CN115991785A

CN115991785A - Bispecific antibody, preparation method and application

Info

Publication number: CN115991785A
Application number: CN202111216317.5A
Authority: CN
Inventors: 路慧丽; 王紫嫣; 杨慧
Original assignee: Shanghai Miaoju Biotechnology Co ltd; Shanghai Jiaotong University
Current assignee: Shanghai Zeyin Biotechnology Co ltd; Shanghai Jiaotong University
Priority date: 2021-10-19
Filing date: 2021-10-19
Publication date: 2023-04-21

Abstract

The embodiment of the application discloses a bispecific antibody, a preparation method and application. The bispecific antibody comprises: a first protein functional region that specifically binds to a stem cell surface antigen, the first protein functional region being an antibody or antigen-binding fragment thereof that is directed against the stem cell surface antigen; and a second protein functional region that specifically binds to a chondrocyte surface antigen, the second protein functional region being an antibody or antigen-binding fragment thereof that is directed against the chondrocyte surface antigen. The bifunctional antibody disclosed by the embodiment of the application realizes the targeted implantation of stem cells at the damaged part of the articular cartilage, and plays a synergistic effect with the stem cells to improve the arthritis treatment effect.

Description

Bispecific antibody, preparation method and application

Technical Field

The present application relates generally to the field of pharmaceutical technology, and in particular to a bispecific antibody, a preparation method and use thereof, and more particularly, to a bispecific antibody, a nucleic acid, an expression vector, a host cell, a method for preparing a bispecific antibody, a pharmaceutical composition, and use of a bispecific antibody, a nucleic acid, an expression vector, a host cell or a pharmaceutical composition in the preparation of a medicament for treating or preventing arthritis.

Background

Arthritis is one of the diseases seriously jeopardizing human health in the world today, and conventional therapies such as physical therapy, drug therapy, immunotherapy or surgical therapy which are clinically adopted at present have serious limitations although the conditions are relieved to some extent.

Matrilin-3 (MATN 3) is a non-collagenous extracellular matrix protein, is mainly secreted by chondroblasts, is specifically distributed on cartilage, and is the only member of the Matrilin family found today that causes cartilage disease by mutation of a gene. Studies have shown that increased MATN3 expression in osteoarthritis, and positively correlated with joint injury severity, are favorable targets for the study of arthritis. However, there is a lack of drugs against MATN3 targets in the prior art.

Disclosure of Invention

The present application has been completed based on the following findings by the inventors:

the stem cells have the effect of promoting the repair and regeneration of the articular cartilage, and can quickly and effectively repair the damage when acting on the damaged part of the articular cartilage, so that the stem cells can be used for preparing medicines for treating or preventing arthritis.

The inventor finds that the specific implantation of the stem cells to the cartilage injury part can be realized by designing a bispecific antibody targeting the surface antigen of the stem cells and the surface antigen of the cartilage cells, and the repairing effect of the stem cells on the cartilage injury is remarkably improved.

In one aspect of the present application, embodiments provide a bispecific antibody comprising a first protein functional region that specifically binds a stem cell surface antigen and a second protein functional region that specifically binds a chondrocyte surface antigen, the first protein functional region being an antibody or antigen binding fragment thereof directed against a stem cell surface antigen, the second protein functional region being an antibody or antigen binding fragment thereof directed against a chondrocyte surface antigen. The bispecific antibody can guide stem cells to target and fix to cartilage injury parts, thereby playing a role in synergistic cartilage repair with the stem cells.

According to an embodiment of the present application, the stem cells are hematopoietic stem cells.

According to an embodiment of the present application, the stem cell surface antigen is CD34.

According to an embodiment of the present application, the chondrocyte surface antigen is MATN3.

According to embodiments of the present application, the light chain variable region of the first protein functional region comprises CDRs as set forth in SEQ ID nos. 1-3, and the heavy chain variable region comprises CDRs as set forth in SEQ ID nos. 4-6; the light chain variable region of the second protein functional region comprises CDRs as shown in SEQ ID Nos. 7-9, and the heavy chain variable region comprises CDRs as shown in SEQ ID Nos. 10-12.

According to an embodiment of the present application, the first protein functional region is selected from the group consisting of full-length immunoglobulins, fab ', (Fab') ₂ Fv, scFv or VHH; and/or the number of the groups of groups,

the second protein functional region is selected from the group consisting of full-length immunoglobulins, fab ', (Fab') ₂ Fv, scFv or VHH.

According to an embodiment of the present application, the first protein functional region is a full length IgG1 immunoglobulin and the second protein functional region is an scFv.

According to embodiments of the present application, the scFv is a light chain variable region-a first linker-a heavy chain variable region, the light chain variable region N-terminus or the heavy chain variable region C-terminus of the scFv being linked to the C-terminus or N-terminus of the full length IgG1 immunoglobulin light chain and/or heavy chain, respectively, by a second linker; or alternatively, the first and second heat exchangers may be,

the scFv is a heavy chain variable region-a first linker-a light chain variable region, the heavy chain variable region N-terminus or the light chain variable region C-terminus of the scFv is linked to the C-terminus or N-terminus of the full length IgG1 immunoglobulin light chain and/or heavy chain, respectively, by a second linker.

According to embodiments of the present application, the scFv is two in number, each symmetrically linked to the C-terminus of the full length IgG1 immunoglobulin light chain.

According to embodiments of the present application, the first linker and the second linker each independently comprise a repeat selected from GS, GGSG, GGGS and GGGGS sequences.

According to embodiments of the present application, the constant region of the full-length immunoglobulin is derived from IgG or IgG mutants.

According to embodiments of the present application, the constant region comprises amino acid substitutions of L234A, L235A and P329G compared to the wild type IgG1 constant region.

In other aspects of the present application, embodiments of the present application provide nucleic acids encoding the bispecific antibodies of the present application, expression vectors, host cells, methods of making, a pharmaceutical composition, and uses of the bispecific antibodies of the present application in the treatment or prevention of arthritis.

The bifunctional antibody disclosed by the embodiment of the application realizes the targeted implantation of stem cells at the damaged part of the articular cartilage, enhances the targeted implantation capacity of the stem cells, ensures that more stem cells can survive for a long time at the damaged part, and improves the treatment effect of arthritis.

Drawings

Other features, objects and advantages of the present application will become more apparent upon reading of the detailed description of non-limiting embodiments, made with reference to the following drawings, in which:

FIG. 1 is a schematic structural diagram of bispecific antibodies of various embodiments of the present application;

FIG. 2 is a schematic structural diagram of a bispecific antibody in the form of an IgG-scFv according to one embodiment of the present application;

FIG. 3 shows the result of PCR identification of amplified fragments, wherein A is the PCR identification of Anti-CD34-V _L Fragment, C _L Fragments; b is PCR identification of Anti-CD34-L and Anti-MATN3-scFv fragments;

FIG. 4 is a PCR identification of pIL-15 vector cleavage;

FIG. 5C by PCR _H Amino acid mutation of a region;

FIG. 6 PCR identification of Anti-CD34-V _H 、C _H Fragments;

FIG. 7 is a PCR identification result of amplified fragments, wherein A is Anti-CD34-V obtained by overlay PCR _H -C _H ' fragment, B is Anti-CD34-V obtained by primer extension _H -C _H Fragments;

FIG. 8 is a diagram showing the detection of the mass ratio of Anti-CD34 XMATN 3-H to Anti-CD34 XMATN 3-L by WB to determine the optimal co-transfection ratio, A for non-reduced samples and B for reduced samples;

FIG. 9 is a HiTrap MabSelect SuRe affinity purification chromatogram of CD34 MATN 3;

FIG. 10 is SDS-PAGE identification of CD34 XMATN 3 bispecific antibody after purification, A is a non-reducing sample, B is a reducing sample;

FIG. 11 is a chart showing the affinity results of Biacore detection antibodies with CD34-Fc antigen, wherein A is the interaction of Anti-CD34-scFv with CD34-Fc antigen and B is the interaction of CD34 XMATN 3 with CD34-Fc antigen;

FIG. 12 shows the affinity results of Biacore detection antibodies with MATN3-Fc antigen, A being the interaction of Anti-MATN3-scFv with MATN3-Fc antigen, B being the interaction of CD34 XMATN 3 with MATN3-Fc antigen;

FIG. 13 shows the affinity of the flow-test antibody for chondrocytes, A as a negative control, B as Anti-MATN3-scFv for chondrocytes, C as CD34 MATN3 for chondrocytes, and D as a comparison of monoclonal antibody, CD34 MATN3 for chondrocytes;

