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WO2019035241A1 - Agent permettant de faire régresser une leucémie - Google Patents

Agent permettant de faire régresser une leucémie Download PDF

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Publication number
WO2019035241A1
WO2019035241A1 PCT/JP2018/014494 JP2018014494W WO2019035241A1 WO 2019035241 A1 WO2019035241 A1 WO 2019035241A1 JP 2018014494 W JP2018014494 W JP 2018014494W WO 2019035241 A1 WO2019035241 A1 WO 2019035241A1
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Prior art keywords
leukemia
antibody
amino acid
endoglin
acid sequence
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English (en)
Japanese (ja)
Inventor
精昭 薗田
龍哉 藤岡
由和 松岡
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KANSAI MEDICAL UNIVERSITY EDUCATIONAL Corp
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KANSAI MEDICAL UNIVERSITY EDUCATIONAL Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid

Definitions

  • the present invention relates to a leukemia improving agent, a leukemia cell marker and the like.
  • the leukemic cell population is stratified in the same manner as normal hematopoietic stem cells (HSCs) and contains a small number of leukemic stem cells (LSCs). Since its discovery, LSC has been thought to be present in the CD34 positive CD38 negative cell fraction as well as HSC (Non-patent Document 1).
  • antibody drugs approved in Japan, the US, and Europe for leukemia use include anti-CD33 antibody for acute myeloid leukemia, anti-CD52 antibody for B-cell chronic lymphocytic leukemia, B-cell acute lymphatic
  • anti-CD20 antibodies against leukemia and anti-CCR4 antibodies (developed in Japan) against adult T-cell leukemia.
  • the recurrence rate is high because LSC is not targeted for treatment.
  • Majeti R, et al .: CD47 is an adverse prognostic factor and therapeutic antibody on human acute myeloid leukemia stem cells.
  • Non-patent Document 2 a very undifferentiated CD34 negative HSC exists in human umbilical cord blood by developing a bone marrow direct transplantation method. Then, based on a new hierarchical model of normal HSCs (Fig. 1), we hypothesized that CD34 negative LSCs exist as leukemic counterparts of CD34 negative HSCs, and conducted experiments to transplant human leukemia cells into severe immunodeficient mice. . As a result, we have demonstrated the presence of CD34 negative LSC in acute myelogenous leukemia, acute lymphocytic leukemia and chronic myelogenous leukemia.
  • CD34 negative LSCs are more likely to be cells that produce undifferentiated, ie CD34 positive LSCs, than previously thought CD34 positive LSCs, and in the development of a cure aimed at eradication a method to eradicate CD34 negative LSCs It is necessary to devise.
  • TIM3 non-patent document 3
  • CD47 non-patent document 4
  • CD123 non-patent document 5
  • CD44 non-patent document 5
  • CLL-1 non-patent document 7
  • CD96 non-patent document 8
  • CD32 non-patent document 9
  • CD25 non-patent document 9
  • An object of the present invention is to provide a leukemia improving agent using an antibody.
  • An object of the present invention is to provide a leukemia improving agent targeted to a leukemia stem cell, more preferably a CD34 negative leukemia stem cell.
  • the present invention includes the following aspects: Item 1. Leukemia improving agents containing anti-endoglin antibodies. Item 2. Item 6. The leukemia ameliorating agent according to item 1, wherein the anti-endoglin antibody has binding to leukemia cells. Item 3. The leukemia ameliorating agent according to item 1 or 2, wherein the anti-endoglin antibody has a binding property to leukemia stem cells. Item 4. The leukemia ameliorating agent according to any one of Items 1 to 3, wherein the anti-endoglin antibody has a binding property to a CD34 negative leukemia stem cell. Item 5.
  • the anti-endoglin antibody comprises a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 2, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6.
  • a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10 a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 12, and a light chain comprising the amino acid sequence shown in SEQ ID NO: 14
  • the leukemia ameliorating agent according to any one of claims 1 to 4, which is an antibody comprising a light chain variable region comprising CDR3. Item 6. 6.
  • Leukemia ameliorating agent Item 7.
  • a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 2, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, and a heavy chain variable region comprising the heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6 And / or a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14
  • Anti-endoglin antibodies including: Item 9. Anti-Endoglin antibodies for use in ameliorating leukemia. Item 10. Use of an anti-endoglin antibody for producing a leukemia ameliorating agent. Item 11. A method for ameliorating leukemia, comprising administering an anti-endoglin antibody.
