Use of Anti-CCR7 mabs for prevention or treatment of graft-versus-host disease (GVHD)

Abstract

To provide pharmaceutical and therapeutic methods for the prevention and treatment of GVHD, particularly for improving the survival rate in allogeneic HSCT.SOLUTION: The present invention provides a novel use and method comprising an antibody or an antigen-binding fragment thereof that binds to a CCR7 receptor, for use as a novel therapeutic agent for the prevention and / or treatment of graft-versus-host disease (GVHD), preferably in hematopoietic stem cell transplantation (HSCT), and more preferably in allogeneic hematopoietic stem cell transplantation. The GVHD of the present invention may be acute (aGVHD) and / or chronic (cGVHD), and is preferably acute. The antibody and antigen-binding fragment are capable of selectively depleting immune cells expressing CCR7 ex vivo or in vitro, and are capable in vivo of selectively killing immune cells expressing a CCR7 receptor and of impairing and / or blocking migration and activation of the immune cells involved in onset and progression of GVHD.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates generally to the fields of medicine and pharmacy, particularly to the field of biologics for use in organ, tissue, or cell transplantation and transplantation procedures. More particularly, the present invention relates to anti-CCR7 receptor antibodies useful for preventing and treating graft-versus-host disease. [Background technology]

[0002] In recent years, hematopoietic stem cell transplantation (HSCT) has been widely performed to treat various blood diseases, such as hematopoietic tumors, leukemia, or aplastic anemia. Moreover, cell transplantation is a useful treatment method in the medical field. HSCT is classified according to differences in stem cell source or donor selection. Common stem cell sources include bone marrow harvested from the iliac crest (Aschan. J. Br Med Bull. 2006; 77-78: 23-36), granulocyte-colony stimulating factor (G-CSF) or plerixafor mobilized peripheral blood stem cells (Bacigalupo et al., Haematologica. 2002 August; 87 (Suppl. No. 8): 4-8), and umbilical cord blood (Kestendjieva et al., Cell Biol Int. 2008 July; 32 (7): 724-32). HSCT can be autologous, where the stem cells are derived from the patient himself, or allogeneic, where the stem cells are derived from healthy individuals, including genotypically identical individual related donors sharing major and minor histocompatibility identities, human leukocyte antigen (HLA)-identical sibling donors, HLA-matched donors among extended family members, HLA-identical unrelated donors, mismatched related donors, mismatched unrelated donors, mismatched cord blood donors, and haplotype-mismatched related donors.

[0003] However, despite the use of highly sophisticated therapeutic approaches, HSCT is still associated with a significant mortality caused by several complications, such as graft-versus-host disease (GvHD), infectious diseases, venous occlusive disease, donor graft rejection, and relapse of the underlying disease; among these complications, GvHD is the most frequent and severe complication after allogeneic HSCT that must be addressed as it affects up to 30-70% of patients and is associated with significant morbidity and mortality.

[0004] GVHD is classically divided into acute and chronic forms. Acute GVHD (aGVHD) typically occurs from the time of engraftment to 100 days after transplantation, whereas chronic GVHD (cGVHD) occurs more than 100 days after HSCT. Both types of GVHD are further subdivided into multiple degrees depending on the clinical severity of the disease. However, this temporal distinction is becoming blurred due to new therapeutic approaches, and GVHD includes overlapping syndromes that share features of both. (Ferrara, JL et al., Lancet, 2009.373(9674):1550-61; Filipovich, AH et al., Biol Blood Marrow Transplant, 2005.11(12):945-56). Furthermore, GVHD is often considered as a single disease and divided into two phases: the acute phase of GVHD, which occurs early after HSCT, and the chronic phase, in which GVHD appears later in the course of transplantation (MacDonald et al., Blood. 2017;129(1):13-21).

[0005] Acute GVHD primarily affects the skin, gastrointestinal tract, and liver. Skin lesions typically consist of a maculopapular rash that may blister and ulcerate in the most extreme cases, with blisters and toxic epidermal necrolysis mimicking Stevens-Johnson syndrome. Gastrointestinal findings include abdominal cramps and pain, diarrhea, bloody stools, ileus, anorexia, nausea, and vomiting. Liver disease is due to damage to the bile canaliculi resulting in cholestasis and therefore hyperbilirubinemia and elevated alkaline phosphatase.

[0006] Chronic GVHD often resembles autoimmune diseases such as systemic sclerosis, with sclerosis and fibrosis commonly affecting the skin, eyes, mouth, stomach, liver, lungs, joints, and genitourinary system. Typical skin findings are sclerosis and poikiloderma, as well as lichenoid lesions. In the case of the lungs, bronchiolitis obliterans is the result of damage and obstruction of the bronchioles, resulting in high mortality rates.

[0007] The hematopoietic system is also commonly affected by thymic injury and cytopenias, both acute and chronic.

[0008] Some methods for preventing, treating, or suppressing GVHD are through the use of immunosuppressants such as calcineurin inhibitors (cyclosporine A and tacrolimus (FK506)), antiproliferative agents (methotrexate and mycophenolate mofetil), mTOR inhibitors (sirolimus or rapamycin), and steroids such as prednisone. Recent approaches involve in vivo removal of mature T cells from the transplant cell population (graft), and are directed to preventing or limiting activation and / or proliferation of autoreactive T or B lymphocytes using cyclophosphamide or antithymocyte globulin (ATG), and other treatments such as extracorporeal photochemotherapy, monoclonal antibodies such as rituximab, kinase inhibitors that interfere with B cell signaling, expansion of regulatory T cells, and the like. However, despite the promising development of these approaches, glucocorticoids still constitute the standard first-line therapy, despite substantial side effects of long-term use due to the non-specific and broad immunosuppressive effects of the drugs, and the toxicity of glucocorticoids is high, and thus infectious diseases due to compromised immune systems or tumor relapse have become problematic (Zeiser and Blazard. N Engl J Med 2017;377:2565-79. DOI:10.1056 / NEJMra1703472). Therefore, there is still a current need for the development of effective treatment or prevention methods and drugs for more selectively avoiding GVHD. Thus, there remains a need in the art for alternative and improved therapeutic approaches that do not suffer from the serious disadvantages of the prior art methods.

[0009] Human CC motif receptor 7 (hereafter referred to as "CCR7") is a seven-transmembrane domain G protein-coupled receptor (GPCR) that was initially found to be lymphocyte-selectively expressed upon EBV infection (Birkenbach et al., 1993, J. Virol. 67:2209-2220). CCR7 selectively binds two chemokines, designated CCL19 and CCL21. In homeostasis and inflammation, CCR7 is expressed on naive T and B lymphocytes, central memory T cells (TCM), some subsets of natural killer cells (NK cells), semi-mature and mature DCs, and plasmacytoid DCs (Forster R et al., Cell 1999;99:23-33; Comerford I et al., Cytokine Growth Factor Rev. 2013 June;24(3):269-83). In these leukocyte subsets, CCR7 regulates migration, organization, and activation.

[0010] Some publications have reported that donor T cells expressing CCR7 may be associated with the pathogenesis of GVHD (Portero et al., 2014, Blood 124:3930; Portero-Sainz et al., 2017, Bone Marrow Transplant. 52, 745-752; Coghill et al., 2010, Blood. 115(23):4914-22). However, none of this literature discloses that targeting CCR7 can be effectively used for the prevention or treatment of GVHD without the disadvantages associated with side effects of the prior art methods.

[0011] It is therefore an object of the present invention to provide pharmaceutical and therapeutic approaches for preventing and treating GVHD that overcome the disadvantages of prior art approaches. In particular, it is an object of the present invention to improve survival rates of allogeneic HSCT. Summary of the Invention [Means for solving the problem]

[0012] In a first aspect, the present invention relates to an anti-CCR7 antibody or an antigen-binding fragment thereof for use in preventing or treating graft-versus-host disease (GVHD) in a recipient of a transplant comprising donor cells. Preferably, the anti-CCR7 antibody has an IC50 of 100 nM or less for inhibiting at least one of CCR7-dependent intracellular signaling and CCR7 receptor internalization by at least one CCR7 ligand selected from CCL19 and CCL21. More preferably, the anti-CCR7 antibody inhibits CCR7-dependent intracellular signaling without substantial agonistic effects. Most preferably, the anti-CCR7 antibody has a Kd for the N-terminal extracellular domain of human CCR7 that is up to 20-fold higher than the Kd of a reference anti-CCR7 antibody, the reference anti-CCR7 antibody being a murine anti-CCR7 antibody whose heavy chain variable domain amino acid sequence is SEQ ID NO:1 and whose light chain variable domain amino acid sequence is SEQ ID NO:2.

[0013] In one embodiment, the anti-CCR7 antibody or antigen-binding fragment thereof for use in the present invention is a chimeric, humanized or human antibody. Preferably, the anti-CCR7 antibody is an antibody having the HVR of an anti-human CCR7 antibody in which the amino acid sequence of the heavy chain variable domain is SEQ ID NO: 1 and the amino acid sequence of the light chain variable domain is SEQ ID NO: 2.

[0014] In one embodiment, the anti-CCR7 antibody or antigen-binding fragment thereof for use in the present invention is an anti-CCR7 antibody that achieves at least one of killing CCR7-expressing cells in a recipient, inducing apoptosis of the cells, inhibiting migration of the cells, inhibiting activation of the cells, inhibiting proliferation of the cells, and inhibiting dissemination of the cells.

[0015] In the method or use of the present invention for preventing or treating GVHD in a recipient, the transplant comprising donor cells is preferably a transplant comprising one or more of organs, tissues, progenitor cells, stem cells, and hematopoietic cells. More preferably, the transplant comprising donor cells is a transplant comprising hematopoietic stem or progenitor cells. Most preferably, the recipient suffers from a malignant disorder, and preferably, the prevention or treatment of GVHD maintains or promotes a graft-versus-tumor effect or a graft-versus-leukemia effect.

[0016] In the method or use of the present invention for preventing or treating GVHD in a recipient, preferably, the prevention or treatment of GVHD comprises at least one of the following: a) administration of an anti-CCR7 antibody to the recipient before the recipient receives a graft comprising donor cells; b) administration of an anti-CCR7 antibody to the recipient after the recipient receives a graft comprising donor cells, and preferably before the recipient shows symptoms of GVHD or the recipient is diagnosed with GVHD; c) administration of an anti-CCR7 antibody to the recipient after the recipient receives a graft comprising donor cells, and preferably after the recipient shows symptoms of GVHD or the recipient is diagnosed with GVHD; d) administration of an anti-CCR7 antibody to the recipient of a graft comprising donor cells, the graft prepared prior to transplantation by ex vivo incubation with an anti-CCR7 antibody or antigen-binding fragment as defined above; and e) administration of an anti-CCR7 antibody to the recipient after relapse of GVHD.

[0017] In a further aspect, the present invention relates to an ex vivo method for preparing a donor-derived organ, tissue or cell preparation for transplantation into a recipient, comprising the steps of: a) incubating a donor-derived organ, tissue or cell preparation with an anti-CCR7 antibody or an antigen-binding fragment thereof as defined above, whereby the anti-CCR7 antibody achieves at least one of: i) reducing the number and ii) inhibiting the activity of CCR7-expressing donor cells in the organ, tissue or cell preparation; and b) optionally removing at least one of the anti-CCR7 antibody and the CCR7-expressing donor cells from the organ, tissue or cell preparation. Preferably, in the method, the anti-CCR7 antibody is included in a preservation solution used to preserve the organ, tissue or cell preparation before transplantation. More preferably, in the method, the organ or tissue is perfused or washed with a preservation solution comprising the anti-CCR7 antibody. Most preferably, in the method, the anti-CCR7 antibody and the CCR7-expressing donor cells are removed from the cell preparation by affinity purification of the anti-CCR7 antibody and the CCR7-expressing donor cells bound thereto.

[0018] The ex vivo method of the invention is preferably used in preparing the graft used in step d) of the method for use of the invention described above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] [Description of the Invention] definition As used herein, "GVHD" is defined as a disease in which lymphocytes in a graft transplanted into a host recognize and attack the host tissue as a foreign tissue. In this case, the term "recipient" or "host" as used herein refers to a subject (transplant patient) receiving a transplanted or implanted cell, tissue, or organ. These terms may refer to a subject receiving, for example, donor bone marrow, donor spermatozoa hematopoietic precursors, donor peripheral blood, donor umbilical cord blood, donor T cells, or a pancreatic islet graft. The transplanted tissue may be derived from a syngeneic or allogeneic donor. The term "donor" as used herein refers to a subject from which a tissue is obtained that is transplanted or implanted into a recipient or host. For example, a donor may be a subject from which bone marrow, peripheral blood, umbilical cord blood, T cells, or other tissue is derived that is administered to a recipient or host. The present invention is primarily targeted to humans and is preferably used in human patients. However, the present invention may also be used in non-human animals in which at least antibody formation due to an immune response is observed. The term human identifies any subject as an adult subject and a pediatric population, the term pediatric population being intended to be that portion of the population from birth to eighteen (18) years of age.

[0020] The term "antibody" is used in the broadest sense and specifically includes, for example, antagonists, neutralizing antibodies, single anti-CCR7 monoclonal antibodies, including full-length or complete monoclonal antibodies, anti-CCR7 antibody compositions with polyepitopic specificity, polyclonal antibodies, multivalent antibodies, single-chain anti-CCR7 antibodies, and fragments of anti-CCR7 antibodies, including Fab, Fab', F(ab')2, and Fv fragments, diabodies, single domain antibodies (sdAbs) (see below), so long as they exhibit the desired biological and / or immunological activity. The term "immunoglobulin" (Ig) is used interchangeably with antibody herein. Antibodies may be human and / or humanized antibodies.