FIG. 14 shows the affinity of flow-detection antibodies with HSCs, A being the negative control-PE, B being the negative control-APC, C being the positive control, D being the binding of varying amounts of CD34×MATN3 to HSCs;

FIG. 15 is a construction of a pool of 293T-GFP and 293T-Anti-MATN3-scFv-GFP stable cells, A being 293T-GFP white light, B being 293T-GFP fluorescent light, C being 293T-Anti-MATN3-scFv-GFP white light, D being 293T-Anti-MATN3-scFv-GFP fluorescent light;

FIG. 16 is a flow assay for the expression of the GFP gene and Anti-MATN3-scFv gene from a pool of 293T-Anti-MATN3-scFv-GFP stable cells, wherein A is the expression of the GFP gene and B is the expression of the Anti-MATN3-scFv gene;

FIG. 17 shows the expression of MATN3 antigen of cartilage pieces, A & D for two cartilage pieces of the blank control group, B & E for two cartilage pieces of the 293T-GFP group, and C & F for two cartilage pieces of the 293T-Anti-MATN3-scFv-GFP group;

FIG. 18 is a CD34 XMATN 3 mediated HSCs targeted cartilage pieces, A & B was 40-fold and 100-fold enlarged from the blank; c & D is CFSE treated HSCs control cartilage pieces amplified 40-fold and 100-fold, E & F is CD34 x MATN3 modified experimental cartilage pieces amplified 40-fold and 100-fold;

FIG. 19 is a graph showing knee wear in rats following drug administration;

FIG. 20 is the results of safranin-fast green staining of knee joints in rats after drug administration treatment;

FIG. 21 shows the result of blue staining of toluidine in knee joints of rats after drug administration treatment;

FIG. 22 is COL2 expression after treatment administration;

FIG. 23 is COL10 expression following treatment with drug;

FIG. 24 shows MMP-13 expression following treatment with drug;

FIG. 25 shows MATN3 expression following treatment with drug;

FIG. 26 is a graph of repair results of HSCs-CD34xMATN3, HSCs and PBS after perforation of the trochanteric sulcus of the rat knee.

Detailed Description

The present application is described in further detail below with reference to examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other.

It should be noted that endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and that such range or value should be understood to include values approaching such range or value. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The application provides a bispecific antibody, which comprises a first protein functional region and a second protein functional region, wherein the first protein functional region is an antibody or an antigen binding fragment thereof for resisting a stem cell surface antigen, and can specifically bind to the stem cell surface antigen, and the second protein functional region is an antibody or an antigen binding fragment thereof for resisting a chondrocyte surface antigen, and can specifically bind to the chondrocyte surface antigen. Thus, the bispecific antibodies of the present application can recognize and bind to both stem cells and chondrocytes, thereby directing targeted engraftment of stem cells to the cartilage injury site.

In some embodiments of the present application, the stem cell is a mesenchymal stem cell or a hematopoietic stem cell, the hematopoietic stem cell surface antigen is selected from the group consisting of CD29, CD44, CD73, CD90, CD105, and CD106, and the hematopoietic stem cell surface antigen is CD34 or Try-1 antigen. In some embodiments of the present application, preferably the stem cells are hematopoietic stem cells and the stem cell surface antigen is CD34.

According to an embodiment of the present application, the first protein functional region and the second protein functional region each comprise a light chain variable region and a heavy chain variable region, the light chain variable region of the first protein functional region comprises CDRs as shown in SEQ ID nos. 1 to 3 (CDRs in turn ₁ 、CDR ₂ And CDR ₃ ) The heavy chain variable region comprises CDRs as shown in SEQ ID Nos. 4 to 6 (CDRs in turn ₁ 、CDR ₂ And CDR ₃ ) The method comprises the steps of carrying out a first treatment on the surface of the The light chain variable region of the second protein functional region comprises a polypeptide as set forth in SEQ ID No.7-9 (CDRs in turn) ₁ 、CDR ₂ And CDR ₃ ) The heavy chain variable region comprises CDRs as shown in SEQ ID Nos. 10 to 12 (CDRs in turn ₁ 、CDR ₂ And CDR ₃ )。

Further, the amino acid sequence of the light chain variable region of the first protein functional region is shown as SEQ ID No.13, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.14, the amino acid sequence of the light chain variable region of the second protein functional region is shown as SEQ ID No.15, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 16.

According to an embodiment of the present application, the first protein functional region and the second protein functional region are each independently selected from the group consisting of full-length immunoglobulins, fab ', (Fab') ₂ Fv, scFv or VHH, i.e.the anti-CD 34 antibody or antigen-binding fragment thereof and the anti-MATN 3 antibody or antigen-binding fragment thereof are each independently selected from the group consisting of full length immunoglobulins, fab ', (Fab') ₂ The possible structure of the bispecific antibodies of the present application is shown in FIG. 1 (cited from doi:10.1016/j. Molimm. 2015.01.003.).

To design bispecific antibodies that are simple in process and retain effective activity, in some embodiments, the bispecific antibodies of the present application are in the form of a structure resembling normal IgG, specifically, separate light and heavy chains are designed, respectively, light and/or heavy chain variable regions are designed at the N-terminus of the light and/or heavy chains that are capable of targeting both targets, and share the same heavy chain Fc region. In other embodiments, the antibody molecule of one target is attached to one end of the light chain or heavy chain of the intact antibody of another target in the form of an scFv, e.g., the first protein domain is an intact antibody, the second protein domain is an scFv, or the first protein domain is an scFv, and the second protein domain is an intact antibody, so that it is possible to avoid that expression of different heavy chain fcs results in non-uniform expression products, e.g., fc in Knob form and Fc in Hole form co-express non-uniform Fc-Fc pairing, affecting expression levels and making purification difficult; the possible effects of light and heavy chain partial region exchange designs on structural activity can also be avoided.

According to an embodiment of the present application, the first protein functional region is a full length IgG1 immunoglobulin and the second protein functional region is an scFv, i.e. the bispecific antibody is in an IgG1-scFv structure (fig. 2).

Illustratively, the scFv may be a light chain variable region-linker-heavy chain variable region structure, may be that the N-terminus of the light chain variable region of the scFv is linked to the C-terminus of the full length IgG1 immunoglobulin light or heavy chain, or that the C-terminus of the heavy chain variable region of the scFv is linked to the N-terminus of the full length IgG1 immunoglobulin heavy or light chain; alternatively, the scFv may be a heavy chain variable region-linker-light chain variable region structure, which may be that the N-terminus of the heavy chain variable region of the scFv is linked to the C-terminus of the full length IgG1 immunoglobulin light chain or heavy chain, or that the C-terminus of the light chain variable region of the scFv is linked to the N-terminus of the full length IgG1 immunoglobulin heavy chain or light chain.

According to some embodiments of the present application, the scFv is a light chain variable region-a first linker-a heavy chain variable region, the light chain variable region N-terminus or the heavy chain variable region C-terminus of the scFv being linked to the C-terminus or N-terminus of the full length IgG1 immunoglobulin light chain and/or heavy chain, respectively, by a second linker; or alternatively, the first and second heat exchangers may be,

Illustratively, the scFv is a heavy chain variable region-a first linker-a light chain variable region, the heavy chain variable region N-terminus of the scFv being linked to the full length IgG1 immunoglobulin light chain C-terminus by a second linker. Thus, the bispecific antibody comprises at least a first protein chain and a second protein chain, the first protein chain having the following formula from N-terminus to C-terminus:

light chain variable region-light chain constant region-second linker-heavy chain variable region-first linker-light chain variable region;

the second protein chain has the formula from N-terminus to C-terminus:

heavy chain variable region-heavy chain constant region (C _H -1) -hinge region-Fc region (C) _H -2+C _H -3)；

Illustratively, the bispecific antibody comprises two identical first protein chains as described above and two identical second protein chains as described above, the light chain variable region-light chain constant region of the first protein chain and the heavy chain variable region-heavy chain constant region of the second protein chain being joined by disulfide bonds to form a protein chain set.

It will be appreciated by those skilled in the art that the number of scFvs linked to an immunoglobulin may also be four, six or eight.

