WO2018150066A1 - Use of cd69 function modulators for mobilising and proliferating haematopoiesis precursors - Google Patents

Abstract

The present invention relates to the use of modulators that inhibit the function of the CD69 molecule, preferably an anti-CD69 antibody, to prepare a medicinal drug that causes the proliferation and output or mobilisation of haematopoietic stem cells from the bone marrow of a patient, and is therefore useful for preventing and/or treating leukocytopenia, thrombocytopenia and/or pancytopenia.

Description

 USE OF CD69 FUNCTION MODULATORS FOR MOBILIZATION AND PROLIFERATION OF HEMATOPOYETIC PRECURSORS

DESCRIPTION

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.

STATE OF THE TECHNIQUE

Many animals, including humans, under certain conditions exhibit an inability to provide the necessary or beneficial amounts of blood elements, either due to certain diseases or by therapeutic treatments. For example, leukopenia is a blood disorder characterized by a decrease in the number of leukocytes in the blood that can be caused by certain treatments such as chemotherapy or radiotherapy. Blood cell production is maintained by hematopoietic stem / progenitor cells (HSPC) that reside in specialized niches within the bone marrow (MO). One of the main mechanisms that HSPCs retain in their MO niches involves the interaction of the CXCR4 receptor with the stromal-derived akyokine factor 1 (SDF-1 / CXCL12). 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. The most commonly used in clinical practice is the granulocyte colony stimulating factor (G-CSF), which is combined in certain cases with the compound AMD3100 (Phenylenebis (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. However, with G-CSF alone, 35% of patients are unable to mobilize a sufficient number of HSPC cells to ensure grafting and sustained hematopoietic recovery. Thus, it is of particular interest to identify new agents that alone or in combination with the previous ones lead to more efficient mobilization strategies, especially in those patients who are at risk of mobilization failure. Other mobilizers are currently being tested: modulators of CXCL12 / CXCR4, inhibitors of the vascular cell adhesion molecule-1 / very late antigen 4 (VCAM / VLA-4), parathyroid hormone, proteosome inhibitors, Grop and stabilizers of HIF However, all these treatments have side effects (Hopman RK et al. 2014 Blood Rev 28 (1): 31-40). Similarly, among the developing strategies are the use of S1 P receptor modulators, such as the FTY720 agonist, also used for multiple sclerosis and which has side effects, such as heart damage.

The most recent studies focus on the composition of mobilized progenitor cells in terms of stem cells and leukocyte progenitors, and how the different mobilization strategies according to the mobilized cells for hematopoietic precursor grafts (autotransplants and allotransplants) can influence The result of the patients. The pharmacological treatment for the mobilization of HSPCs, their blood collection and their implantation, is a field of intense study. 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. In the context of HPSCs cell transplantation, in particular, 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. However, today there is a problem that the mobilization of hematopoietic progenitor cells or HPSCs, especially in some patients, is poor and for a correct treatment or prevention of the indicated pathologies more efficient mobilization strategies are required. On the other hand, due to the success in the application of stem cells for the treatment of hematopoietic disorders, researchers from other clinical specialties are looking for a source of stem cells that can be used safely and effectively for the treatment of damaged organs (for example , heart, spinal cord or liver). Thus, interest in efficient treatment strategies continues using cells with potential for wide differentiation such as those obtained from hematopoietic tissues. Therefore, a method is needed that achieves a more efficient output of hematopoietic precursors from bone marrow for both therapeutic and preventive applications, for example against leukopenia.

DESCRIPTION OF THE INVENTION The new strategy described in this invention for the mobilization of hematopoietic progenitor cells from bone marrow to the blood and lymphatic system is based on the modulation of the CD69 leukocyte molecule. Here it is demonstrated that the modulation of CD69 can be used to regulate the mobility of hematopoietic progenitor cells from their niche in the bone marrow to the blood, lymph and lymphatic organs, to subsequently treat a variety of medical situations that require transplantation of these HSPCs. 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. In addition, this treatment allows the expansion of hematopoietic progenitor cells while preserving their regenerative capacity.

The use of the CD69 molecule as a target for the proliferation and mobilization of hematopoietic precursors from bone marrow is a new strategy, since the use of modulators of the CD69 molecule with the proliferation and mobilization of hematopoietic precursors has not been linked to date. Thus, it has been described that 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). No differences were found in the precursor populations analyzed in bone marrow, although the number of stem hematopoietic progenitors was not examined in depth between CD69 (- / -) and CD69 (+ / +). In addition, these studies showed that lymphocyte maturation processes were not altered in the CD69 (- / -) mice and thus, the functions of the NK and CTL cells of the CD69-deficient mouse showed a cytotoxic activity similar to that of the mouse wild and lymphocytes deficient in CD69 had a normal proliferative response to stimuli of both T and B cells. 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.