[0021] The term "anti-CCR7 antibody" or "antibody that binds to CCR7" refers to an antibody capable of binding to CCR7 with sufficient affinity to be useful as a diagnostic and / or therapeutic agent in targeting CCR7. Preferably, the extent of binding of the anti-CCR7 antibody to unrelated non-CCR7 proteins is less than about 10% of the binding of the antibody to CCR7, as measured, for example, by radioimmunoassay (RIA) or ELISA. In certain embodiments, an antibody that binds to CCR7 has a dissociation constant (K d In certain embodiments, the anti-CCR7 antibody binds to an epitope of CCR7 that is conserved among CCR7 from different species.

[0022] An "isolated antibody" is one that has been identified and separated and / or recovered from a component of its natural environment.

[0023] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains (IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called the J chain, thus containing 10 antigen-binding sites, and secretory IgA antibodies can polymerize to form multivalent assemblies containing two to five basic four-chain units and a J chain). For IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked to a H chain by one covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain contains a variable domain (V H ), followed by three constant domains (C H ), and four C for μ and ε isotypes. H Each L chain has a variable domain (V L ), followed by a constant domain (C L ) V L is V Hand C L is the first constant domain of the heavy chain (C H 1). Certain amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain. H and V L Together, the pairing of these two amino acids forms a single antigen-binding site. Regarding the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th ed., Daniel P. Stites, Abba I. Terr, and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, Chapter 6, p. 71.

[0024] The L chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of the constant domain. The heavy chain constant domain (C H Depending on the amino acid sequence of the immunoglobulins, they can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are H They are further divided into subclasses based on relatively minor differences in sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0025] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain of an antibody. The variable domain of the heavy chain is designated "V H The variable domain of the light chain is sometimes called the "V L These domains are generally the most variable parts of the antibody and contain the antigen-binding sites.

[0026] The term "variable" refers to the fact that certain segments of the variable domains differ widely in sequence among antibodies. The V domains mediate antigen binding and define the specificity of a particular antibody for a particular antigen. However, the variability is not evenly distributed across the 110 amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter highly variable regions called "hypervariable regions" (HVRs), each 9-12 amino acids long. Native heavy and light chain variable domains each contain four FRs connected by three hypervariable regions, mostly adopting a β-sheet configuration, which form loops that connect and in some cases form part of the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not directly involved in binding the antibody to an antigen, but are involved in various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP).

[0027] A "complete" antibody is one that comprises the antigen binding site, the C and at least the heavy chain constant domain, i.e., H 1. C H 2, and C H 3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Preferably, the intact antibody has one or more effector functions.

[0028] A "naked antibody" for purposes herein is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[0029] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, an antibody fragment comprises the antigen-binding site of the intact antibody and thus retains the ability to bind antigen.

[0030] The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of an antibody are determined by sequences in the Fc region, which is also the portion recognized by Fc receptors (FcR) found on certain types of cells.

[0031] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site, in contrast to polyclonal antibody preparations which include different antibodies, each directed against a different determinant (epitope). Monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" should not be construed to require production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention may be prepared by the hybridoma method first described in Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic, or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0032] Monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0033] "Humanized" forms of non-human (e.g. rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate that has the desired specificity, affinity, and capacity of the antibody. In some cases, some framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. In general, humanized antibodies may contain typically two variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody may optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol., 1:105-115 (1998); Harris, Biochem. Soc. Transactions, 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech., 5:428-433 (1994).

[0034] The term "hypervariable region," "HVR," as used herein, refers to the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops responsible for antigen binding. Generally, antibodies contain six hypervariable regions, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Several hypervariable region delineations are in use and are encompassed herein. The hypervariable regions generally consist of amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., around residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the VL and around residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the VH, as numbered according to the Kabat numbering system; see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), and / or amino acid residues from the "hypervariable loops" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in VL and 26-32 (H1), 52-56 (H2), and 95-101 (H3) in VH, as numbered according to the Chothia numbering system; Chothia and Lesk, J. Mol. B iol. 196:901-917 (1987)), and / or amino acid residues from the "hypervariable loops" / CDRs (e.g., residues 27-38 (L1), 56-65 (L2), and 105-120 (L3) in VL and 27-38 (H1), 56-65 (H2), and 105-120 (H3) in VH, as numbered according to the IMGT numbering system; Lefranc, MP et al., Nucl. Acids Res. 27:209-212 (1999); Ruiz, M. et al., Nucl. Acids Res. 28:219-221 (2000)).Optionally, the antibody has symmetric insertions at one or more of the following points: 28, 36 (L1), 63, 74-75 (L2), and 123 (L3) in VL, and 28, 36 (H1), 63, 74-75 (H2), and 123 (H3) in VH, when numbered according to Honneger, A. and Plunkthun, AJ (Mol. Biol. 309:657-670 (2001)). The hypervariable regions / CDRs of the antibodies of the invention are preferably defined and numbered according to the IMGT numbering system.

[0035] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0036] A "blocking" or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0037] An "agonist antibody," as used herein, is an antibody that mimics at least one functional activity of a polypeptide of interest.

[0038] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity between a molecule X and a partner Y is generally determined by the dissociation constant (K d) Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens faster and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention. Specific illustrative embodiments are described below.

[0039] "K d " or "K d "Values" can be measured by using a surface plasmon resonance assay using a BIAcore™-2000 or Biacore™-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with an immobilized antigen CM5 chip of approximately 10-50 response units (RU). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of bound protein. After injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of antibody or Fab (0.78 nM to 500 nM) are injected at a flow rate of approximately 25 μl / min in PBS containing 0.05% Tween 20 (PBST) at 25 °C. The association rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software, version 3.2). d ) to k off / k onSee, e.g., Chen, Y. et al. (1999) J. Mol Biol 293:865-881. If the on-rate is 10 6 M -1 S -1 If the on-rate exceeds , the on-rate can be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) at 25 °C of 20 nM anti-antigen antibody (Fab form) in PBS pH 7.2 in the presence of increasing concentrations of antigen as measured in a spectrometer, for example a stop-flow equipped spectrophotometer (Aviv Instruments) or an 8000 series SLM-Aminco spectrophotometer with a stirred cuvette (ThermoSpectronic).

[0040] The "on-rate" or "rate of association" or "association rate" or "k on " can also be determined using the same surface plasmon resonance techniques described above using a Biacore™-2000 or Biacore™-3000 (BIAcore, Inc., Piscataway, NJ) as described above.

[0041] An antibody that "binds" to an antigen of interest, e.g., a polypeptide CCR7 antigen target, is an antibody that binds to the antigen with sufficient affinity to be useful as a therapeutic agent in targeting cells or tissues expressing the antigen and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody to a "non-target" protein may be less than about 10% of the binding of the antibody to its particular target protein, as determined by fluorescence-activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). With respect to the binding of an antibody to a target molecule, the term "specific binding" or "specifically binding to" or "specific for" a particular polypeptide or epitope of a particular polypeptide target refers to binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule relative to the binding of a control molecule, which is generally a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target, e.g., an excess of unlabeled target. In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. The term "specific binding," or "specifically binding to" or "specific for" a particular polypeptide or epitope of a particular polypeptide target, as used herein, refers to, for example, at least about 10 -4 M, or at least about 10 -5 M, or at least about 10 -6 M, or at least about 10 -7 M, or at least about 10 -8 M, or at least about 10 -9 M, or at least about 10 -10 M, or at least about 10 -11 M, or at least about 10 -12 K against M or more targets d(which can be determined as described above). In one embodiment, the term "specific binding" refers to binding when a molecule binds to a particular polypeptide or epitope of a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes.

[0042] Antibody "effector functions" refer to biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region) and which vary with antibody isotype. Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cell-mediated phagocytosis (ADCP); down-regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0043] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is usually defined to extend from an amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a complete antibody composition may include an antibody population with all K447 residues removed, an antibody population with none of the K447 residues removed, and an antibody population with a mixture of antibodies with and without the K447 residue.

[0044] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor, BCR), and the like. Such effector functions generally require that the Fc region be associated with a binding domain (e.g., an antibody variable domain) and can be assessed, for example, using the various assays disclosed in the definitions herein.

[0045] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies "arm" the cytotoxic cells and are absolutely required for such killing. NK cells, the primary cells involved in mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as those described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest may be assessed in vivo, for example in an animal model, such as those disclosed in Clynes et al. (USA) 95:652-656 (1998). WO 2000 / 42072 (Presta) describes antibody variants with improved or diminished binding to FcR. See also, e.g., Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).

[0046] A "human effector cell" is a leukocyte that expresses one or more FcRs and exerts effector function. Preferably, the cell expresses at least FcγRIII and exerts ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and NK cells being preferred. Effector cells may be isolated from a natural source, such as blood.

[0047] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that binds to a cognate antigen. To assess complement activation, a CDC assay may be performed, e.g. as described in Gazzano-Santoro et al. (1996, J. Immunol. Methods 202:163). Antibody variants with altered Fc region amino acid sequences (antibodies with variant Fc regions) and with improved or reduced C1q binding ability are described, e.g., in U.S. Pat. No. 6,194,551 (B1) and WO 1999 / 51642. See also, e.g., Idusogie et al. (2000, J. Immunol. 164:4178-4184). One substitution that improves C1q binding and thereby improves CDC activity is the E333A substitution, which can be advantageously applied to the antibodies of the present invention.

[0048] "Sequence identity" is defined herein as the relationship between sequences determined by comparing two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences. In the art, "identity" also means the degree of sequence relatedness between such sequences, as determined by the match between strings of amino acid or nucleic acid sequences, as the case may be. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutes with the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods. The term "sequence identity" or "sequence similarity" means that two (poly)peptide or two nucleotide sequences, when optimally aligned, preferably over their entire length (at least of the shortest sequence in the comparison), share at least a certain percentage of sequence identity, as defined elsewhere herein, when maximized and minimized by, for example, the ClustalW (1.83), GAP, or BESTFIT programs using default parameters. GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps. Generally, the following GAP default parameters are used: gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides, the default score matrix used is nwsgapdna, and for proteins, the default score matrix is ​​Blosum62 (Henikoff and Henikoff, 1992, PNAS 89, 915-919). A preferred multiple alignment program for aligning the protein sequences of the present invention is ClustalW (1.83), using the blosum matrix and default settings (gap opening penalty: 10, gap extension penalty: 0.05).Sequence alignment and scores for sequence identity percentage can be determined using computer programs such as the GCG Wisconsin Package, version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software such as the programs "needle" (using the global Needleman Wunsch algorithm) or "water" (using the local Smith Waterman algorithm) in EmbossWIN, version 2.10.0, using the same parameters as for GAP above or using the default settings (for both "needle" and "water", the initial gap opening penalty is 10.0 and the initial gap extension penalty is 0.5 for both protein and DNA alignments; the initial score matrix is ​​Bloom62 for proteins and DNAFull for DNA). When sequences have substantially different overall lengths, local alignments are preferred, such as local alignments using the Smith Waterman algorithm. Alternatively, the percentage of similarity or identity may be determined by searching public databases using algorithms such as FASTA, BLAST, etc.

[0049] [Detailed Description of the Invention] The present invention is based on the finding that the CCR7 receptor is highly expressed on some lymphoid cells and antigen-presenting cells (APCs). In said cells, CCR7 plays a key role in the entry into lymphoid tissues, including lymph nodes (LNs), a process that underlies the onset and progression of GVHD. The inventors have surprisingly found that anti-CCR7 antibodies produce remarkable therapeutic effects in a mouse GVHD model. GVHD can be suppressed without significant side effects by administration of anti-CCR7 antibodies to graft recipients. In vivo models show how CCR7 targeting with antibodies prevents the disease and ameliorates GVHD if it occurs, thus making the CCR7 receptor an interesting target for mAb therapy in both acute and chronic GVHD. Monoclonal antibodies (mAbs) against CCR7, i.e. antibodies that recognize an epitope on the CCR7 receptor and are preferably capable of inhibiting CCR7-dependent intracellular signaling, can be used to inhibit CCR7. + While it is possible in vivo to kill donor and recipient immune cells and / or to block their migration, activation, and / or proliferation, and / or dissemination, CCR7 - It does not affect immune cells and therefore, for example, maintains GVL and improves GVHD symptoms and survival in vivo.

[0050] Thus, in a first aspect, the present invention relates to an anti-CCR7 antibody or an antigen-binding fragment thereof for use in preventing and / or treating GVHD in a recipient of a transplant comprising donor cells. Preferably, at least one of the recipient cells and the donor cells are human cells. The transplant comprises donor cells, preferably comprising immune cells, more preferably immune-competent cells (e.g. mature T cells) capable of initiating an immune response against the recipient tissue to mediate GVHD. The GVHD may be acute or chronic GVHD. Preferably, the GVHD is acute GVHD. "Treating" GVHD, as used herein, is understood to mean suppressing GVHD, reducing the % incidence of GVHD, treating GVHD, ameliorating or attenuating one or more clinical manifestations of GVHD, and improving the survival rate of the subject being treated. "Preventing" GVHD, as used herein, is understood to mean "prophylaxis". Prevention in vivo means inhibiting the onset of GVHD, delaying the onset of GVHD, reducing the percentage of GVHD occurring, or alleviating one or more clinical signs of GVHD if it occurs.

