CN116925220A - IL20RB neutralizing antibody and medical application thereof - Google Patents

IL20RB neutralizing antibody and medical application thereof Download PDF

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CN116925220A
CN116925220A CN202210333483.1A CN202210333483A CN116925220A CN 116925220 A CN116925220 A CN 116925220A CN 202210333483 A CN202210333483 A CN 202210333483A CN 116925220 A CN116925220 A CN 116925220A
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antibody
variable region
chain variable
heavy chain
light chain
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胡国宏
贺云飞
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Shanghai Institute of Nutrition and Health of CAS
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Shanghai Institute of Nutrition and Health of CAS
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Abstract

The invention provides an IL20RB neutralizing antibody and medical application thereof. Specifically, the antibody has high affinity to IL20RB, and can specifically recognize IL20RB and inhibit the interaction of IL 19; in addition, animal experiments prove that the antibody can inhibit lung cancer or breast cancer bone metastasis caused by IL20RB, and has high safety.

Description

IL20RB neutralizing antibody and medical application thereof
Technical Field
The invention relates to the field of medicines, in particular to an IL20RB neutralizing antibody and medical application thereof.
Background
Various tumors such as lung cancer and breast cancer are multi-directional bone metastasis, and most of them are osteolytic metastasis. During bone metastasis, tumor cells that disseminate into bone induce differentiation of mononuclear precursors, called osteoclasts, by producing osteoclast stimulators or by interacting with other cells (e.g., osteoblasts); in turn, osteoclasts release large amounts of growth factors, such as transforming growth factor beta (TGF-beta) insulin-like growth factor (IGF) and calcium, by digesting the bone matrix to promote the growth of tumor cells, forming metastases.
The development and clinical use of osteoclast inhibitors (such as bisphosphonates and dirac) further support the important role of osteoclasts in tumor bone metastasis. However, the function of osteoclasts in bone metastasis is not yet fully understood. In addition to digesting bone matrix, osteoclasts may also act on other stromal cells or tumor components in the bone. For example, it has recently been found that osteoclasts inhibit T cell mediated cytotoxicity, thereby indirectly promoting survival of multiple myeloma cells. In addition, osteoclasts can secrete pro-tumor lipid factors to regulate breast cancer bone metastasis. These data indicate that in addition to digesting bone matrix, osteoclasts can also affect the growth of tumor cells in bone in other ways.
There are still many disadvantages to the prior art drugs used to inhibit tumor bone metastasis. Thus, there remains a need in the art to develop new drugs capable of inhibiting tumor bone metastasis.
Disclosure of Invention
The invention aims to study the influence of osteoclast on tumor metastasis and provide a drug for inhibiting the growth and metastasis of tumor cells on the basis. The tumor is preferably lung cancer or breast cancer.
Specifically, the invention discovers that the osteoclast can act on IL20RB factors expressed by tumor cells through secreting factors IL-19, so that the growth of the tumor cells is promoted to form metastatic cancers, and provides a neutralizing antibody for inhibiting the interaction of IL19 and IL20RB, which is used for inhibiting the metastasis of tumors to bones, and can also inhibit the growth and metastasis of tumors under other conditions caused by IL20 RB.
The invention also aims to provide an IL20RB neutralizing antibody and medical application thereof.
In a first aspect of the invention, there is provided a heavy chain variable region of an anti-IL 20RB antibody, said heavy chain variable region comprising the following three complementarity determining region CDRs:
HCDR1 as shown in SEQ ID No.:1,
HCDR2 as shown in SEQ ID No.:2, and
HCDR3 as shown in SEQ ID No.: 3.
In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence that 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 is capable of retaining IL20RB binding affinity.
In another preferred embodiment, the IL20RB is derived from human IL20RB or a non-human mammal (e.g., mouse, rabbit).
In another preferred embodiment, the IL20RB is the extracellular domain of human IL20RB (XP_ 011511212).
In another preferred embodiment, the heavy chain variable region further comprises an FR region of human origin or an FR region of murine origin.
In another preferred embodiment, the heavy chain variable region comprises the following four FR regions:
HFR1 as shown in SEQ ID NO. 7,
HFR2 as shown in SEQ ID NO. 8,
HFR3 as set forth in SEQ ID NO: 9, and
HFR4 as shown in SEQ ID NO. 10.
In another preferred embodiment, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO. 15.
In a second aspect of the invention there is provided a heavy chain of an anti-IL 20RB antibody, said heavy chain having a heavy chain variable region as described in 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 heavy chain constant region is of human, murine or rabbit origin.
In a third aspect of the invention, there is provided a light chain variable region of an anti-IL 20RB antibody, said light chain variable region comprising the following three complementarity determining region CDRs:
LCDR1 as shown in SEQ ID No.:4,
LCDR2 as shown in SEQ ID No.:5, and
LCDR3 as shown in SEQ ID No. 6.
In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence that 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 is capable of retaining IL20RB binding affinity.
In another preferred embodiment, the light chain variable region further comprises an FR region of human origin or an FR region of murine origin.
In another preferred embodiment, the light chain variable region has the amino acid sequence set forth in SEQ ID NO. 16.
In another preferred embodiment, the light chain variable region comprises the following four FR regions:
LFR1 shown in SEQ ID No.:11,
LFR2 shown in SEQ ID No.:12,
LFR3 as shown in SEQ ID No.:13, and
LFR4 shown in SEQ ID NO. 14.
In a fourth aspect of the invention, there is provided a light chain of an anti-IL 20RB antibody, said light chain having a light chain variable region as described in 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 of human, murine or rabbit origin.
In a fifth aspect of the invention, there is provided an anti-IL 20RB antibody having:
(1) A heavy chain variable region according to the first aspect of the invention; and/or
(2) A 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 as described in the fourth aspect of the invention.
