Application of homoharringtonine targeted therapy

Abstract

This invention provides a novel monoclonal antibody against human CD79b and its conjugate with homoharringtonine for use in anticancer or tumor treatment.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202010675125.X. Technical Field

[0002] This invention belongs to the field of pharmaceutical and antibody technology. Specifically, this invention relates to novel CD79 antibodies and their conjugates with homoharringtonine for anticancer or tumor treatment. Furthermore, this invention also relates to methods for preparing the aforementioned chemical products, pharmaceutical compositions, and pharmaceutical applications. Background Technology

[0003] Omacetaxine mepesuccinate (HHT) is an effective anticancer component extracted from plants of the Cephalotaxus genus, and it has a significant inhibitory effect on the growth of various cancer cell lines. It was approved as a commonly used anticancer drug by the Chinese Ministry of Health as early as the 1990s. In recent years, its anticancer effects have also gained international recognition; in 2012, the US FDA approved omacetaxine injection (Synribo) for the treatment of chronic myeloid leukemia.

[0004] CD79b protein is a B-cell surface antigen expressed in a variety of cancer cells, particularly in over 90% of non-Hodgkin's B-cell lymphomas. Therefore, antibody development targeting this protein has received widespread attention, as documented in international patent applications such as WO2014011519, WO2014011521, WO2014177615, WO2016040856, WO2016090210, WO2016205176, WO2016021621, and WO2017009474.

[0005] However, among the publicly disclosed CD79b-targeting monoclonal antibodies, the number of highly effective ones is very limited, which cannot meet the needs of cancer and tumor treatment. Given the unpredictable availability of highly effective CD79b-targeting monoclonal antibodies in the existing technology, through arduous efforts and a bit of luck, the inventors unexpectedly screened a murine monoclonal antibody and optimized its humanized antibody using their proprietary platform. These antibodies exhibit high affinity for CD79b, and especially after conjugation with homoharringtonine, the resulting antibody-drug conjugate effectively inhibits tumor cell proliferation. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide novel CD79 antibodies and their conjugates with anticancer drug compounds such as homoharringtonine, for use in anticancer or tumor treatment. Furthermore, this invention also relates to methods for preparing the aforementioned chemical products and their pharmaceutical applications.

[0007] Specifically, in a first aspect, the present invention provides a monoclonal antibody against human CD79b or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region are shown in SEQ ID NO: 2, 3, and 4, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 contained in the light chain variable region are shown in SEQ ID NO: 7, 8, and 9, respectively. In this document, the amino acids of the complementarity determinant clusters (CDRs) of the heavy chain and light chain variable regions are determined and annotated using the Chothia numbering system.

[0008] Preferably, in the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the antibody is a murine antibody, a chimeric antibody, a human antibody, or a humanized antibody. For example, the antibody is a murine antibody, whose light chain may include the FR region and / or constant region of the light chain of murine κ, λ chains or variants thereof, and whose heavy chain may include the FR region and / or constant region of the heavy chain of murine IgG1, IgG2, IgG3, IgG4 or variants thereof. In a specific embodiment of the present invention, the amino acid sequence of the variable region of the heavy chain of the murine antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 6.

[0009] For example, the antibody may be a humanized antibody, whose light chain may include the FR region and / or constant region of the human κ, λ chain or its variants, and whose heavy chain may include the FR region and / or constant region of the heavy chain of human IgG1, IgG2, IgG3 or IgG4 or its variants. In one specific embodiment of the present invention, the amino acid sequence of the variable region of the heavy chain of the humanized antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 10.

[0010] In this document, the antigen-binding fragment of an antibody is any fragment that retains the antigen-binding activity of the complete antibody. Preferably, in the antibody or antigen-binding fragment of the first aspect of the invention, the antigen-binding fragment can be Fab, (Fab')2, Fv, linear antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody, or multispecific antibody (e.g., bispecific antibody, diabody, triabody, etc.), preferably Fab, (Fab')2, Fv, or scFv.

[0011] In a second aspect, the present invention provides a polynucleotide encoding an antibody or antigen-binding fragment thereof of the first aspect of the invention. The polynucleotide of the present invention may be in DNA or RNA form, preferably in DNA form.

