mL/min until all proteins flow down. Use a 1.5 mL EP tube with 100 μL of neutralizing buffer solution added to collect the eluate in separate tubes. After mixing, check the pH of the eluate with pH test paper. If the pH is lower than 7, use the neutralization buffer to adjust to about pH 7.4 to prevent antibodies denaturation. Add 10 mL of elution buffer solution, pH 1.9, into the column, and collect the eluate until Aλ280 nm<0.008 according to the method. The protein content in each tube was determined using a spectrophotometer.
Example 10: Blocking Effect Comparison Between SEMG2 (497-509) and Full-Length SEMG2 as Immunogens in Antibody Preparation
[0134] Because SEMG2 (497-509) sequence fragment is the key epitope of SEMG2 binding to CD27, and has a relatively short sequence, so SEMG2 (497-509) was used as an immunogen to prepare antibodies, which is theoretically easier to obtain functional antibody molecules with the function of blocking the binding between SEMG2 and CD27 than using full-length SEMG2 to prepare antibodies. For direct comparison, the differences in effective concentration of producing antibody by the two methods were verified using ELISA in the examples. Antibodies produced with SEMG2 (497-509) as immunogen and antibodies produced with full-length SEMG2 were added into the enzyme-linked immunosorbent assay (ELISA) reaction system at different concentrations (10{circumflex over ( )}-2, 10{circumflex over ( )}-1, 10{circumflex over ( )}0, 10{circumflex over ( )}1, 10{circumflex over ( )}2, 10{circumflex over ( )}3, 10{circumflex over ( )}4 μg/mL), and the ELISA binding values were measured. The specific steps of enzyme-linked immunosorbent assay are as follows: (1) Dissolve SEMG2 protein antigen with 50 mM carbonate coating buffer (pH 9.6) to make the antigen concentration 10 μg/mL, and add into 96-well ELISA plate (purchased from Corning) at 100 μL/well, place at 4° C. overnight. (2) After discarding the coating solution on the next day, wash with PBST for three times, add 150 μL of 1% BSA to each well, and block for 2 hours at 37° C. (3) After washing for 3 times with PBST, add the indicated antibodies (polyclonal antibody produced with SEMG2 (497-509) as immunogen, polyclonal antibody produced with full-length SEMG2 as immunogen) to each well to make different final concentrations as shown in
[0135] The experimental results are shown in
Example 11: Preparation of Mouse Monoclonal Antibody Using SEMG2 (497-509) Epitope Peptide and SEMG2 Full-Length Protein
[0136] The SEMG2 (497-509) sequence was used to synthesize a polypeptide and coupled to a VLP carrier for immunization; HEK293 cells were used to express the full-length SEMG2 protein, and the purity was tested to reach 92%, and the binding activity of SEMG2 protein to CD27 was verified by ELISA. The protein and polypeptide antigens were used to immunize 10 mice respectively, and multiple immunizations were performed to enhance the effect: (1) The first immunization, 50 μg/mice of antigen, multiple subcutaneous injections together with Freund's complete adjuvant, with an interval of 3 weeks; (2) the second immunization; the same dosage and route as above, with incomplete Freund's adjuvant, and with an interval of 3 weeks; (3) the third immunization, the same dosage as above, without adjuvant, intraperitoneal injection with an interval of 3 weeks; (4) the booster immunization, the dose is 50 μg, intraperitoneal injection. 3 days after the last injection, blood was collected to measure its titer and the immune effect, and mice with higher titers were selected for hybridoma fusion screening. After subcloning, the binding of the monoclonal antibodies to the target antigens was detected by ELISA, and the function of different monoclonal antibodies in blocking the binding between SEMG2 and CD27 was measured by ELISA.
[0137] The monoclonal antibodies produced by hybridomas were screened by ELISA. Among the monoclonal antibodies prepared with SEMG2(497-509) as immunogen, 19 strains had blocking function (inhibiting the binding between SEMG2 and CD27) in the first batch of 27 stains of antibodies, as shown in
[0138] Therefore, preparing monoclonal antibodies using SEMG2 (497-509) epitope peptide as immunogen significantly improved the efficiency of finding blocking antibodies. The subtypes (Table 2) and sequences (Table 3) of murine monoclonal antibodies are shown in following tables.
