Improved stem cell populations for allogeneic therapy

Abstract

The present invention relates to improved stem cell populations for allogeneic stem cell therapy, particularly for the treatment and re-treatment of presensitized patients. Further, methods for obtaining said stem cell populations are provided. Furthermore, the present invention relates to pharmaceutical compositions comprising said stem cell populations and their use in allogeneic stem cell therapy.

Description

[Technical field]

[0001] The present invention relates to an improved stem cell population for allogeneic stem cell therapy, in particular for the treatment and re-treatment of presensitized patients.Furthermore, a method for obtaining said stem cell population is provided.Furthermore, the present invention relates to pharmaceutical compositions comprising said stem cell population and their use in allogeneic stem cell therapy. [Background technology]

[0002] 2. Background of the Invention Stem cells for use in research and medical applications can be derived from embryonic, fetal or adult tissues, including embryonic stem cells (ESCs), umbilical cord stem cells, induced pluripotent stem cells (iPSCs) and adult stem cells from different sources. Numerous clinical trials are currently underway or have been successfully completed for the treatment of fistulas, leukemia, lymphoma, neurodegenerative diseases, brain and spinal cord injuries, heart disease, blindness and vision impairment, pancreatic beta cell loss of function, cartilage repair, osteoarthritis, musculoskeletal diseases, wounds, infertility, autoimmune diseases and inflammatory diseases, such as inflammatory bowel disease. An overview of the current medical applications of different types of stem cells can be found in Mahla RS, International Journal of Cell Biology. 2016 (7): 1-24.

[0003] Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into a variety of cell types, including osteoblasts, chondrocytes, myocytes and adipocytes.

[0004] The use of autologous and allogeneic MSCs to treat various pathologies has been evaluated in several clinical trials (Galipeau and Sensebe, (2018), Cell Stem Cell 22, 824-833, especially Table S1), including those focused on the treatment of damaged gastrointestinal tract, sepsis and graft-versus-host disease as well as several autoimmune and inflammatory diseases.

[0005] Adult MSCs can be found in almost all tissues and are mainly located in the perivascular microenvironment. The majority of clinical trials have been conducted using bone marrow MSCs (BM-MSCs), but an increasing number of trials are using MSCs from other sources, such as adipose tissue. BM-MSCs and adipose-derived mesenchymal stem cells (ASCs) share similar potential and replication ratios. However, ASCs have the advantage of being more easily collected and present in large quantities in samples for culture (100 times more in 1 gram of adipose tissue compared to BM tissue).

[0006] Standard immunological dogma holds that any foreign tissue will elicit an immune response in the organism. This concept is clearly prominent in solid organ and hematopoietic transplants, where aggressive immunosuppression is the standard to protect the allograft from rejection. The strongest evidence for the role of antibodies in graft rejection is the hyperacute rejection of mostly vascularized organs, e.g., the kidney and the heart. In recipients exhibiting these reactions, high titers of antidonor antibodies can be demonstrated. These antibodies bind to HLA antigens on endothelial cells, followed by complement fixation and accumulation of polymorphonuclear cells. Then, presumably as a result of enzymes released from polymorphonuclear leukocytes, endothelial damage occurs, followed by platelet accumulation and thrombus formation, resulting in renal cortical necrosis or myocardial infarction. To avoid this anti-HLA antibody-mediated reaction, HLA typing is performed in clinical organ and tissue transplants to match donors and recipients as closely as possible to avoid graft rejection.

[0007] As the field of cell-based therapy develops, it has become evident that various cell types, of which mesenchymal stem cells (MSCs) are the prototype, may have the ability to evade and / or suppress the immune system, and as a result, in some trials, MSCs have been applied without immunosuppression.

[0008] Griffin et al., Immunol. & Cell Biol. (2012), 1-12, review experimental data to determine whether there is good in vivo evidence supporting the “immune privileged” status of allo-MSCs. The authors considered published studies on the immunogenicity of allo-MSCs after activation by inflammatory stimuli or after differentiation. The authors summarize that the majority of these studies document specific cellular (T cell) and humoral (B cell / antibody) immune responses to donor antigens after administration of unmanipulated IFN-γ-activated and differentiated allo-MSCs, with subsequent rapid rejection of the allogeneic transplant and apparently promoting donor-specific tolerance. In several of these studies, rapid rejection of the graft or administered MSCs (see Tables 1 and 2 in Griffin et al.) was observed. The authors recommended revisiting the concept of the immune privileged nature of allo-MSCs and more precisely examining the scope of clinical implications of anti-donor immune responses elicited by allo-MSCs. Therefore, anti-donor immune responses may have implications for efficacy and / or adverse effects resulting from improved clearance of allo-MSCs in human and animal allogeneic therapies.