[0012] In a third aspect, the present invention provides a vector containing the polynucleotides described in the second aspect of the invention. The terms "recombinant expression vector," "expression vector," or "vector" as used herein are interchangeable and refer to bacterial plasmids, phages, yeast plasmids, plant cell viruses, animal viruses, and various other viral vectors commonly used in the art.

[0013] In a fourth aspect, the present invention provides cells containing the polynucleotides described in the second aspect of the invention. These cells can be obtained by transformation or transfection using the vectors described in the third aspect of the invention. The cells can be prokaryotic or eukaryotic cells, such as bacterial cells, yeast cells, plant cells, insect cells, mammalian cells, etc. Preferred cells may be *Escherichia coli*, *Pichia pastoris*, Chinese hamster ovary cells (CHO), or human embryonic kidney (HEK) 293 cells.

[0014] In a fifth aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, comprising culturing the cells described in the fourth aspect of the present invention under conditions suitable for protein expression, and then isolating the antibody or antigen-binding fragment thereof described in the first aspect of the present invention from the culture.

[0015] In a sixth aspect, the present invention provides an antibody-drug conjugate comprising an anticancer drug compound and the antibody or antigen-binding fragment thereof described in the first aspect of the present invention. Preferably, the anticancer drug compound and the antibody or antigen-binding fragment thereof described in the first aspect of the present invention are linked by a linker, preferably covalently.

[0016] Preferably, in the sixth aspect of the invention, the anticancer drug compound may be selected from toxins, chemotherapeutic agents, antibiotics, radioisotopes, and lysozymes. Preferably, in a specific embodiment of the invention, the anticancer drug compound is homoharringtonine.

[0017] In a seventh aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in the first aspect of the invention or the antibody-drug conjugate described in the sixth aspect of the invention, and a pharmaceutically acceptable carrier. Unless otherwise specified, the terms "pharmaceutical composition," "drug," or "product" as used herein refer to a pharmaceutical composition, drug, or product for human use. A pharmaceutically acceptable carrier as used herein refers to a non-toxic filler, stabilizer, diluent, or other pharmaceutical excipient. Those skilled in the art can formulate the pharmaceutical composition into various dosage forms according to the therapeutic purpose and route of administration (e.g., injection), preferably in unit dose form, such as a lyophilized preparation or an injectable solution. In unit dose form, the amount of the antibody or antigen-binding fragment thereof described in the first aspect of the invention or the antibody-drug conjugate described in the sixth aspect of the invention can be from 0.1 mg to 2000 mg, such as from 1 mg to 1000 mg.

[0018] In an eighth aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention or the antibody-drug conjugate described in the sixth aspect of the present invention in the preparation of a medicament for anticancer or tumor treatment.

[0019] Preferably, in the eighth aspect of the invention, the cancer or tumor is lymphoma or leukemia. Exemplary lymphomas include diffuse large B-cell lymphoma, non-Hodgkin lymphoma (NHL), small lymphocytic lymphoma, or mantle cell lymphoma, preferably non-Hodgkin lymphoma, such as aggressive NHL, relapsed aggressive NHL, relapsed painless NHL, refractory NHL, or refractory painless NHL. Exemplary leukemias include chronic lymphocytic leukemia, hairy cell leukemia, or acute lymphoblastic leukemia.

[0020] The beneficial effects achieved by this invention are as follows: it provides a high-affinity anti-CD79b antibody and its highly efficient anticancer conjugate with homoharringtonine, with effects exceeding the expectations of the prior art and possessing broader drug development prospects.

[0021] For ease of understanding, this invention cites publicly available documents that are used to describe the invention more clearly, and all of their contents are incorporated herein by reference.

[0022] The present invention will now be described in detail through specific embodiments. It should be particularly noted that these descriptions are merely exemplary and do not constitute a limitation on the scope of the invention. Many variations and modifications of the invention will be apparent to those skilled in the art based on the discussion herein. Detailed Implementation

[0023] The following examples will describe the present invention in detail. If there are any omissions, please refer to the methods described in experimental manuals such as "Molecular Cloning: A Laboratory Manual", "Antibody Engineering", and "Cellular Experimentation Manual", or refer to the instructions or manuals provided by the manufacturers of the reagents and instruments used in the experiment.

