CN117843781A - CD138 antibody and application thereof - Google Patents
CD138 antibody and application thereof Download PDFInfo
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- CN117843781A CN117843781A CN202211229423.1A CN202211229423A CN117843781A CN 117843781 A CN117843781 A CN 117843781A CN 202211229423 A CN202211229423 A CN 202211229423A CN 117843781 A CN117843781 A CN 117843781A
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Abstract
The present invention relates to CD138 antibodies and uses thereof. Specifically, the invention provides a specific CD138 antibody, which can be combined with a CD138 antigen with high specificity, has high affinity and can be used for preparing and detecting CD138 related diseases. The invention also provides a corresponding detection method and a detection kit.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a CD138 antibody and application thereof.
Background
Multiple Myeloma (MM) is a malignant disease (Kumar, s.k., et al, multiple myela. Nat Rev Dis Primers, 2017.3:p.17046) with abnormal proliferation of clonal plasma cells, and common symptoms include increased blood calcium, renal cell damage, anemia, bone disease, and secondary amyloidosis. With the continuous advent of new drugs and the improvement of detection means, the diagnosis and treatment of MM have been continuously improved and perfected. Although the treatment of new drugs has increased protease inhibitors (bortezomib), immunomodulatory drugs (lenalidomide, pomalidomide) and monoclonal antibodies (up to Lei Tuoyou mab) (Chim, c.s., et al Management of relapsed and refractory multiple myeloma: novel agents, anti-ibodies, immunotherapies and beyond. Leukemia,2018.32 (2): p.252-262.), there is still resistance to most patients, with a potential risk of relapse. Thus, there is an urgent need to develop a new tolerogenic immunotherapeutic approach to treat early stage MMs and further improve patient quality of life.
CD138 (Syndecan-1, SDC 1) is one of the transmembrane proteins in the family of multimeric proteoglycans, is mainly expressed on plasma cells in the blood system, is a marker antigen of plasma cells, and is highly expressed on the surface of Multiple Myeloma (MM) cells (Ren, Z., et al, syndecan-1 proteins Wnt/beta-catenin signaling in multiple myeloma by presenting Wnts and R-spondins. Blood,2018.131 (9): p.982-994.). Meanwhile, CD138 can mediate cell adhesion through interaction with a heparan binding molecule, and is involved in cell proliferation, migration and cytoskeletal organization (Beauvais, D.M., et al, syndecan-1 (CD 138) Suppresses Apoptosis in Multiple Myeloma by Activating IGF1 Receptor: prevention by SynstatinIGF R inhibitors Tumor growth. Cancer Res,2016.76 (17): p.4981-93; khotskaya, Y.B., et al, syndecan-1is required for robust growth,vascularization,and metastasis of myeloma tumors in vivo.J Biol Chem,2009.284 (38): p.26085-95). CD138 expression is closely related to the occurrence and development of diseases (Aref, S., T.Goda, and M.El-Shermanny, syndecan-1in multiple myeloma:relationship to conventional prognostic factors.Hematology,2003.8 (4): p.221-8.), so that CD138 can be used as a brand-new target hopefully becoming the field of MM tumor treatment, thereby developing anti-CD 138-based antibody immunotherapy and providing a new idea for MM patient treatment.
There are many therapeutic drugs developed against the CD138 target, BT062 developed by Biotest (CD 138 antibody-drug conjugate in murine/human chimeric form), ADC drugs against the CD138 target currently in clinical trials in the second phase, used in combination with lenalidomide (lenalidomide), with a response rate up to 83% in MM patients and with reasonable safety (Jagannath, s., et al, indatuximab Ravtansine (BT 062) Monotherapy in Patients With Relapsed and/or Refractory Multiple myela. Clin Lymphoma Myeloma Leuk,2019.19 (6): p.372-380; kelly, k.r., et al, indatuximab ravtansine plus dexamethasone with lenalidomide or pomalidomide in relapsed or refractory multiple myeloma: a multiccent, phase 1/2a study.Lancet Haematol,2021.8 (11): p.e794-e 807). The lyenberg comprehensive cancer center picks up the baton of BT062, and converts the treatment means: CD138-CAR molecules were developed using single chain variable fragment (scFv) sequences derived from BT062 chimeric antibodies, CD 138-based CAR-T cells showed significant anti-MM activity both in vitro and in vivo in preclinical studies, and had no cytotoxic effect on normal epithelial or endothelial cells (Sun, c., et al, safety and efficacy of targeting CD138 with a chimeric antigen receptor for the treatment of multiple myela. Oncotarget,2019.10 (24): p.2369-2383.). Recently, the Jerome Lipper multiple myeloma center reported a novel monoclonal antibody VIS832 targeting CD138 that was effective in inducing killing of human multiple myeloma and further synergistically acting with lenalidomide or bortezomib to deplete MM cells in vitro and in vivo (Yu, t., et al, VIS832, a-novel CD138-targeting monoclonal antibody, potently induces killing of human multiple myeloma and further synergizes with IMiDs or bortezomib in vitro and in vivo.blood Cancer J,2020.10 (11): p.110.). Compared to BT062, VIS832 binds more strongly to all MM cell lines, mainly because the epitope recognized by VIS832 is the membrane proximal region of CD138, which targeted binding confers higher target binding and enhanced Fc effector function, resulting in improved immune synapse formation, ultimately leading to stronger MM targeting and excellent cytotoxicity.