In a first aspect, 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. That is, 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). As used herein, CD69, also known as "very early activation protein", "activation induction molecule" and "gp34 / 28", refers to mammalian CD69 protein, preferably human CD69 protein. The term "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 astringent conditions, preferably highly astringent conditions. In a preferred embodiment, the CD69 is a natural variant or allele of CD69. Examples of sequences of the CD69 polypeptide that are within the scope of the present invention, apart from SEQ ID NO: 1, are, but are not limited to, AR380696 (sequence 1241 of US6607879), AX774846 (sequence 162 of WO03038129), CS500461 ( sequence 162 of EP1767656), FB715394 (sequence 19 of WO200714001 1).

The person skilled in the art knows that 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. et al., 2005, Trends Immunol., 26 (3): 136-40, PM ID: 15745855), its expression is much more intense and persistent in leukocyte infiltrates including those of various chronic inflammatory diseases such as rheumatoid arthritis and chronic viral hepatitis, in the white blood cells responsible for transplant rejection, the white blood cells involved in the allergic response, immune cells in atherosclerotic lesions, lymphocytes that infiltrate tumors or during persistent infections. There is evidence that CD69 is involved in the activation of bone marrow derived cells (Testi R. et al., 1994, Immunol Today, 15 (10): 479-83, PMID: 7945773). However, hematopoietic development and maturation of T lymphocytes are almost normal in CD69-deficient mice under physiological conditions (Lauzurica et al., 2000 Blood Apr 1 95 (7): 2312-20). However, as explained above, the use of the CD69 molecule as a target for the proliferation and mobilization of hematopoietic precursors from bone marrow is a new strategy, since the use of modulators of the CD69 molecule has not been linked to date. with the proliferation and mobilization of hematopoietic precursors. The term "identical" or "substantially 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. In the case of antibodies, 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) . Ideally, 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. Thus, amino acid residues or nucleotides of both chains are compared in corresponding positions. When there is an exact match for both sequences in a given position, then both sequences are identical in that position (the term "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. Ideally, 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.

In the present invention, "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. CD34 + and high expression of the c-Kit marker (c-Kit ^hl ) (Grant A. Challen et al Cytometry A. 2009 Jan; 75 (1): 14-24). In addition, these hematopoietic precursor cells can be classified according to the expression of the FLT3 and CD34 markers in: LT-HSC or long term HSC (KSL CD34 ^neg FLT3 ^neg ), ST-HSC or short term HSC (KSL CD34 ⁺ FLT3 ^neg ) or MPP or multipotent parents (KSL CD34 ⁺ FLT3 ⁺ ) (Merchant A, Blood 2010, 115 (12): 2391-2396).

In the present invention, 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. Mostly, 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.

In a preferred embodiment of the first aspect of the invention 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. Therefore, the present invention relates to a CD69 modulator for use in the prevention and / or treatment of hematopoietic disorders associated with a deficient production of blood cells, preferably of leukopenia, thrombopenia or pancytopenia in a subject. Preferably the leukopenia is neutropenia, lymphopenia, eosinopenia or monocytopenia. Thus, this prevention and / or treatment occurs in the same individual where a proliferation and mobilization of hematopoietic precursors has been previously induced as indicated in the present invention.

In another preferred embodiment of the first aspect of the invention 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.

The transplant can be in the same individual where the proliferation and mobilization of hematopoietic precursors (autologous) or in another individual (allogeneic or heterologous) has been induced. The allogeneic transplant can be syngeneic, allogeneic of HLA (histocompatibility antigen) identical, haploidentical or unrelated donor. Therefore, when the transplant is allogeneic, the prevention and / or treatment of hematopoietic disorders associated with a deficient production of blood cells, preferably of leukopenia, thrombopenia or pancytopenia, It occurs in an individual who has been transplanted the hematopoietic precursors obtained (extracted) after their proliferation and mobilization in the first individual.

 In the present invention, "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.

Examples of CD69 modulators include, but are not limited to, CD69 antisense, preferably mRNA antisense, RNAi (interference ribonucleic acid) to interfere with the CD69 messenger, an anti-CD69 antibody molecule, and other compounds identified by some method described here, for example, compounds that interact with CD69, small molecules that bind to CD69 and antagonize their activity, and modulating anti-CD69 antibody molecules. Examples of 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. Examples of other 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.

The term "inhibitor," as used herein, 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 term "blocker", as used herein is a molecule, such as an antibody, that binds to CD69 without causing reaction but prevents other molecules, for example other antibodies, from binding to CD69.