  • the present invention it is possible to provide a leukemia improving agent using an antibody, preferably a leukemia improving agent targeting leukemia stem cells, more preferably a leukemia improving agent targeting CD34 negative leukemia stem cells.
  • the leukemia improving agent of the present invention can exert an improving effect on a plurality of leukemias.
  • Fig. 7 shows the results of immunoprecipitation of Example 1.
  • 2 shows the results of the isotyping of Example 1.
  • the result (histogram) of the flow cytometry of Example 2 is shown.
  • the top of the histogram shows the cell line name used.
  • the horizontal axis shows PE signal, and the vertical axis shows the number of cells.
  • the peak on the left shows the case where anti-endoglin antibody is not allowed to act, and the peak on the right shows the case where anti-endoglin antibody is allowed to act.
  • the result (histogram) of the flow cytometry of Example 3 is shown.
  • (A) shows the results when acute myeloid leukemia cells (sample No. UPN 439) were used
  • (B) shows the results when acute lymphocytic leukemia (sample No. UPN 303) was used
  • (C) shows chronic The result at the time of using myeloid leukemia (sample number UPN605) is shown.
  • the leftmost histogram shows the results of fractionating leukemia cells using CD34 and CD38 as indices.
  • the three histograms on the right are obtained by examining the positive rate of anti-endoglin antibody-bound cells for the whole fraction or the CD34 + fraction and the CD34 + CD38- fraction, respectively.
  • the positive rate of anti-endoglin antibody-bound cells in the CD34-CD38- fraction is also shown.
  • the horizontal axis indicates the PE signal
  • the vertical axis indicates the number of cells.
  • the peak on the left shows the case where anti-endoglin antibody is not allowed to act, and the peak on the right shows the case where anti-endoglin antibody is allowed to act.
  • 16 shows the leukemic cell increase rate and the survival rate measured in Example 4.
  • the graph of (A) shows the leukemic cell increase rate (vertical axis)
  • the graph of (B) shows the leukemic cell survival rate (vertical axis).
  • Example 5 shows an outline of a test method of Example 5.
  • the spleen photographs taken in Example 5 are shown in (A) to (C).
  • the “healthy mouse” in (A) represents a control mouse not transplanted with leukemia cells
  • the “leukemia transplanted mouse” in (B) is a control mouse implanted with leukemia cells and administered with a mouse IgG isotype antibody.
  • (C) “Anti-Endoglin antibody-administered mouse” is a mouse transplanted with leukemia cells and administered with an anti-Endoglin antibody. One scale of the scale in each photograph shows 1 mm.
  • (D) illustrates the average spleen weight of each group of (A) to (C). In Example 5, the result of having analyzed the leukemia cell in mouse
  • (A) shows the number of leukemia cells in the bone marrow of the left tibia transplanted by the intramedullary direct transplantation (vertical axis), and (B) shows the number of leukemia cells in the contralateral tibia and bilateral femurs (vertical axis)
  • (C) shows the number of leukemia in the spleen (vertical axis)
  • (D) shows the concentration of leukemia cells in peripheral blood (number in 1 ⁇ l of blood) (vertical axis).
  • the antibody administration group indicates a group to which anti-endoglin antibody has been administered
  • the control group indicates a group to which a mouse IgG isotype antibody has been administered instead of the anti-endoglin antibody.
  • conservative substitution means that an amino acid residue is substituted with an amino acid residue having a similar side chain.
  • substitution with amino acid residues having basic side chains such as lysine, arginine and histidine corresponds to conservative substitution.
  • amino acid residues having acidic side chains such as aspartic acid and glutamic acid
  • amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine
  • Amino acid residues having nonpolar side chains such as proline, phenylalanine, methionine and tryptophan
  • amino acid residues having ⁇ -branched side chains such as threonine, valine and isoleucine
  • aromatic side chains such as tyrosine, phenylalanine, tryptophan and histidine
  • CDR is an abbreviation of C omplementarity D etermining R egion, also called complementarity determining regions.
  • the CDRs are regions present in the variable region of immunoglobulin and are regions deeply involved in specific binding to the antigen possessed by the antibody.
