CN118027199A

CN118027199A - Antibodies to CD138 and uses thereof

Info

Publication number: CN118027199A
Application number: CN202410098634.9A
Authority: CN
Inventors: 熊冬生; 王建祥; 卢杨; 国承彩; 王敏; 张砚君; 范冬梅
Original assignee: Institute of Hematology and Blood Diseases Hospital of CAMS and PUMC
Current assignee: Institute of Hematology and Blood Diseases Hospital of CAMS and PUMC
Priority date: 2024-01-23
Filing date: 2024-01-23
Publication date: 2024-05-14

Abstract

The invention discloses an isolated antibody which specifically binds to human CD138 protein, wherein the antibody comprises an amino acid sequence of CDRH1 of a heavy chain variable region shown as SEQ ID No.1, an amino acid sequence of CDRH2 of a heavy chain variable region shown as SEQ ID No.2, an amino acid sequence of CDRH3 of a heavy chain variable region shown as SEQ ID No.3, an amino acid sequence of CDRL1 of a light chain variable region shown as SEQ ID No.4, an amino acid sequence of CDRL2 of a light chain variable region shown as SEQ ID No.5 and an amino acid sequence of CDRL3 of a light chain variable region shown as SEQ ID No. 6. The antibodies of the disclosure have high affinity with CD138 protein, and have strong binding specificity, and no cross reaction with negative cells. Can be used for detecting cells expressing CD138, and can be used in tumor immunotherapy alone or in combination with other methods.

Description

Antibodies to CD138 and uses thereof

Technical Field

The present disclosure relates to the field of biological medicine, in particular, the disclosure relates to an anti-CD 138 antibody and uses thereof.

Background

CD138 (Syndecan-1, SDC 1) is a transmembrane proteoglycan, a transmembrane protein receptor of the extracellular matrix (ECM) that mediates cell adhesion through interactions with heparan binding molecules, involved in cell proliferation, migration and tissue regulation of the cytoskeleton. CD138 is overexpressed in patient's malignant myeloma cells compared to normal plasma cells and is associated with disease progression and prognosis. Thus, CD138 is an ideal detection or therapeutic target for plasmacytomas.

The current drugs developed based on CD138 targets for multiple myeloma treatment are BT062 (indatuximab ravtansine), a mouse/human chimeric form of CD138 antibody-drug conjugate linked to maytansine drug DM4 via disulfide bonds, with primary safety in relapse refractory multiple myeloma (rrMM) patients in clinical trials with lenalidomide or pomalidomide in combination with low doses of dexamethasone, while Indatuximab ravtansine single drug also showed some anti-MM activity, but overall remission rate was poor. Furthermore, the Jerome Lipper multiple myeloma center reports a novel CD138 monoclonal antibody VIS832, which recognizes that the epitope is located in the membrane proximal region of CD138, has a higher affinity than BT062, and shows stronger MM targeting and cytotoxic effects. In addition, in clinical experiments, CD 138-targeting chimeric antigen receptor T cells showed significant killing effects on MM cells both in vitro and in vivo, while not having cytotoxic effects on normal epithelial and endothelial cells. However, current therapeutic agents targeting CD138 have potential risks or limitations such as: dose-limiting toxicity, cytokine release syndrome/immune effector cell-related neurotoxic syndrome, limited and non-persistent therapeutic responsiveness, excessive therapeutic costs, and the like.

Therefore, although CD138 is an ideal therapeutic target for various tumors, no monoclonal antibody targeting CD138 and related immunotherapeutic drugs are currently being formally used in clinic, and neither the current antibodies nor the related drugs achieve an ideal therapeutic effect, suggesting that further development of antibodies targeting CD138 is required.

Disclosure of Invention

The technical problems to be solved are as follows:

In one aspect of the present disclosure, an isolated antibody is provided that addresses the deficiencies in the prior art that lack an antibody that targets CD138 with desirable efficacy.

The technical scheme is as follows:

An isolated antibody that specifically binds human CD138 protein, the antibody comprising the amino acid sequence of CDRH1 of the heavy chain variable region as set forth in SEQ ID No.1, the amino acid sequence of CDRH2 of the heavy chain variable region as set forth in SEQ ID No.2, the amino acid sequence of CDRH3 of the heavy chain variable region as set forth in SEQ ID No.3, the amino acid sequence of CDRL1 of the light chain variable region as set forth in SEQ ID No.4, the amino acid sequence of CDRL2 of the light chain variable region as set forth in SEQ ID No.5, and the amino acid sequence of CDRL3 of the light chain variable region as set forth in SEQ ID No. 6.

In certain embodiments of the present disclosure, the antibody comprises the amino acid sequence of the heavy chain variable region as set forth in SEQ ID No.7 and the amino acid sequence of the light chain variable region as set forth in SEQ ID No. 8.

