CN111434691A

CN111434691A - PD-L2 monoclonal antibody and application thereof

Info

Publication number: CN111434691A
Application number: CN201910032439.5A
Authority: CN
Inventors: 万晓春; 陈有海; 李俊鑫; 刘绿艳; 李映梅
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2019-01-14
Filing date: 2019-01-14
Publication date: 2020-07-21

Abstract

The invention relates to the field of biotechnology, in particular to a PD-L monoclonal antibody and application thereof, and provides an amino acid sequence of three CDR regions of a heavy chain of the monoclonal antibody, an amino acid sequence of three CDR regions of a light chain of the monoclonal antibody, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of the amino acid sequence, or an amino acid sequence with the same or similar function with the shown amino acid sequence, or an amino acid sequence with at least 80% homology with the sequence.

Description

PD-L2 monoclonal antibody and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a PD-L2 monoclonal antibody and application thereof.

Background

Hybridoma technology (Hybridoma technology) is the most widely used monoclonal antibody technology. In 1975, Koehler and Milstein demonstrated that the hybridoma cell lines formed by fusing myeloma cells with immunized animal spleen cells can produce monoclonal antibodies with strong specificity for an antigen and can also be amplified continuously. The hybridoma technology utilizes the characteristics that myeloma cells can be continuously passaged in vitro and lymphocytes can secrete specific antibodies, and effectively exerts the functions of the two cells, so that the large-scale application of the preparation of the monoclonal antibody becomes possible.

Programmed death receptor (PD-1) is a member of CD28 super family, is an important immunosuppressive molecule and is mainly expressed on activated T cells and B cells, and ligands PD-L s (Programmed death-1ligands) of PD-1 mainly comprise PD-L1 (B7-H1/CD274) and PD-L2 (B7-DC/CD273), wherein PD-L1 is a main ligand, and the current clinically applied antibody medicament for blocking PD-1/PD-L1 is ineffective for PD-L1 negative patients, and the development of monoclonal antibodies of PD-L2 is urgently needed.

PD-L protein is I-type transmembrane protein containing 274 amino acid residues and encoded by PDCD 1L G2 gene, the homology with PD-L reaches 40%, but the difference is also certain, PD-L is only expressed on the membrane surface of macrophage, dendritic cell and some B cell subclasses in vivo tissues, but the research shows that PD-L can be induced to express in other various immune cells and non-immune cells under the stimulation of specific microenvironment in recent years, such as lung cancer, breast cancer, thymoma and other tumor cells, a large number of researches show that PD-L2 can be used as an important biomarker to play an important role in the diagnosis, target treatment and 865 of various tumors, the superiority of PD-L monoclonal antibody determines the sensitivity and specificity of various immunoassay methods (such as immunohistochemistry, enzyme-linked immunosorbent assay, flow cytometry assay and the like), determines the safety and effectiveness of target treatment, the antibody aiming at PD 1/PD-L1 is widely used for cancer treatment, the antibody aiming at poor PD-4 is lack of PD-L specificity, and the antibody has high affinity detection significance for detecting PD-L.

Disclosure of Invention

The PD-L antibody provided by the invention has high biological activity and a unique antibody variable region sequence, and has a good anti-tumor effect.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a monoclonal antibody of PD-L2,

(I) the amino acid sequences of the three CDR regions of the heavy chain have the amino acid sequences shown as SEQ ID NO 5, 6 and 7 respectively; and is

(II) the amino acid sequences of the three CDR regions of the light chain have the amino acid sequences shown in SEQ ID NO 8, 9 and 10 respectively; or

(III), (I) or (II) the amino acid sequence obtained by substituting, deleting or adding one or more amino acids, and the amino acid sequence has the same or similar function with the amino acid sequence shown in (I) or (II); or

(IV) and an amino acid sequence having at least 80% homology with the sequence of (I), (II) or (III).

In some embodiments of the invention, the monoclonal antibody,

(V) the heavy chain variable region has an amino acid sequence shown as SEQ ID NO: 2; and is

(VI), the light chain variable region has an amino acid sequence shown in any one of SEQ ID NO 4; or

(VII), (V) or (VI) and an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence and has the same or similar function with the amino acid sequence shown in (V) or (VI); or

(VIII) an amino acid sequence which is at least 80% homologous with the sequence described in (V), (VI) or (VII).

In some embodiments of the invention, the plurality of monoclonal antibodies is 2, 3, 4 or 5.

The invention also provides the nucleotide for coding the monoclonal antibody.