[0009] In view of this, there exists a need for means suitable for allogeneic therapy of patients, said means exhibiting reduced immunogenicity and / or improved compatibility in presensitized patients and / or in allogeneic therapy for re-treatment.Furthermore, methods for obtaining them and their medical uses shall also be provided. Summary of the Invention

[0010] The technical problem underlying the present invention is solved by the provision of the subject matter defined in the claims.

[0011] According to a first aspect, there is provided an in vitro method for selecting a stem cell (SC) population suitable for allogeneic therapy, in particular for the treatment of a presensitized patient or for the re-treatment of a patient with allogeneic therapy, comprising the following steps: a) culturing a sample of the SC population in the presence of a concentration of IFN-γ capable of inducing maximal HLA-class I expression in said SC population (test sample) and separately culturing a sample of the SC population in the absence of IFN-γ (control sample); b) under conditions such that HLA-class I antibodies bind to HLA-class I expressed in the test sample and the control sample. , different The range of concentrations HLA-class I antibodies Test sample and Control sample To To make contact; c) adding complement to the test and control samples so that bound HLA-class I antibodies are saturated with complement and complement-dependent cytotoxicity (CDC) is induced; d) determining CDC for a range of different concentrations of HLA-class I antibodies by measuring induced cell lysis in test and control samples; e) 50% of maximum CDC (EC 50 determining the concentration of HLA-class I antibodies in the test sample and the control sample that induces a β-glucose level (β-glucose level); and f1) EC of test sample 50 EC of control samples versus values 50 Selecting the SC population for allogeneic therapy if the ratio of values ​​is less than 1.25, preferably less than 1.0, more preferably less than 0.5; particularly preferably less than 0.25; or f2) EC of test sample 50 Selecting the SC population for allogeneic therapy if the value is at least 3.5 ng / ml, preferably at least 9 ng / ml, more preferably at least 15 ng / ml, particularly preferably at least 20 ng / ml of HLA-class I antibodies. A method is provided comprising:

[0012] According to a second aspect, there is provided a population of SCs suitable for allotherapy, in particular for the treatment of presensitized patients or for re-treatment with allotherapy, said population having the following characteristics: i) EC of the test sample 50EC of control samples versus values 50 The ratio of the EC values ​​is less than 1.25, preferably less than 1.0, more preferably less than 0.5, and particularly preferably less than 0.25, wherein the EC 50 The value is determined as set forth in the method of the present invention; ii) EC of the test sample 50 a value of at least 3.5 ng / ml HLA-class I antibodies, preferably at least 9 ng / ml, more preferably at least 15 ng / ml, particularly preferably at least 20 ng / ml HLA-class I antibodies, wherein said EC 50 The value is determined as set forth in the method according to the invention; and / or iii) the ratio of CD46 expression in the test sample to the CD46 expression in the control sample is greater than 2.0, preferably greater than 2.5, particularly preferably greater than 3.0, wherein said CD46 expression is determined as set out in the method of the invention; The present invention provides a population of SCs having any of the following:

[0013] According to a third aspect, there is provided a pharmaceutical composition comprising a population of SCs according to the invention and optionally a pharma- ceutically acceptable carrier.

[0014] According to a fourth aspect, there is provided a method for preparing a pharmaceutical composition comprising the steps of: a) carrying out the method of the present invention; and b) formulating the selected SC population with at least one pharma- ceutically acceptable carrier; A method is provided comprising:

[0015] According to a fifth aspect, there is provided the use of the SC population according to the invention in allogeneic stem cell therapy in a patient in need thereof, preferably in a presensitized patient or a patient undergoing re-treatment.

[0016] According to a sixth aspect, there is provided a method of allogeneic stem cell therapy comprising administering the SC population of the present invention to a patient in need thereof, preferably a presensitized patient or a patient undergoing re-treatment.