In certain embodiments of the present disclosure, the antibody may be a mammalian-derived antibody, e.g., murine, rabbit, sheep, horse, monkey, pig, camel, shark, chicken, and the like. In other embodiments of the present disclosure, the antibody may be a chimeric antibody, a humanized antibody, or a fully human antibody.

In certain embodiments of the disclosure, the antibody may be IgG, igA, igM, igD or IgE. Preferably, in certain embodiments of the present disclosure, the antibody may be of the type IgG. Further, in certain embodiments of the present disclosure, the antibody may be one or more selected from IgG1, igG2, igG3, or IgG 4. Preferably, the antibody may be IgG1.

In certain embodiments of the disclosure, the antibody is a monoclonal antibody.

In certain embodiments of the disclosure, the antibody comprises an Fc portion. Preferably, in certain embodiments of the present disclosure, the Fc portion of the antibody is modified or engineered to enhance its ADCC activity, CDC activity or ADCP activity.

In another aspect of the disclosure, there is provided an isolated antigen binding portion that specifically binds human CD138 protein, the antigen binding portion comprising one or more amino acid sequences selected from the group consisting of: the amino acid sequence of CDRH1 of the heavy chain variable region as shown in SEQ ID No.1, the amino acid sequence of CDRH2 of the heavy chain variable region as shown in SEQ ID No.2, the amino acid sequence of CDRH3 of the heavy chain variable region as shown in SEQ ID No.3, the amino acid sequence of CDRL1 of the light chain variable region as shown in SEQ ID No.4, the amino acid sequence of CDRL2 of the light chain variable region as shown in SEQ ID No.5 or the amino acid sequence of CDRL3 of the light chain variable region as shown in SEQ ID No. 6.

In certain embodiments of the present disclosure, the antigen binding portion comprises the amino acid sequence of the heavy chain variable region as set forth in SEQ ID No.7 and/or the amino acid sequence of the light chain variable region as set forth in SEQ ID No. 8.

In certain embodiments of the present disclosure, the antigen binding portion may be a Fab, fab ', F (ab') 2, fd, FCL, dAb, single chain antibody scFv, chimeric antibody, humanized antibody, or fully human antibody.

In another aspect of the present disclosure, there is provided a multivalent antibody, the monomer of which is the above antibody or the above antigen binding portion. The multivalent antibody may be, for example, bivalent, trivalent, tetravalent, hexavalent, nine-valent, and the like. The multivalent antibodies can be prepared using methods appropriate in the art.

In another aspect of the disclosure, an isolated polynucleotide encoding the above antibody, the above antigen binding portion, or the above multivalent antibody is provided. In certain embodiments of the present disclosure, the polynucleotide further encodes one or more selected from the amino acid sequences set forth in SEQ ID nos. 1,2, 3, 4,5, 6, 7, or 8, wherein the amino acid sequences or combinations thereof constitute the antibodies, the antigen binding portions, or the multivalent antibodies described above.

In another aspect of the disclosure, there is provided a vector comprising the polynucleotide described above.

In another aspect of the disclosure, there is provided a cell comprising the above antibody, the above antigen binding portion, the above multivalent antibody, or the above polynucleotide. In the present disclosure, the cell may be any suitable host cell as a means for producing a protein of interest. For example, SP2/0, YB2/0, IR983F, human myeloma Namalwa, PERC6 or CHO cell lines, insect cells, E.coli cells.

In another aspect of the present disclosure, there is provided a method of producing an anti-CD 138 antibody or antigen-binding portion, which is obtained directly or indirectly after protein expression of the above-described cells.

In another aspect of the present disclosure, there is provided a multispecific antibody which selectively binds at least human CD138, the multispecific antibody comprising an antibody as described above or an antigen-binding portion as described above; the multispecific antibody is a monovalent antibody or a multivalent antibody. The multispecific antibodies may be, for example, bispecific, trispecific. The multivalent antibody may be, for example, bivalent, trivalent, tetravalent, hexavalent, nine-valent, and the like. The multivalent antibodies can be prepared using methods appropriate in the art.

In another aspect of the present disclosure, there is provided a pharmaceutical composition comprising the above antibody, the above antigen-binding portion, the above multivalent antibody, the above polynucleotide, the above cell, or the above multispecific antibody, and a pharmaceutically acceptable carrier. In order to achieve better therapeutic effects, in certain embodiments of the present disclosure, other therapeutic agents may also be included in the pharmaceutical compositions.

In another aspect of the present disclosure, there is provided an immunoconjugate comprising: a) The antibody, the antigen-binding portion, the multivalent antibody or the multispecific antibody; and b) a therapeutic agent or detectable label; and c) a linker of the two parts a) and b); wherein the therapeutic agent comprises a drug, enzyme, toxin, cytokine or radionuclide.