In some embodiments of the invention, the nucleotide,

(IX), the heavy chain thereof has the nucleotide sequence shown in SEQ ID NO: 1; and is

(X) the light chain has the nucleotide sequence shown in SEQ ID NO. 3; or

(XI), (IX) or (X) wherein the nucleotide sequence is modified, substituted, deleted or added with one or more bases;

(XII), a sequence having at least 80% homology to the nucleotide sequence of (IX), (X) or (XI);

(XIII), the complement of the nucleotide sequence described In (IX), (X), (XI) or (XII).

The invention also provides an expression vector comprising nucleotides encoding the monoclonal antibody.

On the basis, the invention also provides a host cell for transforming or transfecting the expression vector.

The invention also provides a preparation method of the monoclonal antibody, which comprises the steps of culturing the host cell and inducing the expression of the monoclonal antibody of PD-L2.

Furthermore, the invention provides a conjugate comprising said monoclonal antibody chemically or biologically labeled.

On the basis of the research, the invention also provides a conjugate prepared by coupling the monoclonal antibody or the conjugate with a solid medium or a semisolid medium.

The invention also provides application of the monoclonal antibody or the conjugate in preparing a medicament for inducing cancer cell apoptosis and preventing and/or treating autoimmune diseases or in preparing products for Western blot detection, E L ISA detection and flow cytometry detection.

The invention also provides a medicament, which comprises the monoclonal antibody or the conjugate and pharmaceutically acceptable auxiliary materials.

In addition, the invention also provides a kit which comprises the monoclonal antibody or the conjugate and acceptable auxiliary agents.

The invention provides a monoclonal antibody of PD-L2, wherein (I) the amino acid sequences of three CDR regions of the heavy chain have the amino acid sequences shown in SEQ ID NO. 5, 6 and 7 respectively, and (II) the amino acid sequences of three CDR regions of the light chain have the amino acid sequences shown in SEQ ID NO. 8, 9 and 10 respectively, or (III), (I) or (II) the amino acid sequence obtained by substituting, deleting or adding one or more amino acids has the same or similar function with the amino acid sequence shown in (I) or (II), or (IV) the amino acid sequence with at least 80% homology with the sequence shown in (I), (II) or (III).

The invention widens the possibility of blocking the antibody therapy of PD1/PD 1ligand, and compared with the existing PD-L monoclonal antibody, the 3F4 antibody has unique amino acid sequence and CDR region sequence, high affinity and high specificity, can be used for E L ISA, Western blot and flow cell detection, and has wide application.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows that monoclonal hybridoma cell strain 3F4 capable of secreting PD-L2 antibody is obtained by screening E L ISA;

FIG. 2 shows a subtype identification map of the 3F4 antibody;

FIG. 3 shows the use of the PD-L2 monoclonal antibody for Western Blot;

FIG. 4 shows a diagram of the flow assay Daudi cells with PD-L23F 4 antibody;

FIG. 5 shows the introduction of the PD-L23F 4 antibody detection vector into 293T cells expressing PD-L2.

Detailed Description

The present invention discloses a PD-L2 monoclonal antibody and its application, which can be realized by those skilled in the art by appropriately modifying the process parameters based on the teachings herein, it is particularly noted that all such similar substitutes and modifications which are obvious to those skilled in the art are deemed to be included in the present invention.

The invention provides an amino acid sequence of a PD-L2 monoclonal antibody, which can obtain a new amino acid sequence by substituting, deleting or adding one or more amino acids, and has an amino acid sequence with the same or similar function with the amino acid sequence provided by the invention, or an amino acid sequence with at least 80 percent of homology with the disclosed sequence can be used as an alternative scheme.

The present invention provides nucleotide sequences of the PD-L2 monoclonal antibody, the complement of which or which differ from the nucleotide sequences disclosed herein due to the degeneracy of the genetic code but which encode the same protein, or sequences which are at least 80% homologous to the sequences disclosed herein, as alternatives.

The invention provides a nucleotide sequence of a PD-L2 monoclonal antibody, a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences, and a nucleotide sequence with the same or similar functions as the nucleotide sequence disclosed by the invention can be used as an alternative scheme.

The invention adopts mouse ascites to produce antibody, and can also adopt large-scale culture hybridoma cells secreting the antibody or CHO cells stably expressing antibody genes or 293F cells to produce the antibody.

The invention provides a variable region amino acid sequence and a nucleotide sequence of a mouse anti-human PD-L2 monoclonal antibody, and a human-mouse chimeric antibody and a humanized antibody of an anti-human PD-L2 can be obtained by antibody humanization transformation, the human-mouse chimeric antibody of the anti-human PD-L2 connects a variable region of a mouse-derived monoclonal antibody gene and a human constant region together and then is expressed and generated in a mammalian cell, and the humanized antibody of the anti-human PD-L2 is produced by converting an FR region of the variable region into a human source in addition to the constant region of the antibody into the human source, so that the immunogenicity is reduced.

