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WO2018150066A1 - Utilisation de modulateurs de la fonction de cd69 pour la mobilisation et la prolifération de précurseurs hématopoïétiques - Google Patents

Utilisation de modulateurs de la fonction de cd69 pour la mobilisation et la prolifération de précurseurs hématopoïétiques Download PDF

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Publication number
WO2018150066A1
WO2018150066A1 PCT/ES2018/070103 ES2018070103W WO2018150066A1 WO 2018150066 A1 WO2018150066 A1 WO 2018150066A1 ES 2018070103 W ES2018070103 W ES 2018070103W WO 2018150066 A1 WO2018150066 A1 WO 2018150066A1
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antibody
cells
human
hematopoietic
inhibitor
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Spanish (es)
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Laura NOTARIO MUÑOZ
Pilar LAUZURICA GÓMEZ
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Instituto de Salud Carlos III
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Instituto de Salud Carlos III
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the present invention demonstrates that the use of CD69 modulators causes the mobilization of hematopoietic precursors and their accumulation in blood and peripheral lymphoid organs. Mobilized cells can be drawn from blood to be subsequently retransplanted to the patient. Therefore, such modulators are useful for the prevention and treatment of primary or secondary leukopenias, such as those derived from chemotherapy or radiotherapy treatments that destroy hematopoietic cells.
  • the present invention can therefore be framed in the field of medicine within the pharmacological sector for its application in the health sector.
  • HSPCs While CXCR4 is expressed on the surface of HSPCs, SDF-1 is expressed on the surface of cells that line the HSPC niches. There are other factors involved such as those that show chemotactic activity of HSPCs. HSPCs respond to sphingosine-1-phosphate (S1 P) gradients through their S1 P1 receptor. The agonists of this receptor mobilize cells while the antagonists act in the opposite way (Curr Opin Hematol 2013, 20: 281-288). There are treatments with different factors that affect the interaction of HSPCs with your niche.
  • S1 P sphingosine-1-phosphate
  • G-CSF granulocyte colony stimulating factor
  • AMD3100 Phhenylenebis (methylene) bis [1, 4,8,1 1-tetraazacyclo tetradecane ) that interferes with the interaction of CXCR4 and CXCL12 / SDF-1.
  • These treatments cause the exit of HSPCs from bone marrow niches and peripheral blood trafficking, a process called 'mobilization'.
  • the mobilization phenomenon is used clinically to acquire HSPC for self-transplantation and allogeneic transplantation.
  • HSPCs are used for cell therapy in regenerative medicine for patients with acute myocardial infarction, spinal cord injury, and strokes, among others, as well as for hematopoietic transplantation for reconstitution, after treatments such as ionizing radiation or Chemotherapy, blood cells in many hematological malignancies and various types of solid tumors.
  • a low number of them results in a low transplant efficiency, which can significantly affect to the survival of patients undergoing it. Therefore, expanding the number of transplanted cells has been a long-sought goal.
  • Hematopoietic cells are also required to shorten the time of neutropenia after cytotoxic chemotherapy prior to transplants.
  • the invention also describes how modulation of CD69 in bone marrow induces the proliferation of hematopoietic precursors, mainly those that include stem cells, in addition to inducing proliferation in lymphoid and myeloid cells, superior to that induced by the AMD3100 mobilizer.
  • the present invention specifically demonstrates that the use of monoclonal antibodies specific for human CD69 constitutes an effective therapy for the proliferation and mobilization of hematopoietic progenitor cells from the bone marrow to the peripheral blood and the lymphatic system.
  • this treatment allows the expansion of hematopoietic progenitor cells while preserving their regenerative capacity.
  • CD69 (- / -) mice have a generally normal hematopoietic cell development with normal leukocyte subpopulations in peripheral blood (Lauzurica et al., 2000, Blood, 95 (7): 2312-20, PMID : 10733501; Esplugues et al., 2003, J Exp Med, 197 (9): 1093-106, PMID: 12732655).
  • the present invention demonstrates the role of CD69 as a modulator of the proliferation and mobilization of HSPCs, indicating that the use of this molecule as a target in obtaining blood precursor cells for autologous or allogeneic transplants prior to the removal of hematopoietic cells by treatments such as chemotherapy and others necessary to eliminate different pathologies.
  • the manipulation of the CD69 molecule as a regulator of the proliferation and mobilization of hematopoietic progenitor cells can boost the development of new treatments to obtain precursors to reconstitute the damaged hematopoietic system, including the combination of CD69 regulators with established precursor mobilization treatments. In the medical clinic.
  • the present invention relates to the use of a CD69 modulator to cause or induce the proliferation of hematopoietic precursors in bone marrow and their exit or mobilization from the bone marrow, in vitro or in vivo in a subject; or for the preparation of a medicament, wherein said medicament is preferably used to cause or induce the proliferation of hematopoietic precursors and their exit or mobilization from bone marrow in a subject.
  • the present invention relates to a CD69 modulator for use as a medicament, preferably where said medicament is to cause or induce the proliferation of hematopoietic precursors and their exit (mobilization) from bone marrow in a subject.
  • the induction of precursor proliferation produced by the CD69 modulator will improve the recovery (i.e. mobilization and collection) of an adequate number of said precursors, and therefore facilitate a rapid disposition of the subject to successive precursor mobilizations, in case necessary.
  • CD69 belongs to the family of type C lectins and its gene is located in the gene region of the NK complex (GenBank accession number: Q07108).
  • CD69 also known as "very early activation protein”, “activation induction molecule” and “gp34 / 28", refers to mammalian CD69 protein, preferably human CD69 protein.