Illustratively, when the number of scFv is four: (1) The four scfvs are all light chain variable region-first linker-heavy chain variable region structures, wherein the C-terminal ends of the heavy chain variable regions of two scfvs are respectively and symmetrically connected to the N-terminal ends of the two heavy chain variable regions of the immunoglobulin, and the C-terminal ends of the heavy chain variable regions of the other two scfvs are respectively and symmetrically connected to the N-terminal ends of the two light chain variable regions of the immunoglobulin; (2) Four scfvs are all heavy chain variable region-linker-light chain variable region structures, wherein the N-terminal ends of the heavy chain variable regions of two scfvs are symmetrically connected to the C-terminal ends of two light chains of the immunoglobulin respectively, and the N-terminal ends of the heavy chain variable regions of the other two scfvs are symmetrically connected to the C-terminal ends of two heavy chains of the immunoglobulin respectively; (3) Wherein the two scfvs are light chain variable region-linker-heavy chain variable region structures, the C-terminal ends of the heavy chain variable regions of which are symmetrically linked to the N-terminal ends of the two heavy chain variable regions or the light chain variable region of the immunoglobulin, respectively; the other two scfvs are heavy chain variable region-linker-light chain variable region structures, the N-termini of the heavy chain variable regions of which are symmetrically linked to the C-termini of the two light or heavy chains of the immunoglobulin, respectively.

The above-mentioned C-terminal connection to the light chain or heavy chain refers to connection to the C-terminal of the light chain constant region or heavy chain constant region.

According to embodiments of the present application, the first and second linkers each independently comprise a repeat selected from GS, GGSG, GGGS and GGGGS sequences.

According to embodiments of the present application, the constant region of the full length immunoglobulin is derived from an IgG or an IgG mutant, preferably from IgG1, igG2, igG3 or IgG4, more preferably from IgG1.

According to embodiments of the present application, the constant region comprises amino acid substitutions of L234A, L235A and P329G compared to the wild type IgG1 constant region such that the bispecific antibody reduces or even eliminates ADCC or CDC function, extending half-life to maintain therapeutic efficacy over a long period of time.

In this example, C is specifically the Fc region _H The amino acid of the-2 region is mutated, which corresponds to the Fc region comprising amino acid substitutions L14A, L A and P109G, wherein the amino acid sequence of the wild-type IgG1Fc region is shown in SEQ ID No.17 and the amino acid sequence of the amino acid mutated IgG1Fc region is shown in SEQ ID No. 18.

The application also provides nucleic acids encoding bispecific antibodies, corresponding expression vectors and host cells.

The nucleic acid of the present application may be a DNA molecule or an RNA molecule, or may be a nucleic acid analog. The nucleic acid molecules in the present application may comprise naturally occurring nucleic acid residues or artificially generated nucleic acid residues. The nucleic acid molecules of the present application may be single-or double-stranded, linear or circular, natural or synthetic, and without any size limitation if not otherwise indicated. The nucleic acid molecule may also comprise a promoter, which may be homologous or heterologous.

Preferably, in some embodiments, the nucleotide sequence encoding the light chain variable region of the first protein functional region is shown in SEQ ID No.19, the nucleotide sequence encoding the heavy chain variable region of the first protein functional region is shown in SEQ ID No.20, the nucleotide sequence encoding the light chain variable region of the second protein functional region is shown in SEQ ID No.21, and the nucleotide sequence encoding the heavy chain variable region of the second protein functional region is shown in SEQ ID No. 22.

The nucleic acid molecules of the present application may be cloned into vectors. "vectors" in this application include plasmids, cosmids, viruses, bacteriophages and other vectors commonly used in genetic engineering. In some embodiments, these vectors are suitable for use in transforming cells, eukaryotic cells such as fungal cells, cells of microorganisms such as yeast or prokaryotic cells. In a preferred embodiment, these vectors are suitable for stable transformation of bacterial cells, for example to transcribe nucleic acid molecules of the present application.

The vector of the present application may be an expression vector. Suitable expression vectors that have been widely described in the literature are all useful in this application. In one embodiment, the expression vector may contain a marker gene and an origin of replication that ensures replication in the selected host, as well as a promoter and transcription termination signal. Between the promoter and the termination signal, there is preferably at least one restriction site capable of inserting a nucleic acid sequence/molecule for which expression is desired. Preferably, the expression vector of the present application is selected from pET series expression vector, pGEX series expression vector, pcDNA series expression vector, more preferably the expression vector of the present application is pIL-15 expression vector.

In one embodiment, the bispecific antibody is expressed in a host cell, followed by isolation of the antibody and purification to a generally pharmaceutically acceptable purity. For protein expression, the nucleic acid encoding its protein is inserted into an expression vector by standard methods. Expression is carried out in a suitable stable host cell and the protein is recovered from the cell (supernatant or lysed cells).

In another embodiment, the nucleic acid molecules of the present application and/or vectors containing the nucleic acid molecules of the present invention therein may be transduced, transformed or transfected or otherwise introduced into a host cell. For example, the host cell is a eukaryotic or prokaryotic cell, preferably a eukaryotic cell. As a non-limiting example, the host cell is a mammalian cell. The host cell of the present application may be a human, yeast or fungal cell, such as chinese hamster ovary Cell (CHO), baby hamster kidney cell (BHK, ATCC CCL 10), baby celetoli cell (seltoli cells), monkey kidney cell (COS cell), monkey kidney CVI cell transformed by SV40 (COS-7, ATCC CRL 1651), human embryonic kidney cell (HEK-293), monkey kidney cell (CVI, ATCC CCL-70), african green monkey kidney cell (VERO-76, ATCC CRL-1587), human cervical cancer cell (HELA, ATCC CCL-2) or the like. Preferably, the host cell of the present application is a human HEK293 cell.

The present application also provides a method of preparing a bispecific antibody as described above, comprising the steps of: the host cells of the present application are cultured under conditions suitable for expression of the bispecific antibody, and the bispecific antibody is recovered from the cells or cell culture supernatant. In one embodiment, a method of making a bispecific antibody of the present application comprises the steps of: constructing an expression vector containing a gene encoding the bispecific antibody, constructing a host cell containing the expression vector by a method of transiently transfecting the host cell, culturing the host cell, and collecting cell supernatant to purify the bispecific antibody.

Suitable conditions for expressing the antibody should be known to those skilled in the art, who can empirically select a suitable medium for culture under conditions suitable for growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time. The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell.

Methods for isolating and purifying bispecific antibodies are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant, centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high performance liquid chromatography, and other various liquid chromatography techniques and combinations of these methods.

The present application also provides pharmaceutical compositions comprising the bispecific antibodies, nucleic acids, expression vectors, and/or host cells of the present application.

In some embodiments of the present application, the pharmaceutical composition further comprises stem cells. Illustratively, the bispecific antibodies of the present application are incubated with stem cells, the bispecific antibodies are modified on the surface of the stem cells, and the bispecific antibodies exert synergistic repair effects with the stem cells by mediating stem cell-targeted engraftment into chondrocytes. In some embodiments of the present application, preferably the stem cells are hematopoietic stem cells.

In some embodiments of the present application, the pharmaceutical compositions of the present application may further comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers refer to conventional pharmaceutical carriers in the pharmaceutical arts, for example: diluents, excipients, water and the like, fillers such as starch, sucrose, lactose, microcrystalline cellulose and the like; binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, hydroxypropyl cellulose, croscarmellose, agar, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as cetyl alcohol, sodium lauryl sulfate; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium and magnesium stearate, silica gel micropowder, polyethylene glycol, etc. Other adjuvants such as flavoring agent, sweetener, etc. can also be added into the composition.

Examples of suitable pharmaceutically acceptable carriers are well known in the art. Pharmaceutical compositions comprising such carriers can be formulated by well-known conventional methods. In some embodiments, other therapeutically active ingredients may also be included in the pharmaceutical compositions of the present application.

The present application also provides the use of the bispecific antibodies, nucleic acids, expression vectors, host cells and pharmaceutical compositions described herein in the manufacture of a medicament for the treatment or prevention of arthritis.

Arthritis is selected from the group consisting of osteoarthritis, rheumatoid, ankylosing, reactive, gouty, rheumatic, suppurative arthritis. Joints include, but are not limited to, joints in the hand, wrist, toe, neck, back, knee, and hip areas.

The pharmaceutical composition or the medicine can be prepared into suspension or gel or liquid tissue engineering materials, and when in use, the pharmaceutical composition or the medicine is injected into the joint cavity to be repaired or is embedded into a suitable biological material for tissue engineering to prepare a hard tissue engineering material, and the hard tissue engineering material is implanted into the joint cavity to be repaired in a surgical mode.

In this application, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, immunological laboratory procedures used herein are all conventional procedures widely used in the corresponding field. Meanwhile, for better understanding of the present application, definitions and explanations of related terms are provided below.