[0051] The anti-CCR7 antibody or antigen-binding fragment thereof for use in the present invention may be any antigen-binding protein that specifically binds to CCR7. The antigen-binding protein of the present invention that binds to CCR7 is preferably an anti-CCR7 antibody in the broadest sense defined herein above, including, for example, anti-CCR7 antibodies, antibody fragments, antibody derivatives, antibody muteins, and antibody variants. The anti-CCR7 antibody of the present invention is preferably an isolated antibody. Preferably, the anti-CCR7 antibody of the present invention binds to primate CCR7, more preferably human CCR7. Reference amino acid sequences of human CCR7 are, for example, NP_001288643, NP_001288645, NP_001288646, NP_001288647, NP_001829, NP_001288642, and NP_031745. Amino acids 1-24 of this sequence comprise a membrane translocation signal peptide that is cleaved during expression. Amino acids 25-59 of human CCR7 are Y32 and Y 41 The CCR7 antibody for use in the present invention preferably specifically binds to the N-terminal extracellular domain of CCR7, preferably human CCR7.

[0052] Anti-CCR7 antibodies for use in the present invention are preferably neutralizing antibodies that inhibit CCR7-dependent intracellular signaling, CCR7-dependent function, and / or CCR7 receptor internalization by at least one CCR7 ligand selected from CCL19 and CCL21. The anti-CCR7 antibodies preferably have an IC of 150, 100, 80, 50, 30, 25, 20, 15, 10, 5, or 3 nM or less, as can be determined, for example, in an assay described in the Examples herein, with respect to inhibiting CCR7-dependent intracellular signaling and / or CCR7 receptor internalization by at least one CCR7 ligand selected from CCL19 and CCL21. 50 Alternatively, the antibody has the highest IC 50 is the IC of a reference anti-CCR7 antibody when tested in the same assay 50 Thus, preferably, the anti-CCR7 antibody of the present invention is defined by reference to the IC of the reference anti-CCR7 antibody. 50 IC up to 10, 5, 2, 1.5, 1.2, 1.1, or 1.05 times higher than 50 and the reference anti-CCR7 antibody is a murine anti-CCR7 antibody, the amino acid sequence of the heavy chain variable domain of which is SEQ ID NO: 1 and the amino acid sequence of the light chain variable domain of which is SEQ ID NO: 2.

[0053] The anti-CCR7 antibodies of the present invention preferably inhibit the CCR7-dependent intracellular signaling described above, as can be determined, for example, in the assays described in the Examples herein, without a substantial agonist effect, more preferably without a detectable agonist effect.

[0054] Anti-CCR7 antibodies for use in the present invention preferably have a minimum affinity for the N-terminal extracellular domain of CCR7, preferably human CCR7. The minimum affinity of an antibody is preferably defined herein as the K d Thus, preferably, the anti-CCR7 antibody of the present invention is defined by reference to the K of the reference anti-CCR7 antibody against the N-terminal extracellular domain of human CCR7. d K for the N-terminal extracellular domain of human CCR7 up to 100, 50, 20, 10, 5, 2, 1.5, 1.2, 1.1, or 1.05 times higher than d and the reference anti-CCR7 antibody is a murine anti-CCR7 antibody whose heavy chain variable domain has the amino acid sequence SEQ ID NO: 1 and whose light chain variable domain has the amino acid sequence SEQ ID NO: 2. d Up to 10 times higher than d It is understood herein that an antibody having an affinity at least 1 / 10 that of the reference antibody. Thus, if the reference antibody has an affinity of 1×10 -9 K of M d If the antibody has a -8 K below M d has.

[0055] Examples of anti-CCR7 antibodies having one or more of the above-defined characteristics and suitable for use in the present invention include, for example, the monoclonal antibodies described in U.S. Pat. No. 8,865,170, WO 2009 / 139853, WO 2014 / 151834, and WO 2017 / 025569, all of which are incorporated herein by reference.

[0056] A preferred anti-CCR7 antibody for use in the present invention is an antibody that specifically binds to an epitope comprising or consisting of the amino acid sequence "ZxLFE", where Z is sulfated tyrosine, x can be any amino acid, and F can be replaced with a hydrophobic amino acid. Thus, the antibody of the present invention preferably specifically binds to an epitope comprising or consisting of the amino acid sequence "ZTLFE" at positions 41-45 of the N-terminal extracellular domain of human CCR7. The antibody is preferably specific for human CCR7. Such a preferred anti-CCR7 antibody preferably has a minimum affinity for human CCR7 or a synthetic antigen comprising the "ZTLFE" epitope, preferably the synthetic antigen SYM1899 described in the Examples herein. Thus, preferably, the anti-CCR7 antibody has a minimum affinity of 1×10 to the synthetic antigen SYM1899, preferably. -8 M, 5×10 -9 M, 2×10 -9 M, 1.8×10 -9 M, 1×10 -9 M, 1×10 -10 M, or 1×10 -11 K below M d Alternatively, the minimum affinity of an antibody may be determined by the K of a reference anti-CCR7 antibody when tested in the same assay. d Thus, preferably, the anti-CCR7 antibody of the present invention is defined by reference to the K of the reference anti-CCR7 antibody against human CCR7 or a synthetic antigen comprising the "ZTLFE" epitope (preferably the synthetic antigen SYM1899 described in the Examples herein). d K for that antigen that is up to 10, 5, 2, 1.5, 1.2, 1.1, or 1.05 times higher than d and the reference anti-CCR7 antibody is a murine anti-CCR7 antibody whose heavy chain variable domain has the amino acid sequence SEQ ID NO: 1 and whose light chain variable domain has the amino acid sequence SEQ ID NO: 2. d Up to 10 times higher than d It is understood herein that an antibody having an affinity at least 1 / 10 that of the reference antibody. Thus, if the reference antibody has an affinity of 1×10 -9 K of M dIf the antibody has a -8 K below M d has.

[0057] Anti-CCR7 antibodies for use in the present invention preferably have a maximum k off The anti-CCR7 antibodies of the present invention bind to human CCR7 or a synthetic antigen comprising the "ZTLFE" epitope (preferably the synthetic antigen SYM1899, SEQ ID NO: 3, described in the Examples herein) with a rate constant of 1×10 -3 , 1×10 -4 , or 1 × 10 -5 s -1 The following k off The antibody has a maximum k off The rate constant is the k of a reference anti-CCR7 antibody when tested in the same assay. off Thus, preferably, the anti-CCR7 antibodies of the present invention are defined by reference to a rate constant k of a reference anti-CCR7 antibody against human CCR7 or a synthetic antigen comprising the "ZTLFE" epitope (preferably the synthetic antigen SYM1899 described in the Examples herein). off The reference anti-CCR7 antibody binds to its antigen at a rate constant that is up to 10, 5, 2, 1.5, 1.2, 1.1, or 1.05 times higher than the rate constant, and the reference anti-CCR7 antibody is a murine anti-CCR7 antibody whose heavy chain variable domain has the amino acid sequence of SEQ ID NO:1 and whose light chain variable domain has the amino acid sequence of SEQ ID NO:2.

[0058] One such preferred antibody for use in the present invention is an antibody having HVRs of a reference mouse anti-human CCR7 antibody, the heavy chain variable domain of which has the amino acid sequence of SEQ ID NO:1 and the light chain variable domain of which has the amino acid sequence of SEQ ID NO:2, as defined in WO 2017 / 025569, which is incorporated herein by reference.

[0059] The anti-CCR7 antibody for use in the present invention may be a chimeric antibody, such as a mouse-human antibody, however, preferably the antibody is a humanized or human antibody.

[0060] A humanized antibody for use in the present invention preferably induces little or no immunogenic response against the antibody in a subject to which the antibody is administered. For example, a humanized antibody for use in the present invention induces and / or is expected to induce a substantially reduced level of human anti-mouse antibody response (HAMA) in a host subject compared to an original mouse antibody, e.g., comprising the sequences of SEQ ID NOs: 1 and 2. Preferably, a humanized antibody induces and / or is expected to induce minimal or no human anti-mouse antibody response (HAMA) and / or is expected to induce no human anti-mouse antibody response (HAMA). Most preferably, an antibody of the present invention induces an anti-mouse antibody response at or below a clinically acceptable level.

[0061] Humanization can essentially be performed by substituting hypervariable region sequences for the corresponding sequences of a human antibody according to the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)). In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues, and possibly some framework region (FR) residues, are substituted by residues from analogous sites in rodent antibodies. The choice of human variable domains, both light and heavy, to be used in making the humanized antibody is crucial to reduce immunogenicity and retain specificity and affinity for the antigen. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent sequence is then accepted as the human framework region (FR) for the humanized antibody (Suns et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

[0062] It is further important that the antibody be humanized while retaining high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a method of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. The humanized anti-CCR7 antibody of any of the above embodiments of the invention preferably comprises a heavy chain constant region that is an IgG1, IgG2, IgG3, or IgG4 region. The humanized anti-CCR7 antibody of any of the above embodiments of the invention preferably comprises a functional Fc region that possesses at least one effector function selected from the group consisting of C1q binding, complement dependent cytotoxicity; Fc region binding, antibody-dependent cell-mediated cytotoxicity, and phagocytosis.

[0063] A preferred humanized antibody for use in the present invention is, for example, an antibody described in WO 2017 / 025569, in which the amino acid sequence of the heavy chain variable domain is SEQ ID NO: 4 and the amino acid sequence of the light chain variable domain is SEQ ID NO: 5.

[0064] As an alternative to humanization, human antibodies may be generated. By "human antibody" is meant an antibody containing fully human light and heavy chains and constant regions, produced by any of the known standard methods. For example, transgenic animals (e.g., mice) are available that are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production upon immunization. For example, it has been described that homozygous deletion of the antibody heavy chain joining region PH gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array into such germline mutant mice can result in the production of human antibodies after immunization. See, for example, Jakobovits et al., Proc. Nat. Acad. Sci. USA, 90:255 1 (1993); Jakobovits et al., Nature, 362:255-258 (1993). Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to generate human antibodies and antibody fragments in vitro from donor-derived immunoglobulin variable (V) domain gene repertoires. According to this technique, antibody V domain genes are cloned in frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those functional properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats; for a review of formats, see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-57 1 (1993). Human antibodies can also be generated by in vitro activated B cells or by SCID mice whose immune system has been reconstituted with human cells.Once a human antibody is obtained, its encoding DNA sequence can be isolated, cloned and introduced into an appropriate expression system, i.e., preferably a cell line of mammalian origin, which then expresses and releases the human antibody into the culture medium from which the antibody can be isolated.

[0065] A preferred human antibody for use in the present invention is, for example, an antibody described in WO 2014 / 151834, in which the amino acid sequence of the heavy chain variable domain is SEQ ID NO: 6 and the amino acid sequence of the light chain variable domain is SEQ ID NO: 7 or 8.

[0066] Functional fragments of antibodies that bind to the CCR7 receptor included for use within the present invention retain at least one binding and / or regulatory function of the full-length antibody from which the fragment is derived. Preferred functional fragments retain the antigen-binding function of the corresponding full-length antibody (e.g., the ability to bind to a mammalian CCR7 receptor). Particularly preferred functional fragments retain the ability to inhibit one or more functions characteristic of a mammalian CCR7 receptor, such as binding activity and / or blocking signaling activity and / or stimulation of a cellular response. For example, in one embodiment, the functional fragment can inhibit the interaction of CCR7 with one or more of its ligands and / or inhibit one or more receptor-mediated functions.

[0067] In some embodiments, the anti-CCR7 antibody of the present invention comprises a light chain and / or a heavy chain antibody constant region. Any antibody constant region known in the art can be used. The light chain constant region can be, for example, a kappa or lambda type light chain constant region, for example, a human kappa or lambda type light chain constant region. The heavy chain constant region can be, for example, an alpha, delta, epsilon, gamma, or mu type heavy chain constant region, for example, a human alpha, delta, epsilon, gamma, or mu type heavy chain constant region. Thus, the anti-CCR7 antibody of the present invention can have a constant region of any isotype, i.e., a constant region including IgG, IgM, IgA, IgD, and IgE constant regions, as well as IgG1, IgG2, IgG3, or IgG4 constant regions. In one embodiment, the light or heavy chain constant region is a fragment, derivative, variant, or mutein of a naturally occurring constant region. Techniques for deriving antibodies of different subclasses or isotypes from an antibody of interest, i.e., subclass switching, are known. Thus, an IgG antibody may be derived, for example, from an IgM antibody, or vice versa. Such techniques allow the preparation of new antibodies that retain the antigen-binding properties of a given antibody (the parent antibody), but also exhibit biological properties associated with an antibody isotype or subclass different from that of the parent antibody. Recombinant DNA techniques may be used. Cloned DNA encoding a particular antibody polypeptide, for example DNA encoding the constant domain of an antibody of a desired isotype, may be used in such procedures. See also Lantto et al. (2002, Methods Mol. Biol. 178:303-16). Thus, the anti-CCR7 antibodies of the present invention include antibodies comprising one or more variable domain sequences, for example as disclosed herein, and having a desired isotype (e.g., IgA, IgG1, IgG2, IgG3, IgG4, IgM, IgE, and IgD), as well as Fab or F(ab')2 fragments thereof. Furthermore, if IgG4 is desired, it may also be desirable to introduce a point mutation (CPSCP→CPPCP) in the hinge region to mitigate the tendency to form inter-heavy chain disulfide bonds that could introduce heterogeneity into IgG4 antibodies, as described in Bloom et al. (1997, Protein Science 6:407).