  • human CD69 refers to a polypeptide that has (or is homologous to) at least 80, 81, 82, 83, 84 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 , 95, 96, 97, 98, 99 or 100% identity with an amino acid sequence SEQ ID NO: 1, or which is encoded by: (a) a nucleic acid sequence encoding the human CD69 protein (for example , a nucleic acid sequence encoding human CD69 according to SEQ ID NO: 2); (b) a degenerate nucleic acid sequence to a natural human CD69 sequence; (c) a nucleic acid of sequence homologous to (for example, at least about 85%, 90%, 95% identical to) the nucleic acid sequence for natural human CD69, preferably to SEQ ID NO: 2; or (d) a nucleic acid sequence that hybridizes with any of the nucleic acid sequences indicated above under astring
  • CD69 is a molecule that expresses itself rapidly and transiently during the activation of leukocytes after an immune challenge. CD69 it is expressed in all hematopoietic lineages except erythrocytes, and although it is detected in vivo in some subtypes of T and B lymphocytes in peripheral lymphoid tissues (Testi R. et al., 1994, Immunol Today, 15 (10): 479-83, PMID: 7945773; Sancho D.
  • identical refers to a first amino acid or nucleotide sequence containing a sufficient number of identical or equivalent amino acids or nucleotides (ie, with similar side chains, conserved amino acid substitutions, etc.) a a second amino acid or nucleotide sequence, such that the first and second sequences have similar activities.
  • the second antibody has the same specificity and at least 50% of the affinity demonstrated with the first.
  • Identity calculations between two sequences can be carried out as follows: the sequences are aligned to make an optimal comparison (it is possible to introduce gaps in one or both sequences for optimal alignment and non-identical sequences can be discarded) .
  • the length of a reference sequence aligned for sequence comparison is at least 30% of the total sequence, although it is all the better the higher the percentage.
  • amino acid residues or nucleotides of both chains are compared in corresponding positions.
  • identity is used.
  • the percentage of identity between two sequences is a function of the number of identical positions found in both sequences, taking into account the number of gaps whose introduction is required for optimal alignment, as well as their length.
  • Sequence comparison and determination of the percentage of identity between two sequences can be performed using mathematical algorithms.
  • the Needleman and Wunch algorithm (Needleman and Wunsch (1970), J. Mol. Biol. 48: 444-453) is used, which has been implemented in the GAP program of the GCG software package, using a Blossum matrix 62, either a PAM250 and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5 or 6.
  • Another suitable way to calculate the percentage of identity is to use the GAP program of the GCG software, using an NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the group of reference parameters (which should be used if the researcher is not sure of the parameters that should be applied to determine if a molecule is within the identity limitation of the invention) is constituted in a Blossum 62 matrix with a gap penalty of 12, an extension of the gap penalty of 4 and a frame gap penalty of 5.
  • hematopoietic precursor or “multi-line precursor cells” or “hematopoietic stem cells” means hematopoietic cells that do not express markers of mature hematopoietic (lin-) lineages and which preferably include, but are not limited to, SCA + cells.
  • the outflow or mobilization of hematopoietic precursors from the bone marrow is mostly to the blood, lymph and peripheral lymphoid organs of the same subject, similar to the distribution of hematopoietic cells in baseline state, that is in the absence of externally induced mobilization as proposed in the present invention.
  • the mobilization towards organs is towards lymphatic tissues such as, for example, but without limitation, spleen and ganglia, and in a smaller proportion towards non-lymphoid, mucous, skin and other internal organs.
  • the proliferation and exit of hematopoietic precursors from the bone marrow serves for the prevention and / or treatment of hematopoietic disorders associated with a deficient production of blood cells, such as for example but not limited to, leukopenia, thrombopenia or pancytopenia in a subject, that is, the medicament referred to in the present invention prevents and / or treats hematopoietic disorders associated with a deficient production of blood cells, preferably said diseases.
  • the proliferation and exit of hematopoietic precursors from the bone marrow serves to obtain said precursors for a transplant. That is, the present invention relates to the use of a CD69 modulator for obtaining said precursors for a transplant. Since the administration of the CD69 modulator induces the proliferation of these precursors, it is possible to perform successive rounds of obtaining said precursors.
  • modulator means a substance of any nature that in any way modifies the function of CD69 and includes, but not exclusively, blockers, inhibitors, antagonists and / or agonists.
  • the activity of CD69 can be modulated by the modification of the levels and / or the activity of the CD69 protein, or by the modification of the levels at which the genes encoding CD69 are transcribed, so that the activity levels of The CD69 protein in the cell are modulated.
  • small molecules include, but are not limited to, peptides, peptidomimetics (eg peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, organic and inorganic compounds (including heteroorganic and organometallic compounds) with molecular weight less than about 5000 g / mol, and pharmacologically acceptable salts, esters and other forms of such compounds.
  • peptides eg peptoids
  • amino acids amino acid analogs
  • polynucleotides polynucleotide analogs
  • organic and inorganic compounds including heteroorganic and organometallic compounds
  • CD69 modulators include, but are not limited to: CD69 receptor modulators (antagonists, agonists and blockers), polypeptides, peptides, peptidomimetics, soluble forms of CD69R, fusion proteins modulating CD69R, for example , fusion proteins of CD69R modulators with whey proteins (for example CD69R fusion proteins with immunoglobulins, or CD69R with human serum albumin) or other forms of CD69R modulating fusion proteins designed to increase the half-life in the serum and / or multivalence.