In general, the patentability of CD138 antibodies is not known to a great extent, and at the same time, VIS832 cases also reveal that the screening of specific antibodies against different antigenic determinants is also an important consideration, so the search for more effective antibodies is still the key point of current research and development.
Thus, there remains a need in the art to develop a CD138 antibody that has high affinity and high specificity.
Disclosure of Invention
It is an object of the present invention to provide a CD138 antibody with high affinity and high specificity.
It is another object of the invention to provide the use of antibodies to CD 138.
In a first aspect of the invention there is provided a heavy chain variable region of a CD138 antibody, said heavy chain variable region comprising the following three complementarity determining region CDRs:
(1) A complementarity determining region CDR1, wherein the amino acid sequence of the complementarity determining region CDR1 is shown in SEQ ID NO. 3;
(2) A complementarity determining region CDR2, wherein the amino acid sequence of said complementarity determining region CDR2 is shown in SEQ ID NO. 4; and
(3) And a complementarity determining region CDR3, wherein the amino acid sequence of the complementarity determining region CDR3 is shown in SEQ ID NO. 5.
In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence which is optionally added, deleted, modified and/or substituted with at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and which is capable of retaining the binding affinity of CD 138.
In another preferred embodiment, the heavy chain variable region further comprises four framework regions FR, wherein the four framework regions FR are derived from the four framework regions FR corresponding to those in SEQ ID No. 2.
In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 2.
In a second aspect of the invention there is provided a heavy chain of a CD138 antibody, said heavy chain having a heavy chain variable region of an antibody according to the first aspect of the invention.
In another preferred embodiment, the heavy chain of the antibody further comprises a heavy chain constant region.
In another preferred embodiment, the constant region of the heavy chain is murine.
In a third aspect of the invention there is provided a light chain variable region of a CD138 antibody, said light chain variable region comprising the following three complementarity determining regions CDR':
(1) A complementarity determining region CDR1', wherein the amino acid sequence of said complementarity determining region CDR1' is shown in SEQ ID NO. 8;
(2) A complementarity determining region CDR2', wherein the amino acid sequence of said complementarity determining region CDR2' is shown in SEQ ID NO. 9; and
(3) Complementarity determining region CDR3', the amino acid sequence of said complementarity determining region CDR3' is shown in SEQ ID NO. 10.
In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence which is optionally added, deleted, modified and/or substituted with at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and which is capable of retaining the binding affinity of CD 138.
In another preferred embodiment, the light chain variable region further comprises four framework regions FR, wherein the four framework regions FR are derived from the four framework regions FR corresponding to those in SEQ ID No. 7.
In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID NO. 7.
In a fourth aspect of the invention there is provided a CD138 antibody light chain having the light chain variable region of the antibody of the third aspect of the invention.
In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.
In another preferred embodiment, the light chain constant region is murine.
In a fifth aspect of the invention, there is provided a CD138 antibody having:
(1) A heavy chain variable region according to the first aspect of the invention; and/or
(2) The light chain variable region according to the third aspect of the invention.
Alternatively, the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention.
In another preferred embodiment, the antibody further has a heavy chain constant region and a light chain constant region.
In another preferred embodiment, the antibody has the heavy chain variable region shown in SEQ ID NOs 3, 4 and 5; and/or the light chain variable regions as shown in SEQ ID NOS.8, 9 and 10.
In another preferred embodiment, the heavy chain of the antibody has the amino acid sequence shown in SEQ ID No. 2
In another preferred embodiment, the light chain of the antibody has the amino acid sequence shown in SEQ ID No. 7.
In another preferred embodiment, the antibody is a humanized antibody.
In another preferred embodiment, the antibody specifically binds CD138.
In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.
In another preferred embodiment, the antibody is a monoclonal antibody.
In another preferred embodiment, the antibody is a bispecific antibody.
In another preferred embodiment, the antibody is in the form of a drug conjugate.
In a sixth aspect of the present invention, there is provided a recombinant protein having:
(i) A heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; and
(ii) Optionally a tag sequence to assist expression and/or purification.
In another preferred embodiment, the tag sequence comprises a 6His tag.
In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.
In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.
In a seventh aspect of the invention, there is provided an antibody preparation comprising:
(a) An antibody according to the fifth aspect of the invention; and
(b) A carrier, said carrier comprising: buffering agents, sterile water, optionally surfactants.
In another preferred embodiment, the concentration of the antibody in the formulation is 5-100mg/mL; preferably 10-70mg/mL, more preferably 20-60mg/mL.
In another preferred embodiment, the buffer is selected from the group consisting of: a PBS buffer system, a citrate buffer system, a histidine buffer system, or a combination thereof.
In another preferred embodiment, the pH of the formulation is in the range of 5.0 to 7.5, preferably 5.5 to 7.
In another preferred embodiment, the formulation is an injectable formulation.
In an eighth aspect of the invention there is provided a kit comprising an antibody preparation according to the seventh aspect of the invention, and a container for containing the antibody preparation.