In another preferred embodiment, the antibody is a specific anti-IL 20RB antibody, preferably a specific anti-human anti-IL 20RB antibody; more preferably an antibody specific for the extracellular domain of human IL20 RB.
In another preferred embodiment, the antibody is selected from the group consisting of: an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.
In another preferred embodiment, the antibody is a blocking antibody or a non-blocking antibody, preferably a blocking antibody.
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, or a polyclonal antibody.
In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.
In another preferred embodiment, the antibody is in the form of a drug conjugate.
In another preferred embodiment, the heavy chain variable region sequence of the antibody is set forth in SEQ ID NO. 15; and the light chain variable region sequence of the antibody is shown as SEQ ID NO. 16.
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, a GGGS sequence, a FLAG 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 another preferred embodiment, the recombinant protein specifically binds to human IL20RB, preferably to the extracellular domain of human IL20 RB.
In a seventh 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 human IL20RB, and which scFv has a heavy chain variable region as set forth in the first aspect of the invention and a light chain variable region as set forth in the third aspect of the invention.
In an eighth aspect of the invention, there is provided a recombinant immune cell expressing an exogenous CAR construct according to the seventh 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 a ninth aspect of the invention, there is provided an immunoconjugate 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 conjugate is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like proteins (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticle, etc.
In another preferred embodiment, the antibody moiety is coupled to the coupling moiety via a chemical bond or linker.
In another preferred embodiment, the radionuclide comprises:
(i) A diagnostic isotope selected from the group consisting of: tc-99m, ga-68, F-18, I-123, I-125, I-131, in-111, ga-67, cu-64, zr-89, C-11, lu-177, re-188, or combinations thereof; and/or
(ii) A therapeutic isotope selected from the group consisting of: lu-177, Y-90, ac-225, as-211, bi-212, bi-213, cs-137, cr-51, co-60, dy-165, er-169, fm-255, au-198, ho-166, I-125, I-131, ir-192, fe-59, pb-212, mo-99, pd-103, P-32, K-42, re-186, re-188, sm-153, ra223, ru-106, na24, sr89, tb-149, th-227, xe-133 Yb-169, yb-177, or combinations thereof.
In another preferred embodiment, the coupling moiety is a drug or a toxin.
In another preferred embodiment, the drug is a cytotoxic drug.
In another preferred embodiment, the cytotoxic agent is selected from the group consisting of: an anti-tubulin drug, a DNA minor groove binding agent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folic acid antagonist, an antimetabolite, a chemosensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.
Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs including, for example, auristatins (auristatins), camptothecins (camptothecins), duocarmycin/duocarmycin (duocarmycins), etoposides (etoposides), maytansinoids (maytansines) and maytansinoids (maytansinoids) (e.g., DM1 and DM 4), taxanes (taxanes), benzodiazepines (benzodiazepines), or benzodiazepine-containing drugs (benzodiazepine containing drugs) (e.g., pyrrolo [1,4] benzodiazepines (PBDs), indoline benzodiazepines (indoxazepines) and oxazolobenzodiazepines (oxybenzodiazepines)), vinca alkaloids (vilos), or combinations thereof.
In another preferred embodiment, the toxin is selected from the group consisting of:
auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), aureomycin, mestaneol, ricin a-chain, combretastatin, docamicin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide (tenoposide), vincristine, vinblastine, colchicine, dihydroxyanthrax, diketo, actinomycin, diphtheria toxin, pseudomonas Exotoxin (PE) A, PE, abrin a chain, a-chain of jezosin, α -octacocin, gelonin, mitogellin, restrictocin (retproctrocin), phenol, enomycin, curcin, crotonin, calicheamicin, saporin (Sapaonaria officinalis), glucocorticoids, or combinations thereof.
In another preferred embodiment, the coupling moiety is a detectable label.
In another preferred embodiment, the conjugate is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like proteins (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticle, etc.
In another preferred embodiment, the coupling moiety of the immunoconjugate is a diagnostic isotope.
In another preferred embodiment, the agent is one or more agents selected from the group consisting of: isotope tracer, contrast agent, flow detection reagent, cell immunofluorescence detection reagent, nano magnetic particle and imaging agent.
In a tenth 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, or a combination thereof, is used for the preparation of a medicament, a reagent, a assay plate or a kit.
In another preferred embodiment, the reagent, assay plate or kit is used to detect IL20RB protein.
In another preferred embodiment, the agent is for the treatment or prevention of growth and/or metastasis of cancer or tumor.
In another preferred embodiment, the agent is for the treatment or prevention of bone metastasis of cancer or tumor.
In another preferred embodiment, the cancer or tumor is selected from the group consisting of: lung cancer, breast cancer, kidney cancer.
In another preferred embodiment, the agent is for treating or preventing lung cancer bone metastasis or breast cancer bone metastasis.
In another preferred embodiment, the reagent comprises a chip, an immune microparticle coated with an antibody.
In an eleventh 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 eighth aspect of the invention, an antibody drug conjugate according to the ninth aspect of the invention, or a combination thereof; and
(ii) A pharmaceutically acceptable carrier.
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:
(1) Preventing and/or treating bone metastasis of cancer or tumor;
(2) Preventing and/or treating the growth of tumor cells and tumor formation;
(3) Blocking binding of IL19 to IL20 RB;
(4) Inhibit the interaction of IL20RB with IL 19;
(5) Inhibiting the promotion of IL19 recombinant protein on tumor cell forming organoids; and/or
(6) Inhibiting activation of the JAK1-STAT3 signaling pathway.
In another preferred embodiment, the cancer or tumor is selected from the group consisting of: lung cancer, breast cancer, kidney cancer.
In a twelfth 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;
(3) A CAR construct according to the seventh aspect of the invention.