  • the "light chain CDR” is a CDR present in the variable region of the immunoglobulin light chain
  • the “heavy chain CDR” is a CDR present in the variable region of the immunoglobulin heavy chain.
  • variable region means a region including CDR1 to CDR3 (hereinafter simply referred to as "CDRs 1-3") described above.
  • the arrangement order of these CDRs 1 to 3 is not particularly limited, but preferably, the N-terminal to C-terminal direction, in the order of CDR 1, CDR 2 and CDR 3 or in the reverse order, a frame which is continuous or described later
  • work region FR
  • the “heavy chain variable region” is a region in which the above-described heavy chain CDRs 1-3 is disposed
  • the “light chain variable region” is a region in which the above-described light chain CDRs 1-3 is disposed.
  • the regions other than the above CDR1 to 3 of each variable region are referred to as framework regions (FR) as described above.
  • FR framework regions
  • the region between the N-terminus of the variable region and the CDR1 is FR1
  • the region between CDR1 and CDR2 is the FR2
  • the region between CDR2 and CDR3 is FR3
  • the region between CDR3 and the C-terminus of the variable region Each is defined as FR4.
  • the FR also has a function as a linker sequence connecting the CDRs 1-3 particularly important as the antigen recognition sequence described above, and is a region that contributes to the formation of the three-dimensional structure of the entire variable region.
  • the anti-endoglin antibody is an antibody having binding to endoglin, and is not particularly limited in this regard. In other words, the anti-endoglin antibody recognizes an epitope in endoglin or binds to a region in endoglin.
  • the anti-endoglin antibody is preferably an antibody having specific binding to endoglin.
  • "having specific binding to endoglin” is not particularly limited, but it is detected, for example, when Western blotting is performed using a gel obtained by electrophoresis of whole proteins of cells.
  • the ratio (percentage) of the density value of the band indicating endoglin to the total value of the density of all bands is, for example, 30% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% % Or more and 99% or more.
  • Endoglin is a dimeric type I transmembrane protein highly expressed in vascular endothelial cells and expressed as a component of the TGF- ⁇ receptor system.
  • the origin species of endoglin is not particularly limited, and various mammals such as humans, monkeys, mice, rats, dogs, cats, cats, rabbits, pigs, horses, cattle, sheep, goats, deer, etc. are preferable. Human is mentioned.
  • amino acid sequences of various biological species-derived endoglin can be easily obtained using known databases.
  • Endoglin may have amino acid mutations such as substitutions, deletions, additions, insertions and the like as long as it is endogenous cell endoglin, particularly as long as it is endogenous endoglin that can be expressed in leukemia cells.
  • the mutation preferably includes substitution, more preferably conservative substitution, from the viewpoint that the activity is less likely to be impaired.
  • the epitope of the anti-endoglin antibody is not particularly limited, and may be a linear epitope or a steric epitope.
  • the epitopes are contiguous amino acid sequences.
  • the epitope is a conformational epitope, the epitope may be a continuous amino acid sequence or a plurality of non-consecutive amino acid sequences.
  • the number of amino acid residues constituting the epitope is not particularly limited, and is, for example, 40 or less, 35 or less, 6 to 30, 6 to 25, 6 to 20, 6 to 15, or 6 to 10.
  • Anti-endoglin antibodies preferably have binding to leukemia cells.
  • the anti-endoglin antibody will have binding to the extracellular domain of endoglin.
  • the extracellular region of endoglin is published on various databases, for example, published on the National Center of Biotechnology Information (NCBI).
  • Leukemic cells are cancerous hematopoietic cells and are not particularly limited in this regard.
  • the anti-endoglin antibody preferably has binding to more undifferentiated leukemia cells, specifically, leukemia stem cells (eg, CD34 positive leukemia stem cells, CD34 negative leukemia stem cells, etc.), and more preferably is particularly undifferentiated. Binding to CD34 negative leukemia stem cells considered to be
  • Antibody A A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 2, A heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4 and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6 And / or a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10, A light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14
  • An antibody A comprising a light chain variable region comprising
  • the sequence of the entire heavy chain variable region (a sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are arranged in this order) from antibody N is preferably the amino acid sequence shown in SEQ ID NO: 8
  • the amino acid sequence represented by SEQ ID NO: 16 is mentioned as the sequence of the sequence in which the light chain variable region is arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the N-terminal side of the entire light chain variable region.