  • CD69 receptor modulators antagonists, agonists and blockers
  • polypeptides peptides
  • peptidomimetics soluble forms of CD69R
  • fusion proteins modulating CD69R for example , fusion proteins of CD69R modulators with whey proteins (for example CD69R fusion proteins with immunoglobulins, or CD69R with human serum albumin) or other forms of CD69R modulating fusion proteins designed to increase the half-life in the serum
  • inhibitor primarily refers to a molecule that lowers the level of activity of the CD69 protein or gene (or mRNA) in a cell.
  • the inhibitory agents may be substances that are capable of binding to a receptor and eliciting a response in the cell based on a decrease in the CD69 activity, as well as substances that not only do not activate the receptor, but actually block their activation by agonists.
  • the CD69 modulator is selected from the list consisting of: inhibitor, agonist, antagonist, blocker or an interfering RNA.
  • the modulator is a monoclonal antibody (said antibody may have inhibitory, agonist, antagonist or blocking activity, for example) or a mixture of monoclonal antibodies.
  • the monoclonal antibody is selected from the list consisting of: humanized antibody, human antibody, chimeric antibody, immunized antibody, antibody fragments and nanobody (or nanobodies, single domain antibodies or V H H antibodies).
  • CD69 modulating molecules include, but are not limited to, antibody molecules that bind CD69 and interfere with the binding of CD69 and a polypeptide that binds to CD69, for example, a human anti-CD69 antibody, for example, a human anti-CD69 antibody analogous to the anti-CD69 2.8 monoclonal antibody described herein or to any anti-CD69 antibody known in the literature that can act as an antagonist or blocker or agonist (or an antibody molecule based thereon, for example, a fragment of antibody, or a chimera, humanized or deimmunogenic antibody) or an antibody molecule that binds to the epitope bound by such an antibody, or a molecule that competes for binding with such an antibody, or an antibody molecule that binds to or interferes with the binding of another antibody or ligand to one or more of the amino acid residues of human CD69, preferably to residues Glu 140, Asp171, Glu 180, Glu 185, Glu 187, Phe 175, Met 184, Le
  • the antibody is an antibody against human CD69. More preferably the antibody is the so-called 2.8 monoclonal antibody, which specifically binds to human CD69, wherein said antibody comprises a heavy chain comprising the variable regions CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NO: 3 , SEQ ID NO: 4 and SEQ ID NO: 5, respectively, and a light chain comprising the variable regions CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
  • This anti-CD69 2.8 monoclonal antibody comprises, more preferably, a heavy chain comprising SEQ ID NO: 9 and a light chain comprising SEQ ID NO: 10, which comprise the aforementioned CDR variable domains.
  • the anti-CD69 2.8 monoclonal antibody which specifically recognizes the human CD69 molecule, was generated by fusion of NS-1 myeloma cells with spleen cells of a CD69 (- / -) mouse that had previously been immunized 3 times with cells pre-B 300-19 expressing the human CD69 molecule for having been transfected with the specific cDNA. Specificity is defined by the recognition of human CD69 + cells, but not of CD69 (+ / +) or CD69 (- / -) mouse cells. Thus, anti-CD69 2.8 recognizes the human CD69 molecule but not the mouse.
  • An anti-CD69 antibody molecule is an antibody molecule that interacts with (for example, binds to) CD69, preferably human CD69 protein.
  • the anti-CD69 antibody molecule binds to the extracellular domain of CD69 (for example, to an epitope of CD69 located outside the cell).
  • monoclonal antibodies against human CD69 include, but are not limited to, a human anti-CD69 antibody molecule analogous or homologous to human anti-CD69 antibody 2.8, a human anti-CD69 antibody molecule analogous or homologous to anti-human antibody.
  • Examples of human anti-CD69 antibodies known in the literature and that can be used in the present invention include: TP1 / 8, TP1 / 22, TP 1/28, TP 1/33, TP 1/55 (as described, for example, in Cebrián, et al, 1988, J Exp Med., 168 (5): 1621-37); CH / 4, CH / 1, CH / 2, FAB / 1 (as described for example in Sánchez-Mateos, Sánchez-Madrid, 1991, Immunol., 21 (10): 2317-25); L78, MLR3, FN61, FN50 (as described, for example, in Schwarting, R. et al.
  • binding agent preferably the antibody
  • CD69 for example, a human CD69 protein
  • affinity of at minus 1x10 7 M "1
  • CD69 for example, the human CD69 protein
  • a non-specific antigen eg, bovine serum albumin, casein
  • the antibody molecules can be full length (eg, an IgG1 or IgG4 antibody) or can include only an antigen binding fragment (eg, a Fab, F (ab ') 2, Fv or a single strand of a fragment Fv).
  • an antigen binding fragment eg, a Fab, F (ab ') 2, Fv or a single strand of a fragment Fv.
  • antigen refers to a molecule, such as a peptide, a carbohydrate, a glycolipid, a glycoprotein or a molecule that is recognized and binds to an antibody.
  • the part of the antigen that is the target of the antibody binding corresponds to the antigenic determinant.
  • the antigen is a CD69 peptide, preferably human CD69.
  • the antibody is preferably an antibody molecule designed by methods known to the person skilled in the art, for example, a humanized antibody.
  • Antibodies, or other agents described herein, can be evaluated for their ability to act as CD69 modulators.
  • antibody molecule refers to a molecule that includes a sufficient number of complementarity determining regions (CDRs), preferably 6, presented in an arrangement that allows the binding of the CDRs to the known antigen.
  • CDRs complementarity determining regions
  • the term includes complete antibodies (including natural antibodies and those designed by Molecular Biology), and antigen-binding fragments of natural or designed antibodies.
  • the term includes several types of antibodies or antibody molecules, including monospecific, monoclonal, recombinant, human, and non-human, for example, murine. Also included are single chain antibodies, intrabodies and bivalent antibodies.