In a thirteenth aspect of the invention there is provided a vector comprising a polynucleotide according to the twelfth aspect of the 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 fourteenth aspect of the invention there is provided a genetically engineered host cell comprising a vector or genome according to the thirteenth aspect of the invention having incorporated therein a polynucleotide according to the twelfth aspect of the invention.
In a fifteenth aspect of the present invention, there is provided a method of detecting IL20RB protein in a sample, the method comprising the steps of:
(1) Contacting the sample with an antibody according to the fifth aspect of the invention;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of IL20RB protein in the sample.
In another preferred embodiment, the detection is for non-therapeutic non-diagnostic purposes.
In a sixteenth 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 seventeenth aspect of the present invention, there is provided a method of preventing and/or treating cancer or tumor, 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.
In another preferred embodiment, the cancer or tumor is selected from the group consisting of: lung cancer, breast cancer, kidney cancer; preferably lung cancer or breast cancer; more preferably lung cancer.
In another preferred embodiment, the preventing and/or treating comprises: delaying/preventing the onset/progression of cancer symptoms, reducing the severity of cancer symptoms, reducing the survival/growth/invasion/metastasis of cancer cells, reducing the number of cancer cells, and/or increasing the survival rate of a subject.
In another preferred embodiment, the metastasis is bone metastasis.
In another preferred embodiment, the tumor is clinically manifested as carcinoma in situ or metastasis; preferably a transfer; more preferably bone metastasis.
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.
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FIG. 1 shows that LTMA1G11 specifically recognizes IL20RB and inhibits its interaction with IL 19. (A) Ascites fluid from mice vaccinated with LTMA1G11 hybridoma cells and coomassie brilliant blue staining of antibodies purified from the ascites fluid. (B) And performing immunoblotting identification on IL20RB over-expression or protein of control group A549 cells by using the purified LTMA1G11 antibody. (C) Co-immunoprecipitation experiments were performed with IL19 and IL20RB in the presence of LTMA1G 11. His-tagged IL19 and Flag-tagged IL20RB were overexpressed in HeLa cells, cells were treated with LTMA1G11 or control IgG, cell lysates were immunoprecipitated with Flag antibodies, and the products were immunoblotted with antibodies to IL19 and IL20 RB.
FIG. 2 shows LTMA1G11 blocks activation of signaling pathways within tumor cells and formation of tumor organoids caused by IL19-ILRB interactions. Wherein, the liquid crystal display device comprises a liquid crystal display device,
(A, B) analysis of IL20RB overexpression or control of JAK1/STAT3 phosphorylation levels in A549 cells after 24 hours of LTMA1G11 (5 mg/mL) and/or osteoclast conditioned medium treatment (A), and STAT3 luciferase reporter plasmid activity (B);
(C, D) analysis of JAK1/STAT3 phosphorylation levels in IL20RB over-expressed or control a549 cells after LTMA1G11 and/or IL19 recombinant protein treatment (C), and STAT3 luciferase reporter plasmid activity (D);
(E) After 24 hours of LTMA1G11 (5 mg/mL) and/or osteoclast conditioned medium treatment, IL20RB overexpression or control A549 cell tumor organogenesis ability was analyzed;
(F) After LTMA1G11 (5 mg/mL) and/or IL19 recombinant protein treatment, IL20RB overexpression or tumor organogenesis ability of control A549 cells was analyzed;
(G) After treatment with LTMA1G11 and/or IL19 recombinant proteins, breast cancer cells MCF7 were analyzed for organogenesis.
p-values were obtained by a two-tailed unpaired t-test (B, C, E-G), ns representing non-significant.
FIG. 3 shows that LTMA1G11 inhibited IL20 RB-induced bone metastasis from lung cancer. Wherein, the liquid crystal display device comprises a liquid crystal display device,
(A) Bioluminescence numerical statistics of weekly mouse tumor burden;
(B) Performing in-vitro bioluminescence numerical statistics on hind limbs;
(C) Representative images of whole body and hind limb bioluminescence, micro-CT analysis of hind limb (arrows point to osteolytic areas of leg);
(D) Counting the number of EdU positive tumor cells;
(E) Representative pictures of immunofluorescent staining and H & E staining.
Scale, 100mm. The p-value was obtained by Mann-Whitney U test (A, B) and two-tailed unpaired t test (A), ns representing non-significance.
FIG. 4 shows a safety analysis of LTMA1G11 antibodies. Wherein, the liquid crystal display device comprises a liquid crystal display device,
(A) Body weight of healthy mice injected with LTMA1G11 antibody or control IgG;
(B-D) blood fraction of healthy mice injected with LTMA1G11 antibody or control IgG. (B) WBCs, white blood cells; (C) RBCs, erythrocytes; (D) platelets.
p-values were obtained by two-tailed unpaired t-test (a, B, C, D); ns stands for insignificant.
Detailed Description
The present inventors have studied extensively and intensively, and developed for the first time a monoclonal antibody LTMA1G11 which specifically binds to human IL20 RB. The antibody has high affinity to IL20RB, and can specifically recognize IL20RB and inhibit the interaction of IL 19; the antibody can inhibit the growth of tumor cells caused by IL20 RB; in addition, animal experiments prove that the antibody disclosed by the invention can inhibit lung cancer bone metastasis caused by IL20RB and has high safety. The present invention has been completed on the basis of this finding.
IL20RB (Interlukin-20 receptor subunit beta, interleukin 20receptor beta subunit)
IL20RB is a heterodimer that can be formed with IL20RA or IL22R and bind to cytokines of the IL20 family. After binding to the ligand, it is able to activate the JAK1-STAT3 signaling pathway downstream, promoting cell proliferation.
The inventor discovers through research that IL19 secreted by the osteoclast is combined with IL20RB expressed by the lung cancer cell, activates JAK1-STAT3 signal pathway, promotes proliferation of the lung cancer cell in bone, and helps lung cancer to transfer to bone. Whereas blocking IL19 binding to IL20RB with IL20R antibodies may inhibit lung cancer bone metastasis.