  • the antibody obtained in Example 1 is preferable as the antibody A.
  • the amino acid sequence may be mutated.
  • a sequence having an identity of preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, still more preferably 99% or more to the above-mentioned preferred example (SEQ ID NO: 8 or 16) This can be adopted as the sequence of the entire heavy chain variable region of antibody A, and the sequence of the entire light chain variable region.
  • the mutation site may be optional, but is preferably a site other than CDR.
  • the anti-endoglin antibody may be either a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody from the viewpoint of Kd value, specificity and the like.
  • the molecular weight of the anti-endoglin antibody is not particularly limited, but the lower limit is, for example, 20,000, preferably 50,000, preferably 100,000, more preferably 120,000, and the upper limit is, for example, 1,000,000, preferably 500,000, more preferably 200,000. .
  • the structure of the anti-endoglin antibody is not particularly limited.
  • the antibody of the present invention may contain a constant region or may not contain a constant region. When including a constant region, it may include all of heavy chain constant regions (CH1, CH2 and CH3) and light chain constant regions (CL), or any one or more of them.
  • CH1, CH2 and CH3 heavy chain constant regions
  • CL light chain constant regions
  • anti-endoglin antibody examples include immunoglobulin, Fab, F (ab ') 2 , minibody, scFv-Fc, Fv, scFv, diabody, triabody, A tetrabody (tetrabody) etc. are mentioned. Among these, preferred are immunoglobulins from the viewpoint of the effects of the present invention.
  • An immunoglobulin has a combined structure of one heavy chain having a heavy chain variable region and a heavy chain constant region, and one light chain having a light chain variable region and a light chain constant region.
  • the Fab comprises a heavy chain fragment comprising a heavy chain variable region and CH1 in the heavy chain constant region, and a light chain comprising a light chain variable region and a light chain constant region (CL), wherein the heavy chain variable region and the light chain It has a structure in which the chain variable region is associated by the noncovalent intermolecular interaction described above or is linked by a disulfide bond.
  • CH1 and CL may be disulfide-bonded to each other at the thiol group of the cysteine residue present in each.
  • F (ab ′) 2 has a structure in which two pairs of the above-mentioned Fabs are obtained, and CH1s are disulfide-bonded to each other by the thiol groups of the cysteine residues contained in them.
  • the minibody is a structure in which two fragments in which CH3 is linked to the heavy chain variable region constituting the scFV described below are associated by non-covalent intermolecular interaction between CH3.
  • scFv-Fc two antibody fragments containing the following scFv, CH2 and CH3 are associated by non-covalent intermolecular interaction between CH3 as in the above minibody, and the cysteine residue contained in each CH3 It is the structure which carried out the disulfide bond by the thiol groups of group.
  • Fv is also referred to as the minimum structural unit of an antibody, and is a structure in which a heavy chain variable region and a light chain variable region are associated by noncovalent intermolecular interaction.
  • thiol groups of cysteine residues present in the heavy chain variable region and the light chain variable region may be disulfide-bonded to each other.
  • the scFv has a structure in which the C terminus of the heavy chain variable region and the N terminus of the light chain variable region are linked by a linker, or the N terminus of the heavy chain variable region is linked by a linker to the C terminus of the light chain variable region It is a structure, also called single chain antibody.
  • the above scFv form dimers, trimers and tetramers, respectively, and as in Fv etc., non-covalent intermolecular interactions between variable regions, etc. Is a structure that is associated in a structurally stable state.
  • the anti-endoglin antibody is an immunoglobulin
  • its isotype is not particularly limited.
  • the isotype include IgA, IgD, IgE, IgG, IgM and the like.
  • Preferred isotypes include, for example, IgG, preferably IgG1 and more preferably IgG1 ⁇ .
  • the origin of the anti-endoglin antibody is not particularly limited.
  • the antibody of the present invention may be, for example, a human-derived antibody, a mouse-derived antibody, a rat-derived antibody, a rabbit-derived antibody, a monkey-derived antibody, a chimpanzee-derived antibody and the like.