In a ninth aspect of the invention there is provided a CAR construct, the scFv fragment of the antigen binding region of which is a binding region that specifically binds to CD138 and which has a heavy chain variable region as described in the first aspect of the invention and a light chain variable region as described in the third aspect of the invention.
In a tenth aspect of the invention, there is provided a recombinant immune cell expressing an exogenous CAR construct according to the ninth aspect of the invention.
In another preferred embodiment, the immune cells are selected from the group consisting of: NK cells, T cells.
In another preferred embodiment, the immune cells are derived from a human or non-human mammal (e.g., a mouse).
In an eleventh aspect of the present invention, there is provided an antibody drug conjugate comprising:
(a) An antibody moiety selected from the group consisting of: a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, or a combination thereof; and
(b) A coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, enzyme, or a combination thereof.
In another preferred embodiment, the antibody moiety is coupled to the coupling moiety via a chemical bond or linker.
In a twelfth aspect of the invention there is provided the use of an active ingredient selected from the group consisting of: the heavy chain variable region according to the first aspect of the invention, the heavy chain according to the second aspect of the invention, the light chain variable region according to the third aspect of the invention, the light chain according to the fourth aspect of the invention, or the antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, the immune cell according to the tenth aspect of the invention, the antibody drug conjugate according to the eleventh aspect of the invention, or a combination thereof, the active ingredient being for:
(a) Preparing a detection reagent or a kit;
(b) Preparing a medicament or preparation for preventing and/or treating CD 138-related diseases; and/or
(c) Preparing medicine or preparation for preventing and/or treating cancer or tumor.
In another preferred embodiment, the tumor is selected from the group consisting of: hematological tumors, solid tumors, or combinations thereof.
In another preferred embodiment, the hematological tumor is Multiple Myeloma (MM).
In another preferred embodiment, the tumor is a tumor that highly expresses CD 138.
In another preferred embodiment, the medicament or formulation is for the manufacture of a medicament or formulation for the prevention and/or treatment of a disease associated with CD138 (positive expression).
In another preferred embodiment, the antibody is in the form of A Drug Conjugate (ADC).
In another preferred embodiment, the detection reagent or kit is used for diagnosing CD 138-related diseases.
In another preferred embodiment, the detection reagent or kit is used to detect CD138 protein in a sample.
In another preferred embodiment, the detection reagent is a detection chip.
In a thirteenth aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) An active ingredient selected from the group consisting of: a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, an immune cell according to the tenth aspect of the invention, an antibody drug conjugate according to the eleventh aspect of the invention, or a combination thereof; and
(ii) A pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition further comprises a second active ingredient that is anti-tumor.
In another preferred embodiment, the second active ingredient is selected from the group consisting of: cytotoxic drugs, toxins, cytokines, enzymes, antibodies, or combinations thereof.
In another preferred embodiment, the pharmaceutical composition is a liquid formulation.
In another preferred embodiment, the pharmaceutical composition is an injection.
In another preferred embodiment, the pharmaceutical composition is for the treatment of tumors.
In another preferred embodiment, the tumor is a tumor that highly expresses CD 138.
In a fourteenth aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:
(1) A heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; or (b)
(2) A recombinant protein according to the sixth aspect of the invention; and/or
(3) A CAR construct according to the ninth aspect of the invention.
In a fifteenth aspect of the present invention there is provided a vector comprising a polynucleotide according to the fourteenth aspect of the present invention.
In another preferred embodiment, the carrier comprises: bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.
In a sixteenth aspect of the invention there is provided a genetically engineered host cell comprising a vector according to the fifteenth aspect of the invention or a polynucleotide according to the fourteenth aspect of the invention integrated in the genome.
In a seventeenth aspect of the invention, there is provided a method of detecting CD138 protein in an in vitro sample (including diagnostic or non-diagnostic), the method comprising the steps of:
(1) Contacting the sample with an antibody according to the fifth aspect of the invention in vitro;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of CD138 protein in the sample.
In an eighteenth aspect of the present invention, there is provided a detection plate comprising: a substrate (support) and a test strip comprising an antibody according to the fifth aspect of the invention or an antibody drug conjugate according to the eleventh aspect of the invention.
In a nineteenth aspect of the present invention, there is provided a kit comprising:
(1) A first container comprising an antibody according to the fifth aspect of the invention; and/or
(2) A second container comprising a second antibody against an antibody according to the fifth aspect of the invention;
alternatively, the kit contains a detection plate according to the eighteenth aspect of the invention.
In a twentieth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:
(a) Culturing a host cell according to the fourteenth aspect of the invention under conditions suitable for expression;
(b) Isolating the recombinant polypeptide from the culture, said recombinant polypeptide being an antibody according to the fifth aspect of the invention or a recombinant protein according to the sixth aspect of the invention.
In a twenty-first aspect of the invention, there is provided a method of treating a CD 138-associated disease, the method comprising: administering to a subject in need thereof an antibody according to the fifth aspect of the invention, an antibody-drug conjugate of the antibody, or a CAR-T cell expressing the antibody, or a combination thereof.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 is an identification of the binding capacity of a screened murine antibody to the CD138 antigen.
FIG. 2 is an identification of expression of chimeric antibodies.
FIG. 3 is an identification of the binding capacity of chimeric antibodies to the CD138 antigen.