In another aspect of the disclosure, there is provided the use of the above antibody, the above antigen binding portion, the above multivalent antibody, the above polynucleotide, the above cell, the above multispecific antibody, the above pharmaceutical composition, or the above immunoconjugate for the preparation of a medicament for treating a hematological tumor. Preferably, in certain embodiments of the present disclosure, the hematological neoplasm is plasmacytoma. More preferably, in certain embodiments of the present disclosure, the hematological tumor is multiple myeloma.

In another aspect of the disclosure, there is provided the use of the above antibody, the above antigen binding portion, the above multivalent antibody, or the above multispecific antibody in the preparation of a product for detecting the presence or level of CD138 in a sample.

The beneficial effects are that:

The anti-human CD138 antibody provided by the disclosure has high affinity with CD138 protein, strong binding specificity and no cross reaction with negative cells Molm, kg1a and MV-4-11. Based on the characteristics, the antibody can be used for detecting cells expressing CD138, can be used in tumor immunotherapy alone or in combination with other methods, namely, can be effectively applied to preparing medicines for treating tumors, infectious diseases, autoimmune diseases, anti-immune rejection and the like.

Drawings

FIG. 1 is a graph showing the detection results of a detection reagent for the 5G2 subtype by a Roche mouse monoclonal antibody subtype in the examples of the present disclosure;

FIG. 2 is a diagram showing SDS-PAGE results of 5G2 pure products in examples of the present disclosure, wherein 1: crude pure 5G2 product; 2: pure 5G 2; 3: crude pure 5G2 product; 4: pure 5G 2;

FIG. 3 is a graph showing the results of an affinity constant analysis of 5G2 and K562/CD138 ⁺ cells in the examples of the present disclosure;

FIG. 4 is a graph showing the binding results of antibody 5G2 to CD138 positive cell lines H929, MM1S, 8266 in the examples of the present disclosure;

FIG. 5 is a graph showing the binding results of antibody 5G2, commercial antibody MI15, and CD138 ⁺ cell line U266 in the examples of the present disclosure;

FIG. 6 is a graph showing the cross-reaction results of antibody 5G2 with CD138 negative cell line Molm, MV-4-11, kg1a in the examples of the present disclosure.

DESCRIPTION OF THE SEQUENCES

SEQ ID No.1 is the amino acid sequence of CDRH1 of the heavy chain variable region of the antibody of the present disclosure;

SEQ ID No.2 is the amino acid sequence of CDRH2 of the heavy chain variable region of the antibody of the present disclosure;

SEQ ID No.3 is the amino acid sequence of CDRH3 of the heavy chain variable region of the antibody of the present disclosure;

SEQ ID No.4 is the amino acid sequence of CDRL1 of the light chain variable region of the antibody of the disclosure;

SEQ ID No.5 is the amino acid sequence of CDRL2 of the light chain variable region of the antibody of the disclosure;

SEQ ID No.6 is the amino acid sequence of CDRL3 of the light chain variable region of the antibody of the disclosure;

SEQ ID No.7 is the amino acid sequence of the heavy chain variable region of the antibody of the present disclosure;

SEQ ID No.8 is the amino acid sequence of the light chain variable region of the antibodies of the present disclosure.

Detailed Description

The invention discloses an antibody of CD138 and application thereof, and a person skilled in the art can properly improve the technological parameters by referring to the content of the text. It is to be particularly pointed out that all similar substitutes and modifications apparent to those skilled in the art are deemed to be included in the invention and that the relevant person can make modifications and appropriate alterations and combinations of what is described herein to make and use the technology without departing from the spirit and scope of the invention.

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components. The term "a," "an," and "the") includes plural referents. The term "plurality" refers to two (species) or more. The terms "such as," "for example," and the like are intended to refer to exemplary embodiments and are not intended to limit the scope of the present disclosure.

In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. The definition of common terminology in molecular biology can be found in Lewis's GENES XII, jocelyn E, krebs/Elliott S.Goldstein/Stephen T.Kilpatrick, published Jones & Bartlett,2018.

Isolated antibodies:

The term "isolated" in this disclosure refers to a substance or entity that is separated from its natural environment or from the environment that existed prior to isolation. For example, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it originated. The proportion of separation may be, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%. The separated materials may have different levels of purity relative to the materials prior to their separation.

The term "antibody" in the present disclosure refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). VH and VL regions can also be subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH and VL is prepared from the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antibody binding sites, respectively. 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 (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies. In certain embodiments of the disclosure, the antibody may be IgG, igA, igM, igD or IgE. Preferably, in certain embodiments of the present disclosure, the antibody may be of the type IgG. Further, in certain embodiments of the present disclosure, the antibody may be one or more selected from IgG1, igG2, igG3, or IgG 4. Preferably, the antibody may be IgG1.