  • Chimeric antibody molecules are also included, with a CDR distinct grafted, humanized, disinmunogenic, as well as others that have been designed to reduce immunogenicity, for example, those with CDRs derived from a non-human source, for example, from a non-human animal such as the mouse, and / or derived from the partial or totally random generation of sequences, for example, using a phage selection method.
  • a non-human source for example, from a non-human animal such as the mouse
  • Such non-human fragments can be inserted into human, humanized molecules, or other arrangements that make them less antigenic when administered to a human.
  • an antibody molecule may have CDRs from a non-human source, for example, from a non-human antibody, for example, from a mouse immunoglobulin or other non-human immunoglobulin, from a consensus sequence, or from a sequence generated by selection of phages, or any other method to generate diversity; and having an arrangement that is less antigenic in a human than non-human structure, for example, in the case of CDRs of a non-human immunoglobulin, less antigenic than the non-human structure from which non-human CDRs were taken.
  • a non-human source for example, from a non-human antibody, for example, from a mouse immunoglobulin or other non-human immunoglobulin, from a consensus sequence, or from a sequence generated by selection of phages, or any other method to generate diversity
  • having an arrangement that is less antigenic in a human than non-human structure for example, in the case of CDRs of a non-human immunoglobulin, less antigenic than the non-human structure from
  • the structure of the immunoglobulin can, for example, be human, non-humanized, for example, of a mouse, of modified structure to reduce antigenicity in humans, or a synthetic structure, for example, a consensus sequence or a method of In vitro diversity generation.
  • each VH and VL of an antibody molecule is composed of three CDRs and four FRs, arranged from the amino-terminal to the carboxy-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the CDRs and FRs can come from different sources.
  • the VH or VL chain of an antibody molecule may include all or part of a constant region of the light or heavy chain.
  • the antibody molecule is a tetramer of two heavy and two light chains of immunoglobulins, where heavy and light chains are interconnected by, for example, disulfide bridges.
  • the constant region of the heavy chain is composed of three domains, CH1, CH2 and CH3.
  • the constant region of the light chain is composed of a domain, CL.
  • the variable region of the heavy and light chains contains a binding domain that interacts with the antigen.
  • Constant regions of the antibodies typically mediate the binding of the antibody molecule to host tissues or factors, including various cell types of the immune system (eg, effector cells) and the first component (C1q) of the classical pathway of the system of complement.
  • Antibody molecules can include IgA, IgG, IgE, IgD, IgM (as well as all their subtypes), where the light chains can be of the kappa or lambda type.
  • an antigen binding fragment may refer to a portion of an antibody that specifically binds to CD69 (eg, human CD69).
  • binding fragments include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) an F (ab ') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment, which consists of the VH and CHI domains; (iv) an Fv fragment, which consists of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VH domain; and (vi) one or more isolated CDRs with sufficient structure to specifically bind, for example, an antigen-binding portion of a variable region.
  • Antibody fragments can also be produced by chemical methods, for example, by breaking an intact antibody with a protease, such as pepsin or papain, or, optionally, treating the digested product with a reducing agent.
  • useful fragments can be produced using host cells transformed with truncated genes of the heavy and / or light chains.
  • An antigen-binding portion of a variable region of the light chain and an antigen-binding portion of a variable region of the heavy chain for example, the two domains of the Fv fragment, VL and VH
  • VL and VH an antigen-binding portion of a variable region of the heavy chain
  • scFv single chain Fv
  • Such single chain antibodies are also included within the term "antigen binding fragment" of an antibody.
  • the term "monospecific antibody or antibody molecule” refers to an antibody or antibody molecule that shows a single binding specificity and affinity for a particular target, for example, an epitope. This term includes a monoclonal antibody or a composition of monoclonal antibodies.
  • the anti-CD69 antibody in the present invention is monoclonal, more preferably it is a humanized monoclonal antibody.
  • “Monoclonal antibodies” are homogeneous populations of identical antibodies, produced by a hybrid cell resulting from the fusion of a clone of B lymphocytes descending from a single and single stem cell and a tumor plasma cell, which are directed against a single site or determinant antigenic
  • the process for obtaining the monoclonal antibody of the invention can be carried out according to conventional methods, known in the state of the art. Basically, the method consists in immunizing an animal with a conjugate comprising a macromolecule that confers immunogenicity and subsequently extracting cells from the spleen of the immunized animal, which are fused with myeloma cells in the presence of a fusion inducer, such as PEG-1500 by standard procedures.
  • a fusion inducer such as PEG-1500
  • hybridomas are selected and subcloned by dilution.
  • Clones suitable for expansion constitute a hybridoma cell line.
  • said hybridoma cell line is cultured in a culture medium suitable for hybridoma cells to produce antibodies and secrete them into the medium, and the culture medium supernatant containing the produced monoclonal antibodies is subsequently collected.
  • said antibodies can be purified by conventional means, such as affinity chromatography, A-Sepharose protein, hydroxyapatite chromatography, gel electrophoresis or dialysis.
  • the antibody in the present invention can also be a recombinant antibody.
  • antibody or recombinant antibody molecule refers to antibodies or antibody molecules that are prepared, expressed, created or isolated using recombinant methods, such as antibody molecules expressed using a recombinant expression vector transfected into a host cell, antibody molecules isolated from a recombinant organism, a library of combinatorial antibodies, antibody molecules isolated from an animal (eg, a mouse) that is transgenic for human immunoglobulin genes or antibody molecules prepared, expressed, created or isolated by any other means that involves the combination of immunoglobulin gene sequences human with other DNA sequences.