Detailed Description
The present inventors have conducted extensive and intensive studies and, as a result of extensive screening, have unexpectedly obtained CD138 antibodies having excellent affinity and specificity. The antibodies of the invention are capable of binding to CD138 antigen with high specificity. Experimental results show that the chimeric antibody 103E11 provided by the invention has better antigen recognition capability on myeloma cells than BB4 and slightly better antigen CD138 binding capability than 4320 antibody binding capability. On this basis, the present inventors have completed the present invention.
Terminology
As used herein, the term "conjugate" refers to a soluble receptor or fragment or analog thereof, or an antibody or fragment or analog thereof, that is capable of binding to a target. The term "CD138 conjugate" as used herein refers to an antibody or fragment or analog thereof that specifically recognizes CD138 and binds to CD138 antigen.
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that there may be, but need not be, 1, 2, or 3 antibody heavy chain variable regions of a particular sequence.
CD138
CD138 (Syndecan-1, SDC 1) is one of the transmembrane proteins in the family of multimeric proteoglycans, and is mainly expressed on plasma cells in the blood system, is a marker antigen of plasma cells, and is highly expressed on the cell surface of Multiple Myeloma (MM).
Antibodies to
As used herein, the term "antibody" refers to an immunoglobulin that is a tetrapeptide chain structure formed from two identical heavy chains and two identical light chains joined by an interchain disulfide bond. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be assigned to five classes, or different types of immunoglobulins, i.e., igM, igD, igG, igA and IgE, and the heavy chain constant regions corresponding to the different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. IgG represents the most important class of immunoglobulins, which can be divided into 4 subclasses again due to differences in chemical structure and biological function: igG1, igG2, igG3 and IgG4. Light chains are classified as either kappa or lambda chains by the difference in constant regions. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
The sequences of the heavy and light chains of the antibody near the N-terminus vary widely, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable region includes 3 hypervariable regions (HVRs) and 4 FR Regions (FR) that are relatively conserved in sequence. The amino acid sequences of the 4 FRs are relatively conserved and do not directly participate in the binding reaction. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) consists of 3 CDR regions and 4 FR regions, arranged in sequence from amino-to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain, namely the light chain hypervariable region (LCDR), refer to LCDR1, LCDR2 and LCDR3; the 3 CDR regions of the heavy chain, namely heavy chain hypervariable regions (HCDR), refer to HCDR1, HCDR2 and HCDR3. The CDR amino acid residues of the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the invention are in numbers and positions that meet the known Kabat numbering convention (LCDR 1-3, HCDR 2-3), or that meet the numbering convention of Kabat and chothia (HCDR 1). The four FR regions in the natural heavy and light chain variable regions are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form a partially folded structure. The CDRs in each chain are held closely together by the FR regions and form together with the CDRs of the other chain an antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody. The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.
As used herein, the term "antigen binding fragment" refers to a Fab fragment, fab 'fragment, F (ab') 2 fragment, or single Fv fragment having antigen binding activity. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding.
As used herein, the term "epitope" refers to a discrete, three-dimensional spatial site on an antigen that is recognized by an antibody or antigen-binding fragment of the invention.
The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.
In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using DNA recombination techniques well known in the art.
As used herein, the term "monoclonal antibody" refers to an antibody secreted from a clone derived from a single cell source. Monoclonal antibodies are highly specific, being directed against a single epitope. The cells may be eukaryotic, prokaryotic or phage clonal cell lines.
As used herein, the term "chimeric antibody" is an antibody molecule expressed by a host cell by splicing the V region gene of a murine antibody to the C region gene of a human antibody into a chimeric gene, followed by insertion into a vector. The high specificity and affinity of the parent mouse antibody are maintained, and the human Fc segment of the parent mouse antibody can effectively mediate biological effect functions.
As used herein, the term "humanized antibody", a variable region engineered version of the murine antibody of the present invention, has CDR regions derived (or substantially derived) from a non-human antibody (preferably a mouse monoclonal antibody), and FR regions and constant regions substantially derived from human antibody sequences; i.e., grafting murine-resistant CDR region sequences onto different types of human germline antibody framework sequences. Because CDR sequences are responsible for most of the antibody-antigen interactions, recombinant antibodies that mimic the properties of a particular naturally occurring antibody can be expressed by constructing expression vectors. In the present invention, antibodies may be monospecific, bispecific, trispecific, or more multispecific.
In the present invention, the antibodies of the invention also include conservative variants thereof, meaning that up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence of the antibodies of the invention to form a polypeptide. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.
Table A
| Initial residues | Representative substitution | Preferred substitution |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
CD138 antibodies
As used herein, the term "CD138" refers to a CD138 protein, the amino acid sequence of which is shown in SEQ ID No. 1.
The present invention provides a high specificity and high affinity antibody against CD138 comprising a heavy chain variable region (VH) amino acid sequence and a light chain comprising a light chain variable region (VL) amino acid sequence.