In certain embodiments of the present disclosure, the antibodies are produced using mammalian cells. For example, monoclonal antibodies are produced in mammalian cells using hybridoma technology. The monoclonal antibodies can be prepared using the hybridoma preparation method reported by Kohler et al, nature256:495 (1975). Mice or other suitable host animals are first immunized with the immunogen (with adjuvant if necessary).

The immunogen or adjuvant is typically injected subcutaneously or intraperitoneally. Examples of the adjuvant include Freund's adjuvant (Freund's complete adjuvant or Freund's incomplete adjuvant) and MPL-TDM. Upon immunization, the animals produce lymphocytes in vivo that secrete antibodies that specifically bind to the immunogen. The lymphocytes of interest are collected and fused with myeloma cells using a suitable fusion agent (e.g., PEG 4000) to obtain hybridoma cells (Goding, monoclonal Antibodies: PRINCIPLES AND PRACTICE, pp.59-103,Academic Press,1996).

The hybridoma cells prepared as described above are inoculated into a suitable medium containing one or more substances capable of inhibiting the growth of unfused, parent myeloma cells for growth. For example, for a parent myeloma cell lacking hypoxanthine guanine phosphotransferase (HGPRT or HPRT), addition of agents such as hypoxanthine, aminopterin, and thymidine (HAT medium) to the medium will inhibit the growth of HGPRT-deficient cells.

The preferred myeloma cells should have high fusion rate, stable antibody secretion ability, sensitivity to HAT medium, and the like. Among them, myeloma cells are preferred as murine myeloma, such as MOP-21 and MC-11 mouse tumor derivatives (THE Salk Institute Cell Distribution Center, san Diego, calif. USA), and SP-2/0 or X63-Ag8-653 cell lines (AMERICAN TYPE C. Mu. lture Collection, rockville, md. USA). In addition, human myeloma and human and mouse heteromyeloma cell lines can be utilized to prepare human monoclonal antibodies (Kozbor,J.Immunol.,133:3001(1984);Brodeur et al.,Monoclonal Antibody Production Techniques and Applications,pp.51-63,Marcel Dekker,Inc.,New York,1987).

The culture medium in which the hybridoma cells are grown is used to detect the production of monoclonal antibodies directed against the specific antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined using the following method: immunoprecipitation or in vitro binding assays, such as Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA). For example, the affinity of monoclonal antibodies can be determined using the Scatchard assay described by Munson et al in Anal biochem.107:220 (1980).

After determining the specificity, affinity and reactivity of the antibodies produced by the hybridomas, the cell line of interest can be determined by Goding, monoclonal Antibodies: PRINCIPLES AND PRACTICE, pp.59-103,Academic Press,1996. Suitable media may be DMEM or RPMI-1640, and the like. In addition, hybridoma cells can also be grown in animals as ascites tumors.

Monoclonal antibodies secreted by subcloned cells can be isolated from cell culture fluid, ascites fluid or serum by conventional immunoglobulin purification methods, such as protein a sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, to obtain said monoclonal antibodies.

In certain embodiments of the disclosure, the isolated antibody may be a humanized antibody. Antibody humanization may improve the affinity or other characteristics of the antibody. Description of humanized antibodies and methods can be referred to Riechmann,L.,Clark,M.,Waldmann,H.,&Winter,G.(1988).Reshaping human antibodies for therapy.Nature,332(6162),323–327.

In certain embodiments of the disclosure, the antibody Fc (crystallizable region fragment, fc) is engineered to enhance its triggering effector function by binding to Fc receptors or complement (effector functions). These functions may include: complement Dependent Cytotoxicity (CDC) and Antibody dependent cell mediated toxicity (ADCC) and Antibody dependent cellular phagocytosis (ADCP-DEPENDENT CELLULAR PHAGOCYTOSIS). The modification may include: 1) Engineered glycosylation, for example, aspartic acid at position 297 of the Fc region (N297) may be modified with N-acetylglucosamine. Mutation of N297 to alanine (a), glutamine (Q), or glycine (G) all block glycosylation of the antibody, thereby reducing Fc-mediated effector function. The ability of the antibody to induce ADCC or CDC activity after deglycosylation is decreased; sialic acid (SIALIC ACID) modifications reduce binding to fcγriiia, thus resulting in reduced CDC and ADCC activity. 2) Point mutations, e.g., LALA mutations (L234A/L235A), result in alterations in antibody affinity for FcgammaR (elimination of binding to low affinity FcgammaR and reduction of binding to FcgammaRI), thereby significantly reducing its ADCC and CDC activity. In addition, the effector function of antibodies is also modulated by combinations of cross-subtype antibodies.