[0024] Example 1: Preparation of the CD79b monoclonal antibody of the present invention

[0025] Following conventional hybridoma preparation methods, human CD79 protein (NCBI accession number for the human CD79b gene sequence: NP_000617.1) was used as the antigen. Hybridoma cells were generated by fusing SP2 / 0 cells with immunized lymphocytes. After multiple rounds of ELISA and FACS screening, the hybridoma cell line 79B6G7, exhibiting the best binding affinity for secreted antibodies, was ultimately selected. Hybridoma cells 79B6G7 were cultured in a roller flask incubator at 37°C for 10-15 days. The supernatant was purified by Protein A affinity chromatography to obtain the murine monoclonal antibody mAb79B6G7 of this invention. After RNA extraction, amplification, and sequencing identification, the amino acid sequence of the heavy chain variable region of the murine monoclonal antibody mAb79B6G7 is shown in SEQ ID NO: 1, with the amino acid sequences of HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 2, 3, and 4, respectively; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6, with the amino acid sequences of LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 7, 8, and 9, respectively. The heavy chain and light chain variable regions of the murine monoclonal antibody mAb79B6G7 were linked by a (G4S)3 linker peptide to construct a single-chain antibody (named mAb79B6G7-scFv), which was then recombinantly expressed.

[0026] The heavy chain and light chain variable region sequences of the murine monoclonal antibody mAb79B6G7 were compared with the human antibody variable region sequences in the antibody database. Sequences consistent with the murine antibody sequence were retained, while those inconsistent were listed as candidate mutation sites. After preliminary screening using a bioinformatics model, candidate mutation sites in the framework region (especially the region adjacent to the CDR) were mutated. Then, the heavy chain and light chain variable regions were linked by a (G4S)3 linker peptide to construct a single-chain antibody. Their binding affinity was measured, and the humanized antibody hAb60H5 with the best binding affinity was finally selected. The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 5; the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 10.

[0027] Using Roche's polatuzumab as a control, surface plasmon resonance (SPR) assays showed that the binding affinity of the mouse monoclonal antibody mAb79B6G7, its single-chain antibody, and the humanized antibody hAb60H5 of this invention to CD79b protein was far superior to that of existing polatuzumab monoclonal antibodies (see Table 1).

[0028] Table 1. Binding affinity of the antibody of the present invention to CD79b protein.

[0029]

[0030] Example 2 Preparation of the conjugate of the present invention

[0031] Dissolve 5.46 mg of homoharringtonine in an appropriate amount of propylene glycol, then dilute with water to 1 mL. Add 0.76 g of thiourea to 2 mL of 6 mol / L hydrochloric acid, heat to dissolve, and then cool to room temperature (24 °C). Mix with the above homoharringtonine solution for 20 minutes, then add NaOH to adjust the pH to 9.0, let stand for 20 minutes, and add hydrochloric acid to adjust the pH to 7.2. The resulting reaction solution is ready for use.

[0032] Take 5 mg each of Polatuzumab, mAb79B6G7-scFv and hAb60H5 antibodies, dissolve them in 1 ml of PBS (pH 7.2), add 0.1 mL of DMSO solution of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide to each, mix well, and let stand at room temperature for 1 hour. Then put them into dialysis bags and dialyze in PBS (pH 7.2) at 4°C for 12 hours. The obtained dialysate is used for later use.

[0033] The above reaction solution and the dialysis solution of each antibody were mixed thoroughly and allowed to stand at 4°C for 12 hours before being loaded onto Zeba. TM Spin desalting column is used to remove low molecular weight impurities and unreacted substances. The conjugates of each antibody with homoharringtonine are freeze-dried to obtain the conjugates of each antibody of the present invention and the antibody conjugates as controls.

[0034] Example 3: Study on antitumor activity

[0035] The homoharringtonine conjugates of mAb79B6G7-scFv and hAb60H5 antibodies prepared according to Example 2 were used as experimental drugs, and the homoharringtonine conjugate of Polatuzumab antibody was used as a control drug to study the antitumor activity of the drugs of the present invention. Lymphoma BJAB cell lines cultured on RPMI 1640 medium containing 10% fetal bovine serum were diluted with the same medium to a concentration of 1*10. 6 0.1 mL of the drug was added to each well of a 96-well plate and incubated at 37°C for 24 hours. Then, 0.1 mL of the culture medium containing or without serially diluted versions of the drug was added, and the plate was incubated for another 72 hours. The cell count in each well was then measured, and the half-maximal inhibitory concentration (IC50) was calculated. 50 The results are shown in Table 2. Compared with the existing polatuzumab antibody, the antibody of the present invention, after being conjugated with homoharringtonine, has a significantly enhanced inhibitory effect on the proliferation of lymphoma cells.