As used herein, the term "CD34 protein" is a sialylalbumin cell surface transmembrane protein, a specific marker for hematopoietic stem cells, and when referring to the amino acid sequence of CD34 protein, includes the full length of CD34 protein, as well as extracellular fragments of CD 34. It will be appreciated by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally occurring or artificially introduced in the amino acid sequence of the CD34 protein without affecting its biological function. Thus, in the present application, the term "CD34 protein" shall include all such sequences, including natural or artificial variants thereof.

As used herein, the term "MATN3 protein" is human maternal protein 3, is an oligomeric extracellular protein encoded by the MATN3 gene, and when referring to the amino acid sequence of a MATN3 protein, includes the full length of the MATN3 protein. It will be appreciated by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally occurring or artificially introduced in the amino acid sequence of the MATN3 protein without affecting its biological function. Thus, in the present application, the term "MATN3 protein" shall include all such sequences, including natural or artificial variants thereof.

As used herein, a "protein functional region" refers to a region that can specifically interact with a target molecule, such as an antigen, with a high degree of selectivity in the action, and a sequence that recognizes one target molecule is generally unable to recognize other molecular sequences.

As used herein, the term "specific binding" refers to the binding of an antibody to an epitope on a predetermined antigen.

As used in this application, the termAn "antibody" refers to an immunoglobulin molecule that typically consists of two pairs of polypeptide chains, each pair having one light chain (L chain) and one heavy chain (H chain). In a general sense, heavy chains are understood to be polypeptide chains of greater molecular weight in an antibody, and light chains refer to polypeptide chains of lesser molecular weight in an antibody. Each heavy chain consists of a heavy chain variable region (V _H ) And a heavy chain constant region (C) _H ) Composition is prepared. The heavy chain constant region consists of 3 domains (C _H -1、C _H -2 and C _H -3) each light chain consists of a light chain variable region (V _L ) And a light chain constant region (C _L ) The constant region of the light chain consists of one domain C _L Composition, V _H And V _L The region can also be subdivided into regions of high denaturation called Complementarity Determining Regions (CDRs), the variable regions of each heavy/light chain pair (V _H And V _L ) The antibody binding sites are formed separately. The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies may be of different isotypes, for example, igG or a mutant thereof, igA1, igA2, igD, igE or IgM antibodies.

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to an antigen, also referred to as an "antigen-binding portion. Antigen binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.

As used herein, the term "constant region" includes both heavy chain constant regions and light chain constant regions, the heavy chain constant regions including amino acid sequences from immunoglobulin heavy chains. The polypeptide comprising a heavy chain constant region comprises at least one of: c (C) _H -1 domain, hinge (e.g., upper hinge region, middle hinge region, and/or lower hinge region) domain, C _H -2 domain, C _H -3 domain, or variant or fragment thereof. For example, an antigen binding fragment as used herein may comprise a polypeptide having C _H -a polypeptide chain of domain 1; with C _H -1 domain,At least a portion of a hinge domain and C _H -a 2 domain polypeptide; with C _H -1 domain and C _H -a polypeptide chain of domain 3; with C _H -1 domain, at least a portion of a hinge domain and C _H -3 domain polypeptide chain, or having C _H -1 domain, at least part of hinge structure, C _H -2 domain, and C _H -3 domain polypeptide chain. "C _H Fragment "includes C _H -1 domain, hinge domain, C _H -2 domain and C _H -3 domain.

The light chain constant region includes the amino acid sequence from the antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain and a constant lambda domain.

As used herein, the term "hinge region" includes the C-to-C of a heavy chain molecule _H -1 domain is linked to C _H -that part of the 2 domain. The hinge region comprises about 25 residues and is flexible such that the two N-terminal antigen binding regions move independently. The hinge region can be divided into three distinct domains: upper, middle, and lower hinge domains.

As used herein, the term "Fab" is a fragment obtained by treating an antibody molecule with papain (cleaving amino acid residue 224 of the H chain), wherein about half of the N-terminal side of the H chain and the entire L chain are bound together by disulfide bonds.

As used in this application, the term "F (ab') ₂ "is an antibody fragment having a molecular weight of about 100,000Da and comprising two Fab regions linked at the hinge position, which is obtained by digestion of the lower part of the two disulfide bonds in the hinge region of an antibody molecule with pepsin.

As used herein, the term "Fab '" is obtained by cleavage of F (ab') ₂ An antibody fragment obtained by disulfide bonding of the hinge region of (C). Fab 'can be specifically recognized and bound to F (ab') of an antigen by treatment with a reducing agent such as dithiothreitol ₂ To produce.

As used herein, the term "Fv" is the smallest functional fragment of an antibody molecule that retains an antigen-binding site, consisting of a light chain variable region and a heavy chain variable region, which are joined together by a non-covalent bond.

As used herein, the term "scFv" refers to a V comprising an antibody _H And V _L Antibody fragments of domains, heavy chain variable regions (V) linked by linkers (linker) _H ) And a light chain variable region (V _L ) The linker cross-links the two domains to form an antigen binding site, the scFv is typically 1/6 of the size of an intact antibody, preferably an amino acid sequence encoded by a nucleotide chain.

As used herein, the term "VHH" is a single variable region of a heavy chain antibody that has antigen binding capacity.

As used herein, the term "linker" refers to a peptide that connects two polypeptides, typically with some flexibility, without losing the original structure and function of the polypeptide. Linkers are known to those skilled in the art. The linker may be prepared by any method known in the art, and may be, for example, synthetic.

As used herein, the term "IgG" is an abbreviation for immunoglobulin G, and human IgG has four subtypes, depending on the r-chain antigenicity differences in IgG molecules: igG1, igG2, igG3, igG4.

As used herein, the term "Fc region" refers to the crystallizable section, corresponding to C of an antibody molecule _H -2 and C _H -3 domain.

As used herein, the term "treatment" refers to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects caused by the disease. "treating" as used herein encompasses diseases in mammals, particularly humans, including inhibiting the disease, e.g., arresting the development of the disease; or to alleviate a disease, e.g., to alleviate symptoms associated with a disease. As used herein, "treating" or "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administration of a drug as described herein to an individual in need thereof.

As used herein, the term "preventing" refers to preventing a disease (e.g., preventing arthritis) or delaying or preventing the onset of a disorder in an individual that is susceptible to the disease but has not yet been diagnosed with the disease.

Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application.

EXAMPLE 1 construction of expression vectors for CD34×MATN3 bispecific antibody molecules

1. Containing Anti-CD34-V _L Construction of the first plasmid with the Anti-MATN3scFv fragment

Primers were designed based on the target gene sequence and cleavage sites, and Anti-CD34-V was amplified using Polymerase Chain Reaction (PCR), respectively _L Sequence, C _Lκ Sequences (FIG. 3A) and Anti-MATN3scFv sequences (FIG. 3B), and amplification was completed by amplifying Anti-CD34-V _L Sequence and C _Lκ Sequence overlay ligation; wherein C is _Lκ The amino acid sequence of (2) is shown as SEQ ID No. 23.

PCR reaction A PCR amplification system was prepared according to PrimerStar Max enzyme instructions, and the PCR reaction conditions were as follows: pre-denaturation at 95℃for 10 seconds, followed by 35 cycles of denaturation at 95℃for 10 seconds, annealing at 55℃for 5 seconds and extension at 72℃for 10 seconds, with a final extension at 72℃for 5 minutes after the cycle. And (3) after amplification, identifying and separating by 1% agarose gel electrophoresis to obtain a target band, and then carrying out gel recovery on the clear target band by using an AXYGEN gel recovery kit.

Preparing a digestion system according to the conditions described in HindIII and BamHI restriction enzyme specifications, digestion of eukaryotic vector pIL-15 according to the digestion conditions, gel electrophoresis and gel recovery to obtain digestion product, i.e. linearized vector (FIG. 4), and using ClonExpress MultiS One Step Cloning Kit to obtain Anti-CD34-V _L And C _Lκ Homologous recombination of fusion fragment and Anti-MATN3scFv fragment on pIL-15 carrier, transformation, resistance screening, cloning, extracting plasmid, sequencing to obtain the final productSuccessful first plasmid. After the plasmid is sequenced correctly, the plasmid can be transformed, amplified by shaking bacteria, added with sterile glycerol with the final concentration of 20 percent and placed in a refrigerator with the temperature of minus 80 ℃ for preserving the strain.