The invention provides a variable region amino acid sequence and a nucleotide sequence of an anti-human PD-L2 monoclonal antibody, on the basis of which a genetically engineered antibody can be generated, wherein the heavy chain and light chain variable region sequences contained in the genetically engineered antibody are consistent with the variable region sequences disclosed by the invention, the genetically engineered antibody comprises but not limited to a functional fragment Fab of the antibody, or a single chain antibody, or an antibody functional fragment VH-L formed by fusing a heavy chain variable region and a complete light chain, or an antibody functional fragment formed by arranging, connecting in series or combining one or more CDRs of the heavy chain and the light chain, or an antibody-like functional fusion protein formed by connecting, splicing and fusing the antibody and the antibody functional fragment with other various proteins or polypeptides.

The antibody of anti-human PD-L2 disclosed by the invention can be used independently, and can also be used in combination with commonly used tumor radiotherapy and chemotherapy medicaments.

The antibody of anti-human PD-L2 or the gene engineering antibody generated based on the antibody can be used as a targeting part and coupled with radionuclide, chemical drugs or toxins.

The invention discloses a tumor killing effect of an anti-human PD-L2 antibody, which can be used for treating other various tumor types expressed by PD-L2.

The PD-L2 monoclonal antibody and the raw materials and reagents used in the application thereof provided by the invention can be purchased from the market.

The invention is further illustrated by the following examples:

EXAMPLE 1 production of PD-L2 monoclonal antibody

Immunizing female BA L B/c mice (6 weeks old) with PD-L protein antigen (purchased from sino biological, cat # 10292-h08h-1mg), emulsifying the antigen using Freund's complete adjuvant for the first immunization, emulsifying the antigen using Freund's incomplete adjuvant for the second immunization, injecting subcutaneously 5-6 points, injecting the antigen in an amount of-100 ug. per mouse 10 days after the 3 rd immunization, collecting a small amount of blood from the tail of the mouse, performing a serum titer E L ISA test, selecting mice with high antibody titer (>1:100000) for the 4 th immunization, performing an intraperitoneal injection of the antigen protein, injecting 100 ug. per mouse 3-5 days after the 4 th immunization, killing the mice, fusing splenocytes with SP2/0 cells, obtaining stable hybridoma cells by culturing in HAT medium, obtaining stable hybridoma cells, screening by E L, and obtaining a monoclonal antibody cell strain which can be secreted by a liquid nitrogen-liquid nitrogen dilution method (ISA) and screening).

Example 2 sequencing of antibody Gene variable regions of PD-L2 monoclonal antibody hybridoma cells

The monoclonal antibody 3F4 hybridoma cells in logarithmic growth phase are harvested, TRIZO L is cracked for RNA extraction, cDNA is obtained after reverse transcription, heavy chain and light chain variable regions are amplified and obtained, non-functional VK genes are removed, the heavy chain and light chain variable regions are cloned to a pMD18-T vector, sequencing is carried out, an IMGT/V-QUEST database is used for comparing sequencing results, and further analysis is carried out.

The base sequence of the heavy chain is shown as SEQ ID NO. 1:

the heavy chain variable region amino acid sequence is shown as SEQ ID NO. 2:

the base sequence of the light chain is shown as SEQ ID NO. 3:

the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4:

by comparison with the IMGT/V-QUEST database, the heavy chain:

CDR1-IMGT(SEQ ID NO.5)：

CDR2-IMGT(SEQ ID NO.6)：

CDR3-IMGT(SEQ ID NO.7)：

light chain:

CDR1-IMGT(SEQ ID NO.8)：

CDR2-IMGT(SEQ ID NO.9)：

CDR3-IMGT(SEQ ID NO.10)：

example 3 subtype identification of the PD-L2 monoclonal antibody

Monoclonal antibody subtype identification was performed using a commercial mouse monoclonal antibody Isotyping kit (sino biological, SEK003), and the result is shown in fig. 2, where 3F4 is an IgG1 type antibody.

Example 4 anti-PD-L2 monoclonal antibody detection of PD-L2 of Dauti cells

1. Collecting Daudi cells, blowing, counting, and packaging into 5 centrifuge tubes, each tube having a length of 3 × 10⁵The cells were centrifuged at 500g for 5min, the supernatant was removed, and 1ml of PBS was added to the cells, and centrifuged at 500g for 5min, and the supernatant was removed.

2. Reagents were added as in table 1:

TABLE 1

Placing on ice, incubating for 30 min.