  • the antibody of the present invention may be a chimeric antibody (eg, an antibody in which the amino acid sequence of the constant region of an antibody derived from a non-human organism (such as mouse) is replaced with the amino acid sequence of the constant region of a human derived antibody), It may be a fully humanized antibody or the like.
  • the anti-endoglin antibody may be in the form of a complex with other substances useful for the treatment of leukemia, such as anticancer agents, radioactive isotopes and the like.
  • the anticancer agent is not particularly limited.
  • a combination of idarubicin and cytarabine, a combination of daunorubicin and cytarabine, gemtuzumab ozogamicin, tretinoin, a combination of tretinoin and cytarabine daudanorubicin, vincristine and prednisolone examples include combinations with anthracycline antibiotics (eg, daunorubicin, doxorubicin and the like), nelarabine, imatinib, hydroxycarbamide, interferon- ⁇ and the like.
  • the radioactive isotope is not particularly limited, and examples thereof include 131 I, 89 Sr, 99m Tc, 111 In, 32 P, 90 Y, 153 Sm, and 186 Re.
  • the complex of the anti-endoglin antibody and the other substance is formed by binding the anti-endoglin antibody and the other substance directly or indirectly via a linker or the like.
  • the bonding mode is not particularly limited, and examples thereof include covalent bonding, coordinate bonding, ionic bonding and the like. Binding of the antibody of the present invention to a drug can be carried out according to known methods or in a similar manner, depending on the binding mode.
  • Covalent binding can be performed, for example, by reacting a functional group possessed by each of the antibody of the present invention and a drug or a functional group introduced as needed.
  • the functional groups include amino and carboxyl, carboxyl and hydroxyl, maleimide and thiol, thiol and thiol, hydrazide and ketone, hydrazide and aldehyde, amino and aldehyde Groups, thiol groups and carboxyl groups, amino groups and squaric acid derivatives, dienyl aldehyde groups and amino groups, haloesters and thiol groups, azides and alkynes, and the like.
  • proteoliposomes or cells containing endoglin and having endoglin exposed on the surface are used as an immunizing antigen, or endoglin itself is used as an immunizing antigen.
  • it can manufacture by the method according to a fixed method or according to it (manufacturing method 1).
  • the antibody of the present invention is a polyclonal antibody, it is possible to immunize a non-human animal such as a rabbit and the like with an immunizing antigen and obtain it from the serum of the immunized animal according to a conventional method.
  • the proteoliposome which is an immunizing antigen can be prepared, for example, by a known cell-free protein synthesis system using a polynucleotide encoding endoglin.
  • a lipid which comprises a proteoliposome various known lipids which can constitute a liposome can be used.
  • the polynucleotide encoding the anti-endoglin antibody is not particularly limited as long as it contains the antibody of the present invention in an expressible state, and may contain other sequences besides the coding sequence of the antibody of the present invention. Other sequences include the secretion signal peptide coding sequence, promoter sequence, enhancer sequence, repressor sequence, insulator sequence, replication origin, drug resistance gene coding sequence etc. which are arranged adjacent to the antibody coding sequence of the present invention.
  • the polynucleotide encoding the anti-endoglin antibody may be a linear polynucleotide or may be a cyclic polynucleotide (such as a vector).
  • polynucleotide encoding an anti-endoglin antibody include: (I) A polynucleotide comprising a nucleotide sequence encoding at least one selected from the group consisting of heavy chain, heavy chain variable region and heavy chain CDRs 1-3 of the antibody of the present invention, (II) A polynucleotide comprising a nucleotide sequence encoding at least one selected from the group consisting of the light chain, the light chain variable region and the light chain CDRs 1-3 of the antibody of the present invention, (III) A polynucleotide comprising a nucleotide sequence encoding at least one selected from the group consisting of heavy chain, heavy chain variable region and heavy chain CDRs 1-3 of the antibody of the present invention, and light chain of the antibody of the present invention And a light chain variable region, and a polynucleotide comprising a base sequence encoding at least one selected from the group consisting of light chain CDRs 1-3.
  • the host is not particularly limited, and examples include insect cells, eukaryotic cells, mammalian cells and the like. Among them, from the viewpoint of expressing the antibody more efficiently, mammalian cells such as HEK cells, CHO cells, NS0 cells, and SP2 / O cells are preferable.
  • the transformation, culture and recovery methods are not particularly limited, and known methods for antibody production can be employed.