[0017] The inventors investigated whether allosensitization would occur if ASC populations were administered to patients suffering from Crohn's disease and treatment-resistant complex draining perianal fistulas (where the ASC populations were administered intralesionally to allogeneic non-HLA-matched recipients). It was found that at time 0, only 4 patients showed antibody reactivity against HLA-class I molecules, whereas after 12 weeks of treatment, 22 patients showed this antibody reactivity. In the control group, only an increase in reactivity was observed from 6 patients (time 0) to 9 patients after 12 weeks. ASC cell populations from different donors were then investigated. It was found that plasma samples from presensitized patients and patients showing anti-HLA class I reactivity were significantly different in their binding behavior and their ability to induce complement-dependent cytotoxicity (CDC) in IFN-γ-induced ASC populations from different donors. Based on these findings, the inventors further determined that SC populations from different donors differ significantly in their HLA-class I binding and their susceptibility to cell lysis due to activation of classical complement pathway (formation of antibody / complement complex), where SC populations that are more resistant to alloreactivity in vitro are preferred for allogeneic therapy purposes.This is especially true in situations where patients are pre-sensitized to HLA-class I antigens, for example, by early pregnancy, or in re-treatment situations where the same donor SC population is administered multiple times to the same patient.The inventors further determined that the increased resistance to alloreactivity is mediated by CD46 expression, and that inhibition of CD46 expression in ASC cells correlates with increased CDC susceptibility. [Brief description of the drawings]

[0018] [Figure 1]Figure 1 shows the characterization of DSA production in ADMIRE CD1. A. Distribution of Labscreen Mixed / Labscreen Single Antigen (LSM / LSA) (Tait et al. (2013), Transplantation 95: 19-47) results for the indicated visits: pre-treatment (baseline), W12 and W52 in both the placebo (lower chart) and Cx601 (upper chart) arms of the study. A total of 5 (Cx601 group) and 12 (placebo group) patients discontinued the study and therefore LSM / LSA data were unavailable. B. Kinetic curves showing anti-HLA Ab titers expressed as the sum of mean fluorescence intensities (ΣMFI) from each microsphere measured by LSM assay at the indicated time points. The dotted line in each graph indicates the threshold of MFI > 3,000 used to call for positivity. The circle in the top right panel indicates Patient 92 (Pat92). C. Graph showing HLA mismatches between each patient and ASC. For mismatch correlation, the percentage of those who produced DSA was plotted against the number (range) of mismatched eplets (non-shared unique chains of polymorphic residues found at a locus) (Duquesnoy (2002) Hum. Immunol. 63: 339-352). For linear regression, the Pearson test (r2) was applied. P values ​​were determined by Student's t-test. [Diagram 2]Figure 2 shows HLA expression on ASCs and anti-HLA Ab binding in vitro. A. Graph showing correlation between MFI increase and respective concentrations of class I HLA (W6 / 32) and class II HLA (L243) Abs directed against untreated (black circles) and IFNγ preactivated (white squares) ASCs. B. Plots of FcTox (complement dependent cytotoxicity by flow cytometry assay) representing negative control (isotype), positive control (hyperimmune sample, HI pool) and Patient 92 (Pat92, untreated patient who produced a de novo DSA) serum. The bottom left panel shows FACS binding intensity quantification (total number of IgG+ cells) using isotype control (light grey), hyperimmune serum (dark grey) or Pat92 serum (medium dark grey). The bottom right panel shows FACS cell death quantification (total number of 7-AAD+ cells) in control conditions (no rabbit complement, light grey), hyperimmune serum or Pat92 serum (medium dark grey). P values ​​were determined by Student's t-test and r2 was determined by Pearson's test. [Diagram 3] FIG. 3 shows that ADMIRE CD1 plasma samples induce low cytotoxic death in ASCs in vitro. A. Graph showing normalized percentage values ​​of HLA-I binding for 10 presensitized patients (top panel) and 17 de novo DSA+ patients (bottom panel) at the indicated time points (pre-W0 and post-W12 treatment). ASCs from DonA (donor administered in the ADMIRE CD1 study) and DonB were grown for 48 hours in normal (basal) conditions or in the presence of 3 ng / mL IFNγ prior to the binding assay. B. Graph showing normalized percentage values ​​of 7-AAD positive ASCs for 10 presensitized patients (top panel) and 17 de novo DSA+ patients (bottom panel) at the indicated time points. P values ​​were determined by Student's t-test. [Figure 4]Figure 4 shows that ASCs express high levels of mCRP. A. Graph showing MFI values ​​of CD46, CD55 and CD59 in seven ASC donors (black bars) and one BM-MSC donor (white bars) by FACS analysis. Cells were grown for 48 hours in the presence of 3 ng / mL (IFNγ) or left untreated (basal). B. Graph showing different MFI values ​​of CD46, CD55 and CD59 in seven ASC donors determined by FACS analysis. Cells were grown for 48 hours in the presence of 3 ng / mL IFNγ (IFNγ) or left untreated (basal). C. Graph showing antibody binding (upper panel) and cell death (lower panel) depending on antibody concentration for ASCs from donors DonA, DonB, DonC, DonD, DonE, DonF and DonG cultured under basal conditions (left panel) and with IFNγ (right panel). P values ​​were determined by Student's t-test. From the bottom right panel, EC50 values ​​can be determined. [Diagram 5] FIG. 5 shows that CD46 mediates complement cytotoxicity in ASC. A. Graph showing the percentage of 7-AAD positive parental ASC and CD46KO DonB ASC against increasing concentration levels of W6 / 32 Ab. Prior to analysis, parental ASC and CD46KO DonB ASC were grown for 48 hours in the presence of 3 ng / mL IFNγ (IFNγ) or left untreated (basal). B. Sigmoidal curve showing the percentage of 7-AAD positive parental ASC and CD46KO ASC against the concentration of W6 / 32 Ab (linear to log10 converted). P values ​​were determined from a two-way ANOVA test. [Figure 6]Figure 6A-B. Correlation of MFI values ​​of W6 / 32 (A) and CD46, CD55 and CD59 (B) of ASC donors grown for 48 h in the presence of 3 ng / mL IFNγ (white squares) or under basal conditions (black circles). P-values ​​indicate the significance of the slope from the linear regression. R-squared values ​​for the significance of the slope in IFNγ conditions for CD46 and CD55 (B) were 0.74 and 0.71, respectively. C. Graph showing CD46 MFI values ​​in parental (white bars) and CD46KO (black and grey bars) ASCs. Parental and CD46KO ASCs were grown for 48 h with 3 ng / mL IFNγ (IFNγ) or left untreated (basal) prior to analysis. P-values ​​were determined by Student's t-test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Detailed Description of the Invention When the term "comprise" or "comprising" is used in the present description and claims, it does not exclude other elements or steps. For the purposes of the present invention, the term "comprising" is considered to be an optional embodiment of the term "comprising of". When a group is defined below as comprising at least a certain number of embodiments, this should also be understood as disclosing a group consisting of only these embodiments, as an option.