TABLE-US-00003 TABLE 2 Subtypes of murine monoclonal antibodies Antibody clone ID Subtype Light chain MM02 mIgG2b kappa MM05 mIgG1 kappa MM07 mIgG2b kappa MM08 mIgG1 kappa MM13 mIgG1 kappa MM14 mIgG2b kappa MM15 mIgG2a kappa
TABLE-US-00004 TABLE 3 Heavy and light chain variable region sequences of marine monoclonal antibodies VH amino acid sequences are as follows: MM02 QIQLVQSGPEVKKPGETVRISCKASGYTLTTAGIQWVQKMPGKGLKWIGWINTHSGVPEYAEDFKGRFAFF LETSASTAYLQISNLKNEDTATYFCARLGLLGYWGQGTTLTVSS (SEQ ID NO: 35) MM05 QVQLQQPGAELVRPGASVKLSCEASGYTFTSYWMNWVKQRPGQGLEWIGMIDPSDSETHYNQMFKDK ATLTVDKSSSTAYMQLSSLTSEDSAVYYCARYLGGKEGSFDYWGQGTTLTVSS (SEQ ID NO: 36) MM07 MDWLWTLLFLMAAAQSIQAQIQLVQSGPELKKPGETVRISCKASGYTLTTAGMQWVQKIPGKGLKWIGW INTHSGVAEFAEDFKGRFAFSLETSANTAYLQIRNLKNEDTATYFCARLGLLGYWGQGTTLTVSS (SEQ ID NO: 37) MM08 QVQLQQPGAELVRPGASVKLSCKSSDYTFTRYWMNWVKQRPGQGLEWIGMIDPSDSETHYNQMFKDK ATLTVDKSSSTAYMQLSSLTSEDSAVYYCARYLGGKEGSFDYWGQGTTLTVSS (SEQ ID NO: 38) MM13 EVQLQQSGAELVRSGASVKLSCTASGFNIKDYYMHWMKQRPEQGLEWIGWIDPENGDNEYAPKFQGKAT MTADTSSNTAYLQLSSLTSEDTAVYYCNVGGAHYWGQGTTLTVSS (SEQ ID NO: 39) MM14 QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYAVSWVRQPPGKGLEWLGIIWGDGSTNYHSALISRLSISKDN SKSQVFLKLNSLQTDDTATYYCAKQERFSDGYYDGFAYWGQGTLVTVSA (SEQ ID NO: 40) MM15 QVQLKESGPGLVAPSQSLSITCTVSGFSLTRYGVSWVRQTPGKGLEWLGIIWGDGSTNYHSALISRLSISKDN SKSQVFLKLNSLQTDDTATYYCAKQERFSDGYYDGFAYWGQGTLVTVSA (SEQ ID NO: 41) VL amino acid sequences are as follows: MM02 DILLTQSPAILSVSPGERVSFSCRASQSIGTTIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSI NSVESEDIADYYCQQSNSWPWTFGGGTKLEIKRA (SEQ ID NO: 42) MM05 DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGS GSGTDFTLTISSVQAEDLAVYYCKQSYSLPWTFGGGTKLEIKRA (SEQ ID NO: 43) MM07 MVSSAQFLVFLLFWIPASRGDILLTQSPAILSVSPGERVSFSCRASQSIGTTIHWYQQRTNGSPRLLIKYASESI SGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPWTFGGGTKLEIKRA (SEQ ID NO: 44) MM08 DIVLTQSPSSLAVSAGERVTMSCKSSQSLFNSRTRKNYLAWYQQKPSQSPKLLLYWASTRESGVPDRFTGSG SGTDFTLTISSVKTEDLAVYYCKQSYELPWTFGGGTKLEMKRA (SEQ ID NO: 45) MM13 DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSSGSGS GTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIKRA (SEQ ID NO: 46) MM14/ QIVLTQSPAIMSASPGEKVTITCSASSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTI MM15 SAMEAEDAATYYCQQRSSYPFTFGSGTKLEIKRA (SEQ ID NO: 47)
TABLE-US-00005 TABLE 4 CDR amino acid sequences of mouse antibodies VH CDR sequence-IMGT analysis Antibody CDR1 CDR2 CDR3 MM02 GYTLTTAG INTHSGVP ARLGLLGY (SEQ ID NO: 6) (SEQ ID NO: 12) (SEQ ID NO: 17) MM05 GYTFTSYW IDPSDSET ARYLGGKEGSFDY (SEQ ID NO: 7) (SEQ ID NO: 13) (SEQ ID NO: 18) MM07 GYTLTTAG INTHSGVA ARLGLLGY (SEQ ID NO: 6) (SEQ ID NO: 16) (SEQ ID NO: 17) MM08 DYTFTRYW IDPSDSET ARYLGGKEGSFDY (SEQ IO NO: 8) (SEQ ID NO: 13) (SEQ ID NO: 18) MM13 GFNIKDYY IDPENGDN NVGGAHY (SEQ ID NO: 9) (SEQ ID NO: 14) (SEQ ID NO: 19) MM14 GFSLTSYA IWGDGST AKQERFSDGYYDGFAY (SEQ ID NO: 10) (SEQ ID NO: 15) (SEQ ID NO: 20) MM15 GFSLTRYG IWGDGST AKQERFSDGYYDGFAY (SEQ ID NO: 11) (SEQ ID NO: 15) (SEQ ID NO: 20) VL CDR sequence-IMGT analysis Antibody CDR1 CDR2 CDR3 MM02 QSIGTT YA QQSNSWPWT (SEQ ID NO: 21) (SEQ ID NO: 26) (SEQ ID NO: 30) MM05 QSLLNSRTRKNY WA KQSYSLPWT (SEQ ID NO: 22) (SEQ ID NO: 27) (SEQ ID NO: 31) MM05-2 QSLLNSRTRKNY WA QQSYSLPWT (SEQ ID NO: 22) (SEQ ID NO: 27) (SEQ ID NO: 95) MM07 QSIGTT YA QQSNSWPWT (SEQ ID NO: 21) (SEQ ID NO: 26) (SEQ ID NO: 30) MM08 QSLFNSRTRKNY WA KQSYELPWT (SEQ ID NO: 23) (SEQ ID NO: 27) (SEQ ID NO: 32) MM13 QSLVHSNGNTY KV SQSTHVPYT (SEQ ID NO: 24) (SEQ ID NO: 28) (SEQ ID NO: 33) MM14 SSVSY ST QQRSSYPFT (SEQ ID NO: 25) (SEQ ID NO: 29) (SEQ ID NO: 34) MM15 SSVSY ST QQRSSYPFT (SEQ ID NO: 25) (SEQ ID NO: 29) (SEQ ID NO: 34)