  • the antibody of the present invention may be purified as required. Purification can be performed by known methods in antibody production such as chromatography, dialysis and the like.
  • the present invention relates to a leukemia improving agent (also referred to herein as “the agent of the present invention”) containing an anti-endoglin antibody.
  • the leukemia to be treated is not particularly limited, and includes acute myeloid leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, myeloma and the like.
  • “Amelioration” of leukemia means that the symptoms of leukemia are ameliorated. For example, if swelling is a symptom of the spleen, it diminishes to "ameliorate” leukemia, and if an increase in leukemia cells in certain tissues is a symptom, the number of such cells decreases. To be or to be suppressed is to "ameliorate" leukemia.
  • the content of the active ingredient in the agent of the present invention may be appropriately set in consideration of the type of target disease, target therapeutic effect, administration method, treatment period, patient age, patient weight, etc. it can.
  • the content of the active ingredient in the agent of the present invention can be about 0.0001 parts by weight to 100 parts by weight with respect to 100 parts by weight of the entire agent of the present invention.
  • the administration mode of the agent of the present invention is not particularly limited as long as the desired effect is obtained, and oral and parenteral administration (eg, intravenous injection, intramuscular injection, subcutaneous injection, rectal administration, transdermal administration, topical administration) It can be administered to mammals including humans by any route of administration.
  • the preferred mode of administration is parenteral administration, more preferably intravenous injection.
  • Dosage forms for oral and parenteral administration and methods for their preparation are well known to those skilled in the art, and can be manufactured according to a conventional method by mixing the active ingredient with a pharmaceutically acceptable carrier etc. .
  • Dosage forms for parenteral administration include injection preparations (eg, drip injections, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), external preparations (eg, ointments, patches, lotions) Agents, suppository inhalants, eye drops, eye ointments, nasal drops, ear drops, liposomes and the like.
  • the preparation for injection is prepared by dissolving the antibody in distilled water for injection, and if necessary, a solubilizer, buffer, pH adjuster, tonicity agent, soothing agent, preservative, and stabilization An agent etc. can be added.
  • the agent of the present invention can also be a lyophilized preparation for topical preparation.
  • the agent of the present invention may further contain other agents effective for the treatment or prevention of a disease.
  • the agent of the present invention can also be blended with ingredients such as bactericidal agents, anti-inflammatory agents, cell activators, vitamins, and amino acids as necessary.
  • Carriers used for formulating the agent of the present invention include excipients, binders, disintegrants, lubricants, coloring agents, flavoring agents, and, if necessary, stabilizers, emulsifiers, and the like, which are commonly used in the art.
  • Use of absorption accelerators, surfactants, pH adjusters, preservatives, antioxidants, extenders, wetting agents, surface activators, dispersants, buffers, preservatives, solubilizers, soothing agents, etc. be able to.
  • the dose of the agent of the present invention may be, for example, pharmacological information such as administration route, type of disease, degree of symptoms, age, sex, body weight of patient, severity of disease, pharmacokinetics and toxicological characteristics, etc. It can be determined by the clinician based on various factors such as the presence or absence of the use of a drug delivery system, as well as whether it is administered as part of a combination of other drugs.
  • the administration schedule of the agent of the present invention can also be determined in consideration of the same factors as the dose.
  • Example 1 Screening of leukemia cell-bound antibody As an immune source, acute lymphocytic leukemia cells whose leukemia was confirmed to be remodeled by transplantation of CD34 negative cell fraction of leukemia to severe immunodeficiency (NOG) mice were used. . The immunogen was mixed with an adjuvant and the footpads of Balb / c mice were immunized once / weekly for 3 weeks. Lymphocyte suspensions were prepared from mouse inguinal lymph nodes and fused with a mouse myeloma cell line (NS-1) to generate a large number of hybridoma clones.
  • NS-1 mouse myeloma cell line
  • the binding of the antibody in these hybridoma culture supernatants to the leukemia cells used for immunization was screened by FACS to select a hybridoma clone producing a monoclonal antibody capable of specifically binding to the leukemia cells.
  • the monoclonality of the sorted clones was ensured by performing recultivation after single cell sorting by FACS. Furthermore, by carrying out large-scale culture of these clones in serum-free medium, highly pure antibodies free of antibodies derived from fetal bovine serum were purified.