Terminology
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.).
The three-letter and one-letter codes for amino acids used in the present invention are as described in J.biol. Chem,243, p3558 (1968).
As used herein, the term "treatment" refers to the administration of an internal or external therapeutic agent comprising the monoclonal antibodies of the invention directed against IL20RB protein (preferably the extracellular domain protein of human IL20 RB) and compositions thereof to a patient having one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. Typically, the patient is administered an amount of the therapeutic agent (therapeutically effective amount) effective to alleviate one or more symptoms of the disease.
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.
"sequence identity" as used herein refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate substitutions, insertions, or deletions of mutations. The sequence identity between the sequences described in the present invention and sequences with which it has identity may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,100%.
Antibodies to
As used herein, the term "antibody" or "immunoglobulin" is an iso-tetralin protein of about 150000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H), having identical structural features. Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. One end of each light chain is provided with a variable region (VL) and the other end is provided with a constant region; the constant region of the light chain is opposite the first constant region of the heavy chain and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.
As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming a connecting loop, which in some cases may form a partially folded structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). 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.
The "light chain" of a vertebrate antibody (immunoglobulin) can be classified into one of two distinct classes (called kappa and lambda) depending on the amino acid sequence of its constant region. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant region. There are mainly 5 classes of immunoglobulins: igA, igD, igE, igG and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, igG2, igG3, igG4, igA and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody.
The invention also includes monoclonal antibodies having the corresponding amino acid sequences of the anti-IL 20RB protein (preferably human IL20RB extracellular domain protein), monoclonal antibodies having the variable region chains of the anti-IL 20RB protein (preferably human IL20RB extracellular domain protein), and other proteins or protein conjugates and fusion expression products having these chains. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a light chain and a heavy chain comprising a hypervariable region (complementarity determining region, CDR), provided that the hypervariable region is identical or at least 90% homologous, preferably at least 95% homologous, to the hypervariable regions of the light chain and heavy chain of the invention.
Immunoconjugates and fusion expression products include, as known to those of skill in the art: conjugates of drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules in combination with the anti-IL 20RB protein monoclonal antibodies or fragments thereof. The invention also includes cell surface markers or antigens that bind to the anti-IL 20RB protein monoclonal antibodies or fragments thereof.
The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. Fragments of full length antibodies have been shown to be useful for performing the antigen binding function of antibodies. Examples of binding fragments included in the term "antigen-binding fragment of an antibody" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F (ab') 2 A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bridge on the longer chain region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of a single arm of an antibody. 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.
The invention includes not only intact monoclonal antibodies but also immunologically active antibody fragments such as Fab or (Fab') 2 Fragments; antibody heavy chain; an antibody light chain.
The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes typically comprise at least 3,4,5,6,7,8,9,10,11,12,13,14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation.
The terms "specific binding," "selective binding," "selectively binding," and "specifically binding" refer to binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody is present at about less than 10 -7 M, e.g. less than about 10 -8 M、10 -9 M or l0 -10 Affinity (KD) binding of M or less.
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. The term "murine antibody" is herein a monoclonal antibody against the IL20RB protein made according to the knowledge and skill in the art. The term "chimeric antibody (chimeric antibody)" refers to an antibody in which a variable region of a murine antibody is fused to a constant region of a human antibody, and which can reduce an immune response induced by the murine antibody. The term "humanized antibody (humanized antibody)", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting murine CDR sequences into the framework of human antibody variable regions, i.e., the framework sequences of different types of human germline antibodies. Humanized antibodies can overcome the heterologous response induced by chimeric antibodies that carry large amounts of murine protein components. Such framework sequences may be obtained from public DNA databases including germline antibody gene sequences or published references. To avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region framework sequences may be subjected to minimal reverse or back-mutations to maintain activity.
In the present invention, antibodies may be monospecific, bispecific, trispecific, or more multispecific.
As used herein, the term "heavy chain variable region" is used interchangeably with "VH".
As used herein, the term "variable region" is used interchangeably with "complementarity determining region (complementarity determining region, CDR)".
The term "CDR" refers to one of the 6 hypervariable regions within the variable domain of an antibody that contribute primarily to antigen binding. One of the most common definitions of the 6 CDRs is provided by Kabat E.A et al, (1991) Sequences of proteins of immunological interface.
As used herein, the terms "IL20RB neutralizing antibody", "anti-IL 20RB antibody", "antibody that specifically binds to IL20 RB" are used interchangeably and refer to a monoclonal antibody that is capable of specifically binding to IL20RB protein and is capable of inhibiting the interaction of IL20RB with IL 19.
In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises the following three complementarity determining regions CDRs:
HCDR1:TYGMS(SEQ ID NO.1)
HCDR2:SISSTGRKIYYPDSVKG(SEQ ID NO.2)
HCDR3:LYFGFAY(SEQ ID NO.3)
in another preferred embodiment, the heavy chain variable region comprises the following four FR regions:
HFR1 as set forth in SEQ ID NO. 7: EEQLVESGGGLVKPGGSLKLSCVVSGFTFS the number of the individual pieces of the plastic,
HFR2 as set forth in SEQ ID NO. 8: WGRQTPERRLEWVA the number of the individual pieces of the plastic,
HFR3 as set forth in SEQ ID NO. 9: RFTISRDNAKNTLYLQMSSLRSEDTAMYYCAR, and
HFR4 as set forth in SEQ ID NO. 10: WGQGTLVTVSA.
In another preferred embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 15, wherein the amino acid sequences of the heavy chain variable regions HCDR1, HCDR2, HCDR3 are underlined.