[0139] The ELISA plate was coated with SEMG2 protein, and the serially diluted murine monoclonal antibody was used as the primary antibody, and the anti-mouse secondary antibody was used to detect the binding abilities of the murine monoclonal antibodies to SEMG2. The results are shown in
Example 12: Humanization of Anti-SEMG2 mAb
[0140] The mouse anti-SEMG2 monoclonal antibody MM05 was humanized to reduce immunogenicity when used in human patients. The sequences of the heavy and light chain variable regions (VH and VL) were compared to human antibody sequences in Protein Data Bank (PDB) and homology models were established. The CDRs in the heavy and light chains of mouse mAbs were transplanted to human frame regions that most likely maintain the proper structure required for antigen binding. Reverse mutations or other mutations from human residues to mouse residues were designed when necessary, for example: the amino acid at position 95 of the humanized light chain VL-V2 was mutated from K to Q, and the corresponding CDR3 sequence of the light chain was converted to QQSYSLPWT (SEQ ID NO:95) according to IMGT analysis. Humanized VH and VL regions were fused to the constant regions of heavy chain and K light chain of human IgG1, respectively. Transient transfections were performed in 293E cells using the construction vectors corresponding to mAb sequences, and the binding abilities of the purified mAbs to SEMG2 protein were analyzed using ELISA. Results are shown in absorbance, where higher absorbance indicates a higher level of interaction between the humanized antibody and SEMG2. The amino acid sequences of CDRs, light chain variable regions and heavy chain variable regions, light chains and heavy chains of the 8 humanized antibodies obtained in the present invention are shown in Table 4 and Table 5 below.
TABLE-US-00006 TABLE 5 VH and VL amino acid sequences of MM05 humanized antibody VH amino acid sequences are as follows: VH_V1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGKGLEWVGMIDPSDSETHYNQMFKD RVTITADKSTSTAYMELSSLRSEDTAVYYCARYLGGKEGSFDYWGQGTLVTVSS (SEQ ID NO: 48) VH_V2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGKGLEWVGMIDPSDSETHYNQMFKD RVTITVDKSTSTAYMELSSLRSEDTAVYYCARYLGGKEGSFDYWGQGTLVTVSS (SEQ ID NO: 49) VH_V3 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWVGMIDPSDSETHYAQKFQG RVTITVDKSTSTVYMELSSLRSEDTAVYYCARYLGGKEGSFDYWGQGTLVTVSS (SEQ ID NO: 50) VH_V4 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWVGMIDPSDSETHYAQKFQG RVTITADKSTSTVYMELSSLRSEDTAVYYCARYLGGKEGSFDYWGQGTLVTVSS (SEQ ID NO: 51) VL amino acid sequences are as follows: VL_V1 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGS GSGTDFTLTISSLQAEDVAVYYCKQSYSLPWTFGGGTKVEIK (SEQ ID NO: 52) VL_V2 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGS GSGTDFTLTISSLQAEDVAVYYCQQSYSLPWTFGGGTKVEIK (SEQ ID NO: 53)
Example 13: Functional Comparison Between SEMG2 (497-509) Epitope-Specific Antibodies and Other Epitope-Specific Antibodies in Blocking SEMG2 and CO27 Binding
[0141] To demonstrate the importance of the SEMG2 (497-509) epitope for the preparation of blocking antibodies, the functions of antibodies against different SEMG2 epitopes in blocking binding between SEMG2 and CD27 were further compared. It is known that the existing commercial antibodies of HPA042767 and HPA042835 (both purchased from Sigma Aldrich Company) are rabbit polyclonal antibodies against epitopes of SEMG2 (354-403) and SEMG2 (563-574), respectively.