3. 1ml of PBS + 5% FBS was added, 500g was centrifuged for 5min, the supernatant was removed, 100ul of DMEM culture solution +1ul of PE goal anti-mouse lgG (minor x-reactivity) antibody (biolegend,405307) was added, and the mixture was incubated on ice for 20 min.

4. 1ml of PBS + 5% FBS was added to each, and the mixture was centrifuged at 500g for 5min to remove the supernatant.

5. 300ul PBS + 5% FBS was added to each tube, resuspended, and examined using a Beckman CytoF L EXCM flow cytometer.

As shown in FIG. 4, the 3F4 antibody can be combined with the PD-L2 protein on the cell surface of Daudi, and is applied to flow cytometry detection, and the detection sensitivity is superior to that of a commercial positive control.

Example 5 anti-PD-L2 monoclonal antibody detection of plasmid vectors 293T cells with PD-L2 introduced

1. 293T cells transfected with PD-L2 expression vector (Sino BiologcalHG 10292)

2. 293T cells were collected, blown up and counted, and divided into 5 centrifuge tubes, 3 × 10 per tube⁵Centrifuging the cells at 500g for 5min,the supernatant was removed, 1ml of PBS was added thereto, and the mixture was centrifuged at 500g for 5min to remove the supernatant.

3. Reagents were added as per table 2:

TABLE 2

Placing on ice, incubating for 30 min.

4. 1ml of PBS + 5% FBS was added, 500g was centrifuged for 5min, the supernatant was removed, 100ul of DMEM culture solution +1ul of PE goal anti-mouse lgG (minor x-reactivity) antibody (biolegend,405307) was added, and the mixture was incubated on ice for 20 min.

5. 1ml of PBS + 5% FBS was added to each, and the mixture was centrifuged at 500g for 5min to remove the supernatant.

6. 300ul PBS + 5% FBS was added to each tube, resuspended, and examined using a Beckman CytoF L EXCM flow cytometer.

As shown in FIG. 5, the 3F4 antibody can be combined with the PD-L2 protein over-expressed on the surface of 293T cells, and can be applied to flow cytometry detection.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

A monoclonal antibody to PD-L2, characterized in that,

(I) the amino acid sequences of the three CDR regions of the heavy chain have the amino acid sequences shown as SEQ ID NO 5, 6 and 7 respectively; and is

(II) the amino acid sequences of the three CDR regions of the light chain have the amino acid sequences shown in SEQ ID NO 8, 9 and 10 respectively; or

(III), (I) or (II) the amino acid sequence obtained by substituting, deleting or adding one or more amino acids, and the amino acid sequence has the same or similar function with the amino acid sequence shown in (I) or (II); or

(IV) and an amino acid sequence having at least 80% homology with the sequence of (I), (II) or (III).
2. The monoclonal antibody of claim 1,

(V) the heavy chain variable region has an amino acid sequence shown as SEQ ID NO: 2; and is

(VI), the light chain variable region has an amino acid sequence shown in any one of SEQ ID NO 4; or

(VII), (V) or (VI) and an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence and has the same or similar function with the amino acid sequence shown in (V) or (VI); or

(VIII) an amino acid sequence which is at least 80% homologous with the sequence described in (V), (VI) or (VII).
3. The monoclonal antibody of claim 1 or 2, wherein the plurality is 2, 3, 4, or 5.
4. A nucleotide encoding the monoclonal antibody of any one of claims 1 to 3.
5. The nucleotide of claim 4,

(IX), the heavy chain thereof has the nucleotide sequence shown in SEQ ID NO: 1; and is

(X) the light chain has the nucleotide sequence shown in SEQ ID NO. 3; or

(XI), (IX) or (X) wherein the nucleotide sequence is modified, substituted, deleted or added with one or more bases; or

(XII), a sequence having at least 80% homology to the nucleotide sequence of (IX), (X) or (XI); or

(XIII), the complement of the nucleotide sequence described In (IX), (X), (XI) or (XII).
6. An expression vector comprising nucleotides encoding the monoclonal antibody of any one of claims 1 to 3.
7. A host cell transformed or transfected with the expression vector of claim 6.
8. The use of the monoclonal antibody of any one of claims 1 to 3 for the preparation of a medicament for inducing apoptosis of cancer cells, preventing and/or treating tumors or for the preparation of products for Western blot detection, E L ISA detection and flow cytometry detection.
9. A medicament comprising the monoclonal antibody of any one of claims 1 to 3 and a pharmaceutically acceptable excipient.
10. A kit comprising a monoclonal antibody according to any one of claims 1 to 3 and acceptable auxiliary agents.