EEQLVESGGGLVKPGGSLKLSCVVSGFTFSTYGMSWGRQTPERRLEWVASISSTGRKIYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARLYFGFAYWGQGTLVTVSA(SEQ ID NO.15)
In a preferred embodiment of the present invention, the heavy chain of the antibody comprises the heavy chain variable region described above and a heavy chain constant region, which may be of murine or human origin.
As used herein, the terms "light chain variable region" and "V L "interchangeably used.
In a preferred embodiment of the invention, the light chain variable region of the antibody according to the invention has complementarity determining regions CDRs selected from the group consisting of:
LCDR1:KSSQSLLDSDGRTYVN(SEQ ID NO.4)
LCDR2:LVSKLDS(SEQ ID NO.5)
LCDR3:WQGTHFPWT(SEQ ID NO.6)
in another preferred embodiment, the light chain variable region comprises the following four FR regions:
LFR1 shown in SEQ ID NO. 11: DVVMTQTPLTLSVTIGQQASISC the number of the individual pieces of the plastic,
LFR2 shown in SEQ ID NO. 12: WLLQRPGQSPKRLMY the number of the individual pieces of the plastic,
LFR3 shown in SEQ ID No.: 13: GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC, and
LFR4 as shown in SEQ ID No.:14: FGGGTKLEIK.
In another preferred embodiment, the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16, wherein the amino acid sequences of the light chain variable regions LCDR1, LCDR2, LCDR3 are underlined.
DVVMTQTPLTLSVTIGQQASISCKSSQSLLDSDGRTYVNWLLQRPGQSPKRLMYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTKLEIK(SEQ ID NO.16)。
In a preferred embodiment of the present invention, the light chain of the antibody comprises the light chain variable region described above and a light chain constant region, which may be murine or human in origin.
Using the antibody variable region genes or Complementarity Determining Region (CDR) genes of the present invention, genetically engineered antibodies of different forms can be engineered and produced in any expression system utilizing prokaryotic and eukaryotic cells.
In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably and refer to an antibody that specifically binds to an anti-IL 20RB protein, preferably a human IL20RB extracellular domain protein, such as a protein or polypeptide having a heavy chain variable region (amino acid sequence shown as SEQ ID No.: 15) and/or a light chain variable region (amino acid sequence shown as SEQ ID No.: 16). They may or may not contain an initiating methionine.
In another preferred embodiment, the antibody is a murine or human murine chimeric monoclonal antibody against the IL20RB protein (preferably a human IL20RB extracellular domain protein), the heavy chain constant region and/or the light chain constant region of which may be a humanized heavy chain constant region or a light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is a heavy chain constant region or light chain constant region of human IgG1, igG2, or the like.
In general, the antigen binding properties of antibodies can be described by 3 specific regions located in the heavy and light chain variable regions, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, the β -sheets formed by the FR therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the 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 variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest because they are involved, at least in part, in binding to an antigen. Thus, the invention includes those molecules having monoclonal antibody light and heavy chain variable regions with CDRs, so long as the CDRs are 90% or more (preferably 95% or more, most preferably 98% or more) homologous to the CDRs identified herein.
The invention includes not only intact monoclonal 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.
As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.
The antibodies of the invention are polypeptides comprising the above-described CDR regions having anti-IL 20RB protein (preferably human IL20RB extracellular domain protein) binding activity. The term also includes variants of polypeptides comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.
The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions with the encoding DNA of an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.
The invention also provides other polypeptides, such as fusion proteins comprising a human antibody or fragment thereof. In addition to nearly full length polypeptides, the invention also includes fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids, preferably at least about 60 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the antibody of the invention.
In the present invention, the antibody of the present invention also includes conservative variants thereof, which means 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 antibody of the present invention to form a polypeptide. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.
Table A
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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. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequence or a degenerate variant.
Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; a coding sequence for a mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the mature polypeptide, and non-coding sequences.
The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences.
The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more.
The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.
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 biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form.
At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.
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. Representative examples are: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; animal cells of CHO, COS7, 293 cells, and the like.
Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl 2 The process is carried out using procedures well known in the art. Another approach is to use MgCl 2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.
The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.
Immunoconjugates
The invention also provides immunoconjugates or antibody conjugated drugs (ADCs) based on the antibodies of the invention.
The antibodies of the invention may be used alone or in combination or coupling with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or enzymes capable of producing a detectable product.
Couplable therapeutic agents include, but are not limited to: insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analog, albumin, antibody fragments, cytokines, and hormones.
Therapeutic agents that may also be bound or conjugated to the antibodies of the invention include, but are not limited to: 1. a radionuclide; 2. biological toxicity; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. a viral particle; 6. a liposome; 7. nano magnetic particles; 8. the prodrug activates the enzyme; 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticle, and the like.
Typically, the immunoconjugate drug comprises the antibody, and an effector molecule to which the antibody is conjugated, and preferably chemically conjugated. Wherein the effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.
The antibody of the invention may be coupled to the effector molecule by a coupling agent. Examples of the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond. The nonselective coupling agent refers to a compound such as glutaraldehyde or the like that forms a covalent bond between the effector molecule and the antibody. The coupling agent using carboxyl can be any one or more of cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and acyl hydrazone coupling agent (the coupling site is acyl hydrazone).
Certain residues on antibodies (e.g., cys or Lys, etc.) are useful in connection with a variety of functional groups, including imaging agents (e.g., chromophores and fluorophores), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. The antibody may be conjugated to a functional agent to form an antibody-functional agent conjugate. Functional agents (e.g., drugs, detection reagents, stabilizers) are coupled (covalently linked) to the antibody. The functional agent may be directly attached to the antibody, or indirectly attached through a linker.