[0142] First, SEMG2(497-509) epitope-specific antibodies (for example, MM02, MM05) were compared with other epitope-specific antibodies (for example, HPA042767) for their function in blocking binding between SEMG2 and CD27 at different concentration ranges. The binding of the above antibodies to SEMG2 (497-509) epitope was confirmed by ELISA: MM02 and MM05 were able to hind to SEMG2(497-509), while HPA042767 could not bind to this epitope in a wide range of concentration, as shown in
[0143] Further, the effects of different antibodies on the binding between SEMG2 and CD27 were compared under the condition of the same antibody concentration. In the ELISA, the same concentration (10 μg/mL) of antibody was used, and the strength of the binding between SEMG2 and CD27 was measured. The results are shown in
Example 14: Effective Comparison Between SEMG2 (497-509) Epitope-Specific Antibodies and Other Epitope-Specific Antibodies in Tumor Cell Killing by Activated PBMC
[0144] In the examples, SEMG2 exerts function of inhibiting activated PBMC from killing tumor cells. Since SEMG2 may play the above role by binding to CD27, and SEMG2 (497-509) epitope is a key site for CD27 binding, SEMG2 (497-509) epitope-specific antibody may neutralize the influence of SEMG2 on tumor cell killing by PBMC.
[0145] To test the above hypothesis, the effects of different epitope-specific antibodies on tumor cell killing by activated PBMC were compared. Specifically, A375 human melanoma and LOVO human colorectal cancer cells highly expressing SEMG2 were seeded in 96-well plate. Human peripheral blood mononuclear cells (PBMC; #70025, Stem Cell) were activated with 100 ng/mL of CD3 antibody, 100 ng/mL, of CD28 antibody, and 1.0 ng/mL of IL2 (#317303; #302913; #589102, BioLegend), respectively, and co-cultured with the above tumor cells at a ratio of 10:1 in presence of fluorescent caspase-3/7 substrate (#4440, Essen Bioscience). After 1.0 hours, cells were observed under fluorescence microscope. The results are shown in
Example 15: Verifying the Correlation Between the Expression Level of SEMG2 and the Promotive Function of Blocking Antibodies in Tumor Cell Killing by PBMC
[0146] Since the expression of SEMG2 is a prerequisite for its inhibition of tumor-specific immunity, the expression of SEMG2 is also a potential condition for the suitability of SEMG2-blocking antibody administration. In theory, tumor cells with high SEMG2 expression will have a relative increase in the tumor cell killing by PBMC after neutralizing of SEMG2 activity; tumor cells that do not express SEMG2 may not rely on SEMG2 to play immune escape function, therefore the tumor cell killing by PBMC may not produce a significant change after neutralizing of SEMG2 activity.
[0147] To verify the above hypothesis, tumor cells with high SEMG2 expression (A375, LOVO) and SEMG2-negative tumor cells (DLD1, NCM460 and NCI-H1975) were selected. Different antibodies (irrelevant mouse IgG antibodies, MM02 or MM05 antibodies) were added during the PBMC killing experiments for the tumor cells. Results are shown in
Example 16: Accurate Definition of Associated Epitopes of Antibody for Blocking the Binding Between SEMG2 and CD27
[0148] To clearly distinguish the binding epitopes of blocking antibodies (i.e., antibodies that can inhibit the binding between SEMG2 and CD27) and non-blocking antibodies, a corresponding ELISA analysis method was established. Specifically, SEMG2 full-length protein (1-582), SEMG2(354-403) fragment, SEMG2(442-453) fragment, SEMG2(497-509) fragment and SEMG2(563-574) fragment were immobilized on the ELISA plate, and the same concentration of antibodies (MM02, MM05, MM07, MM08, MM13, MM14, HPA042767 and HPA042835) were added. Anti-mouse or anti-rabbit secondary antibodies were then used to detect the corresponding bound antibodies. The results are shown in
[0149] To further precisely define the exact epitopes (specific to the level of single amino acid) to which blocking antibodies MM02, MM05, MM07, MM08, MM13, MM14 bind, corresponding ELISA analysis method was established. As shown in
Example 17: Preparation and Screening of Fully Human Antibodies Using SEMG2(497-509) Epitope to Block the Binding Between SEMG2 and CD27 and to Promote the Tumor Cell Killing by PBMC
[0150] Results of the examples showed the importance of SEMG2(497-509) epitope in the preparation of blocking antibodies, and this epitope was applied to the screening of fully human antibodies. Specifically, the preparation of polypeptide antigens and the screening of human natural antibody library were firstly performed. The SEMG2(497-509) polypeptide was synthesized and coupled to BSA and KLH, respectively, and screened in a fully human phage display antibody library. ELISA was used to select clones that bind to antigenic epitopes for preliminary screening. Different unique sequences were obtained after sequencing single colonies, sorted according to affinity sorting, and full-length antibodies were constructed from antigen-binding fragments (Fab) with relatively high affinity. The binding ability and blocking function tests were performed after purification, that is, the effect of the antibody on binding between SEMG2 and CD27 was determined by the ELISA experiment.