A preferred antibody of the invention has the respective CDRs of the heavy chain variable region (VH) amino acid sequence and the light chain variable region (VL) amino acid sequence selected from the group consisting of:
a1 SEQ ID NO. 3 (heavy chain CDR 1);
a2 SEQ ID NO. 4 (heavy chain CDR 2);
a3 SEQ ID NO. 5 (heavy chain CDR 3);
a4 SEQ ID NO. 8 (light chain CDR 1);
a5 SEQ ID NO. 9 (light chain CDR 2);
a6 SEQ ID NO. 10 (light chain CDR 3);
a7 Any one of the above amino acid sequences is a sequence having CD138 binding affinity that is added, deleted, modified and/or substituted with at least one (e.g., 1-5, 1-3, preferably 1-2, more preferably 1) amino acid.
In another preferred embodiment, the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence is preferably an amino acid sequence having a homology of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95%.
The antibody of the present invention may be a double-or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and even more preferably fully humanized antibodies.
The antibody derivatives of the invention may be single chain antibodies, and/or antibody fragments, such as: fab, fab ', (Fab') 2 Or other antibody derivatives known in the art, and IgA, igD, igE, igG and IgM antibodies orAny one or more of the other subtype antibodies.
Wherein the animal is preferably a mammal, such as a mouse.
The antibodies of the invention may be murine antibodies, chimeric antibodies, humanized antibodies, CDR grafted and/or modified antibodies that target human CD 138.
In a preferred embodiment of the invention, any one or several of the sequences of SEQ ID NOs 3, 4 and 5 described above, or a sequence thereof having binding affinity for the N-terminal end of CD138, which has been added, deleted, modified and/or substituted with at least one amino acid, is located in the CDR region of the heavy chain variable region (VH).
In a preferred embodiment of the invention, any one or more of the sequences of SEQ ID NOS.8, 9, 10 described above, or a sequence having binding affinity for the N-terminal end of CD138, in which they have been added, deleted, modified and/or substituted for at least one amino acid, are located in the CDR region of the light chain variable region (VL).
In the present invention, the number of amino acids added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20% of the total amino acids of the original amino acid sequence.
In the present invention, the number of the added, deleted, modified and/or substituted amino acids is usually 1, 2, 3, 4 or 5, preferably 1 to 3, more preferably 1 to 2, most preferably 1.
It will be appreciated that antibodies of the invention also include antibodies that contain one or more mutations in the FR region, with no mutation or only 1 or 2 conservative mutations in the 6 CDR regions, and still retain the N-terminal or C-terminal specific binding and affinity of CD 138.
Preparation of antibodies
Any suitable method for producing antibodies may be used to produce the CD138 antibodies of the invention. For example, animals may be immunized with linked or naturally occurring CD138 or fragments thereof. Suitable immunization methods may be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may be used.
Any suitable form of CD138 may be used as an immunogen (antigen) for the generation of non-human antibodies specific for CD138, and the biological activity of the antibodies is screened. The priming immunogen may be recombinant CD138 or a fragment thereof. The immunogens may be used alone or in combination with one or more immunogenicity enhancing agents known in the art. The immunogen may be purified from a natural source or produced in genetically modified cells. The DNA encoding the immunogen may be genomic or non-genomic (e.g., cDNA) in origin. DNA encoding an immunogen may be expressed using suitable genetic vectors including, but not limited to: adenovirus vectors, adeno-associated virus vectors, baculovirus vectors, mass vectors and non-viral vectors.
An exemplary method of producing an anti-CD 138 antibody of the invention is described in example 1.
Antibodies of the invention may be selected from any class of immunoglobulins of any species, including IgG and IgE. Preferred antibodies are IgG antibodies, such as the IgG1 subtype. Optimization of the necessary constant domain sequences to produce the desired biological activity is readily achieved by screening antibodies using the biological assays described in the examples below.
Also, any type of light chain may be used in the compounds and methods herein. In particular, kappa, lambda chains or variants thereof are useful in the compounds and methods of the present invention.
The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as amplification by PCR or screening of a genomic library. In addition, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Preferred animal cells include (but are not limited to): CHO-S, CHO-K1, HEK-293 cells.
The steps described herein for transforming a host cell with recombinant DNA may be performed using techniques well known in the art. The transformant obtained can be cultured by a conventional method, and the transformant expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultivated in conventional medium under suitable conditions.
Typically, the transformed host cell is cultured under conditions suitable for expression of the antibodies of the invention. The antibodies of the invention are then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.
The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined using immunoprecipitation or in vitro binding assays, such as Radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA).
Coding nucleic acids and expression vectors
The invention also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.
In the present invention, the term "expression vector" refers to a vector, such as a plasmid, viral vector (e.g., adenovirus, retrovirus), phage, yeast plasmid, or other vector, carrying an expression cassette for expression of a particular protein of interest or other substance. Representative examples include, but are not limited to: pTT5, pSECtag series, pCGS3 series, pcDNA series vectors and the like, as well as other vectors for use in mammalian expression systems and the like. Included in the expression vector are fusion DNA sequences linked to appropriate transcriptional and translational regulatory sequences.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.
In the present invention, the term "host cell" refers to a cell suitable for expressing the above expression vector, and may be eukaryotic, such as mammalian or insect host cell culture systems, all of which can be used for expression of the fusion protein of the present invention, CHO (chinese hamster ovary ), HEK293, COS, BHK and derivatives of the above cells are suitable for use in the present invention.
Pharmaceutical composition and application
The invention also provides a pharmaceutical composition. Preferably, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavity injection.