[0068] The anti-CCR7 antibodies of the invention preferably comprise a functional Fc region possessing at least one effector function selected from the group consisting of C1q binding, complement dependent cytotoxicity; Fc receptor binding, antibody-dependent cell-mediated cytotoxicity and phagocytosis.

[0069] The anti-CCR7 antibodies of the present invention can be modified to improve effector function, for example to enhance ADCC and / or CDC of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Preferred substitutions in the Fc region of the antibodies of the present invention are those that improve C1q binding and thereby improve CDC activity, for example as described in Idusogie et al. (2000, J. Immunol. 164:4178-4184). A preferred substitution in the Fc region that improves C1q binding is the E333A substitution.

[0070] Glycosyl groups, which are added to the amino acid backbone of glycoproteins, such as antibodies, are formed by several monosaccharides or monosaccharide derivatives, resulting in a composition that may differ in the same antibody produced in cells from different mammals or tissues. In addition, it has been shown that different compositions of glycosyl groups can affect the efficacy of antibodies in mediating antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). These properties can therefore be improved by studying the glycosylation patterns of antibodies from different sources. One example of such an approach is Niwa et al. (2004, Cancer Res, 64(6):2127-33).

[0071] Alternatively or additionally, cysteine ​​residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in the Fc region. The homodimeric antibody so generated may have improved internalization capability and / or enhanced complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al. (1992, J. Exp Med. 176:1191-1195) and Shopes (1992, Immunol. 148:2918-2922). Homodimeric antibodies with enhanced antitumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al. (1993, Cancer Research 53:2560-2565). Alternatively, antibodies with dual Fc regions may be engineered, thereby having enhanced complement lysis and ADCC capabilities. See Stevenson et al. (1989, Anti-Cancer Drug Design 3:219-230). To increase the serum half-life of an antibody, a salvage receptor binding epitope may be incorporated into the antibody (especially an antibody fragment), for example as described in U.S. Pat. No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the serum half-life of the IgG molecule in vivo.

[0072] A preferred anti-CCR7 antibody of the invention comprises a heavy chain constant region of human allotype G1m17,1 (see Jefferis and Lefranc (2009) MAbs vol. 1, no. 4, pp. 1-7), which comprises the amino acid sequence of SEQ ID NO: 9. More preferably, the heavy chain constant region of human allotype G1m17,1 in the antibody of the invention comprises an E333A substitution and comprises the amino acid sequence of SEQ ID NO: 10.

[0073] Anti-CCR7 antibodies for use in the present invention can be prepared by any of several conventional techniques. Anti-CCR7 antibodies are typically produced in recombinant expression systems using any technique known in the art. See, for example, Shukla and Thoemmes (2010, "Recent advances in large-scale production of monoclonal antibodies and related proteins", Trends in Biotechnol. 28(5):253-261), Harlow and Lane (1988) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual (3rd ed.)", Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, NY. Any expression system known in the art can be used to produce recombinant polypeptides of the present invention. Generally, a host cell is transformed with a recombinant expression vector containing DNA encoding the desired polypeptide.

[0074] In one embodiment, the invention relates to the use of an anti-CCR7 antibody or an antigen-binding fragment thereof as defined herein above for treating and / or preventing GVHD in a recipient of a transplant comprising donor cells, preferably wherein the anti-CCR7 antibody effect comprises at least one of the following, preferably in the recipient: killing of CCR7-expressing cells, inducing apoptosis of said cells, blocking migration and / or blocking dissemination of said cells, blocking activation of CCR7-expressing cells, blocking maturation and differentiation of CCR7-expressing cells. The CCR7-expressing cells on which the anti-CCR7 antibody exerts one or more of these effects are preferably CCR7-expressing immune cells, which may be transplanted immune cells derived from the donor or immune cells derived from the host, i.e. from the recipient. Examples of donor- or host-derived CCR7-expressing immune cells include T cells, both CD4+ and CD8+ T cells, such as naive T cells, central memory T cells, regulatory T cells, helper T cells, and cytotoxic T cells, B cells, such as naive B cells and follicular B cells, and antigen-presenting cells (APCs), such as dendritic cells, including mature dendritic cells (mDCs) and plasmacytoid DCs. For example, cells expressing the CCR7 receptor can be identified by conventional methods, and for example, surface expression of the CCR7 receptor can be analyzed by flow cytometry, which is generally known in the art. Death of cells expressing the CCR7 receptor can be determined by any conventional method, for example, by detecting recipient-derived CCR7. + The absence or clearance of CD45 cells can be determined by determining the absence or clearance of CD45 cells in at least one of the recipient's lymph nodes, peripheral blood, spleen, thymus, and bone marrow, particularly lymphoid organs, or in any of the epithelial target tissues of GVHD in the recipient. + and more preferably, the anti-CCR7 antibody or antigen-binding fragment thereof inhibits or suppresses the infiltration of donor cells into at least one of lymph nodes, peripheral blood, spleen, thymus, and bone marrow in the recipient, particularly into lymphoid organs of the recipient, or into epithelial target tissues of GVHD in the recipient. + , CD45 +Prevent or inhibit donor cell infiltration.

[0075] Without wishing to be bound by theory, the therapeutic use of the anti-CCR7 antibodies of the invention advantageously involves, for example, the prevention of CCR7 in a recipient. + Killing T cells and APCs, and / or CCR7 + Impairing and / or blocking the migration and / or seeding of T cells and APCs, and / or impairing or blocking the activation or differentiation or maturation of CCR7+ T cells and APCs, should allow specific prevention or treatment of GVHD in vivo. In most cases, complement-dependent cytolysis (CDC), antibody-dependent cell-mediated phagocytosis (ADCP), and antibody-dependent cell-mediated cytotoxicity (ADCC) are believed to be responsible for the clinical utility of unconjugated anti-CCR7 antibodies, although induction of apoptosis or cell cycle arrest may also play a substantial role. In the case of application of anti-CCR7 antibodies, impairing and / or blocking the migration of immune cells, and / or impairing or blocking the activation, differentiation, proliferation, or maturation of immune cells are additional relevant mechanisms of action.

[0076] Thus, preferably, in one embodiment, the present invention relates to the use of an anti-CCR7 antibody of the present invention, which inhibits migration of donor and / or recipient cells expressing the CCR7 receptor to secondary lymphoid tissues and / or prevents dissemination of donor cells to secondary lymphoid tissues, including lymph nodes, spleen, and mucosa-associated lymphoid tissues (MALT), such as Peyer's patches.

[0077] The recipient of the transplant in which GVHD is prevented or treated according to the present invention is preferably a recipient of a transplant or graft comprising an organ, progenitor cell, stem cell, hematopoietic cell, hematopoietic progenitor cell, or hematopoietic stem cell. The transplant or graft may be a syngeneic or allogeneic transplant, but is preferably a transplant or graft comprising allogeneic donor cells. The transplant may comprise any type of organ or tissue, including, for example, the heart, lung, kidney, liver, pancreas, intestine, face (or part thereof), cornea, skin, hand, leg, penis, bone, uterus, thymus, etc.

[0078] In a preferred embodiment, the anti-CCR7 antibodies or antigen-binding fragments of the invention are used to prevent or treat GVHD in recipients of hematopoietic cell grafts, more particularly, after allogeneic hematopoietic stem cell transplantation (HSCT).

[0079] The donor cells used in the method of the invention can be whole or purified bone marrow cells, spermatozoic progenitors or stem cells from bone marrow, spermatozoic progenitors or stem cells from peripheral blood, (purified) cord blood cells or peripheral blood cells from an apheresis product enriched in hematopoietic progenitors or stem cells after mobilization of hematopoietic progenitors from bone marrow with growth factors such as G-CSF or anti-CXCR4 agents such as plerixafor. In the method of the invention in which donor T cells are introduced, the cell graft can comprise whole or purified bone marrow cells, cord blood cells, or purified stem cells with additional infusion of T cells. Thus, in one embodiment, the donor cells used according to the invention comprise or are derived from at least one of T cells, spleen from Wharton's jelly, umbilical cord blood, amniotic fluid, and dental pulp cells, placenta-derived cells, hair root-derived cells, and / or adipose tissue-derived cells, cell suspensions comprising lymphocytes, monocytes, and / or macrophages, stem cell-containing tissues, stem cell-containing organs, immune cell-containing tissues, and immune cell-containing organs. In one embodiment, donor cells used in accordance with the present invention are hematopoietic stem cells (also known as hematopoietic progenitor cells) including or derived from bone marrow stem cells, peripheral blood stem cells, umbilical cord blood stem cells, adult stem cells of the bone marrow, such as non-adherent bone marrow-derived cells (NA-BMC), embryonic stem cells, and / or reprogrammed adult stem cells (i.e., induced pluripotent cells).

[0080] The recipient of the hematopoietic (stem) cell graft may have a hematologic or non-hematologic disorder. The hematologic disorder may be a non-neoplastic hematologic disorder or a hematologic malignancy. The non-malignant hematologic disorder, particularly the hematopoietic cell deficiency disorder, may be selected from the group consisting of congenital or acquired immune deficiencies, hemoglobinopathies, enzyme deficiencies, or genetic disorders causing autoimmune diseases, severe aplastic anemia, thalassemia, sickle cell anemia, immunological defects, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), hemophagocytic lymphohistiocytosis (HLH), inborn errors of metabolism, lysosomal storage disorders, disorders of peroxisomal function, autoimmune diseases, rheumatic diseases, and relapses of any of the above. The hematological malignancy may be selected from the group consisting of leukemia, acute myeloid leukemia (AML), promyelocytic leukemia (PML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), chronic myelogenous leukemia (CML), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma (HL), multiple myeloma (MM), and neuroblastoma. The recipient of the hematopoietic (stem) cell graft may have a non-hematologic solid tumor (e.g., renal cell carcinoma, colorectal cancer, etc.).

[0081] The recipient of the hematopoietic (stem) cell graft can preferably be treated in a myeloablative conditioning regimen, or a non-myeloablative conditioning regimen, or a reduced intensity conditioning regimen, or neither, prior to receiving the hematopoietic (stem) cell graft.

[0082] In one embodiment, the invention relates to the use of an anti-CCR7 antibody of the invention, wherein the prevention or treatment of GVHD comprises administration of the anti-CCR7 antibody to a recipient before, (approximately) simultaneously and / or after the recipient receives a transplant comprising donor cells. When the anti-CCR7 antibody is administered (approximately) simultaneously with the recipient receiving a transplant comprising donor cells, it is preferably meant that the anti-CCR7 antibody is administered within 96, 72, 24, 12, 6 or 3 hours of each other. Preferably, the prevention or treatment of GVHD includes at least one of: a) administration of an anti-CCR7 antibody to the recipient before the recipient receives a transplant containing donor cells; b) administration of an anti-CCR7 antibody to the recipient 48, 72, or 96 hours after the recipient receives a transplant containing donor cells; c) administration of an anti-CCR7 antibody to the recipient after the recipient receives a transplant containing donor cells, and preferably after the recipient exhibits symptoms of GVHD or GVHD has been diagnosed in the recipient; and d) administration of an anti-CCR7 antibody to the recipient after a relapse of GVHD.

[0083] Administration of the anti-CCR7 antibody before the recipient receives the transplant may be desirable in that it may pretreat the recipient for receiving the transplant containing donor cells, thus making it possible to prevent GVHD or at least reduce the risk of GVHD occurring. Thus, preferably, the anti-CCR7 antibody is administered at least 5, 10, 20, or 40 minutes, or 1, 2, 4, 8, 12, 24, or 48 hours, before the recipient receives the transplant.

[0084] Administration of anti-CCR7 antibodies after the recipient receives the transplant may be desirable in that it may reduce donor immune attack on the recipient host and further promote the recipient's acceptance of the donor transplant and / or cells. Preferably, the anti-CCR7 antibodies are administered as needed after the recipient receives the transplant for as long as necessary to reduce the incidence of GVHD and / or ameliorate or attenuate one or more symptoms of GVHD. The number and dosage of administration may also depend on the serum half-life of the anti-CCR7 antibodies and may be adjusted accordingly. In a preferred embodiment of the present invention, the anti-CCR7 antibodies are administered both before and after the recipient receives the transplant.

[0085] However, as the examples herein demonstrate, administration of anti-CCR7 antibodies to a recipient after an alloreactive response has occurred in the recipient receiving the transplant remains effective in treating GVHD, at least in terms of improving survival. Thus, in one embodiment of the present invention, anti-CCR7 antibodies are administered to a recipient having a transplant containing donor cells after the recipient shows clinical signs of GVHD and / or a detectable alloreactive response, and / or preferably after GVHD has been diagnosed in the recipient. In such cases, the recipient may not have been previously treated with anti-CCR7 antibodies or previously administered(s) anti-CCR7 antibodies.

[0086] The anti-CCR7 antibody administered to the recipient after the recipient receives the transplant can be administered at least 1, 2, 3, 5, 7, 10, 14, 21, or 28 days after at least one of: i) the recipient receives the transplant or graft, ii) symptoms of GVHD develop in the recipient, iii) an alloreactive response is detected in the recipient, and iv) the recipient is diagnosed with GVHD.