Antibodies can be conjugated to drugs to form Antibody Drug Conjugates (ADCs). Typically, an ADC comprises a linker between the drug and the antibody. The linker may be degradable or non-degradable. Degradable linkers typically degrade readily in the intracellular environment, e.g., the linker degrades at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidase. The peptidyl linker may comprise, for example, a dipeptide, such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH sensitive linkers (e.g., linkers that hydrolyze at a pH of less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide bonds). The non-degradable linker typically releases the drug under conditions where the antibody is hydrolyzed by the protease.
Prior to attachment to the antibody, the linker has reactive groups capable of reacting with certain amino acid residues, the attachment being accomplished through the reactive groups. Thiol-specific reactive groups are preferred and include: such as maleimides, halogenated amides (e.g., iodine, bromine, or chlorine); halogenated esters (e.g., iodine, bromine, or chlorinated); halomethyl ketone (e.g., iodine, bromine, or chlorine), benzyl halide (e.g., iodine, bromine, or chlorine); vinyl sulfone, pyridyl disulfide; mercury derivatives such as 3, 6-di- (mercuromethyl) dioxane, while the counterion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, maleimide attached to the antibody via thiosuccinimide.
The drug may be any cytotoxic, cytostatic or immunosuppressive drug. In embodiments, the linker connects the antibody and the drug, and the drug has a functional group that can bond to the linker. For example, the drug may have an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, or a ketone group that may be bonded to the linker. In the case of a drug directly attached to a linker, the drug has reactive groups prior to attachment to the antibody.
Useful classes of drugs include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. In the present invention, a drug-linker can be used to form an ADC in a single step. In other embodiments, the bifunctional linker compounds may be used to form ADCs in two or more step processes. For example, a cysteine residue is reacted with a reactive moiety of a linker in a first step and in a subsequent step, a functional group on the linker is reacted with a drug, thereby forming an ADC.
Typically, the functional groups on the linker are selected to facilitate specific reaction with the appropriate reactive groups on the drug moiety. As a non-limiting example, an azide-based moiety may be used to specifically react with a reactive alkynyl group on a drug moiety. The drug is covalently bound to the linker by 1, 3-dipolar cycloaddition between the azide and the alkyne group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other attachment strategies, such as described in bioconjugate techniques, second edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that for selective reaction of a drug moiety with a linker, when a complementary pair of reactive functional groups is selected, each member of the complementary pair can be used for both the linker and the drug.
Composition and method for producing the same
The invention also provides a composition. In a preferred embodiment, 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: oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.
The pharmaceutical compositions of the invention can be used directly to bind IL20RB protein molecules and thus can be used to extend the half-life of a drug, and in addition, other therapeutic agents can be used simultaneously.
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 monoclonal antibodies (or conjugates 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 1 microgram per kilogram of body weight to about 10 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
When a pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to the 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 8 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 1 milligram 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
(1) The IL20RB neutralizing antibody is safe and effective, and has the potential of inhibiting tumor growth, especially tumor bone metastasis.
(2) The IL20RB neutralizing antibody provided by the invention can inhibit activation of JAK1-STAT3 signal pathway, thereby inhibiting organogenesis capacity, tumor growth and metastasis of tumor cells.
(3) The IL20RB neutralizing antibody disclosed by the invention can specifically block the interaction between IL20RB and IL19, and has high neutralizing activity.
The invention is further illustrated below in conjunction with specific embodiments. 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, in which the detailed conditions are not noted in the following examples, is generally followed by routine conditions such as 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 by weight unless otherwise indicated.
Experimental method
Production and purification of IL20RB neutralizing antibodies
His-tagged IL20RB truncations containing only the extracellular domain were overexpressed in 293T cells. His-tagged IL20RB truncations (XP_ 011511212) were affinity purified using nickel columns. The purified truncate was used to immunize mice. The immunized mice spleen cells were isolated and fused with SP20 tumor cells. The resulting hybridomas were cultured with HybGro medium (H630 KJ, basal media) and supplements thereof (H460 JV, basal media). BALB/c mice were then injected with pristane (P9622, sigma), 300. Mu.L each. 7 days after pristane injection, 2X 10 6 Hybridoma cells were injected into the abdominal cavity of the mice. After 7-14 days, mice were sacrificed with diethyl ether and ascites were collected. The collected ascites were subjected to antibody purification with protein G resin (C600991, sangon Biotech). The purified antibodies were dialyzed into PBS and concentrated to 1mg/mL. And finally, the antibody is frozen at the temperature of minus 20 ℃ for standby after split charging.
Co-immunoprecipitation
Cells were washed twice with PBS, added 600. Mu.L of Co-IP buffer, lysed on ice for 30 min, scraped off with a spatula, and transferred to a 1.5mL centrifuge tube and centrifuged at 13300rpm for 15 min at 4 ℃. And collecting the supernatant after centrifugation to obtain a protein solution. The protein solution was added to the corresponding antibody or IgG and incubated overnight at 4 ℃. The following day, protein A or protein G beads were added and incubated at 4℃for 2 hours. After incubation, centrifugation was carried out at 1000g for 5 min at 4℃and the supernatant was discarded and washed 3 times with Co-IP buffer. After washing, protein SDS PAGE Loading Buffer is added, and the mixture is heated in a metal bath at 95 ℃ for 10-15 minutes, so that the obtained sample can be directly used for immunoblotting experiments.