[0151] In the same batch of screening, a total of 3 unique sequences of antibodies that bind to SEMG2 (497-509) epitope and inhibit binding of SEMG2 and CD27 were obtained. The three clones were named respectively: H88-93, H88-96 and H88-67, The effect of the corresponding fill-length antibody concentration on binding SEMG2 is shown in
TABLE-US-00007 TABLE 6 Variable region sequences of fully human antibodies VH amino acid sequences are as follows: H88-96 QVQLLESGGGLVQPGGSLRLSCSASGFTFSSYAMHWVRQAPGKGLEYVSAISSNGGSTYYADSVK GRFTISRDNSKNTLYLQMSSLRAEDTAVYYCVIEGGSTTGTTSGAFDIWGQGTMVTVSS (SEQ ID NO: 54) H88-93 QITLKESGPTLVKPTQTLTLTCNFSGFSLTTSGVGVAWIRQPPGKALEWLALIYWDDDQRYSPSLKSR LSVTKHTSKDQVVLTMTNVGPVDTATYYCAHLSYGPGWGYYMDVWGNGTMVTVSS (SEQ ID NO: 55) H88-67 QVQLLESGGGVVQPGRSLRLSCAASGFTFSSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMDGSGSPDYWGQGTLVTVSS (SEQ ID NO: 56) VL amino acid sequences are as follows: H88-96 DIQMIQSPPSVSASVGDTVTIACRANQGIDSWLAWYQQKPGRAPKLLIYSASRLQSGVPSRFSGGG SGTDFALTISNLQPEDFATYYCQQALSLPITFGQGTRLEIK (SEQ ID NO: 57) H88-93 EIVLTQSPGTLSLSPGERASLSCRASQSVRNNYLAWYQQKPGQAPRLLIFGASNRATGIPDTFSGSGS GTDFTLTISRLEPEDFAVYYCQQYGHSPITFGQGTRLEIK (SEQ ID NO: 58) H88-67 DIVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRF SGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPAFGQGTKVEIK (SEQ ID NO: 59)
TABLE-US-00008 TABLE 7 CDR amino acid sequences of human antibodies VH CDR sequence-IMGT analysis Antibody CDR1 CDR2 CDR3 H88-96 GFTFSSYA ISSNGGST VIEGGSTTGTTSGAFD (SEQ ID NO: 60) (SEQ ID NO: 62) (SEQ ID NO: 65) H88-93 GFSLTTSGVG IYWDDDQ AHLSYGPGWGYYMDV (SEQ ID NO: 61) (SEQ ID NO: 63) (SEQ ID NO: 66) H88-67 GFTFSSYA ISYDGSNK ARMDGSGSPDY (SEQ ID NO: 60) (SEQ ID NO: 64) (SEQ ID NO: 67) VL CDR sequence-IMGT analysis Antibody CDR1 CDR2 CDR3 H88-96 QGIDSW SA QQALSLPIT (SEQ ID NO: 68) (SEQ ID NO: 71) (SEQ ID NO: 73) H88-93 SQSVRNNY GA QQYGHSPIT (SEQ ID NO: 69) (SEQ ID NO: 72) (SEQ ID NO: 74) H88-67 QSLVYSDGNTY KV MQGTHWPPA (SEQ ID NO: 70) (SEQ ID NO: 28) (SEQ ID NO: 75)
[0152] The binding abilities of fully human antibodies and murine antibodies to SEMG2 were tested, that is, murine antibodies MM02 and MM05 and concentration gradient diluted filly human antibodies H88-93 were mixed and added as primary antibodies in a 96-well microplate coated with SEMG2. Murine monoclonal antibody bound to SEMG2 was measured using anti-mouse HRP secondary antibody. The blocking percentage is calculated according to the following formula:
Blocking percentage=[1−(A450 of experimental antibody group-Blank control)/(A450 of positive control antibody−A450 of empty control)]×100%
[0153] The result shows that H88-93 competes with MM02 and MM05 for binding to SEMG2, as shown in
[0154] Furthermore, the effect of human antibodies H88-93,H88-96 and H88-67 on blocking the binding between SEMG2 and CD27 was detected by ELISA. All antibodies at a concentration of 10 μg/mL inhibited the binding between SEMG2 and CD27 in varying degrees, as shown in
[0155] To verify the effect of the human antibodies on the function of activated PBMC on tumor cell killing, A375 and LOVO cells were co-cultured with activated PBMC, and H88-93, H88-96, or H88-67 antibody was added at the same time, and the apoptosis ratio of tumor cells were detected. The result is shown in
Example 17: Binding Kinetic Determination of the Monoclonal Antibodies of the Present Invention to Antigens by Bio-Optical Interferometry
[0156] The equilibrium dissociation constant (KD) of the antibody of the present invention binding to human SEMG2 was determined by biolayer interferometry (ForteBio Bltz or Gator instrument). For example, the ForteBio affinity assay was performed according to the existing method, that is, half an hour before start, an appropriate amount of AMQ (Pall, 1506091) (for sample detection) or AHQ (Pall, 1502051) (for positive control detection) sensors were taken and soaked in SD buffer (PBS 1×, BSA 0.1%, Tween-20 0.05%). 100 μl of SD buffer, antibody and SEMG2 were added to a 96-well black polystyrene half area microplate, respectively. Select the sensor location based on the sample location layout. KD values were analyzed using molecular interaction analysis software. In the experiments of the assays, the affinity constants of murine monoclonal antibodies and human antibodies H88-67, H88-93 and H88-96 are shown in Table 8, and the affinity and dissociation curves of SEMG2 and corresponding proteins are shown in