[0087] In another embodiment of the invention, the anti-CCR7 antibody is administered to a recipient of a graft comprising donor cells, the graft having been prepared pre-transplant by ex vivo incubation with the anti-CCR7 antibody, preferably according to the methods described below. The anti-CCR7 antibody administered to the recipient can be, but need not be, the same as the anti-CCR7 antibody used in the ex vivo method for preparing the graft pre-transplant.

[0088] In a preferred embodiment of the invention, the anti-CCR7 antibody or antigen-binding fragment thereof is administered separately from the graft, preferably at least once shortly before or shortly after administration of the graft. "Soon" in this context means within 24 hours, preferably within 8 hours, more preferably within 6 hours, more preferably within 4 hours, more preferably within 2 hours, most preferably within 1 hour. "Separately" means that the administration of the CCR7 antibody or antigen-binding fragment thereof is contained in a separate container, e.g., syringe, from the graft. Preferably, the CCR7 antibody or antigen-binding fragment thereof is administered at least 10 seconds, more preferably at least 1 minute, more preferably at least 10 minutes, most preferably at least 1 hour before administration of the graft. In another preferred embodiment, the graft is administered at least 10 seconds, more preferably at least 1 minute, more preferably at least 10 minutes, most preferably at least 1 hour before administration of the CCR7 antibody or antigen-binding fragment thereof.

[0089] Thus, preferably, the treatment involves at least one administration of an anti-CCR7 antibody or antigen-binding fragment thereof to the recipient separate from the transplant.

[0090] In yet another embodiment of the invention, the anti-CCR7 antibody is administered to the recipient after a relapse of GVHD, and the recipient may not have had prior treatment with or prior administration(s) of an anti-CCR7 antibody.

[0091] In another aspect, the present invention relates to a pharmaceutical composition comprising an anti-CCR7 antibody (or an antigen-binding fragment thereof) as defined herein for use in the present invention. The pharmaceutical composition preferably comprises at least an anti-CCR7 antibody or a pharmaceutical derivative or prodrug thereof together with a pharma- ceutically acceptable carrier, adjuvant, or vehicle for administration to a subject. Said pharmaceutical composition can be used in a method of treatment by administration of an effective amount of the composition to a subject in need thereof, as described herein below. The term "subject" is used interchangeably herein with the term "recipient" and, as used herein, refers to all animals classified as mammals, including, but not limited to, primates and humans. The subject is preferably a human male or female of any age or race.

[0092] The term "pharmaceutically acceptable carrier" as used herein is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration (see, for example, "Handbook of Pharmaceutical Excipients", Rowe et al., 7th ed., 2012, www.pharmpress.com). The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include, for example, buffers, such as phosphate, citric acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, zeolite, or the like. amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0093] The antibodies of the invention may be administered in the same formulation or in different formulations. Administration may be simultaneous or sequential, and may be effective in either order.

[0094] Additional active compounds can also be incorporated into the pharmaceutical composition of the present invention. Thus, in certain embodiments, the pharmaceutical composition of the present invention may also contain two or more active compounds necessary for the particular indication being treated, preferably two or more active compounds with complementary activities that do not adversely affect each other. For example, it may be desirable to provide additional chemotherapeutic agents, cytokines, analgesics, or immunomodulatory agents, such as immunosuppressants or immunostimulants. The effective amount of such other active agents depends, among other things, on the amount of the antibody of the present invention present in the pharmaceutical composition, the type of disease or disorder or treatment, etc.

[0095] In addition to use in the monotherapy or prevention of GVHD, the antibodies and pharmaceutical compositions of the present invention may be used with other drugs to provide combination therapy. The other drugs may form part of the same composition or may be provided as separate compositions for administration at the same or different times. The combination therapy may have a synergistic therapeutic effect on the patient. In certain embodiments, the antibodies of the present invention may be combined with other treatments for medical conditions described herein.Other therapeutic agents include, but are not limited to, alkylating agents (e.g., nitrogen mustards [e.g., mechlorethamine], cyclophosphamide, melphalan, and chlorambucil), alkylsulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, semustine, and streptozocin), triazenes (e.g., dacarbazine), antimetabolites (e.g., folic acid analogs, e.g., methotrexate), pyrimidine analogs (e.g., fluorouracil and cytarabine), purine analogs (e.g., cyclosporine ... anti-inflammatory drugs (e.g., fludarabine, idarubicin, cytosine arabinoside, mercaptopurine, and thioguanine), vinca alkaloids (e.g., vinblastine, vincristine, and vindesine), epipodophyllotoxins (etoposide and teniposide), antibiotics (dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, and mitomycin), dibromomannitol, deoxyspergualin, dimethylmyleran, and thiotepa, proteasome inhibitors (bortezomib) , pentostatin, immunosuppressive drugs such as steroids (e.g., prednisone and methylprednisolone), calcineurin inhibitors (e.g., cyclosporine A, tacrolimus, or FK506), mammalian target of rapamycin (mTOR) inhibitors (sirolimus or rapamycin), mycophenolate mofetil, thalidomide, lenalidomide, azathioprine, monoclonal antibodies (e.g., daclizumab (anti-interleukin (IL)-2), infliximab (anti-tumor necrosis factor), etanercept, ME DI-205 (anti-CD2), abx-cbl (anti-CD147), alemtuzumab (anti-CD52), rituximab (anti-CD20), as well as polyclonal antibodies (e.g., ATG (anti-thymocyte globulin)), antihistamines, chemotherapy, radiation therapy, immunotherapy, surgical procedures, alkylating agents, antimetabolites, antihormones, medications for various conditions such as analgesics, diuretics, antidiuretics, antivirals, antibiotics, cytokines, nutritional supplements, medications for anemia, medications for blood clotting, bone medications, mental and psychological medications, and the like.In addition, the antibodies and pharmaceutical compositions of the invention may be used prior to or about the same time as transplantation in combination with other types of therapy as prophylaxis against GVHD, including, but not limited to, immunosuppressive drugs such as calcineurin inhibitors (e.g., cyclosporine A, tacrolimus, or FK506), mammalian target of rapamycin (mTOR) inhibitors (sirolimus or rapamycin), or antiproliferative agents (e.g., mycophenolate mofetil, methotrexate), thymic irradiation, phototherapy, in vivo depletion of T cells with melphalan, cyclophosphamide, or ATG, or ex vivo depletion of T cells with antibodies (e.g., anti-CD3) to prevent the onset of GVHD. In addition, the antibodies and pharmaceutical compositions of the present invention may be used in combination with other types of therapy as a treatment for GVHD, including, but not limited to, steroids (e.g., prednisone and methylprednisolone), extracorporeal photochemotherapy, pentostatin, kinase inhibitors (e.g., ruxolitinib, ibrutinib), proteasome inhibitors (bortezomib), cell therapy using NK cells or regulatory T cells or mesenchymal stem cells, immunotherapy using monoclonal antibodies (e.g., rituximab, alemtuzumab, tocilizumab, etc.) or fusion proteins (e.g., abatacept, alefacept), inhibitors of T cell migration (e.g., maraviroc), etc.

[0096] It may also be useful to treat the patient with cytokines to upregulate expression of CCR7 or other target proteins on the surface of target cells prior to administration of the antibodies of the invention. Cytokines may also be administered simultaneously with, before, or after administration of the depleting or radiolabeled antibodies to stimulate immune effector function.

[0097] In addition, the use of anti-CCR7 antibodies for the treatment or prevention of GVHD of the present invention may further include the administration of a conditioning regimen to the recipient of the transplant, including myeloablative, non-myeloablative, or reduced-intensity conditioning treatment prior to transplantation. These treatments eradicate the underlying disease and suppress and eradicate the host immune system, allowing donor stem cells to populate the bone marrow without the risk of graft rejection. The administration of myeloablative or reduced-intensity or non-myeloablative treatments may be used to induce mixed hematopoietic chimerism or complete hematopoietic chimerism. Total body irradiation (TBI) regimens and / or chemotherapy regimens using busulfan and / or cyclophosphamide are examples of myeloablative regimens. As used herein, "non-myeloablative" refers to a treatment that kills bone marrow cells but does not cause death from bone marrow failure in a significant number of recipients. This allows donor stem cell engraftment with at least mixed donor / recipient chimerism. Final loss of host hematopoietic development is achieved by the graft-versus-host effect of immune donor cells, ultimately resulting in complete donor chimerism. Low-dose TBI, fludarabine, ATG, reduced-dose busulfan, or a combination thereof are used as non-myeloablative regimens. RIC regimens are intermediate approaches that avoid the high toxicity of myeloablative regimens but achieve sufficient control of underlying disease and immunosuppression to prevent graft rejection. Common RIC regimens include fludarabine and melphalan, but many other agents have been introduced for RIC treatment.

[0098] In some embodiments, the antibody of the present invention is prepared with a carrier that can protect the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems, such as liposomes. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for the preparation of such formulations will be clear to those skilled in the art. Liposomal suspensions, including targeted liposomes, can also be used as pharma-ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811 and WO 2010 / 095940.

[0099] The route of administration of the antibody (or fragment thereof) of the present invention may be oral, parenteral, inhalation, or topical. The term "parenteral" as used herein includes intravenous, intraarterial, intralymphatic, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. The intravenous form of parenteral administration is preferred. By "systemic administration" is meant oral, intravenous, intraperitoneal, and intramuscular administration. Of course, the amount of antibody required for therapeutic or prophylactic effect may vary with the antibody selected, the nature and severity of the condition being treated, and the patient. In addition, the antibody may be suitably administered by pulse infusion, for example with declining doses of the antibody. Preferably, the dosage is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.

[0100] Thus, in certain embodiments, the pharmaceutical composition of the present invention may be in a form suitable for parenteral administration, for example, a sterile solution, suspension, or lyophilized formulation in an appropriate unit dosage form. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy needle passage exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, a pharma- ceutically acceptable polyol, for example, glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating agent such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[0101] Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0102] Sterile injection solution can be prepared by incorporating the required amount of active compound (e.g., polypeptide or antibody) into a suitable solvent with one or a combination of the above-listed components as required, followed by filtration sterilization.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other components required from the above-listed components.In the case of sterile powder for preparing sterile injection solution, the preferred method of preparation is vacuum drying and freeze-drying, which obtains a powder of active ingredient and any additional desired components from its solution that has been previously sterilized and filtered.

[0103] In certain embodiments, the pharmaceutical composition is administered intravenously (IV) or subcutaneously (SC). Suitable excipients, such as bulking agents, buffers, or surfactants, may be used. The mentioned formulations can be prepared using standard methods for preparing parenterally administered compositions, which are well known in the art and described in detail in various sources, including, for example, "Remington: The Science and Practice of Pharmacy" (ed. Allen, LV, 22nd edition, 2012, www.pharmpress.com).

[0104] It is particularly advantageous to formulate pharmaceutical compositions, i.e. parenteral compositions, into dosage unit forms for easy administration and uniformity of dosage.Dosage unit forms, as used herein, refer to physically discrete units suitable as unitary dosages for subjects to be treated, each unit containing a predetermined amount of active compound (the antibody of the present invention) calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier.The specifications for the dosage unit forms of the present invention are dependent on and directly depend on the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the limitations inherent in the technical field of compounding such active compounds for the treatment of individuals.

[0105] In general, the effective dosage of the antibody of the invention may depend on the relative efficacy of the compound selected, the severity of the disorder being treated, and the body weight of the affected individual. However, the active compound may be administered, typically once or more than once per day, for example 1, 2, 3, or 4 times per day, at a typical total daily dosage ranging from 0.001 to 1,000 mg / kg body weight / day, preferably from about 0.01 to about 100 mg / kg body weight / day, and most preferably from about 0.05 to 10 mg / kg body weight / day. More specifically, for the use of the invention, the anti-CCR7 antibody is preferably administered at a dosage of 1 to 1000, 2 to 500, 5 to 200, 10 to 100, 20 to 50, or 25 to 35 mg / kg body weight / day, preferably administered in multiple doses every 1, 2, 4, 7, 14, or 28 days.

[0106] In addition to administering the antibody to a patient, the present application contemplates administering the antibody by gene therapy. WO 96 / 07321 relates to the use of gene therapy to generate intracellular antibodies.

[0107] The pharmaceutical compositions can be included in a container, pack, or dispenser device together with instructions for administration.

[0108] The antibodies and pharmaceutical compositions of the invention may be used together with other drugs to provide combination therapy, which may form part of the same composition or may be provided as separate compositions for administration at the same or different times.

[0109] In a further aspect, the present invention relates to an ex vivo or in vitro method for preparing a donor-derived organ, tissue or cell preparation for transplantation into a recipient, the method preferably comprising the steps of a) incubating a donor-derived organ, tissue or cell preparation with an anti-CCR7 antibody or an antigen-binding fragment thereof as defined herein, whereby preferably the anti-CCR7 antibody achieves at least one of: i) reducing the number and ii) inhibiting the activity of CCR7-expressing donor cells in the organ, tissue or cell preparation, and b) optionally removing at least one of the anti-CCR7 antibody and the CCR7-expressing donor cells from the organ, tissue or cell preparation. Preferably, the anti-CCR7 antibody is incubated with the organ, tissue or cell preparation in an amount and for a time sufficient / effective to reduce the number and / or inhibit the activity of CCR7-expressing donor cells in the organ, tissue or cell preparation to an extent sufficient to reduce the risk of development of GVHD and / or reduce the severity of GVHD in a recipient of the donor organ, tissue or cell preparation. For example, the anti-CCR7 antibody is incubated with the donor organ, tissue, or cell preparation in an amount and time sufficient / effective to substantially inhibit the activity of CCR7-expressing donor cells in the transplant, preferably to reduce the activity by at least 40%, more preferably to reduce the activity by at least 80%, and most preferably to reduce the activity by at least 90%. Or, for example, the anti-CCR7 antibody is incubated with the donor organ, tissue, or cell preparation in an amount and time sufficient / effective to substantially reduce the number of CCR7-expressing donor cells in the transplant, preferably to reduce the number by at least 40%, more preferably to reduce the number by at least 80%, and most preferably to reduce the number by at least 90%. It is understood by this specification that the CCR7-expressing donor cells in the donor organ, tissue, or cell preparation are preferably CCR7-expressing immune cells, more preferably CCR7-expressing immune cells including at least one or more of T lymphocytes, B lymphocytes, NK cells, or APCs.