In a more preferred embodiment, the CD69 modulator is selected from the list consisting of: inhibitor, agonist, antagonist, blocker or an interfering RNA. In a more preferred embodiment, the modulator is a monoclonal antibody (said antibody may have inhibitory, agonist, antagonist or blocking activity, for example) or a mixture of monoclonal antibodies. Preferably 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).

Methods for generating monoclonal antibodies (both human, murine, camelid or other vertebrates), humanizing antibodies, generating chimeric antibodies, deimmunizing antibodies and generating antibody fragments are known to those skilled in the art.

Examples of 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 u 190, Glu 185 and Lys188. Preferably 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. Preferably, the anti-CD69 antibody molecule binds to the extracellular domain of CD69 (for example, to an epitope of CD69 located outside the cell). Examples of 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. Human CD69 2.22 or a human anti-CD69 antibody known in the literature that can act as an agonist, blocker, antagonist or eliminator of CD69 or antibody molecules that recognize epitopes that overlap with the epitope recognized by such antibody or that compete with such antibody for the Union.

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. (Eds) Leukocyte Typing IV, Springer-Verlag, New York, 1989, p. 428); MLR3 (as described, for example, in Corte et al, 1981, Eur J Immunol., 1 1 (2), 162-164); EA-1; Leu 23 (as described, for example, in Lanier, et al, 1988, J Exp Med., 167 (5): 1572-85); and C1.18, E16.5 (as described, for example, in Gerosa, et al, 1991, Mol Immunol., 28 (1-2): 159-68).

Examples of antibody molecules that can be used in the invention include but are not limited to: an antibody molecule that interacts with, for example, binds to, an epitope that includes one or more residues of the neck region of a polypeptide of CD69 (for example, one or more residues of 62-84 of human CD69); an antibody molecule that interacts with, for example, binds to, one or more residues of the external domain (or carbohydrate recognition domain, CRD) of a CD69 polypeptide (eg, one or more residues of residues 82 to 199 of human CD69); an antibody molecule that interacts with, for example, binds to, an epitope that includes one or more residues of the intracellular domain of a CD69 polypeptide (eg, one or more 1-40 residues of human CD69); an antibody molecule that interacts with, for example, binds to, an epitope to which when it binds modulates, for example, increases or decreases, the interaction of cytoplasmic and / or transmembrane CD69 regions with an effector, whose activity can determined by the methods described herein (for example, increased expression of CXCR4 and / or S1 P1 receptors, production of TGF- [beta], MAPK activation, or Ca2 + signaling); an antibody molecule that interacts with, for example, binds to, an epitope (for example, a conformational or linear epitope), which is modulated when the antibody is bound, for example, increases or decreases the formation of CD69 dimeros (for example, an epitope that includes the Cys68 residue of the human CD69 or a residue located near the Cys68); an antibody molecule that can modulate the binding of a CD69 ligand, for example, an antibody that can inhibit, for example, competitively inhibit, or enhance the binding of a ligand to CD69; an antibody molecule that interacts with, for example, binds to, or that can inhibit or enhance the binding of a ligand to one or more amino acid residues of human CD69, preferably to residues Glu 140, Asp171, Glu 180, Glu 185, Glu 187, Phe 175, Met 184, Leu 190, Glu 185 and Lys188.

In a preferred embodiment, the anti-CD69 antibody molecule binds to all or part of the epitope recognized by an antibody described herein as, for example, by a human anti-CD69 antibody known in the literature that can act as an antagonist or a eliminator, or by a human anti-CD69 antibody analogous to a mouse anti-CD69 antibody, for example, antibody 2.2, 2.3, or H1.2F3 (described, for example, in Esplugues et al. 2003 J Exp Med May 5, 197 (9): 1093-106). The anti-CD69 antibody can bind to one or more residues of the described epitopes or compete for binding to an antibody that binds to one of the described epitopes. The anti-CD69 antibody molecule can, for example, competitively inhibit the binding of an antibody described herein, for example, a human anti-CD69 antibody known in the literature as described herein against human CD69. An anti-CD69 antibody molecule can bind to an epitope, for example, a conformational or linear epitope, so that when they interact, the binding of one of the antibodies described herein is prevented, for example, a human anti-CD69 antibody known in the literature as described here against human CD69. The epitope may be very closely or functionally associated, for example, an overlapping or adjacent epitope in a linear or conformational sequence, to one of those recognized by an antibody described herein, for example, a human anti-CD69 antibody known in the literature as the described here against human CD69.

In a preferred example, the interaction, for example, binding, between an anti-CD69 and CD69 antibody molecule occurs with high affinity (eg, affinity constant of at least 3x10 ⁷ M ^"1 , preferably, between 3x10 ⁸ M ^" and 3x10 ¹⁰ M ^{" 1} , or about 3x10 ⁹ M ^" ) and specificity. Preferably, the anti-CD69 antibody molecule modulates the immune response, for example, it acts as a blocker, antagonist or eliminator of CD69.