2. Construction of a second plasmid containing Anti-CD34-VH and Fc region containing L234A, L235A and P329G mutations.

Three-amino acid mutated C is obtained by PCR amplification _H Fragments, respectively C _H 1/2/3 (FIG. 5), C was performed by overlay PCR _H 1/2/3 ligation to mutation successful C _H Fragments; PCR amplification to obtain Anti-CD34-V _H Fragments (fig. 6); wherein C is _H The amino acid sequence of the-1 is shown as SEQ ID No.24, C _H The amino acid sequence of the-2 is shown as SEQ ID No.25, C _H The amino acid sequence of-3 is shown as SEQ ID No. 26.

The CH fragment was reacted with Anti-CD34-V _H The fragments were ligated by overlay PCR to give Anti-CD34-VH-C _H ' fragment (FIG. 7A), and extension of the signal peptide by a primer to give Anti-CD34-VH-C _H Fragment (fig. 7B);

and (3) carrying out double restriction enzyme digestion on pIL-15 by HindIII and BamHI restriction enzymes to obtain a linearization vector, carrying out homologous recombination on the fusion fragment and the pIL-15 vector, and carrying out transformation, AMP screening, monoclonal selection, plasmid extraction and sequencing to obtain a second plasmid which is successfully constructed. After the plasmid is sequenced correctly, the plasmid can be transformed, amplified by shaking bacteria, added with sterile glycerol with the final concentration of 20 percent and placed in a refrigerator with the temperature of minus 80 ℃ for preserving the strain.

EXAMPLE 2 expression of CD34×MATN3 bispecific antibodies

1. Preparing cells: counting HEK293E cell suspension in logarithmic growth phase, centrifuging at 1000rpm at room temperature for 3min, discarding supernatant, re-centrifuging after re-suspending cell precipitate with appropriate amount of Freestole 293 culture medium (this step is to wash off serum); resuspended to a cell density of 6X 10 with Freestole 293 Medium ⁶ And (3) placing the mixture in an incubator for standby.

2. Preparing a plasmid: diluting the first plasmid and the second plasmid to 40 ng/. Mu.L with Freestole 293 medium; at every 10 ⁶ Plasmid was measured for each cell corresponding to 0.5. Mu.g plasmid.

3. Mixing the plasmid with PEI: mixing the plasmids and PEI according to the mass ratio of 1:5, and standing for 15min at room temperature after uniformly mixing.

4. Transfection: adding the mixture of plasmid and PEI into the prepared cells, mixing, and adding 5% CO ₂ Shake cultivation in a shake cultivation box at 125rpm and 37 ℃;

wherein the first plasmid and the second plasmid are co-transfected into the cell in a mass ratio of 3:1, 2:1, 1:1, 1:2 and 1:3, respectively.

5. Supplementing liquid: after 4h, SFM4HEK293 medium was added at the same volume as Freestyle293 medium, G418 at a final concentration of 100. Mu.g/mL and 3.75mM VPA solution; after 24 hours TN1 was added to the culture medium to a final concentration of 0.5%, and 1000 Xanti-clasping agent was added.

6. And (3) sample collection: when the cell viability was measured to be reduced to about 50%, the cell pellet was removed by centrifugation at 4000rpm for 10min, and then the expression supernatants were collected by centrifugation at 7000rpm for 20min, respectively, at 4℃and finally filtered through a 0.45 μm microporous filter membrane for further use.

Collected samples were subjected to SDS-PAGE electrophoresis, and WB detection was performed with HRP-coat-Anti-Human-IgG (H+L) antibody, and the optimal co-transfection ratio was determined based on the target protein band. As can be seen from FIG. 8, the target protein expression level was high when the mass ratio of the first plasmid (L) to the second plasmid (H) was 1:1, the molecular weight of the non-reduced sample was about 250kDa, the molecular weight of the reduced sample was between 50kDa and 70kDa, and the band was single. The theoretical molecular weight of the target protein is about 210kDa, and the actual molecular weight is probably due to the formation of multimers of the antibody, glycosylation of the antibody or the influence of electrophoresis conditions, for which specific reasons are to be studied further.

EXAMPLE 3 purification of CD34×MATN3 bispecific antibodies

Protein A specifically affinities for the Fc region of IgG antibodies, mabSelect SuRe is a Protein A affinity medium where bispecific antibodies were purified using a HiTrap MabSelect SuRe pre-cartridge

1. Sample and solution pretreatment: placing the obtained expression supernatant on ice for later use; the solutions used in this experiment were all filtered using a 0.22 μm microporous filter.

2. A HiTrap MabSelect SuRe purification column having a volume of 1mL was connected to the ethanol flushing line at a flow rate of 1mL/min using the gas bubbles and impurities that may be present in the 20% ethanol flushing line

A protein purification instrument;

3. ddH was used at a flow rate of 1mL/min with 10 column volumes ₂ O flushing system and purifying column to ensure the ethanol to be flushed;

4. the whole chromatography system was equilibrated at a flow rate of 1mL/min to UV280 baseline and steady conductivity using equilibration buffer pH 7.2, 20mM PB,150mM NaCl;

5. loading the expression supernatant into a purification column at a flow rate of 1mL/min to ensure that the pipeline is bubble-free;

6. rinsing with equilibration buffer at a flow rate of 1mL/min until UV280 baseline and conductance values were stable;

7. the removal of the impurity proteins was performed at a flow rate of 1mL/min using a pH 5.0,0.1M citric acid-sodium citrate impurity removal buffer of about 10 column volumes;

8. bispecific antibody was eluted at a flow rate of 1mL/min using pH 3.0,0.1M citric acid-sodium citrate and immediately the pH of the collected proteins was adjusted to neutral using pH 9.0,1m Tris-HCl buffer;

9. the residual protein on the purification column was washed clean with about 10 column volumes of 50mM NaOH at a flow rate of 1 mL/min;

10. ddH was used at a flow rate of 1mL/min with 10 column volumes ₂ O flushing system and purifying column;

11. flushing the system and purification column with 20% ethanol at a flow rate of 1mL/min, leaving the system in a sterile environment, and preserving the purification column with 20% ethanol;

12. the protein sample is ultrafiltered, concentrated and replaced into PBS buffer solution, after the concentration is measured, the protein sample is filtered by a sterile filter membrane, and the protein is split-packaged and stored in a refrigerator at the temperature of minus 80 ℃.

The purification pattern is shown in FIG. 9, and SDS-PAGE identification (FIG. 10) is carried out on the collected proteins after purification, so that the purity of the target protein obtained by purification is about 70%, and the requirements of affinity and in vivo experiments can be met. The molecular weight of the sample subjected to non-reduction treatment is about 250kDa, and the molecular weight of the sample subjected to reduction treatment is between 50kDa and 70kDa, so that the WB detection condition is consistent. After purification to obtain the target protein, the buffer solution is replaced by PBS, the protein concentration is measured, and after aseptic filtration, the protein is split-packed at-80 ℃ for storage, and the yield of the CD34 xMATN 3 bispecific antibody prepared by the preparation method is about 22mg/L.

Example 4 CD34×MATN3 bispecific antibody antigen affinity assay

In this example, antigen/antibody was immobilized on CM5 chip and the antigen-antibody affinity was detected as follows:

1. CM5 chips were removed from PBS and ddH was used ₂ After O is washed, drying the chip by liquid nitrogen, putting the chip into a chip shell, executing a change chip instruction, and putting the chip into the chip according to the indication direction;

2. wiping the buffer inlet pipe with dust-free paper, putting the buffer inlet pipe into freshly prepared PBS, and executing a change buffer instruction;

3. obtaining proper ligand immobilization concentration and pH value: the ligand was diluted to different concentrations with Acetate at ph=4.5, 5.0, 5.5, set the pH rating program: combining 180s, and regenerating with 50mM NaOH for 30s at a flow rate of 30 μL/min (enrichment conditions with RL 0.5-2 times that of the target RL are more suitable);

4. immobilization was performed using the ligand of appropriate pH and concentration obtained in step 3: the immobilized buffer was PBS, and ligand immobilization procedure was set: activating 420s with EDC+NHS1:1 mixture at a flow rate of 10 μl/min, setting a fixation time according to step 3, and sealing with ethanolamine for 420s; (conditions are more suitable when 70% -80% of the RL is 0.5-2 times that of the target RL)

5. Performing Binding test to obtain proper dissociation time and regeneration conditions: program for setting Binding test: glycine with pH=2.5 (the pH value of regenerated Glycine needs to be determined according to different experiments) was regenerated for 30s with a flow rate of 30. Mu.L/min using a flow rate of 10. Mu.L/min in combination with 120s, dissociation 120 s;

6. Kinetic constant measurement experiment: setting a concentration gradient group and a corresponding repeated group, and setting two blank groups at the beginning and the end respectively to observe whether a base line is stable or not; wiping the buffer inlet pipe with dust-free paper, putting the buffer inlet pipe into a freshly prepared HBS-EP, and executing a change buffer instruction; the analyte was diluted using HBS-EP-fold ratio, and kinetic constant determination procedure was set: combining 120s, dissociating 120s, and starting up for 5 cycles at a flow rate of 30 mu L/min; the regeneration reagent is Gly2.5; regeneration time 30s; the flow rate is 30 mu L/min;

7. after the experiment is finished, executing a change chip instruction, changing the chip into a maintenance chip, recording a used channel of the used chip, and soaking the used channel in PBS (phosphate buffer solution); changing Buffer into ultrapure water;

8. data were analyzed using a 1:1 binding model.