  • an antibody can be modified as follows: (i) by destruction of the constant region; (ii) by replacing the constant region with another constant region, for example, a constant region that increases the half-life, stability or affinity of the antibody, or a constant region of another species or class of antibody; or (iii) by the modification of one or more amino acids of the constant region to alter, for example, the number of glycosylation sites, the function of the effector cell, the binding to Fe receptors (FcR), complement fixation, and / or transport through the placenta, among others.
  • the constant region of the antibody can be replaced by another constant region of, for example, a different species.
  • This replacement can be performed using Molecular Biology techniques.
  • the nucleic acid encoding the VL or VH region of an antibody can be converted to a full-length heavy or light chain gene, respectively, by the operational binding of nucleic acids encoding VH or VL to other acids.
  • nucleic encoding the constant regions of heavy or light chains The sequences of the genes of the constant regions of human heavy and light chains are known to the person skilled in the art.
  • the constant region is human, but the constant region of other species, for example, rodents (eg, mouse or rat), primate, camel, rabbit, can also be used.
  • Antibody molecule conjugates :
  • the antibody molecules of the invention can be conjugated, covalently or noncovalently, with other structures, eg, therapeutic agents or signals, eg, toxins (eg, proteins, (eg, diphtheria or ricin) or chemical toxins), therapeutic isotopes , or other therapeutic structures.
  • therapeutic agents or signals eg, toxins (eg, proteins, (eg, diphtheria or ricin) or chemical toxins), therapeutic isotopes , or other therapeutic structures.
  • an early activation anti-polypeptide antibody molecule can be derivatized or bound to another functional molecule (eg, another peptide or protein).
  • Antibodies and antibody portions of the invention include derivatized or modified forms of any form of the antibodies described herein, including immunoadhesion molecules.
  • an antibody or antibody portion of the invention can be functionally linked (by chemical binding, genetic fusion, non-covalent association or otherwise) to one or more molecular entities, such as another antibody, (eg, a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate the association of an antibody or a portion of antibody with another molecule (such as the main streptavidin region or a polyhistidine tail).
  • another antibody eg, a bispecific antibody or a diabody
  • detectable agent e.g., a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate the association of an antibody or a portion of antibody with another molecule (such as the main streptavidin region or a polyhistidine tail).
  • Useful detectable agents with which an antibody of the invention or portion thereof can be derivatized (or labeled) may include fluorescent components, various enzymes, prosthetic groups, luminescent or bioluminescent materials, fluorescent emission metal atoms, eg, europium ( Eu), and other lanthanides, and radioactive materials (described below).
  • fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and others of the same type.
  • An antibody can also be derivatized with a prosthetic group (eg, streptavidin / biotin and avidin / biotin).
  • a prosthetic group eg, streptavidin / biotin and avidin / biotin
  • an antibody can be derivatized with biotin and detected through the indirect measurement of the binding of avidin or streptavidin.
  • suitable fluorescence materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • An example of a luminescent material is luminol; and examples of bioluminescent materials are luciferase, luciferin and aecuorin.
  • Therapeutic agents include, but are not limited to, antimetabolites, (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbacin), alkylating agents (eg, mechlorethamine, thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclotosfamide, busulfan, dibromomanitol, streptotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin); anthracyclines (eg, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthramycin (AMC)), and anti-mitotic agents (eg, vincristine, vin
  • An anti-early polypeptide antibody or an antigen binding fragment thereof can be conjugated to another molecular entity, eg, a structure that modulates immunogenicity and / or half-life.
  • the molecular entity is polyethylene glycol (PEG) or derivatives thereof.
  • PEGylation is a chemical conjugation method that can reduce potential immunogenicity and / or extend the half-life.
  • Several methods of PEGylation of an antibody are known. See, eg, Bhandra et al. (2002) Pharmazie 57 (1): 5-29.
  • CD69 modulators Other reagents binding to an early activation polypeptide (i.e., CD69 modulators):
  • an "early activation polypeptide binding reagent” is defined as an agent that interacts (binds) with the early activation polypeptide, preferably of human origin. The interaction preferably occurs with high affinity (with a binding constant of at least 10 7 M "1 , preferably between 10 8 and 10 10 M "1 ) and specificity. Binding reagents for an early activation polypeptide may be antagonists or eliminators (depleting) of CD69. Examples of binding reagents for early activation polypeptide may be cited antibodies against the activation polypeptide early (as described above), as well as small molecular molecules or peptidomimetics.
  • the amino acids added in each cycle are randomly selected, or they can be selected to obtain a directed library, that is, in which certain parts of the inhibitor are selected by a non-random method, for example, to select inhibitors with structural identity or similarity with a known peptide capable of interacting with an antibody, such as the binding site of antigen of anti-idiotypic antibodies.
  • a directed library that is, in which certain parts of the inhibitor are selected by a non-random method, for example, to select inhibitors with structural identity or similarity with a known peptide capable of interacting with an antibody, such as the binding site of antigen of anti-idiotypic antibodies.
  • This division strategy produces a library of peptides, some of them inhibitors, which can be used to prepare a library of test compounds of the invention.
  • a library of diversomers is generated according to the method of Hobbs DeWitt et al. (Proc. Nati. Acad. Sci. USA. 90: 6909 (1993)).
  • Other methods of synthesis such as that of the Houghten tea bag (see Houghten et al., Nature 354: 84-86 (1991)) can be used to generate libraries of compounds of the subject invention.
  • the compounds can be synthesized in solid resin microspheres following a microsphere-a compound pattern;
  • the compounds can be immobilized in the resin through a photolabile bridge.