As used herein, "specific binding" or "specificity" refers to the property of the binding agent, preferably the antibody, of: (1) binding to CD69, for example, a human CD69 protein, with an affinity of at minus 1x10 ⁷ M ^"1 , and (2) preferably bind to CD69, for example, the human CD69 protein, with an affinity that is at least twice, 50 times, 100 times, 1000 times, or greater than its binding affinity to a non-specific antigen (eg, bovine serum albumin, casein) other than CD69.

As can be seen in the present invention, many types of anti-CD69 antibody molecules, for example, antibodies, or fragments thereof that bind to the antigen, are useful in the methods of this invention. The antibody molecules can be of several isotypes, including: IgG (for example, lgG1, lgG2 (for example, lgG2a, lgG2b), lgG3, lgG4), IgM, lgA1, lgA2, IgD, or IgE. A preferred antibody molecule is of the IgG isotype. 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).

The term "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. In the context of the present invention, 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.

As used herein, the term "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. Thus, 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. Such non-human fragments can be inserted into human, humanized molecules, or other arrangements that make them less antigenic when administered to a human.

Thus, 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.

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.

Preferred antibody molecules may include at least one, and preferably two, variable regions of the heavy chain (VH) or their antigen binding fragments, and at least one or preferably two variable regions of the light chain (VL) or their antigen binding fragments. The VH and VL regions are subdivided into regions of hypervariability, called complementarity determining regions (CDR), interspaced with regions that are more conserved, called structural regions (FR). The extent of the structural regions and the CDRs are known to the person skilled in the art. Preferably, 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. In one example, 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.

As discussed above, the antigen-binding fragments of an antibody molecule are within the term "antibody molecule." An antigen binding fragment, as used herein, may refer to a portion of an antibody that specifically binds to CD69 (eg, human CD69). Examples of 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. Alternatively, 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, can be linked using methods recombinants, by a synthetic union that allows them to constitute a simple chain of protein in which the pair of VL and VH regions form monovalent molecules (known as single chain Fv (scFv)). Such single chain antibodies are also included within the term "antigen binding fragment" of an antibody. These antibody fragments are obtained using known conventional techniques, and the fragments are studied for their usefulness in the same manner as intact antibodies.

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. The hybridomas are selected and subcloned by dilution. Clones suitable for expansion constitute a hybridoma cell line. Then, 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. Optionally, 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. The term "antibody or recombinant antibody molecule", as used herein, 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. Such recombinant antibody molecules include humanized, chimeric, deimmunized CDR antibody molecules generated in vitro (eg, by phage selection), and may optionally include constant regions derived from human germline immunoglobulin sequences. Monoclonal, chimeric and humanized antibodies, which have been modified by, for example, destruction, addition, or substitution of other portions of the antibody, for example, the constant region, can also be used in the present invention. For example, 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.

In a particular embodiment, 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. For example, 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. Preferably, 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. The constant regions of these species are known. Methods for altering the constant region of an antibody are known. Antibodies with impaired function, for example, affinity altered by an effector ligand, such as the FcR in a cell or the C1q component of the complement, can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see, for example, EP 388, 151 A1, US 5,624,821 and US 5,648,260) and can be used in the present invention. Similar types of alterations have been described, so that if applied to murine or other species immunoglobulins, they would reduce or eliminate these functions. In a preferred embodiment, the antibody of the invention is linked to a marking agent that allows its location and / or identification, by spectroscopic, photochemical, biochemical, immunochemical or chemical means.

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.

Accordingly, 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. For example, 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).

One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies).

Suitable crosslinking agents are those that are heterobifunctional, having two distinct reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate).

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). Examples of 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 detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, [beta] -galactosidase, acetylcholine esterase, glucose oxidase and others of the same type. When an antibody is derivatized with a detectable enzyme, it is detected by the addition of reagents that the enzyme uses as substrates to generate a detectable reaction product. For example, when the detectable agent of horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product that is detectable. An antibody can also be derivatized with a prosthetic group (eg, streptavidin / biotin and avidin / biotin). For example, an antibody can be derivatized with biotin and detected through the indirect measurement of the binding of avidin or streptavidin. Examples of 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. An early activation anti-polypeptide antibody or a fragment thereof that binds to the antigen can be conjugated to another molecular entity, typically a brand or therapeutic agent or structure (eg, cytotoxic or cytostatic). A cytotoxin or cytotoxic agent includes any agent that is destructive to the cells with which it interacts. Examples are taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, ethoxide, tenopoxide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin- diona, mitoxantrone, mitramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propanolol, puromycin, maitansinoids, eg, maitansinol (see US 5,208,020), CC-1065 (see US 5, 475, 092, 5, 585, 499, 5,846,545) and their analogues or counterparts. 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, vinblastine, taxol and maitansinoids) . 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. In one example, 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.