Immunoblotting
SDS-PAGE gels were prepared at varying concentrations of 8% -12% depending on the size of the desired identified protein (the smaller the desired identified protein, the higher the gel concentration). At the time of loading, 10-20. Mu.g of protein sample was added to each well. After the sample loading is finished, performing constant-pressure electrophoresis with the voltage of 80V, adjusting the voltage to 120V after the sample strip enters the separation gel, continuing electrophoresis until the bromophenol blue front indicator reaches the bottommost part of the separation gel, and turning off the power supply. SDS-PAGE gels containing sample bands were removed and electrotransport was performed using a sandwich method at a constant pressure of 105V under ice bath conditions, after two hours the electrotransport was completed, at which time the protein bands had been transferred to the NC membrane. The NC membrane was removed and blocked with 5% BSA solution. After blocking for 1 hour, the blocking solution was discarded, and the primary antibody diluted with 5% BSA solution was added and incubated overnight at 4 ℃. The next day the membranes were washed 3 times for 5 minutes with TBST solution. After washing, secondary antibodies with HRP-label diluted with 5% bsa solution were added and incubated for 1 hour at room temperature. After incubation, the membrane was washed 3 times with TBST solution for 5 minutes each. After the washing is finished, the ECL developer kit is used for developing, and the photo analysis is carried out in a multifunctional full-automatic chemiluminescence detector.
Tumor organoid culture experiments
Cells were seeded in Matrigel (BD Biosciences, 356231) and overlaid with organoid medium and osteoclast conditioned medium or IL19 recombinant protein was added in a proportion. The 24-well plate was inoculated 500-1000 fines per well. After 7-14 days of culture, the organoids were counted.
Organoid medium formulation is as follows:
animal experiment
1) Left ventricular injection
Preparing 5-6 weeks oldBALB/c nude mice of (E). After the mice were anesthetized, they were allowed to lie on their back on a plate and fixed with medical tape. Firstly sucking a certain amount of air by a 0.45 mu m injector to balance the air pressure (blood return cannot be observed after the injector penetrates into the left ventricle), then sucking the cell suspension, then inwards inclining the injector at an angle of 45 DEG, penetrating the injector from one rib to two ribs of a mouse, injecting the cell suspension into the left ventricle after blood return, and injecting 100 mu L of the cell suspension into 2X 10 mice each 6 cell/mL cell suspension.
2) In vivo fluorescence imaging
Mice were first anesthetized, then 100 μl of 5mg/mL luciferase substrate was injected through the fundus venous plexus, and finally the mice were placed into an imager (PerkinElmer, IVIS Spectrum CT system) for imaging and analysis of the data.
3) Ex vivo fluorescence imaging
Mice were sacrificed by cervical dislocation and their femur and tibia were removed. The muscle tissue adhering thereto was scraped off, and the blood stain was washed off in PBS. The bones were immersed in 50. Mu.g/mL of luciferase substrate solution for 10 minutes, protected from light. Finally, the bone is placed in an imager (PerkinElmer, IVIS Spectrum CT system) along with the solution for imaging and analysis of the data.
4) Small animal CT
Mice were first anesthetized, then placed in a micro-CT instrument (Scanco, vivaCT 80) for imaging and analysis of the data.
EXAMPLE 1 preparation of monoclonal antibodies
The inventor uses the extracellular domain (amino acids #35-236, XP_01511212) of human IL20RB as antigen to immunize mice, and successfully finds a monoclonal antibody LTMA1G11 with excellent activity and performance from 3 candidate antibodies through functional screening.
Wherein, the amino acid sequence of the variable region of the LTMA1G11 antibody is as follows:
HCDR1:TYGMS(SEQ ID NO.1)
HCDR2:SISSTGRKIYYPDSVKG(SEQ ID NO.2)
HCDR3:LYFGFAY(SEQ ID NO.3)
LCDR1:KSSQSLLDSDGRTYVN(SEQ ID NO.4)
LCDR2:LVSKLDS(SEQ ID NO.5)
LCDR3:WQGTHFPWT(SEQ ID NO.6)
heavy chain variable region of LTMA1G11 (LTMA 1 g11_vh):
EEQLVESGGGLVKPGGSLKLSCVVSGFTFSTYGMSWGRQTPERRLEWVASISSTGRKIYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARLYFGFAYWGQGTLVTVSA(SEQ ID NO.15)
light chain variable region of LTMA1G11 (LTMA 1 g11_vl):
DVVMTQTPLTLSVTIGQQASISCKSSQSLLDSDGRTYVNWLLQRPGQSPKRLMYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTKLEIK(SEQ ID NO.16)
example 2 characterization of antibodies
2.1 LTMA1G11 specifically recognizes IL20RB and inhibits its interaction with IL19
Antibodies purified from ascites fluid from mice vaccinated with LTMA1G11 hybridoma cells (FIG. 1A) were able to specifically detect IL20RB protein in lysates of A549 cells overexpressing IL20RB (FIG. 1B). More importantly, the LTMA1G11 antibody effectively inhibited the interaction between IL19 and IL20RB (fig. 1C).
IL19, upon binding to IL20RB, activates the JAK-STAT3 signaling pathway. After overexpression of IL20RB in a549 cells, osteoclast conditioned medium containing IL19 activated the pro-cancerous signaling pathway JAK1-STAT3 in a549 cells (fig. 2a,2 b). IL20RB also activated the JAK1-STAT3 signal of A549 cells when A549 cells were treated with IL19 recombinant protein (FIGS. 2C, 2D). Importantly, LTMA1G11 was effective in inhibiting activation of JAK1-STAT3 signals in A549 cells by osteoclast conditioned medium and IL19 recombinant protein (FIGS. 2A-D).
This suggests that LTMA1G11 antibodies inhibit the interaction between IL19 and IL20RB, thereby inhibiting downstream signaling pathways in tumor cells.
2.2 LTMA1G11 inhibits IL20 RB-induced tumor cell growth
The inventors tested the effect of LTMA1G11 on IL20RB induced tumor cell growth by tumor organogenesis assays. In lung cancer cell A549 overexpressing IL20RB, osteoclast conditioned medium or IL19 recombinant protein treatment enhanced tumor organoid formation (FIGS. 2E, 2F). However, tumor organoids were significantly inhibited after treatment with LTMA1G11, rendering IL20RB overexpression ineffective (FIGS. 2E, 2F). This suggests that LTMA1G11 is capable of blocking lung cancer cell proliferation caused by IL19-IL20RB, thereby inhibiting tumor formation.