In the present invention, the term "pharmaceutical composition" means that the bispecific antibodies of the present invention can be combined with a pharmaceutically acceptable carrier to form pharmaceutical formulation compositions that provide more stable therapeutic effects, such formulations ensuring the conformational integrity of the amino acid core sequences of the disclosed bispecific antibodies while also protecting the multifunctional groups of the proteins from degradation (including, but not limited to, aggregation, deamination or oxidation).
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the bispecific antibody (or conjugate thereof) of the invention as described above, and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, bispecific antibodies of the invention can also be used with other therapeutic agents.
When a pharmaceutical composition is used, a safe and effective amount of the bispecific antibody or immunoconjugate thereof is administered to a mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
Detection application and kit
The antibodies of the invention may be used in detection applications, for example for detecting samples, thereby providing diagnostic information.
In the present invention, the samples (specimens) used include cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to a person skilled in the art. Thus biopsies used in the present invention may include tissue samples prepared, for example, by endoscopic methods or by puncture or needle biopsy of an organ.
Samples for use in the present invention include fixed or preserved cell or tissue samples.
The invention also provides a kit comprising an antibody (or fragment thereof) of the invention, which in a preferred embodiment of the invention further comprises a container, instructions for use, buffers, etc. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.
The main advantages of the invention include:
(a) The antibody has excellent bioactivity and specificity and high binding force.
(b) The invention provides detection reagents and methods that can specifically detect CD 138.
(c) The binding capacity of the antibody to the CD138 antigen on the U266 surface of the myeloma cell is extremely superior to BB4 and is slightly superior to 4320.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
The experimental materials used in the following examples are illustrated below:
pcDNA3.4, PMD20-T vector: ji Kai organism
Myeloma cells: h929 cells, mm1.R cells, U266 cells, 8226 cells: ji Kai organism
FBS: gibco, cat: 10099-141C
Trypan Blue stain 0.4%: invitrogen, cat#: t10282
96-well round-bottom cell culture plate: corning, cat: CLS3799
Double antibody: hyclone, cat: SV30010
1640: corning, cat: 10-040-CVRC
Goat Anti-human IgG Fc (Dyligt 488): abcam, cat No.: ab97003
Freund's complete adjuvant, freund's incomplete adjuvant: sigma, cat No.: f5881-10ML, F5506-10ML
SDC1 (Mammalian, C-6 His): offshore protein, cat No.: CA71
Human Syndecan-1protein, his Tag: ACRObiosystem, cat No.: SY1-H5225
Goat pAb to Ms IgG(HRP)：Abcam，ab97265
The experimental reagents used in the following examples are illustrated below:
RevertAid First Strand cDNA Synthesis Kit: thermo, cat No.: k1621
EXAMPLE 1 preparation and screening of recombinant murine monoclonal antibodies against CD138
(1) Immune antigen
CD138 (SDC 1) is a single transmembrane protein, the extracellular portion contains 232 amino acid residues (23-254), and this region is presumed to be strongly immunogenic based on immunological principles and experience, so this item directly selects this portion as an immunogen (near-shore protein, CA 71). The antigen sequence is shown as SEQ ID NO.1.
(2) Immunization of animals
2 Balb/c and 3 SJL mice were purchased from Vetong Liwa, females, 6 weeks old in place, and fed one week after in place to accommodate the new environment. On the day of immunization, 200. Mu.g of Human Syndecan-1protein, his Tag (from ACRObiosystems, 400. Mu.g for primary immunization) was added to 5mL of tube, DPBS was added to 1mL, and 1mL of Freund's adjuvant (from sigma) was added to the tube and mixed well. Fixing tube on vortex vibration instrument, and vibrating for 30min to fully emulsify antigen protein. The emulsified antigen protein product was aspirated by syringe and administered by intraperitoneal injection, 200. Mu.L per mouse. After immunization once every 14 days, 200. Mu.g SDC1 (Mammalian, C-6 His) protein and incomplete Freund's adjuvant were emulsified for subsequent 5 immunizations, for a total of 6 immunizations. Blood is taken after 6 times of immunization, serum titer is measured by ELISA method gradient dilution, and the mice with the best immune antibody titer are selected for the next cell fusion.
Results
As can be seen from table 1, there are mice producing antibodies against the antigen, which can be used in subsequent experiments.
(3) Hybridoma cell fusion
Spleen of the mice was taken, ground with a cell screen, and the adherent tissues were removed to prepare spleen cell suspensions. Murine SP2/0 myeloma cells were prepared and counted together with spleen cell suspensions according to 3:1, and then in a centrifuge at 1500rpm for 7min. After discarding the supernatant, the cells were resuspended by addition of Cytofusion Medium C and centrifuged at 1500rpm for 7min. Cells were resuspended with Cytofusion Medium C again and added to the electrode cup of the BTX fusion apparatus for electrofusion. The cell suspension was added to hybridoma incubation (dmem+20% fbs), placed in a 37 ℃ 5% co2 incubator for 30min, and then centrifuged to change the hybridoma medium for further culture.