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.

Useful in this invention include mimetic agents of the early activation polypeptide. These agents, including peptides, semi-peptide or non-peptide compounds (such as organic molecules of small molecular size), are inhibitors of the activity of the early activation polypeptide. In an optimal embodiment, the agent is part of a combinatorial library, for example a collection of organic peptides or molecules, or part of a library of natural products. In these circumstances, a group of test compounds may include 10, 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ or 10 ⁸ compounds, which share structural or functional characteristics.

In its current form, this invention includes libraries of early activation polypeptide binding agents. The genesis of combinatorial libraries is well characterized in the literature and has been thoroughly reviewed. The libraries of compounds of this invention can be prepared according to different methods, some of which have been previously characterized. For example, a strategy called division can be implemented as follows: functionalized polymer substrate microspheres are placed in different reaction vessels; there is a wide variety of polymeric substrates suitable for solid phase peptide synthesis, and some are commercially available (as examples, see M. Bodansky Principles of Peptide Synthesis, 2 edition (1993), Springer-Verlag, Berlin). A solution containing an activated amino acid is added to each aliquot of microspheres, and the reactions are carried out, resulting in a series of immobilized amino acids, each in a reaction vessel. The aliquots of derivatized microspheres are washed and collected (recombination), and this set is again divided, placing each aliquot in a new reaction vessel, adding a new activated amino acid to each aliquot. This cycle is repeated until peptides of the desired length are achieved. 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. Thus, a wide variety of non-peptide peptidomimetic compounds can be obtained.

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. In another illustrative example, 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.

Subsequently, screenings can be carried out to determine which members of the library have a desirable activity, and if so, identify the active substance. Combinatorial analysis methods of library screening have been described (see Gordon et al., J. Med. Chem., Supra). Soluble compound libraries can be identified by affinity chromatography with an appropriate receptor to isolate ligands for the receptor, followed by the identification of ligands isolated by conventional techniques, such as mass spectrometry, NMR and the like. The immobilized compounds can be identified by contacting them with a soluble receptor, preferably coupled to a label (fluorophores, colorimetric enzymes, radioisotopes, luminescent compounds and the like) that can be detected by indicating ligand binding. Alternatively, immobilized compounds can be selectively released, allowing diffusion through a membrane to interact with a receptor. Thus, the interaction of the compounds identified by screening with the early activation polypeptide can be tested by testing the ability of each compound to interact with it, for example, by incubating the investigated compound with the early activation polypeptide and a lysate in a container. suitable reaction, such as a standard 96-well plate. In this situation, the activity of each individual compound can be determined, being used as control a well or wells without the compound tested. After incubation, the activity of each compound can be determined in each well. Therefore, the activities of a plurality of compounds can be determined in parallel. Thus, the binding of large amounts of different compounds can be determined simultaneously. For example, 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. Subsequently, 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.

Combinatorial libraries of compounds can be synthesized with labels that encode the identity of each member of the library. In general, this method includes the use of inert, but easily detectable, markers that bind to solid support or compounds. When an active compound is found (by one of the techniques described above), the identity of said compound is determined by the identification of the accompanying label. This method of marking allows the synthesis of large libraries of compounds that can be identified even at very low levels. Such a mapping scheme can be useful (for example, in the nano-drop screening system), to identify compounds released from microspheres.

On the other hand, it is understood that 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. The 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.

A non-essential residue is defined as one that can be altered with respect to the wild type of the binding reagent (antibody or others), without inhibiting, or better yet, without substantially altering the biological activity, while a residue Essential amino acid is one that results in such changes.

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.

The term "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. In the context of the present invention, 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.

In the present invention, the terms "composition", "pharmaceutical composition", "drug" and "medicament" are used interchangeably.

The term "pharmaceutical composition" here refers to any substance that is used for the prevention, diagnosis, relief, treatment or cure of diseases in humans or animals. The pharmaceutical composition of the invention can be used alone or in combination with other pharmaceutical compositions. In the context of the present invention it refers to a pharmaceutical composition or medicament characterized in that it comprises the modulator of the invention or the polynucleotide that encodes it, which allows its expression in the organism to be treated, in a therapeutically effective amount, such that the modulator of the invention performs its function in the target tissue or cell. In a preferred embodiment the composition also comprises an excipient and / or an acceptable pharmaceutical vehicle.

The term "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. The term "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. When the presentation is liquid, the pharmacologically acceptable carrier is the diluent.

In another preferred embodiment, the pharmaceutical composition also comprises an adjuvant. Here, the term "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.