[0110] The methods of the present invention for preparing a donor-derived organ, tissue or cell preparation for transplantation into a recipient are preferably methods practiced in an in vitro or ex vivo environment, where ex vivo does not exclude treatment with an anti-CCR7 antibody while the donor organ, tissue or cell preparation is still present in the body of a brain-dead donor or a donor who has died by circulatory death, by administration of the anti-CCR7 antibody to the donor's body.

[0111] All of the above disclosures regarding the clinical treatment or prevention of GVHD associated with in vitro or ex vivo environments apply to this practice. Thus, anti-CCR7 antibodies can be included in preservation solutions used to preserve organs, tissues, or cell preparations prior to transplantation. For example, anti-CCR7 antibodies can be added to preservation solutions for organ transplants in an amount sufficient to bind to and inhibit the activity of immune cells in the organ. In addition, anti-CCR7 antibodies can be added to preservation solutions for organ transplants in an amount sufficient to bind to and reduce the number of immune cells in the organ. Such preservation solutions can be suitable for preserving different types of organs, such as heart, kidney, and liver, and tissues derived therefrom. One example of a commercially available preservation solution is Plegisol (Abbott), and other preservation solutions named in relation to their origin include UW solution (University of Wisconsin), Stanford solution, and modified Collins solution (J. Heart Transplant (1988) Vol. 7 (6): 456 4467). The preservation solution may also contain conventional co-solvents, excipients, stabilizers, and / or buffers. The preservation solution or buffer containing anti-CCR7 antibody may also be used to wash or rinse organ transplants before transplantation or storage. Thus, the organ or tissue to be transplanted can be perfused with a preservation solution containing anti-CCR7 antibody, preferably before transplantation. For example, the preservation solution containing anti-CCR7 antibody may be used to flush an isolated heart, which is then stored in the preservation solution at 4°C.

[0112] In another embodiment, the practice of the present invention can be used to pretreat organ or tissue transplants prior to transplantation, where an anti-CCR7 antibody or fragment can be added to a washing buffer to remove activated T lymphocytes, B lymphocytes, NK cells, or APCs from the transplant.

[0113] The concentration of anti-CCR7 antibody or fragment in the storage solution or washing buffer may vary depending on the type of transplant. In the present invention, the incubation step may be carried out for example for 1 minute to 7 days. With regard to removing at least one of the anti-CCR7 antibody (e.g. unbound anti-CCR7 antibody) and the CCR7-expressing donor cells from the organ, tissue or cell preparation in the method and use of the present invention, various means for carrying out said step are known to the skilled person. One exemplary means for removing the antibody from the transplant is washing the transplant. Washing may be performed, for example, by using centrifugation if the transplant constitutes or is a cell suspension. Alternatively, the anti-CCR7 antibody and the CCR7-expressing donor cells may be removed from the cell preparation to be transplanted (e.g. bone marrow cells, peripheral blood cells or umbilical cord blood cells) by affinity purification of the anti-CCR7 antibody and preferably the CCR7-expressing donor cells bound thereto. Thus, preferably, the affinity ligand used for purification does not affect the antigen-binding ability of the anti-CCR7 antibody, and CCR7-expressing donor cells bound to the anti-CCR7 antibody can be co-purified from the cell preparation. Methods for affinity purification are well known in the art, including, for example, methods in which the affinity ligand is immobilized on a solid support material, such as magnetic beads, or solid support materials used in affinity (column) chromatography.

[0114] The amount of antibody used in the incubating step is not particularly limited. The appropriate amount can be easily determined by a person skilled in the art and may depend, for example, on the type of graft used. Preferably, in the present invention, the incubating step is carried out with an antibody amount of 0.1 μg to 100 mg. The selection of a suitable amount of antibody is well within the expertise of a person skilled in the art. In general, a higher amount or concentration of antibody is preferred when the graft comprises or is a tissue, or comprises or is an organ, respectively. Furthermore, the selection of the exact amount or concentration of antibody used may also depend on the size of such tissue or organ, respectively.

[0115] In this document and the claims, the verb "comprise" and its conjugations are used in an open-ended sense to mean that the items following the word are included but not to exclude items not specifically mentioned. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that only one element is present. Thus, the indefinite article "a" or "an" typically means "at least one."

[0116] The terms "about" or "approximately," when used in connection with a numerical value (e.g., about 10), preferably mean that the value may be 0.1% more or less than the stated value (10).

[0117] All patent and literature references cited herein are incorporated by reference in their entirety.

[0118] The present invention is further described by the following examples, which should not be construed as limiting the scope of the invention. [Brief description of the drawings]

[0119] [Figure 1A]Graphs showing that anti-CCR7 antibody is effective in preventing the onset of GVHD. A) Relative weight loss in three experimental groups: control group in which mice were treated with PBS (n=4), isotype control (IC) group in which mice were treated with an irrelevant antibody (n=5), and anti-CCR7 group in which mice were treated with an antibody targeting CCR7 (n=5). Body weight on day 0 was taken as 100%. P value indicates the comparative analysis results between the anti-CCR7 group and other groups. [Figure 1B] B) Kaplan-Meier survival curves for all experimental groups. [Figure 1C] C) Percentage of human CD45+ cells found in serial samples of peripheral blood (PB) obtained from each experimental group. [Figure 1D] D) Percentage of human CD45+ cells found in lymphoid tissues (bone marrow and spleen) harvested at the time of animal euthanasia. [Figure 2A] Graphs showing that anti-CCR7 antibody is effective in treating early stage GVHD. A) Relative weight loss in experimental groups. Isotype control (IC) group (n=5) in which mice were treated with an irrelevant antibody, and anti-CCR7 group (n=5) in which mice were treated with an antibody targeting CCR7. Body weight on day 0 was taken as 100%. P value refers to the comparative analysis results between anti-CCR7 group and other groups. [Figure 2B] B) Kaplan-Meier survival curves for each experimental group. [Figure 2C] C) Percentage of human CD45+ cells found in serial samples of peripheral blood (PB) obtained from each experimental group. [Figure 2D] D) Percentage of human CD45+ cells found in lymphoid tissues (bone marrow and spleen) harvested at the time of animal euthanasia. [Figure 3A]Graphs showing that anti-CCR7 antibodies are effective in treating early and late stage GVHD. A) Relative weight loss in experimental groups. Isotype control (IC) group, where mice were first treated with a non-related antibody on day +3 (n=2), +7 (n=2), or +10 (n=1). Anti-CCR7 group, where mice were first treated with an antibody targeting CCR7 on day +3 (n=5), +7 (n=5), or +10 (n=5). Body weight on day 0 was taken as 100%. P values ​​refer to comparative analysis results between anti-CCR7 group and other groups. [Figure 3B] B) Kaplan-Meier survival curves for each experimental group. All animals from each IC group were grouped into one single group. [Figure 4] Graph showing the selection of anti-CCR7 mAbs. Several commercial antibody clones targeting CCR7 were characterized based on their ability to block CCR7-mediated migration towards CCL19 and CCL21 (A) and their potency to induce complement-mediated target cell killing (CDC) (B). Both migration (% of input, n=2 for basal, CK, 150503, and 2H4; n=1 for 6B3, 3D12, H60) and CDC (% specific lysis, n=2) were tested against CCR7-expressing chronic lymphocytic leukemia cells according to the procedures described in the Materials and Methods section. Bars represent the mean ± SD. Based on these results, clone 150503 was selected to perform in vitro and in vivo proof of concept in GVHD. [Figure 5A]Graphs showing the mechanism of action of neutralizing anti-CCR7 antibodies. A) Blocking CCR7 neutralizes target-mediated cell migration of apheresis-derived TN and TCM cells. Specific antagonism to CCR7-ligand interaction expressed as a reduction in % of input cells migrating is shown for CD4+ and CD8+ T cell subsets. In both cases, serum-starved PBMCs (n=3) isolated from apheresis were pre-incubated with 10 μg / ml anti-CCR7 or the respective isotype control (IC) for 30 min. Chemotaxis induced by 1 μg / ml CCL19 or CCL21 was then assayed in nude transwell chambers (4 h). Basal migration represents spontaneous migration in the absence of chemotactic stimulation. Cells migrated to the lower chamber were stained and counted by flow cytometry. The percentage of migrated cells (% input) was calculated as described in Materials and Methods. [Figure 5B] B) Anti-CCR7 mAb specifically eliminates TN and TCM. Specific elimination of CCR7 positive cells expressed as % of specific lysis mediated by complement activation (CDC) is shown for CD4+ and CD8+ T cell subsets. In both cases, apheresis-derived target cells (n=3) were incubated with 10 μg / ml anti-CCR7 or the respective isotype control (IC) for 30 min and then exposed to rabbit complement for 1.5 h. Cell lysis was determined by quantification of 7-AAD incorporation in each subset by flow cytometry. The percentage of specific lysis was calculated according to the formula given in Materials and Methods. Bars represent mean ± SD. ns, not significant; *, p<0.05; **, p<0.01; ***p<0.001. [Figure 6]Graphs showing that the percentage of infused CCR7+ T cell subpopulations in apheresis does not correlate with CMV or relapse rate. A-B) Percentage of infused CCR7+ T cell subpopulations in apheresis comparing recipient CMV infection status within the first 6 months after transplantation. Apheresis samples were analyzed by flow cytometry and separated into samples infused into patients who exhibited CMV DNA after transplantation (n=60) and those who did not (n=43). Percentages of infused CD4+CCR7+ (A) and CD8+CCR7+ (B) subpopulations in patients with and without CMV are shown. A viral load cutoff of >57 copies / ml was used to determine CMV reactivation.

[0120] C-D) Infused CCR7 in apheresis comparing patients with and without relapsed disease. + Percentage of T cell subpopulations. Apheresis samples were analyzed by flow cytometry and separated into samples infused into patients who relapsed after transplant (n=25) and those who did not relapse (n=78). CD4 + CCR7 + (C) and CD8 + CCR7 + (D) Percentages of subpopulations are shown. [Figure 7]

[0023] Figure 1 shows that the percentage of infused CCR7+ T cell subpopulation in apheresis does not correlate with recurrent disease. Apheresis samples were analyzed by flow cytometry and separated into samples infused in patients who relapsed after transplant (Yes) and those who did not relapse (No). Percentages of CCR7+ T cell (CD4+ or CD8+) subpopulations in apheresis comparing patients with and without relapsed disease: A) Myelodysplastic syndromes (MDS); yes ns CD4+ p=0.4199; CD8+ p=0.2117; B) Acute lymphoblastic leukemia (ALL); yes ns CD4+ p=0.5758; CD8+ p=0.1908; C) Acute myeloid leukemia (AML); yes ns CD4+ p=0.1638; CD8+ p=0.4126; D) Hodgkin lymphoma (HD); yes ns CD4+ p=0.5106; CD8+ p=0.8873; E) Non-Hodgkin lymphoma (NHL); yes ns CD4+ p=0.9926; CD8+ p=0.7369, F) shown for different hematological disorders including multiple myeloma (MM). EXAMPLES

[0121] Example 1: Antibodies to CCR7 as a means to treat GVHD Materials and Methods Samples, Reagents, and Flow Cytometry (FCM) Peripheral blood samples from healthy volunteers were obtained after informed consent. Analysis of CCR7 expression was then performed on normal T and B lymphocytes. Phycoerythrin (PE)-conjugated mouse anti-human CCR7 was purchased from R&D Systems (McKinley Place, MN). In all cases, appropriate isotype controls (IC) were included. Immunofluorescence staining was analyzed on a FACS CANTO II flow cytometer using DIVA software (BD Biosciences). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient centrifugation (Histopaque-1077, Sigma-Aldrich, Madrid, Spain).

[0122] Xenogeneic Mouse Models of GVHD The in vivo GVHD model was developed using NOD / SCID-IL2Rγ null For this purpose, in all models, animals were sublethally irradiated with 2 Gy and, 4 h later, 8 × 10 6 Human peripheral blood mononuclear cells (PBMCs) (in 200 μl PBS) were inoculated intravenously into each irradiated mouse. Both male and female mice aged 6–10 weeks were used for the in vivo proof-of-concept. Experiments were performed in the animal facilities of the Centro de Biologia Molecular Severo Ochoa (CBMSO), in accordance with Spanish law and the CBMSO Ethical Committee guidelines.

[0123] Clinical parameters evaluated in mice included weight loss, hunched posture (kyphosis), skin changes, hind limb paralysis (or reduced mobility), and tachypnea. To study infiltration into peripheral blood (PB), blood samples were taken at different time points during the experiment. To analyze infiltration into different tissues, mice were euthanized and organs / tissues, including spleen and bone marrow (BM), were harvested and disaggregated. In both cases, cells were labeled with a human-specific anti-CD45 FITC-mAb (clone HI30, BD Biosciences, www.bdbiosciences.com) and then analyzed by flow cytometry.