TABLE-US-00009 TABLE 8 Affinity constants (equilibrium dissociation constants) for the detection of antigen-antibody binding by biolayer optical interferometry Antibody KD (M) MM02 1.33 × 10.sup.−9 MM05 5.28 × 10.sup.−9 MM07 1.82 × 10.sup.−9 MM08 2.34 × 10.sup.−9 MM13 .sup. 6.93 × 10.sup.−10 MM14 1.44 × 10.sup.−9 H88-67 2.84 × 10.sup.−8 H88-93 4.60 × 10.sup.−9 H88-96 1.40 × 10.sup.−8
Example 18: Affinity Maturation of Fully Human Monoclonal Antibodies
[0157] Using the plasmids constructed from the VH and VL coding sequences of fully human antibodies H88-96 and H88-67 as templates, the plasmids were obtained by gene synthesis, and then made single-point and double-point saturation mutation. In vitro ligation method was then performed to recombine antibody genes. Finally, the Fab gene sequence of recombinant antibody was inserted into the vector, and then transformed to obtain 4 phage affinity-matured antibody libraries with titer higher than 10.sup.8CFU. The antibody mutant library was screened by the immunotube gradient screening assay, and the mutants with finely improved affinity compared to the wild type were obtained. The full- length affinity matured human antibody was then constructed according to the detected Fab sequence or the recombination of VH and VL sequences in the Fab sequence. The VH and VL sequences derived from H88-67 and the CDR regions of the VH sequence of H88-96 after affinity maturation are shown in Table 9, and CDR regions of the light and heavy chain of the antibody are shown in Table 9.
TABLE-US-00010 TABLE 9 CDR sequences corresponding to affinity matured fully human antibodies VH CDR sequence-IMGT analysis VH ID CDR1 CDR2 CDR3 67-3 GFTFSSYA ISYDGSNK ARMDNHGSPDY (SEQ ID NO: 60) (SEQ ID NO: 64) (SEQ ID NO: 77) 67-6 GFTFSSYA ISYDGSNK ARMDGHGSPDY (SEQ ID NO: 60) (SEQ ID NO: 64) (SEQ ID NO: 78) 67-9 GFTFSSYA ISYDGSNK ARMDSGGSPDY (SEQ ID NO: 60) (SEQ ID NO: 64) (SEQ ID NO: 79) 96-10R GFTFSSRA ISSNGGST VIEGGSTGSTTSGAFDI (SEQ ID NO: 76) (SEQ ID NO: 62) (SEQ ID NO: 80) 96-10V GFTFSSYA ISSNGGST VIEGGSTSSTVSGAFDI (SEQ ID NO: 60) (SEQ ID NO: 62) (SEQ ID NO: 81) VL CDR sequence-IMGT analysis VL ID CDR1 CDR2 CDR3 67-3 QSLVSDGNTY KV MQGTHWPPA (SEQ ID NO: 70) (SEQ ID NO: 28) (SEQ ID NO: 75) 67-4 QSLVYSDGNTY EV MQGTHWPPA (SEQ ID NO: 70) (SEQ ID NO: 83) (SEQ ID NO: 75) 67-5 QSLVYSDGNTY GV MQGTHWPPA (SEQ ID NO: 70) (SEQ ID NO: 84) (SEQ ID NO: 75) 67-6 QSLVYKDGNTY KV MQGTHWPPR (SEQ ID NO: 82) (SEQ ID NO: 28) (SEQ ID NO: 85)
TABLE-US-00011 TABLE 10 VH and VL sequences corresponding to affinity matured fully human antibodies Antibody Number Heavy chain variable region VH Light chain variable region VL 67-3-67-3 QVQLLESGGGVVQPGRSLRLSCAASGFTPS DIVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLN SYAMHWVRQASGKGLEWVAVISYDGSNK WFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDF YYADSVKGRFTISRDNSKNTLYLQMNSLRA TLKISRVEAEDVGVYYCMQGTHWPPAFGQGTKVEIK EDTAVYYCARMDNHGSPDYWGQGTLVTV (SEQ ID NO: 59) SS ((SEQ ID NO: 96) 67-3-67-4 QVQLLESGGGVVQPGRSLRLSCAASGRTFS DIVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLN SYAMHWVRQASGKGLEWVAVISYDGSNK WFQQRPGQSPRRLIYEVSNRDSGVPDRFSGSGSGTDF YYADSVKGRFTISRDNSKNTLYLQMNSLRA TLKISRVEAEDVGVYYCMQGTHWPPAFGQGTKVEIK EDTAVYYCARMDNHGSPDYWGQGTLVTV (SEQ ID NO: 101) SS ((SEQ ID NO: 96) 67-3-67-5 QVQLLESGGGVVQPGRSLRLSCAASGRTFS DIVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLN SYAMHWVRQASGKGLEWVAVISYDGSNK WFQQRPGQSPRRLIYGVSNRDSGVPDRFSGSGSGTDF YYADSVKGRFTISRDNSKNTLYLQMNSLRA TLKISRVEAEDVGVYYCMQGTHWPPAFGQGTKVEIK EDTAVYYCARMDNHGSPDYWGQGTLVTV (SEQ ID NO: 102) SS ((SEQ ID NO: 96) 67-3-67-6 QVQLLESGGGVVQPGRSLRLSCAASGRTFS DIVMTQSPLSLPVTLGQPASISCRSSQSLVYKDGNTYLN SYAMHWVRQASGKGLEWVAVISYDGSNK WFQQRPGQSPRRLIYKVSNRDSGVPDRF5G5GSGTDF YYADSVKGRFTISRDNSKNTLYLQMNSLRA TLKISRVEAEDVGVYYCMQGTHWPPRFGQGTKVEIK EDTAVYYCARMDNHGSPDYWGQGTLVTV (SEQ ID NO: 103) SS ((SEQ ID NO: 96) 67-9-67-3 