[0020] When an indefinite or definite article is used when referring to a singular noun, e.g., "a" or "an", "the", this includes the plural of that noun unless otherwise specified. Conversely, when the plural of a noun is used, it also refers to the singular.

[0021] Moreover, terms such as first, second, third or (a), (b), (c) in this specification and claims are used to distinguish between similar elements and are not necessarily used to describe a sequential or chronological order. The terms so used are interchangeable under appropriate circumstances, and it should be understood that the embodiments of the invention described herein can operate in sequences other than those described or illustrated herein.

[0022] In the context of the present invention, any numerical value indicated generally relates to an interval of accuracy that a person skilled in the art would understand to still ensure the technical effect of the feature in question. As used herein, the deviation from the indicated numerical value is in the range of ±10%, preferably ±5%. The aforementioned deviation from the indicated numerical interval of ±10%, preferably ±5%, is also indicated by the terms "about" and "approximately" used herein in relation to numerical values.

[0023] According to a first aspect, there is provided an in vitro method for selecting a stem cell (SC) population suitable for allogeneic therapy, in particular for the treatment of a presensitized patient or for the re-treatment of a patient with allogeneic therapy, comprising the following steps: a) culturing a sample of the SC population in the presence of a concentration of IFN-γ capable of inducing maximal HLA-class I expression in said SC population (test sample) and separately culturing a sample of the SC population in the absence of IFN-γ (control sample); b) under conditions such that HLA-class I antibodies bind to HLA-class I expressed in the test sample and the control sample. , different The range of concentrations HLA-class I antibodies Test sample and Control sample To To make contact; c) adding complement to the test and control samples so that bound HLA-class I antibodies are saturated with complement and complement-dependent cytotoxicity (CDC) is induced; d) determining CDC for a range of different concentrations of HLA-class I antibodies by measuring induced cell lysis in test and control samples; e) 50% of maximum CDC (EC 50 determining the concentration of HLA-class I antibodies in the test sample and the control sample that induces a β-glucose level (β-glucose level); and f1) EC of test sample 50 EC of control samples versus values 50 Selecting the SC population for allogeneic therapy if the ratio of values ​​is less than 1.25, preferably less than 1.0, more preferably less than 0.5; particularly preferably less than 0.25; or f2) EC of test sample 50 Selecting the SC population for allogeneic therapy if the value is at least 3.5 ng / ml, preferably at least 9 ng / ml, more preferably at least 15 ng / ml, particularly preferably at least 20 ng / ml of HLA-class I antibodies. A method is provided comprising:

[0024] By "stem cell (SC) population" herein is meant any stem cell, including embryonic pluripotent stem cells (ESC), fetal stem cells and adult stem cells. The population can be derived from either a primary cell culture, a cell line or a clone.