[0124] Prophylactic use of anti-CCR7 antibodies in mice To evaluate the efficacy of blocking CCR7 on donor cells, mice were used in a preventive setting. For this purpose, mice were first treated with either purified mouse anti-human CCR7 mAb (n=5 mice; clone 150503, isotype IgG2a, R&D Systems, Minneapolis, MN, USA), or an unrelated isotype control (IC) antibody (n=5 mice; IgG2a, Biolegend, San Diego, CA, USA), or PBS (n=4 mice). Both anti-CCR7 mAb and IC were injected intraperitoneally at approximately 10 mg / kg (approximately 200 μg / mouse). Two hours later, each animal was inoculated with PBMC from a single healthy donor. Animals received 4 or more doses of anti-CCR7, IC, or PBS every 4 days. PB samples were analyzed at +10, +13, +18, and +21 days after transplantation. BM and spleen were analyzed after animals were euthanized.

[0125] Therapeutic use of anti-CCR7 antibodies during the peak of GVHD To study the therapeutic efficacy of anti-CCR7 mAbs, a model was developed to evaluate whether anti-CCR7 mAbs had an effect on the alloreactive population found in PB and whether this approach attenuated GVHD symptoms. In this model, PBMC-bearing mice were first treated with approximately 10 mg / kg (approximately 200 μg / mouse) of anti-CCR7 (n=5) or its corresponding IC (n=5) on day +5 after engraftment. Treatment was repeated every 3 days. PB sampling was performed on days +10, +13, +18, +25 after transplantation. Spleen and BM sampling was performed when the animals were euthanized. The experiment was terminated 33 days after PBMC engraftment.

[0126] Another model assessed the efficacy of using anti-CCR7 at different time points during or after the peak of alloreactivity. For this purpose, 20 human PBMC-bearing mice were treated with anti-CCR7 (n=15) or IC (n=5). In the anti-CCR7 treatment group, 5 mice received their first dose on day +3, 5 mice received their first dose on day +7, and 5 mice received their first dose on day +10. In the IC treatment group, 2 mice were first treated on day +3, 2 mice on day +7, and 1 mice on day +10 after engraftment. The experiment was terminated on day +26.

[0127] Assay for determining inhibition of CCR7-dependent intracellular signaling The ability of anti-CCR7 antibodies to inhibit CCL19- and / or CCL21-mediated intracellular signaling in human CCR7-overexpressing Chinese Hamster Ovary (CHO) cells was determined by a well-established standard β-arrestin recruitment assay (PathHunter™, DiscoverX, Fremont, CA, USA; Southern et al., 2013, J Biomol Screen. 18(5):599-609).

[0128] Assay for determining inhibition of cell migration The ability of anti-CCR7 antibodies to inhibit the migration (chemotaxis) of human T cell lymphoma cells that endogenously express the human CCR7 receptor, induced by the ligands CCL19 and CCL21, was determined in a cell migration assay.

[0129] Cell migration assays were performed using transwell dual chambers (Costar, Cambridge, MA, USA) with 8 μm pore size inserts. The lower chamber contained ligands (CCL19 or CCL21) diluted in HamF12 medium supplemented with 0.5% BSA. CCR7 endogenously expressing cells (T cell lymphoma (HuT-78)) preincubated with CCR7 monoclonal antibody were placed in the inserts and the chamber assembly was incubated at 37° C. The amount of transmembrane migrating cells in the lower chamber was determined by DNA staining (CyQuant GR dye solution, Life Technologies Ltd, UK) after cell lysis.

[0130] Assay for Complement-Dependent Cytotoxicity (CDC) CDC assays were performed as described by Cuesta-Mateos et al. (Cancer Immunol Immunother. 2015, 64:665-76). Briefly, 2 × 10 5 PBMC target cells were plated in 96-well round-bottom plates with the indicated concentrations of purified anti-CCR7, alemtuzumab (anti-CD52), or IC antibodies. After 30 min at 37°C, cells were washed and added with complete RPMI1640 medium containing 25% rabbit complement (Serotec, Oxford, UK) with or without prior heat inactivation (56°C, 30 min). After 1.5 h, cells were stained with anti-CD19-FITC, anti-CD3-PE, and anti-CD5-APC mAbs to distinguish CLL and T cell populations. 7-AAD was used as a viability exclusion dye. The percentage of specific lysis (SL%) was calculated using the formula: 100 x (% dead cells with activated complement -% dead cells with inactivated complement) / (100 -% dead cells with inactivated complement).

[0131] Assay to determine the absence of agonist effects Anti-human CCR7 binding monoclonal antibodies, tested at high concentration (267 nM), were tested using a well-established standard β-arrestin recruitment assay (Pathhunter™, DiscoverX, Fremont, CA, USA; Southern et al., 2013, J Biomol Screen. 18(5):599-609) for the (absence of) induced detectable intracellular agonist effects in human CCR7-overexpressing Chinese Hamster Ovary (CHO) cells (data not shown). An irrelevant IgG2a was used as a negative control and CCL21, the natural ligand of CCR7, was used as a positive control. Anti-human CCR7 binding antibodies are found to lack detectable intracellular agonist effects if they do not induce an intracellular agonist effect as well as the negative control.

[0132] Biacore affinity assay The affinity of the monoclonal antibodies was determined by Biacore measurements under standard conditions. The monoclonal antibodies were immobilized on an appropriate sensor surface, and a solution of the sulfated antigen SYM1899 ((pyroGlu)DEVTDDZIGDNTTVDZTLFESLCSKKDVRNK, SEQ ID NO: 3), which contains residues 19-49 from the N-terminus of human CCR7, was flowed over the sensor surface.

[0133] result Prophylactic administration of anti-CCR7 prevents the onset of GVHD Mice receiving the first preventive dose of anti-CCR7 before engraftment of hPBMCs and four consecutive doses after engraftment did not develop any GVHD clinical signs. In contrast, mice receiving IC or PBS showed clinical signs including weight loss (Figure 1A). Weight differences were observed between days +9 and +12 after transplantation (IC vs. anti-CCR7, p=0.045; and PBS vs. anti-CCR7, p=0.0134). Of note, animals receiving anti-CCR7 rather gained weight throughout the experiment. Thus, anti-CCR7 antibodies extended overall survival (Figure 1B). Animals treated with anti-CCR7 mAb did not develop any clinical signs and survived up to day 32, the time point at which the animals were sacrificed, making this period a bona fide disease-free period. In contrast, control mice exhibiting severe clinical signs were euthanized on days +11, +13, +14, and +18. On days +13, +14, and +18, one animal from the anti-CCR7 treatment group was sacrificed to prepare a comparative analysis of organ infiltration. None of the animals that received anti-CCR7 antibody showed clinical signs and were sacrificed for purely experimental purposes. Thus, during these days, anti-CCR7 treated mice did not develop any clinical signs and gained weight, and therefore the presence of reactive donor cells was not detected in the PB from anti-CCR7 treated mice (Figure 1C). In contrast, the presence of GVHD-inducing cells in the PB of controls increased over time. At the time of sacrifice, infiltration was analyzed in the BM and spleen (Figure 1D). Consistent with the findings in the PB, GVHD-inducing cells were not found in lymphoid tissues from animals treated with anti-CCR7 mAb. On the contrary, there was a constant infiltration of these tissues in the control groups (BM control vs. BM anti-CCR7: 34.6% vs. 0.57%, p=0.002; BM IC vs. BM anti-CCR7: 41.7% vs. 0.57%, p=0.003 / spleen control vs. spleen anti-CCR7: 70.1% vs. 0.17%, p≦0.001; spleen IC vs. spleen anti-CCR7 71.3% vs. 0.17%, p≦0.001). No differences were observed between the control groups (PBS vs. IC: BM, p=0.57 / spleen, p=0.86).

[0134] Anti-CCR7 therapy ameliorates GVHD To demonstrate the therapeutic efficacy of anti-CCR7 antibodies in vivo, a model was used in which animals were treated when alloreactive responses occurred. These responses, occurring on days +3 to +5, are a major contributor in GVHD pathogenesis. Thus, in one model, human PBMCs were engrafted into immunodeficient mice. Animals were treated with either IC (n=5) or anti-CCR7 antibodies (n=5). The first dose of antibody was administered on day +5, with successive doses given every 2 days. In this model, anti-CCR7 antibodies positively affected the weight of the animals (Figure 2A). In contrast, control animals lost weight. Differences were first observed on day +12. In addition, anti-CCR7 therapy extended overall survival (Figure 2B).

[0135] The animals treated with anti-CCR7 mAb did not develop any clinical signs and survived up to 33 days, the time point when the animals were sacrificed, which was considered a genuine disease-free period. In contrast, control mice exhibiting severe clinical signs were euthanized on days +12, +20, and +28. On days +12 and +28, one animal from the anti-CCR7 treatment group was sacrificed to prepare a comparative analysis of organ infiltration. None of these animals that received anti-CCR7 antibody showed clinical signs, and sacrifice had an experimental purpose. Thus, during these days, anti-CCR7-treated mice did not develop any clinical signs and gained weight, and therefore the presence of reactive donor cells was not detected in the PB from anti-CCR7-treated mice (Figure 2C). In contrast, the presence of GVHD-inducing cells in the PB of controls increased over time. Of note, significant differences in PB infiltration were observed from day +10 (control vs. anti-CCR7: 12.6% vs. 2.6%; p=0.02). These differences expanded to day +12 and remained distinct until the end of the experiment (47.3% vs. 6.5%; p<0.001) (Figure 2C). At the time of sacrifice, infiltration was analyzed in BM and spleen (Figure 2D). Consistent with the findings in PB, a small percentage of GVHD-inducing cells was found in lymphoid tissues (BM and spleen) from animals treated with anti-CCR7 mAb. In contrast, there was a constant infiltration of these tissues in the control group (BM control vs. BM anti-CCR7: 27.3% vs. 3.5%; p=0.005 / spleen control vs. spleen anti-CCR7: 59.2% vs. 8.4%; p=0.006).

[0136] In another model, we aimed to evaluate the efficacy of anti-CCR7 antibodies in treating GVHD at different times after the onset of the disease. For this purpose, anti-CCR7 antibodies were administered at different time points after engraftment. Animals were treated on days +3, +7, and +10 of donor PBMC engraftment. 15 mice were treated with anti-CCR7 antibodies (5 on day +3, 5 on day +7, 5 on day +10) and 5 mice were treated with IC (2 on day +3, 2 on day +7, 1 on day +10). All mice received successive doses every 2 days until the end of the experiment. Mice that received the first dose of anti-CCR7 antibodies on day +3 showed increased body weight and prolonged overall survival (Figures 3A and 3B). The animals treated with anti-CCR7 mAb did not develop any clinical signs and survived up to 26 days at the time of sacrifice, which was considered a genuine disease-free period. In contrast, the control mice showed an overall median survival of 14 days. Notably, animals treated with anti-CCR7 antibodies from day +7 or +10 showed the worst outcome compared to mice in which treatment was started on day +3. However, animals in which treatment was only started on day +7 or +10 still showed a better outcome than the respective controls. Some animals that received the first dose on day +7 or +10 lived until day +19, while no animals in the respective control groups survived longer than day +12.

[0137] Anti-CCR7 antibody is a human T N and T CM Impairs in vitro chemotaxis of cells toward CCL19 and CCL21 These results prompted us to evaluate the usefulness of CCR7 not as a biomarker for selecting suitable grafts, but as a targetable receptor for antibody-based therapy. To evaluate the usefulness of CCR7 as a targetable receptor, we selected and used antibodies that are capable of blocking CCR7-ligand interactions and killing target cells via CDC or antibody-dependent cellular cytotoxicity (ADCC) (Figure 4).

[0138] The ability of selected mAbs to inhibit ligand-driven chemotaxis of apheresis-derived hPBMCs was then first evaluated. As expected, when PBMCs were preincubated with IC, the addition of CCL19 or CCL21 to the culture medium inhibited the CCR7 + T N and T CM induced the migration of a subset of T N However, binding of 10 μg / ml of anti-CCR7 mAb reduced migration to basal levels in these cells. EM and T EMRA do not migrate in response to CCR7 ligands, and thus anti-CCR7 inhibits T EM and T EMRA had no effect on the behavior of

[0139] Anti-CCR7 antibodies bind to CCR7 via CDC. + Human T N and T CM Specific removal of cells As previously described (Cuesta-Mateos C. Targeting CCR7 in T-cell Prolymphocytic Leukemia. CONTROL-T: International Conference April 2016 Mature T-cell lymphomas - molecular pathology, modeling of cellular dynamics, and therapeutic approaches. 2016), the selected antibodies were potent enough to kill tumor T cells via CDC, but their targeting of healthy CCR7 was not sufficient. + The effect on T cell subsets has not been addressed previously. Therefore, an in vitro CDC assay was performed using fresh hPBMCs from apheresis. CD4 + T N Or T CM Upon binding to cells, 10 μg / ml of anti-CCR7 mediated potent CDC activity (Figure 5B). Similar results were observed with CD8 + T NIn contrast, anti-CCR7 mAb inhibited CCR7-negative T cells. EM and T EMRA This suggests that anti-CCR7 therapy can preserve effector cells and thus protect against pathogens and maintain the GVL effect. To explore this idea further, we investigated the effector cells in the grafts and their effect on the survival of CCR7. + We investigated whether the number of CD4 + CCR7 + or CD8 + CCR7 + No clear differences were found between patients with and without CMV reactivation, either in percentage (Figures 6A and 6B) or absolute numbers (data not shown). Furthermore, multivariate logistic regression analysis (Table 1) demonstrated that the CCR7 + We confirmed that the percentage of CD4 + (p=0.144);CD8 + (p=0.092)].