An isolated antigen binding portion:

The term "antigen-binding portion" in the present disclosure 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 fragment". See generally Fundamental Immunology, ch.7 (Paul, W., ed., 2 nd edition, RAVEN PRESS, N.Y. (1989). Antigen binding fragments of antibodies may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.

Wherein the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F (ab') ₂ fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region. The term "Fd fragment" means an antibody fragment consisting of VH and CH1 domains; the term "Fv fragment" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.

In certain embodiments of the present disclosure, the antigen binding portions described above are prepared using a protease digestion process, using proteases such as papain, pepsin, and the like. In other embodiments of the present disclosure, the antigen binding portions described above are prepared using a chemical reagent treatment method. In other embodiments of the present disclosure, the antigen binding portions described above are prepared using genetic engineering methods. That is, a fragment containing all or part of the gene sequence of the antigen binding portion is ligated to a suitable vector and expressed. Examples of vectors for expression include bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

Modification of the antibody or antigen binding portion:

In addition to the above modifications to the antibody Fc fragment to improve its performance, amino acid modifications may be made to other portions of the antibody or antigen binding portion to produce functionally equivalent variable regions and/or CDRs and variants with enhanced or reduced activity and/or affinity that do not significantly affect the properties of the antibody. Examples of such modifications include conservative substitutions (substitutions) of amino acid residues, one or more amino acid deletions or additions that do not significantly alter the functional activity of the antibody or mature the affinity of the antibody for its target antigen. Examples of amino acid residues which are conservative substitutions (substitutions) for each other may be amino acids in the group consisting of 1) alanine (a), glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); 6) Phenylalanine (F), tyrosine (Y), tryptophan (W); 7) Serine (S), threonine (T); and 8) cysteine (C), methionine (M).

Multivalent antibodies and multispecific antibodies:

Multivalent antibodies in the present disclosure include at least two antibodies or antigen binding portions described in the present disclosure that are capable of competitively binding to human CD138 molecules and producing a different effect than monovalent antibodies. The multivalent antibodies can be obtained, for example, using protein fusion, addition of linkers, covalent or non-covalent bonds.

Multispecific antibodies, e.g., bispecific antibodies, trispecific antibodies, in the present disclosure, including antibodies or antigen-binding portions described in the present disclosure, which are capable of binding to at least one other different site or molecule of interest in addition to competitively binding to the human CD138 molecule. For example, BCMA, CD38, CD40, IL-6, CS1, SLAMF7, PD-1, GPRC5D, etc. The multispecific antibodies may be linked together by a direct link or by a linker. The multispecific antibodies may be expressed by recombinant methods. The multispecific antibodies may be monovalent or multivalent in order to achieve a better therapeutic effect.

The term "polynucleotide" in the present disclosure is also used interchangeably as "nucleic acid," and refers to a strand of nucleotides of any length, and includes DNA or RNA. It may comprise any known nucleotide analogue or modified nucleotide or base.

The term "vector" in the present disclosure refers to a polynucleotide molecule capable of transporting and/or expressing one or more genes of interest. Examples of vectors may be viral vectors, naked DNA or RNA expression vectors, plasmids, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

Pharmaceutical composition:

The term "pharmaceutical composition" in the present disclosure refers to a composition comprising at least one other substance in addition to the antibody, the antigen-binding portion, the multivalent antibody, the polynucleotide, the vector, the cell, or the multispecific antibody described in the present disclosure. The other substance may be, for example, a pharmaceutically acceptable carrier, excipient, physiological saline, cell culture medium, glucose, water for injection, glycerol, ethanol, and their combinations, stabilizer, surfactant, preservative, isotonic agent, etc. Other therapeutic agents are also possible, for example, chemotherapeutic agents: horse flange, doxorubicin, cyclophosphamide, vincristine, etc.; glucocorticoids: prednisone, dexamethasone, and the like; immunomodulating drugs: thalidomide, lenalidomide, pomalidomide, and the like.

In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail with reference to specific embodiments.

Examples:

Example 1: preparation of anti-human CD138 antibodies

1. Mouse hybridoma monoclonal antibody screening

Immunizing a Balb/c mouse by taking a human CD138 protein positive cell line as an immunogen and adopting an intraperitoneal injection mode, respectively carrying out boosting immunization on the 2 nd week, the 4 th week, the 6 th week and the 8 th week after primary immunization, taking 10 μl of mouse tail blood on the 8 th day after boosting immunization, placing the mouse tail blood in 90 μl PBS, standing at room temperature for 1 hour, centrifuging at a temperature of 4 ℃ for 10 minutes at 12000rpm, and collecting serum; tail blood supernatants were diluted in PBS to different concentrations: 1:200,1:400,1:800,1:1600,1:3200,1:6400,1:12800. collecting CD138 positive cells, washing with PBS for 1 time, counting cells, adding 100 μl of serum with different dilutions to the cells with 1×10 ⁶ cells, incubating the negative control group with CD138 negative cells at 4deg.C for 30min, and washing with PBS twice; adding an APC marked anti-mouse F (ab) ₂ secondary antibody in a ratio of 1:500, incubating for 30min at room temperature in a dark place, and washing twice by PBS; the cells were resuspended in 200 μl PBS buffer, and the flow cytometer detected the percentage of binding of antibodies to cells and the fluorescence intensity in the serum; the average fluorescence intensity is more than twice of that of the negative control, and the effective titer is higher than 6400, so that the fusion can be carried out. The immunized mice were impact immunized by tail vein injection of immunogen 3 days prior to fusion. And (3) taking successfully immunized mice spleen cells and myeloma SP2/0 cells to carry out cell fusion, adding PEG into spleen cells and myeloma cell clusters within 1min under the water bath environment of 37 ℃ during fusion, and then adding 10ml of serum-free 1640 medium within 5 min. The supernatant was centrifuged, the cells were resuspended in semi-solid medium containing HAT and pipetted into a 10cm dish (10 ml/plate). The cells were cultured at 37℃under 5% CO ₂. When the clones were large enough, the single clones were picked up and cultured in 96-well plates with 200. Mu.l of complete medium per empty space for 3-4 days. CD138 positive cell lines were used as test cells. When the cell clones in the 96-well plate are observed to be large enough under a cell microscope, 100 μl of supernatant of the corresponding well is taken and incubated together with CD138 positive cells, and the method is the same as detection titer. Positive wells were selected and subjected to the next cloning culture. And (3) expanding the positive hybridoma clones from the 96-well plate to the 24-well plate for 3-5 days, performing culture supernatant screening detection again, performing subcloning culture on the positive clones, and freezing the residual cells. Hybridoma cells were collected from the 24-well plate, counted, and the cell density was adjusted to 10/mL; spreading the cells into 96-well plates, 200 μl each, culturing at 37deg.C in a 5% CO ₂ incubator; after culturing for about 10 days, forming visible clone, selecting a hole with only a single clone, sucking the culture supernatant, selecting positive clone as before, expanding to 24-hole plate culture, detecting the supernatant again, selecting positive clone to perform secondary subcloning culture, and obtaining stable hybridoma cell strain after multiple rounds of subcloning culture until all detection holes are positive. Positive hybridoma culture supernatants were selected, and antibody subtype detection test paper was used to detect the subtype of the antibody, the monoclonal antibody number in this example being 5G2, murine IgG1 subtype, and light chain being kappa chain (as shown in fig. 1).

2. Ascites preparation and purification

The hybridoma cells were washed with sterile PBS and injected intraperitoneally into liquid paraffin pre-sensitized Balb/c mice at a cell rate of 5X10 ⁶/500. Mu.l/g. Ascites was collected 7 to 10 days later, and the supernatant was collected at room temperature 3000rpm for 10 minutes. The antibody was crude purified with 33% final concentration of saturated ammonium sulfate and desalted by PBS at 4deg.C for 24h, with 3 changes. The crude purified antibody was further purified by using an AKTA Protein purification system, followed by 1ml Protein G purification cartridge, according to the purification manual provided by GE company. The obtained antibody pure product is used for subsequent antibody detection and function experiments. And (3) performing BCA quantitative detection on the pure antibody product to obtain the protein concentration of the pure antibody product. 20ug of protein was subjected to SDS-PAGE, and it was found that the pure antibody had a single band at 150kDa, and after reduction with mercaptoethanol, light and heavy bands at 25kDa and 50kDa, respectively (as shown in FIG. 2).

3. Monoclonal antibody titer detection

Pure 5G2 antibody was incubated with 2X 10 ⁵ K562-CD138 at room temperature for 30min, PBS was used for 2 times, 1 ul/well of APC-labeled anti-mouse F (ab) ₂ secondary antibody was added, incubated at room temperature for 30min in dark place, PBS was used for two times, after washing with PBS for 2 times, and the 5G2 antibody was purified at final concentrations of 200nM, 100nM, 50nM, 25nM, 12.5nM, 6,25nM, 3.2nM, 1.6nM, 0.8nM, 0.4nM, 0.2nM, 0.1nM, 0.05nM, 0.025nM, and 2X 10 ⁵ K562-CD138 secondary antibody was added; cells were resuspended in 200 μl PBS buffer, fluorescence intensity was measured by FACS, and Mean values were counted. The Kd values of the antibodies were calculated using data analysis software GRAPHPAD PRISM. The Kd value of 5G2 is 0.8957 X10 ^-9 M. (the results are shown in FIG. 3)

4. Cloning of 5G2 variable region Gene by RT-PCR method

Total RNA extraction, single-stranded cDNA synthesis:

Total RNA of the 5G2 hybridoma cell line was extracted with TRIzol reagent, and cDNA library was synthesized using reverse transcriptase with RNA as a template. RT-PCR amplification of heavy chain (VH) and light chain (VL) variable region gene fragments of anti-human CD138 antibody, and the primer sequences are shown in Table 1:

TABLE 1

The PCR reaction system (50. Mu.l) was prepared as follows:

2 μl of cDNA; 2. Mu.l of the upstream primer (10. Mu.M); 2. Mu.l of the downstream primer (10. Mu.M); 25 μl of 2 Xpfu DNA polymerase; ddH ₂ O was added to 50. Mu.l. Reaction conditions: pre-denaturation at 95 ℃ for 5min; the following cycle was repeated 35 times: 95 ℃ for 30s,58 ℃ for 30s and 72 ℃ for 1min; finally, the extension is carried out at 72℃for 10min. The VL and VH fragments were separated and recovered by agarose gel electrophoresis. The recovered VL and VH fragments were ligated with pMD19-T (simple) vector (Takara Co.) using T4 ligase (Takara Co.) in the following manner: VL PCR product/VH PCR product 70ng each; 1 μl of pMD19-T (simple) vector; solution I was added to the reaction mixture in an amount of 5. Mu.l; ddH ₂ O was made up to 10. Mu.l. After overnight ligation at 4℃and transformation of the ligation products into E.coli DH 5. Alpha. Competent bacteria, single colonies were picked and sequenced after overnight incubation at 37 ℃.

Example 2: specific binding of 5G2 to cells highly expressing CD138

Binding of antibody 5G2 to H929, MM1S, 8266 surface CD138 protein was detected by FACS: incubating the 5G2 antibody and commercial antibody with H929, MM1S and 8266 cells respectively, taking CD138 commercial antibody (biolegend: MI 15) as positive control, incubating for 30min at room temperature, and washing twice with PBS; 100ul of resuspended cells, 1ul of APC labeled anti-mouse F (ab) ₂ secondary antibody was added and incubated at room temperature for 30min in the absence of light; cells were resuspended in 500 μl PBS buffer and FACS detected. The results are shown in FIG. 4: it can be seen that the 5G2 antibody binds efficiently to both CD138+ cell lines.

FACS method compares the affinity of antibody 5G2 with that of commercial antibody CD138 (biolegend:MI 15): the 5G2 antibody and commercial CD138 antibody MI15 were incubated with CD138 positive cell line U266 at a final concentration of 0.5ug/ml,0.25ug/ml,0.125ug/ml,0.0625ug/ml,0.03125ug/ml for 30min at room temperature, PBS was washed 2 times, and then 1 ul/well of APC-labeled anti-mouse F (ab) ₂ secondary antibody was added, incubated at room temperature in the absence of light for 30min, and PBS was washed twice; cells were resuspended in 200 μl PBS buffer, fluorescence intensity was measured by FACS, and Mean values were counted. The results are shown in FIG. 5: it can be seen that at the same concentration, the average immunofluorescence (MFI) of the 5G2 antibody test group was higher than that of commercial CD138 antibody MI15, i.e., the 5G2 antibody affinity was better than MI15.

Example 3: cross-reaction of 5G2 with CD138 negative cells

The 5G2 antibody and commercial antibody were incubated with 2X10 ⁵ Molm13, MV-4-11, kg1a cell lines (CD 138 negative), respectively, with the commercial antibody of CD138 as a control. Incubation is carried out at room temperature for 30min, and PBS is used for washing twice; 100ul of resuspended cells, 1ul of APC labeled anti-mouse F (ab) ₂ secondary antibody was added and incubated at room temperature for 30min in the absence of light; cells were resuspended in 500 μl PBS buffer and FACS detected. The results are shown in FIG. 6:5G2 has no cross reaction with Molm, MV-4-11 and Kg1a cells, and has good binding specificity.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. An isolated antibody that specifically binds human CD138 protein, comprising the amino acid sequence of CDRH1 of the heavy chain variable region as set forth in SEQ ID No.1, the amino acid sequence of CDRH2 of the heavy chain variable region as set forth in SEQ ID No.2, the amino acid sequence of CDRH3 of the heavy chain variable region as set forth in SEQ ID No.3, the amino acid sequence of CDRL1 of the light chain variable region as set forth in SEQ ID No.4, the amino acid sequence of CDRL2 of the light chain variable region as set forth in SEQ ID No.5, and the amino acid sequence of CDRL3 of the light chain variable region as set forth in SEQ ID No. 6.

2. The isolated antibody of claim 1, wherein the antibody comprises the amino acid sequence of the heavy chain variable region as set forth in SEQ ID No.7 and the amino acid sequence of the light chain variable region as set forth in SEQ ID No. 8.