[0036] Table 2. Antitumor activity of the drug of the present invention

[0037] SEQUENCE LISTING <110> Xi'an Disai Biopharmaceutical Co., Ltd. <120> Application of homoharringtonine targeted therapy <130> CN <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 117 <212> PRT <213> Mus musculus <400> 1 Gln Leu Ala Arg Pro Gly Ala Val Gln Leu Gln Gln Ser Gly Ala Glu 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Tyr Pro Gly Asn Ser Tyr Ile 20 25 30 Gly Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Ser Arg Trp Phe Gly Thr Thr Tyr Tyr Tyr Asn Glu Lys Phe 50 55 60 Glu Asp Lys Ala Thr Leu Thr Ala Gly Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Lys Val Gly Phe Asp Leu Asp Lys Asn Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 2 <211> 7 <212> PRT <213> Mus musculus <400> 2 Tyr Pro Gly Asn Ser Tyr Ile 1 5 <210> 3 <211> 6 <212> PRT <213> Mus musculus <400> 3 Ser Arg Trp Phe Gly Thr 1 5 <210> 4 <211> 8 <212> PRT <213> Mus musculus <400> 4 Val Gly Phe Asp Leu Asp Lys Asn 1 5 <210> 5 <211> 117 <212> PRT <213> Homo sapiens <400> 5 Glu Val Lys Lys Pro Gly Ser Val Gln Leu Val Gln Ser Gly Ala Glu 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Tyr Pro Gly Asn Ser Tyr Ile 20 25 30 Gly Ile Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Ser Arg Trp Phe Gly Thr Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Glu Asp Lys Ala Thr Leu Thr Ala Gly Arg Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Gly Phe Asp Leu Asp Lys Asn Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 6 <211> 112 <212> PRT <213> Mus musculus <400> 6 Asp Phe Leu Thr Gln Met Thr Pro Leu Ser Leu Pro Val Arg Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Thr Glu Leu Gln Thr Phe Ser Ile Val 20 25 30 Lys Asn Ser Gly Tyr His Arg Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Asp Val Gly Asn Ser Arg Trp Gly Val Pro 50 55 60 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Asp Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys His Phe Gln 85 90 95 Pro Gly Ser Ser Gly Phe Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 7 <211> 16 <212> PRT <213> Mus musculus <400> 7 Thr Glu Leu Gln Thr Phe Ser Ile Val Lys Asn Ser Gly Tyr His Arg 1 5 10 15 <210> 8 <211> 7 <212> PRT <213> Mus musculus <400> 8 Asp Val Gly Asn Ser Arg Trp 1 5 <210> 9 <211> 9 <212> PRT <213> Mus musculus <400> 9 His Phe Gln Pro Gly Ser Ser Gly Phe 1 5 <210> 10 <211> 112 <212> PRT <213> Homo sapiens <400> 10 Asp Phe Val Thr Gln Met Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Thr Glu Leu Gln Thr Phe Ser Ile Val 20 25 30 Lys Asn Ser Gly Tyr His Arg Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Asp Val Gly Asn Ser Arg Trp Gly Val Pro 50 55 60 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Asp Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys His Phe Gln 85 90 95 Pro Gly Ser Ser Gly Phe Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110

Claims

1. The application of antibody-drug conjugates in the preparation of drugs for the treatment of diffuse large B-cell lymphoma, wherein, The antibody-drug conjugate comprises an anticancer drug compound and an anti-human CD79b monoclonal antibody or its antigen-binding fragment, wherein the anti-human CD79b monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region are shown in SEQ ID NO: 2, 3 and 4, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 contained in the light chain variable region are shown in SEQ ID NO: 7, 8 and 9, respectively, and the anticancer drug compound is homoharringtonine.

2. The application according to claim 1, wherein, The antibody is a murine antibody, a chimeric antibody, a human antibody, or a humanized antibody.

3. The application according to claim 1, wherein, The antigen-binding fragment is selected from Fab, (Fab')2, Fv, or scFv.

4. The application according to claim 1, wherein, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1 or 5.

5. The application according to claim 1, wherein, The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 6 or 10.