[0140] [Table 1]

[0141] Similarly, CCR7 in grafts + The percentage or absolute number of cells did not correlate with the rate of disease relapse after transplantation, and again, no clear differences were observed between patients who relapsed and those who did not (Figures 6C and 6D and data not shown). Thus, multivariate logistic regression analysis (Table 1) suggested that the expression of CCR7 in grafts was significantly associated with a higher incidence of CCR7+ in grafts than in non-grafts. + We confirmed that the percentage of CD4 + (p=0.702);CD8 + (p=0.362)]. Finally, CCR7 in the grafts +The lack of association between cell percentage and incidence of relapse was further confirmed when patients were grouped according to the underlying disease diagnosis (Figure 7). Taken together, these results preclude the use of CCR7 (in apheresis) as a biomarker to predict CMV infection or relapsed disease, although, concomitantly, this suggests that the presence of CCR7 in the graft may be a predictor of relapse. + It was suggested that any approach aimed at reducing the percentage of cells was not associated with a higher risk of infection or a higher rate of recurrence.

[0142] Anti-CCR7 mAb blocks CCR7 signaling without agonist effects Monoclonal antibodies bearing HVRs of SEQ ID NOs: 1 and 2, tested at high concentration (267 nM), did not show any detectable agonist effect in human CCR7-overexpressing Chinese Hamster Ovary (CHO) cells as determined by a well-established standard β-arrestin recruitment assay (Pathhunter™, DiscoverX, Fremont, CA, USA; Southern et al., 2013, J Biomol Screen. 18(5):599-609) (data not shown).

[0143] Consideration GVHD is a frequent and potentially fatal complication induced after allogeneic transplantation. It has been recently demonstrated that higher CCR7 expression in donor cells correlates with a higher grade of recipient secondary lymphoid organ (SLO) infiltration and thus a higher likelihood of finding alloantigens that can lead to an allogeneic immune response. Our previous data demonstrated that migration to SLO is dependent on CCR7, and a correlation between migration toward CCR7 ligands and the onset and grade of GVHD has been established (Portero-Sainz, I et al., Bone Marrow Transplantation (2017), 1-8). Similarly, other publications propose that naive T cells and TCMs are key players in the development of both aGVHD and cGVHD (Yakoub-Agha, I. et al., Leukemia, 2006.20(9):1557-65; Distler, E. et al., Haematologica, 2011.96(7):1024-32; Cherel, M. et al., Eur J Haematol, 2014.92(6):491-6), but naive T cells have a greater capacity to respond to recipient antigens than TCMs. These data suggest the possibility of using CCR7 as a therapeutic target in immunotherapy, not only because of its high density in naive T cells, TCMs, and some APCs, but also because of its critical role in disease progression and pathogenesis. In this sense, the present inventors have demonstrated in vivo that administration of anti-CCR7 antibodies to NHPs results in selective reduction of naive T cells and TCM cells (data not shown). Furthermore, they have shown that anti-CCR7 antibodies are effective in blocking migration of CCR7-expressing T cells toward CCR7 ligands (data not shown). Finally, anti-CCR7 antibodies are effective in preventing and treating GVHD, as demonstrated using an in vivo mouse model.Notably, the most effective therapeutic approach involved administration of anti-CCR7 antibodies on days +3 to +5, thus overlapping with the therapeutic window in which cyclophosphamide is used to prevent alloreactivity in HSCT (Luznik, L. et al., Biol Blood Marrow Transplant, 2002.8(3):131-8). Taken together, the results of the preclinical use of anti-CCR7 antibodies confirm that removing CCR7-expressing cells (including naive T cells and TCMs) and / or neutralizing their migration to SLOs is a rational approach to prevent and / or treat GVHD. Thus, by removing CCR7-expressing cells and / or blocking their migration to SLOs, alloreactive CCR7-expressing cells will not be activated, thus impairing the development of GVHD.

[0144] Thus, Sasaki et al. (2003, J Immunol, 170(1):588-96) showed that early use of a CCL21 antagonist prevented donor T cell entry into lymph nodes and therefore GVHD development. Dutt et al. showed that removal of naive CD62L cells delayed GVHD onset and extended OS in a preclinical in vivo model (Dutt, S. et al., Blood, 2005.106(12):4009-15). Similarly, in clinical settings, removal of CD45RA-expressing naive T cells from apheresis affected the incidence and development of GVHD (Touzot, F. et al., J Allergy Clin Immunol, 2015.135(5):1303-9 e1-3; Shook, DR et al., Pediatr Blood Cancer, 2015.62(4):666-73; Triplett, BM et al., Bone Marrow Transplant, 2015.50(7):1012). However, in all these papers, in contrast to anti-CCR7 therapy, TCM cells were not targeted. In conclusion, it is important to mention that recent evidence suggests that immunity against infectious diseases is not dependent on CCR7+ cells (Choufi, B. et al., Bone Marrow Transplant, 2014.49(5):611-5), and therefore, removing and / or blocking CCR7-expressing cells is considered a safe approach for recipient patients.

[0145] Example 2: Identification of patients at low risk of GVHD Materials and Methods A cohort of 103 donor-recipient pairs (see Table 2) undergoing allo-HSCT at La Princesa University Hospital, Madrid, Spain was analyzed (Portero-Sainz et al., 2017). The study protocol was approved by the Ethics Committee (reference number PI-624) and was carried out in accordance with the Declaration of Helsinki.

[0146] [Table 2] JPEG2025066696000003.jpg194149

[0147] Phenotype identification Apheresis samples were stained with a seven-color panel of antibodies (Table 3) as previously described (Portero-Sainz et al., 2017). Relative and absolute counts of T cell subsets refer to total white blood cell counts. N , T CM , T EM , and T EMRA Subsets were identified using the following antibodies: CD45RA-FITC, CD62L-PE, CD3-APC, CD4-PB (BD Biosciences, San Jose, Calif.).

[0148] [Table 3]

[0149] therapeutic antibodies Purified mouse anti-human CCR7 mAb (IgG2a isotype) was purchased from R&D Systems (MN, USA) and matched isotype control (IC) was purchased from Biolegend (CA, USA).

[0150] statistical analysis Qualitative variables are presented as relative frequencies (percentages, %) and absolute frequencies (number, n). Quantitative variables are expressed as mean (mean) and degree of dispersion (SD or SEM). Qualitative data between groups were compared by Pearson's χ2 test or Fisher's exact test, as appropriate. Quantitative variables with homogeneity of variance (Levene's test) were analyzed using t-tests or one-way analysis of variance (ANOVA). Mann-Whitney U test or Kruskal-Wallis test were used for heterogeneity of variance.

[0151] Binary logistic regression analysis was performed accordingly in the cohort of patients described by Portero-Sainz et al. (2017) to assess the risk of confounding variables: age, HLA and CMV status, sex, conditioning regimen, infused CD3+ and CD34 + Predictors of GVHD and CMV reactivation (or recurrent disease) adjusting for number of tumors, allosensitization, relapse after HSCT, underlying disease, graft source, and preventive measures were identified.

[0152] Exemplary univariate analyses were performed to investigate variables associated with the dependent variables aGVHD, cGVHD, and CMV infection or recurrent disease (P<0.05). Confounding variables reaching the probability threshold for univariate analysis (P<0.10) were included in the multivariate logistic regression model. The sensitivity ± 95% CI of the aGVHD risk score from our previous model (Portero-Sainz et al., 2017) was estimated by ROC curves. The sensitivity of the CMV infection risk score was calculated similarly. Significance was set at a value of P<0.05. A cutoff value of viral load >57 copies / ml was used to determine CMV reactivation. Statistical analyses were performed using Stata version 13.0 (College Station, TX, USA).

[0153] result CCR7 + Apheresis selection based on cell percentage does not prevent or delay GVHD To establish a potential cut-off point for selecting apheresis with a low risk of developing GVHD, we performed a sensitivity analysis (ROC curve) on our cohort and arbitrarily selected the 25th percentile (<3.6%) to identify patients engrafted with a low percentage of CCR7+ T cells. As seen in Table 4, the percentage within the 25th percentile (<3.6%) of CD4 + CCR7 + 87.88% (75.23%-100%) of patients who received grafts containing CD8 cells did not develop aGVHD. + CCR7 + In our case, selection of <2.2% cells (25th percentile) in apheresis was associated with a sensitivity of 88.57% (76.60%-100%) for cGVHD.

[0154] [Table 4]

[0155] Further embodiments are as follows. [Embodiment 1] An anti-CCR7 antibody or antigen-binding fragment thereof for use in preventing or treating graft-versus-host disease (GVHD) in a recipient of a transplant comprising donor cells. [Embodiment 2] IC of 100 nM or less for inhibiting at least one of CCR7-dependent intracellular signaling and CCR7 receptor internalization by at least one CCR7 ligand selected from CCL19 and CCL21 50 2. The anti-CCR7 antibody or antigen-binding fragment thereof of embodiment 1, comprising: [Embodiment 3] The anti-CCR7 antibody or antigen-binding fragment thereof of embodiment 2, which inhibits CCR7-dependent intracellular signaling without substantial agonistic effects. [Embodiment 4] The anti-CCR7 antibody is a 20-fold increase over the K d K for the N-terminal extracellular domain of human CCR7 is up to 20-fold higher than d and the reference anti-CCR7 antibody is a murine anti-CCR7 antibody, the heavy chain variable domain of which has an amino acid sequence represented by SEQ ID NO: 1 and the light chain variable domain of which has an amino acid sequence represented by SEQ ID NO: 2. [Embodiment 5] The anti-CCR7 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4, which is a chimeric, humanized, or human antibody. [Embodiment 6] The anti-CCR7 antibody or antigen-binding fragment thereof according to embodiment 5, which is an antibody having the HVR of an anti-human CCR7 antibody in which the amino acid sequence of the heavy chain variable domain is SEQ ID NO: 1 and the amino acid sequence of the light chain variable domain is SEQ ID NO: 2. [Embodiment 7] The anti-CCR7 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, which achieves at least one of killing CCR7-expressing cells in a recipient, inducing apoptosis of the cells, inhibiting migration of the cells, inhibiting activation of the cells, inhibiting proliferation of the cells, and inhibiting dissemination of the cells. [Embodiment 8] The anti-CCR7 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 7, wherein the transplant comprising donor cells is a transplant comprising one or more of an organ, a tissue, a progenitor cell, a stem cell, and a hematopoietic cell. [Embodiment 9] The anti-CCR7 antibody or antigen-binding fragment thereof of embodiment 8, wherein the transplant comprising donor cells is a transplant comprising hematopoietic stem or progenitor cells. [Embodiment 10] The anti-CCR7 antibody or antigen-binding fragment thereof of embodiment 9, wherein the recipient is suffering from a malignant disorder, and preferably, the prevention or treatment of GVHD maintains or promotes a graft-versus-tumor or graft-versus-leukemia effect. [Embodiment 11] Prevention or treatment of GVHD a) administration of an anti-CCR7 antibody to the recipient prior to the recipient receiving a transplant containing donor cells; b) administering an anti-CCR7 antibody to the recipient after the recipient receives a transplant comprising donor cells, and preferably before the recipient exhibits symptoms of GVHD or the recipient is diagnosed with GVHD; c) administering an anti-CCR7 antibody to the recipient after the recipient receives a transplant comprising donor cells, and preferably after the recipient exhibits symptoms of GVHD or is diagnosed with GVHD; d) administration of an anti-CCR7 antibody to a recipient of a transplant comprising donor cells, the transplant having been prepared prior to transplantation by ex vivo incubation with the anti-CCR7 antibody or an antigen-binding fragment thereof; and e) Administration of anti-CCR7 antibodies to the recipient after relapse of GVHD The anti-CCR7 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 10, comprising at least one of the following: [Embodiment 12] 1. An ex vivo method for preparing a donor-derived organ, tissue, or cell preparation for transplantation into a recipient, comprising: a) incubating a donor-derived organ, tissue or cell preparation with an anti-CCR7 antibody or antigen-binding fragment thereof according to any one of embodiments 2 to 7, whereby the anti-CCR7 antibody achieves at least one of i) a reduction in the number and ii) an inhibition of the activity of CCR7-expressing donor cells in said organ, tissue or cell preparation; and b) optionally removing at least one of anti-CCR7 antibodies and CCR7-expressing donor cells from said organ, tissue or cell preparation. A method comprising: [Embodiment 13] 13. The method of embodiment 12, wherein the anti-CCR7 antibody is comprised in a preservation solution used to preserve the organ, tissue, or cell preparation prior to transplantation. [Embodiment 14] 14. The method of embodiment 13, wherein the organ or tissue is perfused or washed with a preservation solution comprising an anti-CCR7 antibody. [Embodiment 15] 14. The method of embodiment 12 or 13, wherein the anti-CCR7 antibody and the CCR7-expressing donor cells are removed from the cell preparation by affinity purification of the anti-CCR7 antibody and its bound CCR7-expressing donor cells.

Claims

[Claim 1] An anti-CCR7 antibody or antigen-binding fragment thereof for use in preventing or treating graft-versus-host disease (GVHD) in a recipient of a transplant comprising donor cells.

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