QVQLLESGGGVVQPGRSLRLSCAASGFTFS DIVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLN SYAMHWVRQASGKGLEWVAVISYDGSNK WFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDF YYADSVKGRFTISRDNSKNTLYLQMNSLRA TLKISRVEAEDVGVYYCMQGTHWPPAFGQGTKVEIK EDTAVYYCARMDSGGSPDYWGQGTLVTV (SEQ ID NO: 59) SS ((SEQ ID NO: 97) 67-6-67-6 QVQLLESGGGVVQPGRSLRLSCAASGRTFS DIVMTQSPLSLPVTLGQPASISCRSSQSLVYKDGNTYLN SYAMHWVRQASGKGLEWVAVISYDGSNK WFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDF YYADSVKGRFTISRDNSKNTLYLQMNSLRA TLKISRVEAEDVGVYYCMQGTHWPPRFGQGTKVEIK EDTAVYYCARMDGHGSPDYWGQGTLVTV (SEQ ID NO: 103) SS ((SEQ ID NO: 98) 96-10R-10 QVQLLESGGGLVQPGGSLRLSCSASGFTFS DQMIQSPPSVSASVGDTVTIACRANQGIDSWLAWYQ SRAMHWVRQAPGKGLEYVSAISSNGGSTY QKPGRAPKLLYSASRLQSGVPSRFSGGGSGTDFALTIS YADSVKGRFTISRDNSKNTLYLQMSSLRAE NLQPEQFATYYCQQALSLPITFGQGTRLEIK DTAVYYCVIEGGSTGSTTSGAFDIWGQGT (SEQ ID NO: 57) MVTVSS ((SEQ ID NO: 99) 96-10V-10 QVQLLESGGGLVQPGGSLRLSCSASGFTFS DIQMQSPPSVSASVGDTVTIACRANQGIDSWLAWYQ SYAMHWVRQAPGKGLEYVSAISSNGGSTY QKPGRAPKLLIYSASRLQSGVPSRFSGGGSGTDFALTIS YADSVKGRFTISRDNSKNTLYLQMSSLRAE NLQPEDFATYYCQQALSLPITFGQGTRLEIK DTAVYYCVIEGGSTGSTVSGAFDIWGQGT (SEQ ID NO: 57) MVTVSS ((SEQ ID NO: 100)
[0158] Through the above affinity maturation process, we obtained anti-human SEMG2 monoclonal antibodies with improved affinity, such as 67-3-67-3, 67-3-67-4, 67-3-67-5 and 67-3-67-6 which consist of the combination of the affinity-matured heavy chain numbered 67-3 and the affinity-matured light chain sequence numbered 67-3, 67-4, 67-5 and 67-6, antibody 67-9-67-3 consists of the combination of the heavy chain numbered 67-9 and the light chain numbered 67-3, antibody 67-6-67-6 consists of the combination of the light chain and heavy chain numbered 67-6, and antibodies 96-10R-10 and 96-10V-10 reconstituted by the heavy chains numbered 96-10R and 96-10V and the light chain of H88-96L. The recombinant monoclonal antibodies consist of these light and heavy chains have an affinity more than 10-fold higher for SEMG2 and BSA-S2 (497-509) (i.e., BSA-SP7) than that of the parent antibodies (see
Example 19: Validation of the Anti-Tumor Effect of SEMG2 Antibody in Xenograft Model of PBMC-HIS Model in Human Malignant Melanoma A375 Cells
[0159] 30 male NPSG mouse models aged 6-8 week were weighed. A375 cells (with confirmed endogenous expression of SEMG2) were cultured in vitro to obtain 1.8×10.sup.8 cells. After 30 mice were inoculated with PBMC, A375 tumor cells were inoculated on the 3rd day. After that, the proportion of hCD45+ cells in mouse blood and the body weight were measured once a week. After inoculation, tumor volume was measured once a week, and the proportion of hCD45+ cells in mouse blood was measured when the average tumor volume reached about 40-80 mm.sup.3. Mice were grouped randomly based on tumor volume and the proportion of hCD45+ cells in mouse blood, and the administration was started immediately. The date began the administration was considered day 0. Dosing regimen: SEMG2 antibody (MM05 clone) was injected intraperitoneally at 5 mg/kg three times a week. After the start of administration, the tumor growth status of the mice was observed every week. After the tumor growth, the body weight and tumor volume were measured 3 times a week, and the relative count of hCD45+ cells in mouse blood was monitored by flow cytometry 3 times a week. When the tumor volume reached the end point, blood was collected and the same indexed were detected, and the experiment was ended. The observation of mice includes: daily observation, observation of animal morbidity and death every working day after inoculation. Measurement of tumor volume: after inoculation and before grouping, when tumors were visible, the tumor volume of experimental animals was measured once a week. After inoculation and grouping, the tumor volume of animals in the experiment was measured twice a week. The tumor volume was measured by a bidirectional measurement method. First, the long and short diameters of the tumor were measured with a vernier caliper, and the tumor volume was then calculated using the formula TV=0.5*a*b2, where a is the long diameter of the tumor and b is the short diameter of the tumor. The experimental results are shown in