The binding and dissociation of CD34-Fc antigen to Anti-CD34-scFv and CD34 XMATN 3 were examined and the measured affinity parameters are shown in FIG. 11, table 2; the binding and dissociation of MATN3-Fc antigen to Anti-CD34-scFv and CD34 XMATN 3 were examined and the affinity parameters measured are shown in FIG. 12, table 2.

The obtained CD34×MATN3 has high affinity with corresponding affinity of KD=8.53 nM and KD=8.95 pM respectively with two antigens of CD34-Fc and MATN3-Fc, and can meet the requirement of subsequent experiments.

TABLE 1 affinity parameters of Anti-CD34-scFv and CD34 XMATN 3 with CD34-Fc antigen

TABLE 2 affinity parameters of Anti-CD34-scFv and CD34 XMATN 3 with MATN3-Fc antigen

Example 5 detection of Targeted Capacity of CD34×MATN3

1. And (5) extracting and culturing chondrocytes.

Rats were sacrificed and immersed in 70% ethanol for 5 minutes;

spreading the rat in an ultra-clean bench, cutting the skin and muscle of the leg, cutting the patella ligament and the patella, and exposing the joint cavity; cutting off cartilage with a sterile scalpel, and chopping the cartilage; adding sterile PBS into cartilage, washing twice, and washing off blood; 1mL pancreatin was added to the cartilage and digested on a shaker at 37℃for half an hour; sucking out pancreatin, adding 1mL type II collagenase with concentration of 2mg/mL, and water-bathing at 37 ℃ for 4 hours; 1mL of complete medium (DMEM medium containing 10% FBS and 1% PS) was added and the liquid was sucked out and filtered through a 200 mesh cell filter; centrifugation at 1000rpm for 3min, the supernatant was discarded, and the pellet was resuspended in 1mL of medium and then added to a T25 flask containing 4mL of medium.

Removing non-adherent cells after 48 hr, changing culture medium every three days, sucking original culture medium until the cells are adherent to about 80%, adding sterile PBS, gently washing, adding pancreatin, placing into incubator, digesting for about 3-5min, adding complete culture medium when part of cells are in the form of quicksand, centrifuging at 1000rpm for 3min, and concentrating the cells at 2×10 ⁵ The density of each mL is inoculated in a culture dish for culture.

2. Affinity detection of CD34 XMATN 3 bispecific antibodies to chondrocytes

Taking good-growing third generation chondrocytes, digesting with pancreatin, and taking 5×10 groups of three groups ⁵ The cells were centrifuged at 1000rpm for 3min, the supernatant was discarded, and the cells were resuspended to 100. Mu.L using FACS (PBS containing 2% FBS, pre-chilled in advance of 4 ℃); 10. Mu.g of CD34 XMATN 3 was added to the experimental group, 10. Mu.g of Anti-MATN3-scFv was added to the positive control group, 10. Mu.g of PBS was added to the negative control group, and the mixture was incubated at 4℃for 1 hour; centrifugation at 1000rpm for 3min, supernatant was discarded, 500. Mu.L of FACS was added for resuspension, and after repeated washing twice, the cells were resuspended to 100. Mu.L; mu.L of FITC-Anti-human IgG (H+L) was added to each group and incubated at 4deg.C for 30min in the absence of light;

repeating the cleaning steps; cells were resuspended with 200 μl FACS and detected using flow cytometer FITC channel. The specific detection results are shown in FIG. 13, compared with the negative control, both the Anti-MATN3-scFv and the CD34 xMATN 3 have better affinity with the chondrocytes, and the CD34 xMATN 3 has better affinity with the chondrocytes than the control Anti-MATN3-scFv, which is consistent with SPR affinity detection data.

3. Affinity detection of CD34 XMATN 3 bispecific antibodies to hematopoietic stem cells

Hematopoietic Stem Cells (HSCs) cells were resuscitated and divided into 8 total groups of 5X 104 cells each, treated according to the chondrocyte procedure, with 0.5. Mu.g of CD 34X MATN3 added to group 1, 0.1. Mu.g to group 2, 0.05. Mu.g to group 3, 0.02. Mu.g to group 4, 0.01. Mu.g to group 5, 0.002. Mu.g to

group

6, 10. Mu.g of PE-anti-CD34 to positive control, 10. Mu.g of PBS to negative control, and incubated at 4℃for 1 hour; cleaning and re-suspending according to the steps; 1 mu LAPC-Anti-human IgG (H+L) was added to each group, 1 mu L PE-Anti-CD34 was added to the positive control group, and incubated at 4deg.C for 30min in the absence of light; repeating the cleaning steps; cells were resuspended with 200 μl FACS, the positive rate of HSCs cells was detected using the flow cytometer PE channel, and the affinity of bispecific antibodies to HSCs cells was detected at different concentrations using the APC channel, see fig. 14, which shows that cd34×matn3 has better affinity to HSCs cells and that the affinity of both correlates with the amount of cd34×matn3 added. When 0.1 μg of bispecific antibody was added, more than 95% of HSCs cells had been bound.

4. CD34 xMATN 3 in vitro targeting ability detection

Since the HSCs are modified to CD34 xMATN 3 and are expected to target cartilage sites for repair, 293T-Anti-MATN3-scFv-GFP and 293T-GFP cell lines are constructed by a lentiviral infection technique, and whether 293T-Anti-MATN3-scFv-GFP cells (the 2 nd experimental group) can target cartilage cells or not is observed by taking the 293T-GFP cell line as a control (the 1 st experimental group) to determine the expression of MATN3 antigen at the cartilage sites; furthermore, it was observed whether the cartilage pieces could adsorb HSCs modified with CD34×MATN3, i.e. demonstrate their targeting ability in vitro, as follows.

1) 293T-Anti-MATN3-scFv-GFP cell pool construction

293T cells were 1X 10 on the day prior to lentiviral infection ⁵ The cells were spread evenly in 6-well plates at a density of individual cells/mL and cultured in a constant temperature stationary incubator. When the cell confluency is about 50%, the infection can be carried out, viruses and Polybrene are slowly thawed on ice before the infection, and the results are calculatedVirus is needed: desired virus (μl) =cell number MOI/titer (6 well plate is full of about 2E6 cells, 293T cells MOI value 1-3) Polybrene concentration 1 μg/mL was used. Uniformly mixing DMEM, lentivirus and Polybrene, discarding the original culture medium, adding 500 mu L of the three mixtures, and performing stationary culture in a constant temperature incubator; after 4 hours of infection, 500. Mu.L of complete medium was fed per well. 24 hours after infection, the virus-containing medium was replaced with fresh complete medium in order to reduce damage to the cells. 48 hours after infection, 2. Mu.g/mL of Puromycin was added to the medium to screen the stable cell lines, after which the medium was changed about every 2-3 days until the Blank cells not infected with the target gene were killed, the stable cell pool was continued to be subjected to expansion culture under pressure, and the medium containing 10% DMSO was used to freeze the stable cell pool in a liquid nitrogen tank. In the cell culture process, a flow cytometer is used for detecting the expression condition of the Anti-MATN3-scFv gene, and because the gene can express an antibody, the APC-Anti-human IgG (H+L) secondary antibody can be directly added for detection, and the FITC channel and the APC channel are used for respectively detecting the expression condition of GFP and the Anti-MATN3-scFv gene. As can be seen from FIG. 15, the fluorescence efficiency of both stable cell pools reached more than 90%, meeting the requirements of the subsequent experiments. Almost 100% of cells in the stable cell pool expressed GFP compared to blank 293T (experimental group 1 and 2); as can be seen from FIG. 16, almost 100% of cells in the stable cell pool expressed Anti-MATN3-scFv (group 2), so that the cell pool was constructed successfully and almost 100% of cells successfully integrated the foreign gene.