3. The isolated antibody of claim 1 or 2, wherein the antibody is a chimeric, humanized or fully human antibody.

4. The isolated antibody according to claim 1 or 2, wherein the antibody is one or more selected from the group consisting of IgG1, igG2, igG3 and IgG 4;

preferably, the antibody is IgG1.

5. The isolated antibody of claim 1 or 2, wherein the Fc portion of the antibody is modified or engineered to enhance ADCC activity, CDC activity or ADCP activity thereof.

6. An isolated antigen binding portion that specifically binds human CD138 protein, the antigen binding portion comprising one or more amino acid sequences selected from the group consisting of seq id nos: the amino acid sequence of CDRH1 of the heavy chain variable region as shown in SEQ ID No.1, the amino acid sequence of CDRH2 of the heavy chain variable region as shown in SEQ ID No.2, the amino acid sequence of CDRH3 of the heavy chain variable region as shown in SEQ ID No.3, the amino acid sequence of CDRL1 of the light chain variable region as shown in SEQ ID No.4, the amino acid sequence of CDRL2 of the light chain variable region as shown in SEQ ID No.5 or the amino acid sequence of CDRL3 of the light chain variable region as shown in SEQ ID No. 6.

7. The isolated antigen-binding portion of claim 6, wherein the antigen-binding portion comprises the amino acid sequence of the heavy chain variable region as set forth in SEQ ID No.7 and/or the amino acid sequence of the light chain variable region as set forth in SEQ ID No. 8.

8. The isolated antigen-binding portion of claim 6 or 7, wherein the antigen-binding portion is a Fab, fab ', F (ab') ₂, fd, FCL, dAb, single chain antibody scFv, chimeric antibody, humanized antibody, or fully human antibody.

9. A multivalent antibody, characterized in that the monomer of the multivalent antibody is an antibody as defined in any one of claims 1 to 5, or an antigen binding portion as defined in claims 6, 7, 8.

10. An isolated polynucleotide encoding an antibody according to any one of claims 1 to 5, an antigen binding portion according to claim 6, 7, 8, or a multivalent antibody according to claim 9;

The polynucleotide further encodes one or more of the amino acid sequences selected from the group consisting of SEQ ID nos. 1,2, 3,4, 5, 6, 7 or 8, wherein the amino acid sequences or combinations thereof constitute an antibody as claimed in any one of claims 1 to 5, an antigen binding portion as claimed in claim 6, 7, 8, or a multivalent antibody as claimed in claim 9.

11. A vector comprising the polynucleotide of claim 10.

12. A cell comprising the antibody of any one of claims 1 to 5, the antigen-binding portion of claims 6,7,8, the multivalent antibody of claim 9, the polynucleotide of claim 10, or the vector of claim 11.

13. A method of producing an anti-CD 138 antibody or antigen-binding portion, wherein the anti-CD 138 antibody or antigen-binding portion is obtained directly or indirectly after protein expression of the cell of claim 12.

14. A multispecific antibody which binds at least selectively to human CD138, the multispecific antibody comprising an antibody according to any one of claims 1 to 5 or an antigen-binding portion according to claims 6, 7, 8; the multispecific antibody is a monovalent antibody or a multivalent antibody.

15. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 5, an antigen-binding portion according to claim 6, 7, 8, a multivalent antibody according to claim 9, a polynucleotide according to claim 10, a vector according to claim 11, a cell according to claim 12, or a multispecific antibody according to claim 13, and a pharmaceutically acceptable carrier;

preferably, the pharmaceutical composition further comprises other therapeutic agents.

16. An immunoconjugate, the immunoconjugate comprising:

a) An antibody according to any one of claims 1 to 5, an antigen binding portion according to claim 6, 7, 8, a multivalent antibody according to claim 9 or a multispecific antibody according to claim 13; and

B) A therapeutic agent or detectable label; and

C) A connector between the two parts a) and b);

Wherein the therapeutic agent comprises a drug, enzyme, toxin, cytokine or radionuclide.

17. Use of an antibody according to any one of claims 1 to 5, an antigen binding portion according to claims 6, 7, 8, a multivalent antibody according to claim 9, a polynucleotide according to claim 10, a vector according to claim 11, a cell according to claim 12, a multispecific antibody according to claim 14, a pharmaceutical composition according to claim 15, or an immunoconjugate according to claim 16 for the manufacture of a medicament for the treatment of a hematological tumor;

Preferably, the hematological neoplasm is a plasmacytoma;

More preferably, the hematological neoplasm is multiple myeloma.

18. Use of an antibody according to any one of claims 1 to 5, an antigen binding portion according to claim 6,7, 8, a multivalent antibody according to claim 9 or a multispecific antibody according to claim 14 for the preparation of a product for detecting the presence or level of CD138 in a sample.