Example 20: Knockout of the Corresponding Gene Svs3a in Mice Proves No Significant Side Effect After Function Blockade of SEMG2
[0160] To prove the possible toxic and side effects after functional blockade of SEMG2 as a drug target, the corresponding gene Svs3a in mice was knocked out systemically. The specific scheme was as follows: CRISPR/cas9 technology was adopted in the project, and non-homologous recombination was used to introduce mutation, resulting in a shift in the reading frame and loss of function of Svs3a gene. The brief process is as follows: Cas9 mRNA and gRNA were obtained by in vitro transcription; Cas9 mRNA and gRNA were microinjected into the fertilized eggs of C57BL/6J mice to obtain F0 generation mice. The positive F0 mice verified by PCR amplification and sequencing were mated with C57BL/6J mice to obtain positive F1 mice.
TABLE-US-00012 gRNAs sequence (5′-3′): gRNA1, CAGCCGCAGAGAGGCACTCAGGG; gRNA2, ATGCACCACCAAGAAACACTGGG.
[0161] Sequence alignment before and after knockout:
TABLE-US-00013 Wild-type: TGAGTTCAGGGAGCAGCCGCAGAGAGGCACTCAGGGAGAATGTCCA TAAGGATGCCATGGCAGTGAGAG......AGTGTCTTAGCAAACGGGAG AGCTGTCTGCCCCAGTGTTTCTTGGTGGTGCATGGTGGGCTCCCTGT GCCCGCAGTGC; Mutant: TGAGTTCAGGGAGCAGCCGCAAGAGAGG... (−1006 bp)...GAGGTGCATGGTGGGCTCCCTGTGCCCGCAGTGC.
[0162] Subsequent reproduction: the obtained gene knockout heterozygous mice (gene+/−) were divided into two parts: a part of heterozygous mice was mated with wild-type mice for expansion of more heterozygous mice; a part of heterozygous mice self-bred to obtain gene knockout homozygous mice (gene−/−) for gene knockout effect verification and subsequent phenotype analysis.
[0163] Phenotype analysis: Anticoagulated whole blood was taken from mice for flow cytometry, and the proportion of CD8+, CD4+, CD3+, CD27+ positive cells in blood was analyzed. After the mice rested for 2 days, the anticoagulated whole blood was collected from the inner canthus, and the molecule department would perform the blood routine test. After the mice rested for 3 days, the mice were weighed and anesthetized, and the mouse gross bodies were imaged; the eyeballs of the mice were removed, the blood was collected, and the serum was separated. The molecule department would measure the serum biochemical parameters. After the eyeball was removed and the blood was collected, the mice were euthanized for material collection: brain: the whole brain was removed and divided by the sagittal plane, and the left side was fixed, and the right side was quick-frozen; liver: the whole liver was removed and divided in two, the left lobe was fixed, and the rest were quick-frozen; spleen: the whole spleen was removed and divided in two, half fixed, half quick-frozen; kidney: the left kidney was removed for fixation, the right kidney was removed and quick-frozen; stomach: the whole stomach was removed and divided sagittal, the greater curvature was fixed, and the lesser curvature was quick-frozen; large intestine: the intact large intestine was removed for Swiss roll fixation; small intestine: the whole small intestine was removed and divided into three sections (duodenum, ileum, jejunum) for Swiss roll fixation; lung: the left lung was removed for fixation, and the right lung for removed for quick freezing; heart: the entire heart was removed for fixation after dilation. All fixed samples were sent to pathology for paraffin embedding, wherein 11 organs (brain, heart, lung, kidney, spleen, liver, stomach, duodenum, jejunum, ileum, and colon) of one KO mouse (#98) were sectioned, HE stained, and read for analysis.
[0164] The phenotype analysis results of wild-type (WT) and homozygous knockout mice (KO) are shown in
[0165] The embodiments of the present invention have been described above by the inventors, but the present invention is not limited thereto, and those skilled in the art can understand that modifications and changes can be made within the scope of the purpose of the present invention. The manner of modifications and changes should fall within the scope of protection of the present invention.