(4) Screening of clones
The hybridoma supernatants were tested by ELISA, and positive hybridomas were selected. Transferring the positive hybridomas obtained by screening in the 96-well plate into a 24-well plate for continuous culture for 3 days, continuously detecting the supernatant of the hybridomas by using ELISA experiments, and selecting positive hybridomas capable of reacting with CD 138. Meanwhile, tumor cells expressing CD138 are incubated on the supernatant of the hybridomas in the 24-well plates by using a FACS method, and positive hybridoma cells which can react with the cells are selected. Positive hybridoma cells were finally determined and subcloned according to ELISA and FACS experiments. And finally detecting the supernatant of the hybridoma by an ELISA method, and selecting a positive monoclonal hole to ensure the singleness and stability of the cell strain.
As can be seen from Table 2, 16 positive hybridoma cells were selected in total by the first round of ELISA screening. As can be seen from the ELISA results of Table 3 and the FACS experiment results of Table 4, 6 cells of 103E11, 109D6, 109C10, 90A5, 126G1, 88E5 were selected together again by repeated screening of ELISA and FACS experiments of round 2, and the subsequent subcloning experiments were performed. However, in the subsequent culture, 109D6 died, so that only 5 cells could be subjected to the subsequent ELISA experiments to ensure some but some of the cell lines were stable.
As can be seen from the results in Table 5, each hybridoma found a positive monoclonal and subclone achieved the intended purpose.
TABLE 2
TABLE 3 Table 3
| Platema | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| A | 103E11 | 109D6 | 109C10 | 109G5 | 90A5 | 90H11 | 80A5 | 80H11 | 130A1 | 128E1 | 128C4 | 126F1 |
| B | 126G1 | 126H1 | 124F11 | 88E5 | NC | PC | ||||||
| OD450 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| A | 3.2444 | 3.0342 | 2.8307 | 0.0512 | 0.7126 | 0.0498 | 0.0508 | 0.0512 | 0.0504 | 2.5807 | 0.0528 | 0.0634 |
| B | 2828 | 00506 | 20876 | 07078 | 00719 | 29008 |
TABLE 4 Table 4
| Platemap | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| A | 103E11 | 109D6 | 109C10 | 109G5 | 90A5 | 90H11 | 80A5 | 80H11 | 130A1 | 128E1 | 128C4 | 126F1 |
| B | 126G1 | 126H1 | 124F11 | 88E5 | NC | PC | ||||||
| MFI | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| A | 238815 | 167341 | 1694 | 1358 | 5787 | 1382 | 1361 | 1335 | 1338 | 1329 | 1350 | 3585 |
| B | 2141 | 1562 | 1386 | 4636 | 1367 | 208351 |
TABLE 5
| Platemap | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| A | 103E11F9 | 109C10F3 | 90A5E6 | 88E5D6 | 126G1F3 | NC | PC |
| OD450 | 1 | 2 | 3 | 4 | 5 | NC | PC |
| A | 1.1616 | 2.7773 | 0.3644 | 0.6677 | 1.5568 | 0.0892 | 2.91 |
(5) Production verification of mouse anti-mice
Taking 3×10 7 Hybridoma cells with good growth status were cultured in antibody preparation medium (dmem+10 Ultra low IgG FBS) for 5 days. The culture supernatant was collected and purified with Protein A to give the corresponding antibody. ELISA and FACS experiments were then used to verify whether the murine antibodies produced respond positively to antigen CD 138. Based on ELISA and FACS experimental results, positive clones were determined for subsequent sequencing.
As can be seen from FIG. 1A, ELISA experiments show that 103E11F9, 109C10F3 and 126G1F3 have positive reaction to antigen CD138, while FACS experiments show that 90A5E6, 88E5D6 and 103E11F9 have better affinity to myeloma cell line and high specificity to human kidney epithelial cell line 293T cells. Thus, based on ELISA and FACS results, 90A5E6, 103E11F9, 109C10F3, 126G1F3 and 88E5D6 were finally selected for subsequent sequencing.
(6) Hybridoma sequencing
The cell suspension was collected and total RNA was isolated from hybridoma cells using RNAzol RT Isolation Reagent. The total RNA was reverse transcribed into cDNA using a reverse transcription kit (RevertAid First Strand cDNA Synthesis Kit) and G addition reactions were performed. The variable region of the heavy chain and the variable region of the light chain of the antibody are amplified by using specific primers, and then PCR products of the variable regions of the heavy chain and the light chain of the antibody are cloned on a PMD20-T vector, and sequencing is carried out, so that the sequence of 88E5D6 and the sequence of 90A5E6 are consistent.
Example 3 determination of affinity of anti-CD 138 recombinant murine/human chimeric antibody
This example is an assay for affinity of anti-CD 138 recombinant murine/human chimeric antibodies and uses BB4 (anti-human CD138 antibody in BT 062) and anti-human CD138 antibody 4320 (VIS 832) in US20200392241, which are currently in rapid clinical progression, as positive controls. The measurement method is as follows:
A. expression purification of chimeric antibodies
(1) The variable regions of the heavy and light chains of the murine antibody were linked to the constant regions of the heavy and light chains of human IgG1, respectively, to form the heavy (VH mouse+ch human) and light (VL mouse+cl human) chains of the murine/human chimeric antibody, and the heavy and light chains of the chimeric antibody were constructed on pcdna3.4 vectors, respectively, to raise plasmids.