In a preferred embodiment the inhibitor is an interference RNA, a microRNA or an antisense nucleic acid chain.

In the present invention, in a preferred embodiment, 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.

In another more preferred embodiment, the CD69 modulator or medicament comprising it is also used in combination with at least one additional modulator that is selected from the list consisting of: colony stimulating factor, granulocyte and macrophage stimulating factor, an inhibitor of CXCR4, a c-kit modulator, a CXCL12 / CXCR4 modulator (preferably a CXCL12 / CXCR4 inhibitor), a SIP agonist, a VCAM / VLA-4 inhibitor, parathyroid hormone, a proteasome inhibitor, Grop and / or a HIF stabilizer. This combined use can be simultaneous or sequential.

In an even more preferred embodiment, the CXCL12 / CXCR4 inhibitor is TG-0054, 1, 1 '- [1, 4-Phenylenebis (methylene)] bis [1, 4,8, 1 1-tetraazacyclotetradecane] (AM D3100, plerixaflor or Mozobil), NOX-A12, C28H54N8, pol6326, BKT-140 or T6-0054.

In another even more preferred embodiment, the SIP agonist is SEQ2871.

In another even more preferred embodiment, the proteasome inhibitor is Bortezomib. In another even more preferred embodiment Grop is SB-251353.

In another even more preferred embodiment, the HIF stabilizer is FG-4497.

In the present invention 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. In another preferred embodiment of the first aspect of the invention 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. In a more preferred embodiment, the CD69 modulator or medicament comprising it is preferably administered for 5, 6 or 7 days to the subject. Preferably 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.

In the present invention the term "therapeutically effective or effective amount" (or therapeutically effective dose ") 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.

In another even more preferred embodiment, 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:

 to. administering to a subject a CD69 modulator, preferably an anti-CD69 antibody, or the medicament defined in the present invention; b. collecting from blood, lymph or lymphatic organs the hematopoietic stem cells of the subject of step (a), that is, from a subject who previously received a CD69 modulator, preferably an anti-CD-69 antibody, or the medicament defined herein invention, preferably the apheresis collection is performed; Y

 C. 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:

to. administering to a subject 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

 C. Optionally store the cells obtained in step (b) until use.

Here the terms "amino acids", "amino acid sequence", "polypeptide", "peptide" and "oligopeptide" are used interchangeably.

Here the terms "nucleotides", "nucleotide sequence", "polynucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention. BRIEF DESCRIPTION OF THE FIGURES

The experiments were performed on humanized HU: CD69 +/- mice, which are transgenic mice carrying the human CD69 molecule but deficient for the mouse CD69 molecule (MU: CD69 - / -). In all experiments, the HU: CD69 +/- mice were treated with 500 μg 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 μg 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). FIG. 2. Treatment with human anti CD69 promotes the outflow of lymphoid and myeloid cells from the bone marrow to the periphery. HU: CD69 +/- mice were treated with 500 μg of anti-hCD69 2.8 Ac 24 hours before analysis. A, Percentages in bone marrow and spleen and 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 μg 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. HU: CD69 +/- mice were treated with 500 μg 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 μg 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. B, On the other hand, 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 6. 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 μg 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. 8. Treatment with human anti-CD69 2.8 in HU individuals: CD69 +/- induces an increase in the number of regulatory CD25 + Foxp3 + CD4 + T cells and not Regulatory Surface expression of CD25 and intranuclear FoxP3 factor in spleen, ganglion and thymus cells is shown by flow cytometry of spleen (A), ganglia (B) and thymus (C). HU: CD69 +/- mice were treated 12 and 5 days before analysis. Data set 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 μg 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.

EXAMPLES The invention will now be illustrated by tests carried out by the inventors, which show the effectiveness of the product of the invention.

Example 1: Treatment with human anti-CD69 monoclonal antibody (Acm) 2.8 contributes to the outflow from the bone marrow of hematopoietic cells to the circulation of the blood and lymphatic system.

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. To know the effect that treatment with human anti-CD69 Acm has in vivo, the humanized mouse model HU: CD69 +/-, transgenic carrier of the human CD69 molecule but deficient for the mouse CD69 molecule (MU: CD69 - / -). Thus, the effects seen by the treatments on human CD69 will not be influenced by the presence of the mouse CD69 molecule. In this model, 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.