[0025] The population of stem cells may be a population of pluripotent stem cells or may be a population of mesenchymal stem cells (MSCs), such as bone marrow-derived, umbilical cord tissue-derived, blood-derived (including umbilical cord blood-derived), menstrual blood-derived, dental pulp-derived, placenta-derived or adipose-derived MSCs (Huang et al., J Dent. Res. (2009) 88(9): 792-806; Carvalho et al., Curr. Stem Cell Res. Ther. (2011) 6(3): 221-8; Harris et al., Curr Stem Cell Res Ther. (2013) 8(5): 394-9; Li et al., Ann. N YAcad. Sci. (2016) 1370(1): 109-18). In preferred embodiments, the stem cells are human stem cells (e.g. human ASCs). In preferred embodiments of the invention, the population of stem cells are adipose-derived stromal stem cells (ASCs). The ASC may be an expanded population of ASC.

[0026] Methods for generating and culturing populations of stem cells are well known.

[0027] The stem cell population can be substantially pure. The term "substantially pure" in relation to a stem cell population (e.g., an MSC population, such as an ASC population) refers to a stem cell population that is at least about 75%, generally at least about 85%, more generally at least about 90%, and most generally at least about 95% homogeneous. Homogeneity can be assessed by morphology and / or cell surface marker profile. Techniques for assessing morphology and cell surface marker profile are disclosed herein.

[0028] The stem cells are characterized by their self-renewal capacity in an undifferentiated state and their ability to differentiate into specialized cell types. ESCs are publicly available from clones. Adult stem cells are undifferentiated cells including hematopoietic stem cells, mammary stem cells, intestinal stem cells, mesenchymal stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells and testicular cells. Preferably, the SC population is a mesenchymal stem cell (MSC), more preferably a human MSC population.

[0029] pluripotent stem cells There are two sources of pluripotent stem cells. First, embryonic stem cells (ESCs) are derived from the inner cell mass of preimplantation blastocysts, and pluripotency is controlled by an intrinsic regulatory network of core transcription factors octamer-binding transcription factor 4 (OCT4), sex determining region Y-box2 (SOX2), and Nanog homeobox (NANOG). Second, induced pluripotent stem cells (iPSCs) are induced by ectopic or high expression of four transcription factors essential for induction of pluripotency in somatic cells: OCT4, SOX2, Krüppel-like factor 4 (KLF4), and MYC proto-oncogene (c-MYC).

[0030] Techniques for isolating stable (undifferentiated) cultures of embryonic stem cells, such as human embryonic stem cells, are well established (e.g., US 5,843,780; Thomson et al., Science (1998) 282: 1145-1147; Turksen & Troy (2006) Human Embryonic Stem Cells in Turksen K. (ed.) Human Embryonic Stem Cell Protocols. Methods in Molecular Biology, Vol. 331, Humana Press; Sevilla et al., Stem Cell Research (2017) 25: 217-220; and Mitalipova & Palmarini (2006) Isolation and Characterization of Human Embryonic Stem Cells in Turksen K. (ed.) Human Embryonic Stem Cell Protocols. Methods in Molecular Biology, Vol. 331, Humana Press).

[0031] Techniques for generating iPSCs have been well established since their discovery by the Yamanaka group in 2007 (e.g., Takahashi et al., Cell (2007) 131(5): 861-72). Since then, new and improved methods for iPSC generation have been developed, including non-integrative and feeder-free methods as well as automated high-throughput derivation (Paull et al., Nature Methods (2015) 12(9): 885 892).

[0032] iPSCs are characterized by the expression of a set of pluripotency markers: NANOG, SOX2, SSEA4, TRA1-81, TRA1-60, and the absence of lineage-specific markers. The pluripotency of iPSCs is demonstrated by their ability to differentiate into the three germ layers in embryoid body assays with post-mortem analysis of the three germ layer-derived differentiation markers Tuj1 (ectoderm marker), SMA (mesoderm marker) and SOX17 (endoderm marker) by immunohistochemistry (Paull et al., Nature Methods (2015) 12(9): 885 892).

[0033] Induced pluripotent stem cells (iPSCs) are derived by ectopic or high-level expression of four transcription factors essential for the induction of pluripotency in somatic cells: OCT4, SOX2, Krüppel-like factor 4 (KLF4), and MYC proto-oncogene (c-MYC).