  • the spheres (100,000 or more) can be combined into yeast cells and sprayed in the form of nano-drops, so that each drop includes a single sphere (and therefore a compound). Exposure of the nano-drops to UV light results in the release of the compounds from the drops, resulting in a method that allows rapid screening of large libraries.
  • the anti-CD69 antibodies of this invention may have additional conservative or nonessential substitutions, which do not have a substantial effect on their functionality. It can be determined whether a specific substitution will be tolerable (will not adversely affect the desired biological properties, such as binding activity) as described by Bowie et al. (1990) Science 247: 1306-1310.
  • a conserved amino acid substitution is defined as one in which a residue is replaced by another that has a similar side chain, which is well established in the literature.
  • amino acid families with similar side chains are: basic side chains (lysine, arginine, histidine), acidic (aspartic and glutamic acids), uncharged but polar side chains (glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), [beta] -branched (threonine, valine, isoleucine) and aromatic (tyrosine, phenylalanine, tryptophan, histidine) side chains.
  • An antiserum comprising the antibody that recognizes the CD69, preferably human, of the present invention for the uses described herein can also be used in the present invention.
  • antiserum refers to a serum obtained after immunization of an animal with an immunogen.
  • the antiserum comprises antibodies specific to said immunogen generated after the immune response produced in the animal.
  • the immunogen is CD69 or a fragment of CD69 and the antiserum comprises specific antibodies generated against CD69.
  • a pharmaceutical composition comprising the modulator (preferably the antibody) of CD69, preferably human, of the present invention can also be used in the present invention for the uses described herein.
  • composition in the present invention, the terms “composition”, “pharmaceutical composition”, “drug” and “medicament” are used interchangeably.
  • excipient refers to a substance that aids the absorption of the elements of the composition of the invention and actively stabilizes or aids the preparation of the composition in the sense of giving consistency or flavor. Therefore, carriers may have the function of keeping the ingredients together, as in the case of starches, sugars or celluloses, function of sweeteners, function as a dye, protective function of the composition, such as to isolate the air and / or moisture, filling the role of a tablet, capsule or other form of presentation, such as di-basic calcium phosphate, disintegration function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other excipients that are not mentioned in this paragraph.
  • pharmaceutical carrier or vehicle refers to a substance used in the pharmaceutical composition or medicament to dilute any component of the present invention included therein to a given volume or weight.
  • the function of the vehicle is to facilitate the incorporation of other elements, which will allow a better dosage and administration or give body and form to the composition.
  • the pharmacologically acceptable carrier is the diluent.
  • the pharmaceutical composition also comprises an adjuvant.
  • adjuvant refers to an agent that increases the effect of the modulator of the invention when co-administered or as part of the same treatment protocol.
  • Pharmaceutically acceptable adjuvants and vehicles that can be used in the pharmaceutical composition of the present invention are those known to those skilled in the art.
  • the inhibitor is an interference RNA, a microRNA or an antisense nucleic acid chain.
  • the subject has been or will be treated with chemotherapy and / or radiotherapy and / or with any other treatment that induce a deficient production of blood cells, preferably that induces leukopenia, thrombopenia and / or pancytopenia, in a subject.
  • the CD69 modulator or medicament comprising it is preferably administered orally, parenterally, intra-muscularly, intra-peritoneally, intra-arterially, intravenously, intratracheally, intra-nasally, transdermally, intra-dermal, intra-vaginal, intravesicular, epidural, subcutaneous, cutaneous, topical, otic, ophthalmic, inhalation, sublingual, vaginal, rectal, gastroenteric or mucous.
  • the CD69 modulator or medicament comprising it is preferably administered from 4 to 24 hours (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours) before the proliferation and mobilization of bone marrow cells.
  • the CD69 modulator or medicament comprising it is preferably administered for 5, 6 or 7 days to the subject.
  • the CD69 modulator or medicament comprising it is repeatedly administered in successive doses. every 5 or 7 days (5, 6 or 7), or until the improvement in the condition of the subject is seen or expected.
  • the dose of the modulator administered is a therapeutically effective amount.
  • the term "therapeutically effective or effective amount” refers to the amount of the agent or compound capable of producing a desired effect (in the present invention the modulation of CD69) and is generally determined by characteristics of the compounds, the route and frequency of administration in the same way and other factors, including the age, condition of the patient and the severity of the alteration or disorder.
  • the subject is a human being (male or female of any age).
  • a second aspect of the present invention relates to a method, preferably in vitro, of obtaining hematopoietic precursors useful for a transplant comprising:
  • a CD69 modulator preferably an anti-CD69 antibody, or the medicament defined in the present invention
  • step (b) Store the cells obtained in step (b) until they are used.
  • a third aspect of the present invention relates to the in vitro use of a CD69 modulator, preferably of an anti-CD69 antibody, or of the medicament defined in the present invention, for obtaining hematopoietic precursors.
  • This aspect of the invention relates to the in vitro (ex vivo) administration of the CD69 modulator or the medicament comprising an isolated bone marrow to cause proliferation, and thus allow obtaining hematopoietic precursors.
  • the present invention also relates to a method of obtaining hematopoietic progenitor cells that include SCA + CD34 + cells and high expression of the c-Kit marker (c-Kit hl ) and not expressing markers of mature hematopoietic (lin-) lineages, useful for a transplant comprising:
  • a CD69 modulator preferably an anti-CD69 antibody, or the medicament defined in the present invention
  • b. Collect the leukocyte fraction comprising SCA +, CD34 + cells, high expression of the c-Kit marker (c-Kit hl ) and not expressing markers of mature hematopoietic (lin-) lineages, and
  • step (b) Optionally store the cells obtained in step (b) until use.