(2) 100mL of 293 cells were cultured in OPM-293 (O Pu Mai) and incubated at 37℃and 120rpm with 8% CO 2.
(3) 24. Mu.g of the heavy chain and 36. Mu.g of the light chain were added together to 1mL of OPM-293 medium and mixed well.
(4) Simultaneously, 15 mug of PEI is added into 1mL of OPM-293 culture medium, and after being mixed evenly, the mixture is added into the mixed solution, and the mixture is incubated for 15min at 37 ℃.
(5) Then adding the mixed transfection solution into the cell suspension, shaking while adding, continuously placing into a shaking table for culturing, and collecting the supernatant after one week of expression.
(6) Put into a centrifuge, centrifuged at 8000rpm for 5min.
(7) The supernatant after centrifugation was purified by Protein A affinity chromatography and subjected to SDS-PAGE to verify purity.
As can be seen from fig. 2, either the positive control antibody or the chimeric expressed antibody, appears to be one band in the non-denatured state and two bands in the denatured state, indicating that the heavy and light chains of the antibody are bound together. Meanwhile, the purity of the product reaches more than 95%, and the product can be used for subsequent researches.
B. Binding ability of chimeric antibodies to myeloma cell surface antigen CD138
(1) Tumor cells were 2X 10 per well 5 50. Mu.L was plated into 96-well round-bottomed cell culture plates.
(2) Antibodies were prepared at an initial concentration of 60 μg/mL, then diluted 3-fold in gradient, and a total of 7 concentrations were obtained.
(3) The antibody dilutions were added to the tumor cell suspensions previously spread in the 96 wells above, 50 μl per well, and incubated at 4 ℃ for 1h.
(4) The 96-well plate was placed in a centrifuge at 1000rpm for 5min and washed 2 times.
(5) 50. Mu.L of secondary antibody (Goat anti-Human IgG (FcDylight 488)) diluted 1000-fold with PBS was added, and the mixture was blown on and incubated at 4℃for 1h.
(6) The 96-well plate was placed in a centrifuge at 1000rpm for 5min and washed 3 times.
(7) Cells were resuspended and examined with a flow cytometer.
Results
As a result, as shown in FIG. 3, the ability of chimeric antibody 103E11 to recognize antigen on myeloma cells was slightly better than BB4, and the ability to bind to antigen CD138 was slightly better than that of the 4320 antibody.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Claims (10)
1. A heavy chain variable region of a CD138 antibody, said heavy chain variable region comprising the following three complementarity determining region CDRs:
(1) A complementarity determining region CDR1, wherein the amino acid sequence of the complementarity determining region CDR1 is shown in SEQ ID NO. 3;
(2) A complementarity determining region CDR2, wherein the amino acid sequence of said complementarity determining region CDR2 is shown in SEQ ID NO. 4; and
(3) And a complementarity determining region CDR3, wherein the amino acid sequence of the complementarity determining region CDR3 is shown in SEQ ID NO. 5.
2. A heavy chain of a CD138 antibody, wherein said heavy chain comprises the heavy chain variable region of the antibody of claim 1.
3. A light chain variable region of a CD138 antibody, said light chain variable region comprising the following three complementarity determining regions CDR':
(1) A complementarity determining region CDR1', wherein the amino acid sequence of said complementarity determining region CDR1' is shown in SEQ ID NO. 8;
(2) A complementarity determining region CDR2', wherein the amino acid sequence of said complementarity determining region CDR2' is shown in SEQ ID NO. 9; and
(3) Complementarity determining region CDR3', the amino acid sequence of said complementarity determining region CDR3' is shown in SEQ ID NO. 10.
4. A CD138 antibody light chain, characterized in that said antibody light chain has the light chain variable region of the antibody of claim 3.
5. A CD138 antibody, said antibody having:
(1) The heavy chain variable region of claim 1; and/or
(2) The light chain variable region of claim 3.
Alternatively, the antibody has: the heavy chain of claim 2; and/or the light chain of claim 4.
6. A recombinant protein, said recombinant protein comprising:
(i) A heavy chain variable region according to claim 1, a heavy chain according to claim 2, a light chain variable region according to claim 3, a light chain according to claim 4, or an antibody according to claim 5; and
(ii) Optionally a tag sequence to assist expression and/or purification.
7. A CAR construct, wherein the scFv fragment of the antigen binding region of the CAR construct is a binding region that specifically binds to CD138 and the scFv has the heavy chain variable region of claim 1 and the light chain variable region of claim 3.
8. A recombinant immune cell expressing the CAR construct of claim 7 exogenously.
9. An antibody drug conjugate, comprising:
(a) An antibody moiety selected from the group consisting of: the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5, or a combination thereof; and
(b) A coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, enzyme, or a combination thereof.
10. Use of an active ingredient, characterized in that the active ingredient is selected from the group consisting of: the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5, the recombinant protein of claim 6, the immune cell of claim 8, the antibody drug conjugate of claim 9, or a combination thereof, the active ingredient being for:
(a) Preparing a detection reagent or a kit;
(b) Preparing a medicament or preparation for preventing and/or treating CD 138-related diseases; and/or
(c) Preparing medicine or preparation for preventing and/or treating cancer or tumor.
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