[0034] MSC "Mesenchymal stem cells" (also referred to herein as "MSCs") are multipotent stromal cells. They are generally derived from connective tissue and are non-hematopoietic cells. A population of MSCs (according to Dominici et al. (2006), Cytotherapy 8(4): 315-317) can (1) adhere to plastic under standard culture conditions (e.g., minimum essential medium + 20% fetal bovine serum); (2) express CD105, CD90, CD73 and CD44 (i.e., more than 80% of the population of MSCs); (3) lack expression of CD45, CD14 or CD11b, CD790L or CD19, and HLA-DR (HLA class II) (e.g., less than 5% of the population of MSCs); (4) have the ability to differentiate into osteoblasts, adipocytes and chondroblasts.

[0035] MSCs can be obtained, for example, from bone marrow, umbilical cord tissue and blood, menstrual blood, dental pulp, umbilical cord blood, placenta and adipose tissue using standard methods. Although MSCs obtained from different tissues are similar, they have some differences in phenotypic and functional characteristics. For example, the expression levels of cell surface markers CD54 and CD106 may differ depending on the source / origin of MSCs. These can be measured by flow cytometry. The mRNA levels of some genes, such as SOX2, IL1 alpha, IL1 beta, IL6 and IL8, may be differentially expressed by MSCs from different tissues and can be measured by routine methods. IL6 and PGE2 secretion may also differ between MSCs of different origins, and thus the cells may have different regulatory capabilities (see, for example, Yang et al. PLoS ONE (2013) 8(3) e59354).

[0036] Bone marrow-derived MSCs (BMSCs) Bone marrow mesenchymal stem cells (BM-MSCs) are similar to MSCs from other tissue sources. However, they have some differences in phenotypic and functional characteristics compared to other tissue origin MSCs, such as umbilical cord MSCs, placenta MSCs, dental pulp MSCs, and menstrual blood MSCs. Although they share the same minimum characterization criteria, including their ability to adhere to plastic, minimal surface identity markers, and ability to differentiate into bone, cartilage, tendon, and adipose tissue, they all have some slight differences. These properties include different expression levels of several surface markers such as CD105, different levels of secreted soluble factors associated with their immunomodulatory and regenerative capacities, and generally slightly different functional properties that may make each source or origin more suitable for a particular therapeutic indication (Miura et al., Int J Hematology (2016) 103(2): 122-128; Wuchter et al., Cytotherapy (2015) 17(2): 128-139; Wright et al., Stem Cells (2011) 29(2): 169-178).

[0037] Umbilical cord- and dental pulp-derived MSCs Huang et al. (J. Dent. Res. (2009) 88(9): 792-806) review dental pulp-derived MSCs and compare their characteristics with MSCs from other sources. Carvalho et al. (Curr Stem Cell Res Ther. (2011) 6(3): 221-228) and Harris et al. (Curr Stem Cell Res Ther. (2013) 8(5): 394-399) review umbilical cord-derived MSCs, their characterization (including phenotype and secretome) and their applications.

[0038] ASC Adipose-derived MSCs (ASCs) are usually isolated from subcutaneous adipose tissue, which allows obtaining a large number of ASCs. ASCs proliferate rapidly with high cellular activity, making them an ideal source for obtaining MSCs.

[0039] By "adipose tissue" is meant any adipose tissue. The adipose tissue may be brown or white adipose tissue derived from subcutaneous tissue, omental / visceral tissue, mammary tissue, gonadal tissue, or other adipose tissue sites. Generally, the adipose tissue is subcutaneous white adipose tissue. Such cells may comprise primary cell cultures or immortalized cell lines. The adipose tissue may be derived from any organism that has adipose tissue. Generally, the adipose tissue is mammalian, and most commonly, the adipose tissue is human. A convenient source of adipose tissue is from liposuction, but the source of the adipose tissue or the method of isolation of the adipose tissue is not critical to the present invention.

[0040] The preferred ASCs are human allogeneic adipose-derived stem cells (human eASCs) approved in the product "Darvadstrocel" (trade name "Alofisel®"). These expanded ASCs express the cell surface markers CD29, CD73, CD90 and CD105. The cells can express factors such as vascular endothelial growth factor (VEGF), transforming growth factor-beta 1 (TGF-β1), interleukin 6 (IL-6), matrix metalloproteinase inhibitor-1 (TIMP-1) and interferon-gamma (IFN-γ) and inducible indoleamine 2,3-dioxygenase (IDO). Thus, the population of ASCs is at least about 50% At least about 60%; at least about 70%; at least about 80%; at least about 85%; at least about 90% or at least about 95% or more may be characterized as expressing one or more of CD29, CD73, CD90 and / or CD105. The population of ASCs may be characterized as at least about 50%, at least about 60%; at least about 70%; at least about 80%; at least about 85%; at least about 90% or at least about 95% of the population of cells expressing all of CD29, CD73, CD90 and CD105. Generally, the population of ASCs may be characterized as at least about 80% of the population of cells expressing all of CD29, CD73, CD90 and CD105.