The mobilization of cells from the marrow to the blood and peripheral organs is caused by different treatments demanded in clinical practice. 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 (δθθμς) 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. Correspondingly, but in a slower kinetics, 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. We consider the spleen test as an indirect reading of the number of blood cells. In addition, we observe that 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. According to the significant decrease in total bone marrow cells, a large reduction in the number of cells of the most abundant populations, B lymphocytes, macrophages and neutrophils was observed, while the rest of the populations remain the same and the eosinophils increase ( Fig. 2B). Because the treatment induces a very important neutrophil output, this decrease is detected in the% analysis (Fig. 2A). In spleen, we find the numbers of the majority of the populations analyzed increased (Fig. 2B). Blood cell production is maintained by hematopoietic stem / progenitor cells (HSPC) that reside in specialized niches within the bone marrow. There are treatments with different factors that affect the interaction of HSPCs with your niche. In clinical practice one of the factors used is AMD3100 that interferes with the interaction of CXCR4 with CXCL12 / SDF-1. In this example, the effect of human anti-CD69 2.8 and AMD3100 will be compared. We characterized the effect on the mobilization of hematopoietic precursors of the treatment with human anti-CD69 Acm 2.8 and compared it with that of the CXCR4 inhibitor AMD3100 (Fig. 3). Although we do not know the exact kinetics of mobilization by treatment with human anti-CD69 2.8 Acm (a single dose of 500 μg), we chose to analyze the effect at 24 hrs of the treatment and compare it with that of the CXCR4 inhibitor at 1 hr, when It is known that it reaches the maximum of cells mobilized in blood. The decrease in the total number of bone marrow cells is similar in both treatments and the increase in spleen cells is also similar (Fig. 3A). In the analysis of bone marrow subpopulations of mice treated with human anti-CD69 2.8 (Fig. 3B) it is observed that all cell types are mobilized in a similar way to that induced by AMD3100 although there are different characteristics. In the spleen, differences are observed being the most pronounced in B lymphocytes, neutrophils and macrophages (Fig. 3C). Likewise, in 24-hour treatments with human anti-CD69 2.8 we analyzed the mobilization by studying the pluripotent hematopoietic precursors, characterized by the absence of markers of any of their lineages (T, B, NK lymphocytes, myeloid cells, neutrophils, monocytes, macrophages, neutrophils and dendritic cells), which is called lin-, and classified according to the expression of Sea and c-Kit (fig. 4). We observed that there was a significant increase in cells of the subpopulation lin-, Sca +, cKit ^hl , which contain primitive precursors and an increase in the lin-Sca + cKit ' ^{nt population} that is composed of the common precursors to the lymphoid line (CLP) , both in the bone marrow of treated individuals and in the spleen. Thus, the treatment mobilizes these primitive precursor cells, since they are found in the spleen, but also surprisingly the number of these cells in the bone marrow increased. This data was corroborated by the analysis of primitive progenitor cells with markers that define populations within the Lin- which are considered long-term hematopoietic stem cells (lin-LT-HSC), that is, they are stem cells that remain for a long time giving It leads to the next population, called short-term hematopoietic stem cells, which is shorter and gives rise to multipotent parents (MPP) (Fig. 4C). A very significant increase in the most primitive subpopulations induced by the treatment is observed. When we assess the proliferative capacity in bone marrow and spleen cells in individuals treated with human anti-CD69, we observe that the treatment increases the proliferation capacity of most lymphoid and myeloid cells, and occurs in both bone marrow and spleen (Fig. 5A). We also assess the proliferation capacity induced by treatment with 2.8 in populations of lin- precursor cells and compare it with untreated and treated individuals with a AMD3100 dose (Fig. 5B). The results indicate that the action of the anti-CD69 2.8 Acm in bone marrow induces cell proliferation in the HSCs and MPP populations that more than double that produced by AMD3100 and that observed in the absence of treatment. Thus, 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. But 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. Thus, we measured 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

Similarly, treatment with anti-CD69 induces a large proliferation in the hematopoietic subpopulations of precursors found in the spleen, which is much higher than that induced by treatment with AMD3100 (Fig. 5B). As previously described, 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.

Subsequently, given the kinetics of bone marrow cell output observed in Figure 1, we considered whether it was possible to induce mobilization with two successive treatments with a smaller amount of human anti-CD69 2.8. This type of treatment could be useful in processes that require long-term treatments. When the mice were treated with two doses of antibody (200 μg / dose) at 12 days and 5 days before the analysis, it was observed that in the treated mice compared to the untreated mice the absolute number of bone marrow cells decreased in a way important and increased in spleen and lymph nodes and no significant changes in thymus were observed between treated and untreated mice (Fig. 7A). The spleen weight was increased corresponding to the cellular change observed in this organ, while the thymus did not change (Fig. 7B). Overall, the mobilization induced by the 2 treatments is similar to that observed with a single treatment with 500μg of the antibody. Likewise, the analysis of leukocyte subtypes mobilized with the anti-CD69 2.8 Acm shows that both lymphoid and myeloid cells in the bone marrow decrease and increase in peripheral locations but do not vary in thymus (Fig. 7C-F). In addition, the analysis of lymphoid and myeloid populations in spleen revealed that the majority of lymphoid subpopulations were increased when the mice had been treated with anti-CD69 2.8, while within the myeloid cells only eosinophils were increased (Fig. 7E). Finally, we analyzed the thymus cells and did not observe changes in the observed populations (Fig. 7F). It is important to note that the treatment with the anti-CD69 2.8 Acm induced in the CD25 + CD4 + regulatory T populations an increase proportional to the increases in total cells in peripheral locations, not varying in thymus (Fig. 8). Thus, the presence of regulatory cells that increase proportionally in the spleen (Fig. 8A) and nodes (Fig. 8B) to the increase of non-regulatory FoxP3-CD25 cells - will give greater assurance of a good regulation of the implant process, since in the Recovery after a hematopoietic transplant as crucial is the hematopoietic reconstitution as the immune one.