In addition, we also analyzed the effect of LTMA1G11 on IL19 to promote proliferation of other types of tumor cells (e.g., breast cancer cells MCF 7). The results show that the neutralizing antibody LTMA1G11 provided by the invention can also significantly inhibit the promotion effect of the IL19 recombinant protein on the breast cancer cell MCF7 forming organoids (figure 2G).
2.3 LTMA1G11 inhibits IL20 RB-induced tumor bone metastasis
Further, the inventors tested the effect of LTMA1G11 on the treatment of tumor bone metastasis in vivo. Taking lung cancer cells as an example, IL20RB over-expression or control lung cancer A549 cells were injected into the left ventricle of nude mice, and LTMA1G11 was injected one week later. Every other day, 100 μg LTMA1G11 or IgG was intraperitoneally injected as a control for each animal.
Bioluminescence imaging analysis showed that bone metastasis of a549 cells overexpressing IL20RB was greatly reduced following treatment with LTMA1G11 (fig. 3). Three weeks after treatment, the tumor burden of the hind limbs was reduced by approximately 10-fold (fig. 3b, c). Further analysis found that treatment with LTMA1G11 also inhibited proliferation of tumor cells in bone (fig. 3d, e).
Taken together, these data demonstrate the effectiveness of LTMA1G11 antibodies in treating tumor bone metastases.
2.4 Drug safety assay for LTMA1G11
Further, the inventors also tested the drug safety of the neutralizing antibody LTMA1G11 targeting IL20 RB.
Healthy mice were intraperitoneally injected with the same dose of LTMA1G11 as before with the same frequency as in the previous experiments for 4 weeks.
The results showed that continuous LTMA1G11 treatment had no significant effect on mouse body weight (FIG. 4A) and blood components (FIGS. 4B-D).
Taken together, these results demonstrate the potential of IL20RB neutralizing antibodies for the treatment of tumors.
Discussion of the invention
The inventor discovers for the first time in the research that the osteoclast can act on IL20RB factor expressed by tumor cells through secreting factor IL-19, so that the growth of the tumor cells is promoted to form metastatic cancers.
Based on the influence of osteoclast on tumor metastasis, the invention provides a neutralizing antibody for inhibiting the interaction of IL19 and IL20RB, which can inhibit the growth and metastasis of tumor cells, especially inhibit the metastasis of tumor to bone, and can inhibit the growth and metastasis of tumor under other conditions caused by IL20 RB. The tumor is preferably lung cancer or breast cancer.
The data in examples 2.2-2.3 of the present invention indicate that the antibodies of the present invention inhibit organogenesis (i.e., the ability to inhibit tumor growth) of lung and breast cancer cells under induction of IL19-IL20RB, as well as bone metastasis of lung cancer in vivo.
Further studies by the inventors demonstrate the mechanism of action of IL2RB in mediating tumor growth. After the cells such as lung cancer and breast cancer are spread to bones, the cells can induce the osteoclasts to secrete IL-19, and the IL-19 is combined with a receptor IL20RB expressed on the surface of tumor cells to activate JAK1-STAT3 signal paths in the tumor cells, so that proliferation and growth of the tumor cells are increased, and a metastasis is formed. Since IL20RB is generally expressed only in tumor cells, not normal cells, while IL20RB binding to IL-19 occurs extracellular, neutralizing antibodies that target IL20RB to inhibit its binding to IL-19 can specifically target tumor cells, inhibit proliferation of tumor cells due to IL-19 secreted by osteoclasts or other paracancestor cells, and suppress tumors, especially bone metastases.
All documents mentioned in this disclosure are incorporated by reference in this disclosure 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 application as defined in the appended claims.
Sequence listing
<110> Shanghai nutrition and health institute of China academy of sciences
<120> IL20RB neutralizing antibody and medical use thereof
<130> P2022-0394
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100 105 110

Claims (10)

1. A heavy chain variable region of an anti-IL 20RB antibody, said heavy chain variable region comprising the following three complementarity determining region CDRs:
HCDR1 as shown in SEQ ID No.:1,
HCDR2 as shown in SEQ ID No.:2, and
HCDR3 as shown in SEQ ID No.: 3;
any of the amino acid sequences described above also includes derivative sequences that are optionally added, deleted, modified and/or substituted with at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and are capable of retaining IL20RB binding affinity.
2. A heavy chain of an anti-IL 20RB antibody, wherein said heavy chain has the heavy chain variable region of claim 1.
3. A light chain variable region of an anti-IL 20RB antibody, said light chain variable region comprising the following three complementarity determining region CDRs:
LCDR1 as shown in SEQ ID No.:4,
LCDR2 as shown in SEQ ID No.:5, and
LCDR3 as shown in SEQ ID No.: 6;
any of the amino acid sequences described above also includes derivative sequences that are optionally added, deleted, modified and/or substituted with at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and are capable of retaining IL20RB binding affinity.
4. A light chain of an anti-IL 20RB antibody, wherein said light chain has the light chain variable region of claim 3.
5. An anti-IL 20RB antibody, characterized in that the antibody has:
(1) The heavy chain variable region of claim 1; and/or
(2) A light chain variable region according to 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 of claim 1, a heavy chain of claim 2, a light chain variable region of claim 3, a light chain of claim 4, or an antibody of 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 human IL20RB, and wherein 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 immunoconjugate, said immunoconjugate 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, or a combination thereof, for use in the preparation of a medicament, reagent, assay plate, or kit.
CN202210333483.1A 2022-03-30 2022-03-30 IL20RB neutralizing antibody and medical application thereof Pending CN116925220A (en)

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