2) Affinity detection of 293T-Anti-MATN3-scFv-GFP cells on cartilage pieces

After the rat was sacrificed, it was immersed in 70% ethanol for 5 minutes; spreading the rat in an ultra-clean bench, cutting the skin and muscle of the leg, cutting the patella ligament and the patella, and exposing the joint cavity; several pieces of cartilage were excised, washed twice with PBS, and placed in a dish for use.

Resuscitates 293T-GFP and 293T-Anti-MATN3-scFv stable cell pools, 1X 10 was added to each of the two EP tubes ⁶ A group of cells are additionally arranged, and only culture medium is added without adding the cells to serve as blank control; adding several pieces of cartilage pieces into each tube, incubating at 37deg.C for 30min, and clamping out the cartilage pieces, and adding into the tubeThe samples were washed twice in PBS and the results recorded using a fluorescence inverted microscope. As can be seen from FIG. 17, the cartilage sheet itself has a little green fluorescence, but the fluorescence of the cartilage sheet itself is well differentiated from that of cells, the MATN3 expression of the cartilage sheet is detected by using 293T-Anti-MATN3-scFv-GFP cells, the magnification is 40 times, FIG. 17A&D is two cartilage pieces without cells added, blank, FIG. 17B&E is cartilage sheet with 293T-GFP cells added, and it was found that some cells remained on the cartilage sheet, FIG. 17C&F is the cartilage piece added with 293T-Anti-MATN3-scFv-GFP cells, and compared with the former two groups, the cartilage piece can obviously adsorb more 293T-Anti-MATN3-scFv-GFP cells, thus proving that the cartilage piece does contain more MATN3 antigen.

3) Bispecific antibodies mediate HSCs targeting chondrocyte pellet detection.

After labeling HSCs cells with CFSE dye at a final concentration of 0.5 μm, the cells were resuspended in 1mL medium and split into 2 portions for use on ice; wherein 0.5mL of cells was added with 2. Mu.g of CD34 XMATN 3, and the other 0.5mL of cells was added with 1. Mu.L of PBS, and incubated at 37℃for 30min; centrifugation at 1000rpm for 3min, supernatant was discarded, pellet was resuspended in 0.5mL PBS, several pieces of cartilage pieces were added to each of the two tubes, and a blank control group was set with no cells added only to cartilage pieces, incubated at 37℃for 30min, and after the cartilage pieces were clamped out, washed in PBS and the experimental results were recorded using a fluorescent inverted microscope. FIGS. 18A & B are graphs showing that the cartilage pieces were spontaneously green-fluorescent but different from the fluorescence of cells when no cell group was added, i.e., when the cartilage pieces were amplified 40-fold and 100-fold in the blank group, respectively; FIGS. 18C & D are control groups of HSCs treated with CFSE added, showing very little retention of cells; fig. 18e & f are HSCs experimental groups modified with cd34×matn3, and the cartilage pieces were seen to adsorb significantly more cells than the first two groups, thus demonstrating that cd34×matn3 can mediate HSCs cell targeting to cartilage pieces.

EXAMPLE 6 CD34×MATN3-mediated Stem cell-targeted repair of cartilage injury

1. Cartilage injury model construction

The experimental material is SD male rats weighing 250-300 g. The rats were anesthetized by intraperitoneal injection of sodium pentobarbital and placed on a constant temperature heating pad to maintain body temperature. Removing hair near the knee joint by using a shaver, opening skin along the inner side of the knee joint by using a scalpel, and cutting muscle tissue along the patella ligament; firstly straightening the rat leg, then moving the patella to the outer side of the knee joint to expose the joint cavity; grinding cartilage at the protrusions on two sides of the femur chute of the rat by using a 1mm spherical drill bit; then washing with normal saline, scrubbing with iodophor, straightening the rat leg, and resetting the patella; suturing the wound using 19mm round needle absorbable suture; gentamicin sulfate was injected intramuscularly in the rat leg and placed in a heating pad Wen Suxing. Three days after the operation, gentamicin sulfate was continuously injected intramuscularly. The molding time is one month.

2. Administration treatment

Treatment was performed using 50 μl of cells injected into the joint cavity at a dose of 5×10 per subject ⁵ Individual cells. Four groups of PBS, MSCs, MSCs + HSCs, MSCs+ HSCs-CD34 XMATN 3 were assigned to this study and were administered to the left and right knee joint cavities of each rat. Based on previous flow cytometry experiments, it has been determined that when the number of HSCs is 5X 10 ⁴ When 0.1 mu gCD and 34 XMATN 3 are added, the binding rate of more than 90 percent can be achieved; cd34×matn3 bispecific antibodies will be added in this proportion in this study.

Resuscitates MSCs and HSCs in a water bath at 37 ℃, resuscitates the MSCs and the HSCs with PBS, directly mixes the MSCs and the HSCs in a ratio of 1:1 for the experimental group of MSCs+HSCs, adds a certain amount of CD34 xMATN 3 into the HSCs firstly, incubates the MSCs for 30 minutes at 37 ℃, washes the unbound CD34 xMATN 3 by using PBS, and mixes the HSCs-CD34 xMATN 3 with the MSCs in a ratio of 1:1.

After the rat is inhaled and anesthetized by using diethyl ether, the hair near the knee joint is removed by using a shaver, a joint cavity of the rat is pressed into a stamp by using a 1mL syringe needle, then cells are injected into the cavity by using an insulin syringe, the joint cavity is penetrated without resistance, and the needle is slowly withdrawn after administration so as to avoid the leakage of the cells.

3. Treatment effect detection

One month after the transplantation treatment, the tissues are harvested for pathological staining and immunohistochemical experiments, and the repair effect of the HSCs and the capacity of the CD34 xMATN 3 for guiding the HSCs to target the implantation and repair the damaged cartilage are observed. As can be seen from fig. 19, the surface of the femur of the blank group (cartilage is not damaged) is bright white and glossy, the elasticity is good, and no fissure is found; the femur abrasion of the PBS group is serious, white smooth cartilage is not found on the surface of the femur, roughness is uneven, and obvious cracks are visible; compared with the PBS group, the MSCs treatment group has a certain repairing effect, but the surface is still darker and coarser; MSCs+HSCs treatment group femur surface is bright white, only a small dark area is arranged on the right side, and the whole repairing effect is good; compared with the previous treatment group, the MSCs+HSCs-CD34 xMATN 3 group has obviously thicker cartilage layer, better elasticity, bright color and better repairing effect.

Further, pathological staining of safranin-fast green (FIG. 20) and toluidine blue (FIG. 21) was used to observe tissue sections, and it was found that MSCs+HSCs-CD34×MATN3 group cartilage layers were thicker, cartilage cells were more orderly arranged, and the repair effect was best.

Further immunohistochemistry was used to analyze the expression of common pathological markers in arthritis, and as can be seen from fig. 22, 23, 24 and 25, the expression of COL2 was significantly up-regulated and the expression of COL10, MMP-13 and MATN3 was significantly down-regulated after cartilage repair treatment using stem cells.

Therefore, HSCs have better repairing capability on damaged cartilage, and the CD34 xMATN 3 is added to guide the target cartilage damage part, so that the repairing effect is improved.

EXAMPLE 7 CD34×MATN3 mediated targeting of hematopoietic Stem cells to repair cartilage injury

1. Cartilage injury model construction

The experimental material is SD male rats weighing 250-300 g. The rats were anesthetized by intraperitoneal injection of sodium pentobarbital and placed on a constant temperature heating pad to maintain body temperature. Removing hair near the knee joint by using a shaver, opening skin along the inner side of the knee joint by using a scalpel, and cutting muscle tissue along the patella ligament; firstly straightening the rat leg, then moving the patella to the outer side of the knee joint to expose the joint cavity; punching holes in the femoral pulley groove of the rat by using a 1mm spherical drill bit, wherein the diameter is about 2mm, and grinding away cartilage layers; then washing with normal saline, scrubbing with iodophor, straightening the rat leg, and resetting the patella; suturing the wound using 19mm round needle absorbable suture; gentamicin sulfate was injected intramuscularly in the rat leg and placed in a heating pad Wen Suxing. Three days after the operation, gentamicin sulfate was continuously injected intramuscularly. The molding time is one month.

2. Administration treatment

Treatment was performed using 50 μl of cells injected into the joint cavity at a dose of 5×10 per subject ⁵ Individual cells. A total of three groups PBS, HSCs, HSCs-CD34 XMATN 3 were provided and each rat was dosed in the left and right knee joint cavities. HSCs were resuscitated in a 37 ℃ water bath, resuspended in PBS and modified with an amount of CD34 xmtn 3, and unbound CD34 xmtn 3 was washed away using PBS after incubation at 37 ℃ for 30 minutes.