Example 3: Study of the mobilization of hematopoietic precursors by treatment with the murine anti-CD69 Acm 2.2.

To study whether the mouse CD69 molecule acts as the human CD69 molecule in the mobilization of hematopoietic precursors, we use the mouse anti CD69 2.2 antibody, which specifically recognizes mouse CD69 to treat WT mice and thus expressing CD69. WT CD69 + / + mice were treated with 500 of murine anti-CD69 Ac 2.2 24 h before analysis and the result was analyzed in bone marrow, spleen and ganglia (Fig. 9). The treatment with murine anti-CD69 2.2 has leukocyte mobilization effect from the bone marrow to the periphery measured in spleen and nodules (Fig. 9 A). The size of the spleen increases proportionally (Fig. 9B). The number of cells of different subpopulations of leukocytes in bone marrow decreased in multiple subpopulations (Fig. 9 C), and increased in different subpopulations of spleen (Fig. 9 D). Thus, in this example it is demonstrated that a specific anti-CD69 mouse antibody acting in vivo on the mouse CD69 molecule induces a mobilization of hematopoietic progenitors similar to that observed with the specific human anti-CD69 antibody acting in vivo on the CD69 molecule human This suggests that the function of the CD69 molecule is similar in the 2 species, mouse and human.

Taking into account the 3 examples, 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.

Claims

one . Use of a CD69 inhibitor for the preparation of a drug to cause the proliferation of hematopoietic precursors and their exit from bone marrow in a subject, where the inhibitor is a monoclonal antibody that specifically binds to CD69, an interfering RNA, a microRNA or an antisense nucleic acid chain. 2. Use of a CD69 inhibitor according to claim 1 wherein the medicament is for the prevention or treatment of leukopenia, thrombopenia and / or pancytopenia in a subject. 3. Use according to claim 2 wherein the leukopenia is neutropenia, lymphopenia, eosinopenia and / or monocytopenia. 4. Use according to claim 1 wherein the medicament is for obtaining said precursors for a transplant. 5. Use according to any one of claims 1 to 4 wherein the monoclonal antibody is selected from the list consisting of: humanized antibody, human antibody, chimeric antibody, immunized antibody, antibody fragments and nanobody. 6. Use according to any one of claims 1 to 5 wherein the CD69 inhibitor is monoclonal antibody 2.8 that specifically binds to human CD69, wherein said antibody comprises a heavy chain comprising SEQ ID NO: 9 and a light chain comprising SEQ ID NO: 10. 7. Use according to any of claims 1 to 6 wherein the subject has been or will be treated with chemotherapy and / or radiotherapy. 8. Use according to any one of claims 1 to 7 wherein the medicament is administered orally, parenterally, intra-muscularly, intra-peritoneally, intra-arterially, intra-venously, intra-tracheally, intra-nasally, transdermally, intra- dermal, intra-vaginal, intravesicular, epidural, subcutaneous, cutaneous, topical, otic, ophthalmic, inhalation, sublingual, vaginal, rectal, gastroenteric or mucous. 9. Use according to any of claims 1 to 8 wherein the medicament is administered for 5, 6 or 7 days. 10. Use according to any of claims 1 to 9 wherein the subject is a human being. eleven . In vitro use of a CD69 inhibitor to obtain hematopoietic precursors, where the CD69 inhibitor is a monoclonal antibody that specifically binds to CD69, an interfering RNA, a microRNA or an antisense nucleic acid chain. 12. In vitro method of obtaining hematopoietic precursors useful for a transplant comprising:  to. Collect from a isolated sample of blood, lymph or lymphatic organs the hematopoietic stem cells of a subject who previously received a CD69 inhibitor; Y  b. storing the cells obtained in step (a) until use, where the CD69 inhibitor is a monoclonal antibody that specifically binds to CD69, an interfering RNA, a microRNA or an antisense nucleic acid chain.