  • amino acids amino acids
  • amino acid sequence amino acid sequence
  • polypeptide amino acid sequence
  • peptide amino acid sequence
  • oligopeptide amino acid sequence
  • nucleotides are used interchangeably.
  • mice humanized HU: CD69 +/- mice, which are transgenic mice carrying the human CD69 molecule but deficient for the mouse CD69 molecule (MU: CD69 - / -).
  • MU CD69 - / -
  • the HU: CD69 +/- mice were treated with 500 ⁇ g of anti-hCD69 2.8 Ac 24 hours before analysis, except when times are indicated.
  • FIG. 1 Treatment with human anti-CD69 monoclonal antibody (mAb) 2.8 induces mobilization and exit of hematopoietic precursors from the bone marrow to peripheral organs.
  • the HU: CD69 +/- mice were treated with a dose of 500 ⁇ g of anti-hCD69 2.8 a day 0.
  • A shows the temporal evolution (4 hours, 1 day, 3 days, 6 days or 9 days) of the total number of cells in the bone marrow and spleen and B, shows the expression of CD69 in thymocytes of untreated mice (white) and treated mice (gray).
  • HU CD69 +/- mice were treated with 500 ⁇ g of anti-hCD69 2.8 Ac 24 hours before analysis.
  • A Percentages in bone marrow and spleen
  • B Numbers in bone marrow and spleen of the main lymphoid and myeloid subpopulations that were analyzed by flow cytometry. Data set of two experiments.
  • FIG. 3 Comparison of the effect of human anti-CD69 2.8 with that of the CXCR4 inhibitor, AMD3100.
  • HU CD69 +/- mice were treated with 500 ⁇ g of anti-hCD69 2.8 Ac 24 hours before analysis, or with AMD3100 (150 g / mouse) or PBS i.p. (control) 1 h before analysis. The effect of both treatments is shown on the total number of cells in the indicated organs (A) and on the number of leukocyte subpopulation cells in bone marrow (B) and spleen (C). Data set of two experiments.
  • FIG. 4 Treatment with human anti-CD69 monoclonal antibody (mAb) 2.8 induces the output of early hematopoietic precursors and a large increase in multipotent bone marrow precursors.
  • mAb monoclonal antibody
  • HU CD69 +/- mice were treated with 500 ⁇ g of anti-hCD69 2.8 Ac 24 24 hours prior to flow cytometric analysis of Bone Marrow and Spleen cells.
  • AB The percentage and number of early hematopoietic precursors within precursors that are negative for lineage markers (lin-) are shown.
  • the subpopulation lin-Sca + cKit ' nt represents the precursors common to the lymphoid line (CLP) and the lin-, Sca +, cKit hl (KSL) the primitive precursors containing the stem cells in bone marrow A and spleen BC LT-HSC, ST-HSC and MPP subpopulations were classified according to their expression of CD34 and FLT3, selected by their expression Sca +, C-kit hl . Percentages and cell numbers of the subpopulations indicated in bone marrow. Data set of three experiments.
  • FIG. 5 An increase in the proliferation rate is observed in HuCD69 +/- mice treated with anti-human-CD69 in vivo.
  • Mice were treated with 500 ⁇ g of anti-hCD69 2.9 iv, PBS iv (control) or AMD3100 ip as appropriate and sacrificed one day later. The mice received 1 mg of BrdU intraperitoneally and three hours later the mice were sacrificed. Bone marrow and spleen cells were collected. Total cells were classified into Lin + and Lin- cells and the percentage of BrdU in both subpopulations was analyzed. A, Within Lin +, Brdu incorporation was measured in lymphoid and myeloid populations present in bone marrow and spleen.
  • KSL Lin-cells were also stained with Sea and c-Kit to differentiate two subpopulations of hematopoietic stem cells: KSL (Lin-Sca1 + c-kit hi) and CLP cells (common lymphoid progenitors: Lin-Sca1 + c-kit ' nt ).
  • KSL cells were analyzed according to CD34 and FLT3 expression: LT-HSC or long-term HSC (KSL CD34neg FLT3neg), ST-HSC or short-term HSC (KSL CD34 + FLT3neg) and MPP or multipotent progenitors (KSL CD34 + FLT3 + ).
  • the cell proliferation rate was evaluated in all subpopulations in the bone marrow and in the spleen by flow cytometry.
  • C Number of Colony Forming Units obtained by plating 10 5 Bone Marrow cells in Complete Methyl Cellulose Medium, counted after 10 days of culture. It has been carried out once compared to AMD3100, but it is a representative experiment of three comparing treatment with human anti-CD69 with untreated mice.
  • FIG. Expression of CXCR4 in mice treated with human anti-CD69 2.8. Surface detection of CXCR4 in spleen and bone marrow cells is shown by flow cytometry. HU: CD69 +/- mice were treated with 500 ⁇ g of anti-hCD69 2.8 Ac 24 24 hours prior to flow cytometric analysis. A, expression measured in percentage (%) and in Geometric Mean (GM) in Bone Marrow and in Spleen as indicated. B, Shows the number of cells expressing CXCR4.
  • FIG. 7. Repeating treatment with human anti-CD69 2.8 maintains the leukocyte mobilization effect from the bone marrow.
  • HU CD69 +/- mice were treated 12 and 5 days before analysis. The total number of cells in the indicated organs (A), organ weight (B), percentages of leukocyte subpopulations in bone marrow (C) and number of leukocyte subpopulation cells in bone marrow (D) are shown in spleen (E) and thymus (F). An independent experiment of two experiments.
  • FIG. 9 Treatment with murine anti-CD69 2.2 also has leukocyte mobilization effect from the bone marrow in the WT mouse.