3. Treatment effect detection

One month after treatment, the tissues were harvested for pathological staining and immunohistochemical experiments to observe the repair effect of HSCs and the ability of cd34×matn3 to direct HSCs to target engrafted repair damaged cartilage. As can be seen from fig. 26, the wear of the PBS group was not effectively repaired, and the damaged round hole was still visible; compared with the PBS group, the HSCs treatment group has obvious repairing effect, but the surface of the repairing area is still darker and coarser; compared with the previous treatment group, the cartilage layer of the HSCs-CD34 XMATN 3 group is obviously thicker, has better elasticity, bright color and better repairing effect. According to the result, the CD34 XMATN 3 bispecific antibody effectively mediates the targeting of HSCs at cartilage injury sites, thereby promoting the regenerative repair of cartilage tissue.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present application have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the present application, and that modifications, alternatives, and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the present application.

SEQUENCE LISTING

<110> Shanghai university of transportation

<120> A bispecific antibody, preparation method and use

<130> 0000

<160> 26

<170> PatentIn version 3.5

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Arg Ala Ser Gln Asp Ile Gly Ser Ser Leu Asn

1 5 10

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Ala Thr Ser Ser Leu Asp Ser

1 5

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<211> 9

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Leu Gln Tyr Ala Ser Ser Pro Tyr Thr

1 5

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Gly Tyr Ser Phe Thr Ser Tyr Tyr Met His

1 5 10

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Phe Ile Asp Pro Phe Asn Gly Gly Ile Thr Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 6

<211> 14

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Cys Tyr Tyr Asn Tyr Asp Asp Glu Gly Arg Ala Met Asp Tyr

1 5 10

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Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 8

<211> 7

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<400> 8

Asp Ala Ser Asn Leu Glu Thr

1 5

<210> 9

<211> 9

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<400> 9

Gln Gln Tyr Asp Asn Leu Pro Leu Thr

1 5

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<211> 8

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<400> 10

Gly Gly Thr Phe Ser Ser Tyr Ala

1 5

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<211> 8

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Ile Ile Pro Ile Phe Gly Thr Ala

1 5

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Ala Arg Gly Gln Gly Tyr Trp Phe Asp Pro

1 5 10

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser

20 25 30

Leu Asn Trp Leu Gln Gln Gly Pro Asp Gly Thr Phe Lys Arg Leu Ile

35 40 45

Tyr Ala Thr Ser Ser Leu Asp Ser Ser Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 14

<211> 123

<212> PRT

<213> artificial sequence

<400> 14

Glu Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Met Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Lys Gln Ser Gln Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Phe Ile Asp Pro Phe Asn Gly Gly Ile Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met His Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Cys Tyr Tyr Asn Tyr Asp Asp Glu Gly Arg Ala Met Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

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<400> 15

Asp Val Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

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Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gln Gly Tyr Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

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<211> 227

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<400> 17

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

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<400> 18

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 19

<211> 324

<212> DNA

<213> artificial sequence

<400> 19

gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggacattggt agtagcttaa actggcttca gcaggggcca 120

gatggaactt ttaaacgcct gatctacgcc acatccagtt tagattctag tgtccccaag 180

aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240

gaagattttg tagactatta ctgtctacaa tatgctagtt ctccgtacac gttcggaggg 300

gggaccaagc tggaaatcaa acga 324

<210> 20

<211> 369

<212> DNA

<213> artificial sequence

<400> 20

gagatccagc tgcagcagtc tggacctgag ctgatgaagc ctggggcttc agtgaagata 60

tcctgcaagg cttctggtta ctcattcact agctactaca tgcactgggt gaagcagagc 120

caaggaaaga gccttgagtg gattggattt attgatcctt tcaatggtgg tattacatac 180

aaccagaaat tcaagggcaa ggccacattg actgtagaca gatcttccag cacagcctac 240

atgcatctca gaagcctgac atctgaggac tctgcagtct attactgtgc aagatgctac 300

tataattacg acgacgaggg gagggctatg gactactggg gtcaaggaac ctcagtcacc 360

gtctcatca 369

<210> 21

<211> 324

<212> DNA

<213> artificial sequence

<400> 21

gacgtccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc aggcgagtca ggacattagc aactatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctacgat gcatccaatt tggaaacagg ggtcccatca 180

aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 240

gaagatattg caacatatta ctgtcaacag tatgataatc tcccgctcac tttcggcgga 300

gggaccaagc tggagatcaa acgt 324

<210> 22

<211> 351

<212> DNA

<213> artificial sequence

<400> 22

gaggtccagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggaggg atcatcccta tctttggtac agcaaactac 180

gcacagaagt tccagggcag agtcacgatt accgcggaca aatccacgag cacagcctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagaggccaa 300

gggtattggt tcgacccctg gggccaggga accctggtca ccgtctcctc a 351

<210> 23

<211> 107

<212> PRT

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<400> 23

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 24

<211> 98

<212> PRT

<213> artificial sequence

<400> 24

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val

<210> 25

<211> 110

<212> PRT

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<400> 25

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

100 105 110

<210> 26

<211> 105

<212> PRT

<213> artificial sequence

<400> 26

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

1 5 10 15

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

65 70 75 80

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro

100 105

Claims

1. A bispecific antibody comprising:

a first protein functional region that specifically binds to a stem cell surface antigen, the first protein functional region being an antibody or antigen-binding fragment thereof that is directed against the stem cell surface antigen;

a second protein functional region that specifically binds to a chondrocyte surface antigen, the second protein functional region being an antibody or antigen-binding fragment thereof that is directed against the chondrocyte surface antigen.

2. The bispecific antibody of claim 1, wherein the stem cells are hematopoietic stem cells.

3. The bispecific antibody of claim 2, wherein the stem cell surface antigen is CD34.

4. The bispecific antibody of claim 1, wherein the chondrocyte surface antigen is MATN3.

5. The bispecific antibody according to claim 1, characterized in that,

the light chain variable region of the first protein functional region comprises CDRs shown as SEQ ID No.1-3, and the heavy chain variable region comprises CDRs shown as SEQ ID No. 4-6;

the light chain variable region of the second protein functional region comprises CDRs as shown in SEQ ID Nos. 7-9, and the heavy chain variable region comprises CDRs as shown in SEQ ID Nos. 10-12.

6. The bispecific antibody according to claim 1, characterized in that,

the first protein functional region is selected from the group consisting of full-length immunoglobulins, fab ', (Fab') ₂ Fv, scFv or VHH; and/or the number of the groups of groups,

7. The bispecific antibody of claim 6, wherein the first protein functional region is a full length IgG1 immunoglobulin and the second protein functional region is a scFv.

8. The bispecific antibody of claim 7, wherein the scFv is a light chain variable region-a first linker-a heavy chain variable region, the light chain variable region N-terminus or the heavy chain variable region C-terminus of the scFv being linked to the C-terminus or the N-terminus of the full length IgG1 immunoglobulin light chain and/or heavy chain, respectively, by a second linker; or alternatively, the first and second heat exchangers may be,

9. The bispecific antibody of claim 8, wherein the scFv is two in number and is symmetrically linked to the C-terminus of the full length IgG1 immunoglobulin light chain, respectively.

10. The bispecific antibody of claim 8, wherein the first linker and the second linker each independently comprise a repeat selected from GS, GGSG, GGGS and GGGGS sequences.

11. The bispecific antibody of claim 6, wherein the constant region of the full length immunoglobulin is derived from IgG or an IgG mutant.

12. The bispecific antibody of claim 11, wherein the constant region comprises amino acid substitutions of L234A, L235A and P329G compared to a wild type IgG1 constant region.

13. A nucleic acid encoding the bispecific antibody of any one of claims 1-12.

14. An expression vector comprising the nucleic acid of claim 13.

15. A host cell comprising the nucleic acid of claim 13 or the expression vector of claim 14.

16. A method of preparing the bispecific antibody of any one of claims 1-12, comprising the steps of:

culturing the host cell of claim 15 under conditions suitable for expression of the bispecific antibody;

the bispecific antibody was collected and purified.

17. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-12, the nucleic acid of claim 13, the expression vector of claim 14, or the host cell of claim 15.

18. The pharmaceutical composition of claim 17, further comprising stem cells; preferably, the stem cells are hematopoietic stem cells.

19. Use of the bispecific antibody of any one of claims 1-12, the nucleic acid of claim 13, the expression vector of claim 14, the host cell of claim 15 in the manufacture of a medicament for the treatment or prevention of arthritis.