  • the target antigen of the obtained antibody was examined by immunoprecipitation and mass spectrometry. Specifically, immunoprecipitation was performed using the obtained antibody and a leukemia cell line capable of binding to the obtained antibody, and an antigen-derived band was prepared by polyacrylamide electrophoresis. Mass spectrometry (LC-MS / MS analysis) was performed using a sample from which an antigen-derived band was cut out to identify a target antigen of each antibody. In addition, antibody isotypes were determined using Thermo Fisher Scientific Rapid ELISA Mouse mAb Isotyping Kit (Product No. 37503).
  • one clone antibody (303-23) is an IgG1 ⁇ type antibody whose endoglin is a target antigen.
  • the results of immunoprecipitation are shown in FIG. 2, and the results of isotyping are shown in FIG.
  • the positive rate for each cell line was THP-1: 98.19%, F-36P: 98.28%, NALM-6: 99.44%, KU812: 84.83%.
  • Example 3 Evaluation of Binding of Anti-Endoglin Antibody to Patient-Derived Leukemia Cells
  • leukemia cells acute myeloid leukemia cells, acute lymphocytic leukemia cells, and chronic myelogenous leukemia cells
  • leukemia cells collected from the patient are used for leukemia. Except measuring the positivity of the whole cell fraction or each fraction (specifically, CD34 + fraction, CD34 + CD38 ⁇ fraction, and CD34 ⁇ CD38 ⁇ fraction) on the basis of CD34 and CD38, The same procedure as in Example 2 was performed. A histogram showing the analysis result is shown in FIG.
  • Tables 1 to 3 Table 1: acute myeloid leukemia cells
  • Table 2 acute lymphocytic leukemia cells
  • Table 3 chronic myelogenous leukemia cells
  • anti-Endoglin antibodies have binding to all acute myeloid leukemia, acute lymphocytic leukemia, and chronic myelogenous leukemia cells.
  • anti-endoglin antibody showed binding to all types of FAB classified M1 to M7 against acute myeloid leukemia.
  • the anti-endoglin antibody also showed binding to each cell fraction (CD34 + CD38 ⁇ , CD34 ⁇ CD38 ⁇ ) where leukemia stem cells are considered to be present at a high ratio. From the above, it has been suggested that anti-endoglin antibodies can exert an improving effect on leukemia regardless of the type and disease type of leukemia.
  • Example 4 Evaluation of anti-leukemia effect of anti-endoglin antibody 1 The present inventor has already successfully established CD271 + SSEA 4 + positive mesenchymal stem cells (DP-MSCs) with higher capacity for supporting CD34 + HSC and CD34-HSC than human bone marrow-derived Lin-CD45-cells. (Non-Patent Document 17). This DP-MSC was co-cultured with patient-derived acute lymphocytic leukemia cells (Sample No. UPN 303), anti-endoglin antibody was added thereto, and the number of viable cells was counted 7 days after the addition.
  • DP-MSCs positive mesenchymal stem cells
  • the results are shown in FIG.
  • Example 5 Evaluation of anti-leukemia action of anti-endoglin antibody 2 The outline of the test method of this example is shown in FIG.
  • the leukemia cells 1.0 ⁇ 10 5
  • the leukemia cells were transplanted into the severe immunodeficiency (NSG) mice by the intramedullary direct transplantation method, and on the next day, administration of the antibody by intraperitoneal injection of anti-Endoglin antibody was started.
  • the dose was 300 ⁇ g, and once weekly administration was continued for 10 weeks.
  • Mouse IgG isotype antibody was administered to the control group in the same manner. At 10 weeks, leukemia cells in the mouse were analyzed.
  • Analyzes are as follows: (1) spleen, (2) bone marrow of left tibia transplanted, (3) bone marrow of contralateral tibia and bilateral femur, and (4) peripheral blood, surface of acute lymphoid leukemia cells An antibody against the marker CD19 antigen was used and flow cytometry was performed. The spleen was also imaged and weighed.
  • FIG. Fig. 8A shows the spleen of a comparison control mouse not transplanted with leukemia cells
  • Fig. 8B shows the spleen of a comparison control mouse transplanted with leukemia cells and administered a mouse IgG isotype antibody
  • Fig. 8C shows a transplanted leukemia cell and anti-Endoglin antibody. It is the spleen of the administered mouse.