[0041] According to Bourin et al. (Cytotherapy (2013) 15(6): 641-648), a population of ASCs may be defined as positive for expression of CD13, CD29, CD44, CD73, CD90 and CD105, and negative for expression of CD31 and CD45. In the population of ASCs, at least about 50%, at least about 60%; at least about 70%; at least about 80%; at least about 85%; at least about 90% or at least about 95% of the population of cells may express CD13, CD29, CD44, CD73, CD90 and CD105, and less than about 5%, about 4%, about 3% or about 2% of the population of ASCs may express CD31 and CD45. Generally, in the population of ASCs, at least about 80% of the population of cells may express CD13, CD29, CD44, CD73, CD90 and CD105, and less than about 5% of the population of ASCs may express CD31 and CD45.

[0042] ASCs can adhere to plastic under standard culture conditions. Expanded ASCs (eASCs) display a fibroblast-like morphology in culture. Specifically, these cells are large and have the morphological characteristics of a shallow cell body with few long, thin cell processes. The nuclei are large and round with prominent nucleoli that give them a distinct appearance. Most of the eASCs display this spindle-shaped morphology, but some of the cells usually acquire a polygonal morphology (Zuk et al. Tissue Eng (2001) 7(2): 211-228).

[0043] ASCs may be positive for surface markers HLA-I, CD29, CD44, CD59, CD73, CD90, and CD105. In some embodiments, the population of ASCs may be characterized in that at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%; at least about 90% or at least about 95% of the population of ASCs express the surface markers HLA-I, CD29, CD44, CD59, CD73, CD90, and CD105. Generally, at least about 80% of eASCs express the surface markers HLA1, CD29, CD44, CD59, CD73, CD90, and CD105.

[0044] ASCs may be negative for surface markers HLAII, CD11b, CD11c, CD14, CD45, CD31, CD80 and CD86. In some embodiments, the population of ASCs may be characterized in that less than about 5% of the population of ASCs expresses the surface markers HLAII, CD11b, CD11c, CD14, CD45, CD31, CD80 and CD86. More typically, less than about 4%, 3% or 2% of the population of ASCs express the surface markers HLAII, CD11b, CD11c, CD14, CD45, CD31, CD80 and CD86. In one embodiment, less than about 1% of the population of ASCs expresses the surface markers HLAII, CD11b, CD11c, CD14, CD45, CD31, CD80 and CD86.

[0045] In some examples, in a population of ASCs, at least about 80% of the population of cells express all of CD29, CD73, CD90 and CD105, and less than about 5% of the population of ASCs expresses surface markers HLAII, CD11b, CD11c, CD14, CD45, CD31, CD80 and CD86.

[0046] In some embodiments, the population of ASCs may express one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or 7) of HLAI, CD29, CD44, CD59, CD73, CD90, and CD105. In some embodiments, eASCs may not express one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or 8) of HLAII, CD11b, CD11c, CD14, CD45, CD31, CD80. In some embodiments, eASCs express four or more of HLAI, CD29, CD44, CD59, CD73, CD90, and CD105, and do not express four or more of HLAII, CD11b, CD11c, CD14, CD45, CD31, CD80.

[0047] Expression of CD34 may be negative or at a low level, e.g., expressed by 0 to about 30% of the population of ASCs. Thus, in some examples, the ASCs may express CD34 at a low level, e.g., about 5 to about 30% of the population. Alternatively, in other examples, the ASCs do not express CD34, e.g., less than about 5% of the population of ASCs expresses CD34.

[0048] In some embodiments, the population of ASCs (e.g., at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%; at least about 90% or at least about 95% of the population of cells) may express one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more (e.g., up to 13)) of the markers CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90, and CD105. For example, the ASCs may express one or more (e.g., two, three or all) of the markers CD29, CD59, CD90, and CD105, e.g., CD59 and / or CD90.

[0049] In some embodiments, the population of ASCs may not express one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more (e.g., up to 15)) of the markers Factor VIII, alpha-actin, desmin, S-100, keratin, CD11b, CD11c, CD14, CD45, HLAII, CD31, CD45, STRO-1, and CD133, e.g., the ASCs do not express one or more (e.g., 2, 3 or all) of the markers CD45, CD31, and CD14, e.g., CD31 and / or CD45.

[0050] In certain embodiments, the ASCs (i) do not express markers specific to antigen-presenting cells (APCs); (ii) do not constitutively express IDO; and / or (iii) do not constitutively express significant MHCII. In general, the expression of IDO or MHCII can be induced by stimulation with IFN-γ. In certain embodiments, the ASCs do not express Oct4.