  • WT CD69 + / + mice were treated with 500 ⁇ g of murine 2.2 anti-CD69 Ac 24 hours before analysis.
  • the total number of cells in the indicated organs (A), spleen weight (B), number of leukocyte subpopulation cells in bone marrow (C) and number of leukocyte subpopulation cells in spleen (D) are shown.
  • the described anti-CD69 monoclonal antibodies that recognize the human CD69 molecule do not recognize the mouse molecule and vice versa, that is, they are species specific.
  • the humanized mouse model HU: CD69 +/-, transgenic carrier of the human CD69 molecule but deficient for the mouse CD69 molecule MU: CD69 - / -.
  • MU CD69 - / -
  • the effects seen by the treatments on human CD69 will not be influenced by the presence of the mouse CD69 molecule.
  • different injection patterns of the human anti-CD69 2.8 of the IgG1 isotype were performed, whose Fe does not react with either the complement system or the Fe-leukocyte cell Fe receptors.
  • Figure 1 demonstrates the ability of the anti-CD69 Acm to mobilize cells from the medulla that is.
  • the results of the analysis of hematopoietic cells of HU: CD69 +/- mice, untreated (control) and treated with human anti-CD69 2.8 with single dose ( ⁇ ) were presented and the kinetics of marrow precursors were examined. that is.
  • a decrease in the number of bone marrow cells was observed, reaching its maximum between 4 and 24 hours (4 hours was the first time examined), increasing the number of cells gradually from 48 hours to 9 days.
  • the decrease in the number of cells in the bone marrow at 24 hours is greater than 25% of the initial.
  • the spleen experienced an increase in the number of cells whose maximum is reached 3 days after treatment, (Fig. 1A), subsequently decreasing with slow dynamics.
  • Fig. 1A we consider the spleen test as an indirect reading of the number of blood cells.
  • the action of the Acm affects the expression of CD69, not being detected in the rudder of HU: CD69 +/- mice treated with human anti-CD69 2.8, while it is high in those not treated (Fig. 1B).
  • the main lymphoid and myeloid populations of the bone marrow and spleen were analyzed 24 hours after treatment with the human anti-CD69 antibody.
  • HSPC hematopoietic stem / progenitor cells
  • AMD3100 that interferes with the interaction of CXCR4 with CXCL12 / SDF-1.
  • human anti-CD69 2.8 and AMD3100 will be compared.
  • anti-CD69-induced proliferation is largely due to an intrinsic effect of the antibody in HSCs and not to the homeostatic process derived from the output of bone marrow cells, since AMD3100 also induces anti-precursor output similar to anti -CD69 but the proliferation induced by AMD3100 is much smaller.
  • the large increase in the proliferation of HSPCs induced by anti-CD69 is compatible with the increase in these precursors found in the bone marrow despite observing that the treatment also mobilizes these cells, as we observe them in the spleen.
  • the induction of proliferation of HSPCs, especially in the most primitive LT-HSCs and ST-HSCs has the risk of inducing loss of stem cell capacity.
  • this capacity by means of the production test of colony forming units (Fig. 5C), finding that the number of HSCs cells was correlated with the capacity to produce colonies, which demonstrates that anti-CD69-induced proliferation does not lose colony forming capacity
  • CXCR4 is the most relevant chemokine in the mobilization of hematopoietic cells due to its interaction with its CXCL12 ligand, an interaction that balances the balance between cell output and retention from the bone marrow to the periphery. CXCR4 expression was measured in both bone marrow and spleen (Fig. 6).
  • the graphs show both the percentage and the Geometric Mean (geometric mean), with an increase in the expression of CXCR4 both in the bone marrow and in the spleen of mice treated with human anti-CD69 2.8, as well as in the number of cells that express CXCR4 in the spleen with treatment, with no changes in bone marrow.
  • This observation points to the regulation of CXCR4 by way of CD69 affecting the mobilization of precursors. Together, therefore, the CD69 molecule is a drug target for the mobilization and proliferation of hematopoietic precursors.
  • Example 2 Study of the mobilization of hematopoietic precursors by two successive treatments with the human anti-CD69 Acm 2.8.
  • the results indicate that the action on the CD69 molecule with specific antibodies induces an output of hematopoietic precursors and an increase of these that include HSCs to the peripheral circulation.
  • Anti-CD69 induces a proliferation of HSCs that rapidly increases this cell population without losing its colony forming capacity. During this mobilization a change in the expression of CXCR4 is induced, potentially the route of action to induce precursor output. Together it is shown that the CD69 molecule acts as a target for the mobilization of hematopoietic precursors.

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Abstract

La présente invention concerne l'utilisation de modulateurs qui inhibent la fonction de la molécule CD69, de préférence d'un anticorps anti-CD69, pour l'élaboration d'un médicament conçu pour provoquer la prolifération et la sortie ou la mobilisation de cellules hématopoïétiques depuis la moëlle osseuse chez un sujet, de manière à être utile pour prévenir et/ou traiter des leucopénies, thrombopénies et/ou pancytopénies.
PCT/ES2018/070103 2017-02-20 2018-02-13 Utilisation de modulateurs de la fonction de cd69 pour la mobilisation et la prolifération de précurseurs hématopoïétiques Ceased WO2018150066A1 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2024112867A1 (fr) 2022-11-23 2024-05-30 University Of Georgia Research Foundation, Inc. Compositions et méthodes d'utilisation de celles-ci pour augmenter les réponses immunitaires

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Publication number Priority date Publication date Assignee Title
WO2024112867A1 (fr) 2022-11-23 2024-05-30 University Of Georgia Research Foundation, Inc. Compositions et méthodes d'utilisation de celles-ci pour augmenter les réponses immunitaires

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