  • FIG. 8D shows the weight of spleen obtained from each mouse. The average weight of each was 44.5 mg, 269.3 mg, 43.8 mg (FIG. 8D). As shown in FIGS.
  • the spleens of control mice receiving leukemia and receiving mouse IgG isotype antibody are significantly enlarged, whereas the spleens of mice receiving anti-Endoglin antibody show leukemia. It was comparable to the non-transplanted control mice, and the swelling of the spleen due to leukemia cells was significantly suppressed.
  • the spleen weight of the anti-endoglin antibody-administered mice was statistically significantly lower than the spleen weight of the comparison control mice administered the IgG isotype antibody.
  • the absolute number of leukemic cells and peripheral blood in the bone marrow of the left tibia transplanted by the intramedullary direct transplantation (FIG. 9A), the contralateral tibia and bilateral femurs (FIG. 9B), and the spleen (FIG. 9C)
  • the concentration of leukemia cells (number in 1 ⁇ l of blood, FIG. 9D) was clearly lower in mice receiving anti-endoglin antibody as compared to control mice receiving any mouse IgG isotype antibody.
  • the number of leukemia cells was about 300-fold in peripheral blood and about 3000-fold in spleen.
  • Example 6 Determination of Amino Acid Sequence of Anti-Endoglin Antibody From genomic information of the hybridoma clone cloned in Example 1, the amino acid sequence of the antibody was determined. The amino acid sequence of each region of the heavy chain is shown in Table 4, and the amino acid sequence of each region of the light chain is shown in Table 5.

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Abstract

La présente invention aborde le problème de la fourniture d'un agent permettant de faire régresser une leucémie qui utilise un anticorps. Le problème est résolu par un agent permettant de faire régresser une leucémie contenant un anticorps anti-endogline.
PCT/JP2018/014494 2017-08-18 2018-04-04 Agent permettant de faire régresser une leucémie Ceased WO2019035241A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013506677A (ja) * 2009-09-30 2013-02-28 トラコン ファーマシューティカルズ、インコーポレイテッド エンドグリン抗体
JP2017506234A (ja) * 2014-02-06 2017-03-02 オンコマトリックス バイオファーマ，エス．エル． 抗体−薬物複合体及び免疫毒素

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013506677A (ja) * 2009-09-30 2013-02-28 トラコン ファーマシューティカルズ、インコーポレイテッド エンドグリン抗体
JP2017506234A (ja) * 2014-02-06 2017-03-02 オンコマトリックス バイオファーマ，エス．エル． 抗体−薬物複合体及び免疫毒素

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Title
DOURADO, K. M. C . ET AL.: "Endoglin ( CD105) in AML: a potential novel target for therapeutic intervention", BLOOD, vol. 128, 2016, pages 5211, ISSN: 0006-4971, Retrieved from the Internet <URL:http://www.bloodjournal.org/content/128/22/5211> [retrieved on 20180621] *
DOURADO, K. M. C. ET AL.: "Endoglin: a novel target for therapeutic intervention in acute leukemias revealed in xenograft mouse models", BLOOD, vol. 129, no. 18, May 2017 (2017-05-01), pages 2526 - 2536, XP055575174, ISSN: 0006-4971 *
GOUGOS, A. ET AL.: "Identification of distinct epitopes of endoglin, an RGD-containing glycoprotein of endothelial cells, leukemic cells, and syncytiotrophoblasts", INT. IMMUNOL., vol. 4, no. 1, 1992, pages 83 - 92, XP000886023, ISSN: 0953-8178 *
POREBA, M. ET AL.: "Expression of CD105 antigen in patients with acute leukemia, malignant lymphoma, and multiple myeloma in active phase of the disease", ADV. CLIN. EXP. MED., vol. 15, no. 6, January 2006 (2006-01-01), pages 1023 - 1028, XP055575182, ISSN: 1230-025X *
VRBACKY, F. ET AL.: "Prognostic relevance of angiopoietin-2, fibroblast growth factor-2 and endoglin mRNA expressions in chronic lymphocytic leukemia", NEOPLASMA, vol. 61, no. 5, 2014, pages 585 - 592, XP055575183, ISSN: 0028-2685 *

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