[0051] Methods for preparing populations of ASCs Methods of isolating and culturing ASCs to provide eASCs and populations of stem cells of the present invention, as well as compositions comprising populations of stem cells of the present invention, are known in the art. ASCs are generally prepared from the stromal fraction of adipose tissue and selected for their adhesiveness to a suitable surface, e.g., plastic. Thus, the method of cryopreservation of stem cells disclosed herein may comprise the initial steps (before step (a) of any one of the methods) of (i) isolating a population of ASCs from the stromal fraction of adipose tissue obtained from a patient, and (ii) culturing said population of ASCs. ASCs can optionally be selected in step (i) for their adhesiveness to a suitable surface, e.g., plastic. Optionally, the phenotype of ASCs can be evaluated during and / or after the culturing step (ii).

[0052] Methods for preparing populations of ASCs Methods of isolating and culturing ASCs to provide eASCs and populations of stem cells of the present invention, as well as compositions comprising populations of stem cells of the present invention, are known in the art. ASCs are generally prepared from the stromal fraction of adipose tissue and selected for their adhesiveness to a suitable surface, e.g., plastic. Thus, the method of cryopreservation of stem cells disclosed herein may comprise the initial steps (before step (a) of any one of the methods) of (i) isolating a population of ASCs from the stromal fraction of adipose tissue obtained from a patient, and (ii) culturing said population of ASCs. ASCs can optionally be selected in step (i) for their adhesiveness to a suitable surface, e.g., plastic. Optionally, the phenotype of ASCs can be evaluated during and / or after the culturing step (ii).

[0053] ASCs can be obtained by any means standard in the art. Generally, the cells are obtained from a source tissue (e.g., lipoaspirate or adipose tissue) by isolating the cells, generally by treating the tissue with a digestive enzyme such as collagenase. The digested tissue material is then generally filtered through a filter of about 20 microns to 1 mm. The cells are then isolated (generally by centrifugation) and cultured on an adherent surface (generally a tissue culture plate or flask). Such methods are known in the art and are disclosed, for example, in U.S. Pat. No. 6,777,231.

[0054] According to this method, lipoaspirate tissue is obtained from adipose tissue and cells are derived therefrom. During this method, cells may be washed, preferably with PBS, to remove contaminating debris and red blood cells. Cells are digested with collagenase in PBS (e.g., 0.075% collagenase; type I, Invitrogen, Carlsbad, CA, for 30 minutes at 37°C). To remove residual red blood cells, digested samples can be washed (e.g., with 10% fetal bovine serum), treated with 160 mmol / L NH4Cl, and finally suspended in DMEM complete medium (DMEM containing 10% FBS, 2 mM glutamine, and 1% penicillin / streptomycin). Cells can be filtered through a 40 μm nylon mesh.

[0055] Cultured human ASCs have been described in DelaRosa et al. (Tissue Eng Part A. (2009) 15(10): 2795-806), Lopez-Santalla et al. (Stem cells (2015) 33: 3493-3503). In one embodiment (as described in Lopez-Santalla et al. (2015) cited above), human lipoaspirate tissue from a healthy donor was washed twice with phosphate-buffered saline and digested with 0.075% collagenase (type I; Invitrogen). The digested samples were washed with 10% fetal bovine serum (FBS), treated with 160 mM NH4Cl to remove residual red blood cells, and suspended in culture medium (Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS). Cells were seeded (2-3 × 10) in tissue culture flasks. 4 pieces / cm 2 ) and cultured (37°C, 5% CO2) with changing the culture medium every 3 to 4 days. When the cells reached 90% confluence, they were transferred to a new flask (10 3 pieces / cm 2 ). Cells were expanded in duplicate up to 12–14 times and frozen.

[0056] In another embodiment (as described by DelaRosa et al. (2009), Tissue Eng Part A 15(10): 2795-806), lipoaspirate tissue obtained from human adipose tissue from healthy adult donors was washed twice with PBS and digested with 18 U / mL type I collagenase in PBS at 37°C for 30 min. One unit of collagenase liberated 1 mM L-leucine equivalent from collagen in 5 h at 37°C, pH 7.5 (Invitrogen, Carlsbad, CA). The digested samples were washed with 10% fetal bovine serum (FBS), treated with 160 mM NH4Cl, suspended in culture medium (DMEM containing 10% FBS), and filtered through a 40 mm nylon mesh. Cells were seeded (2-3 × 10 4 pieces / cm 2 ) and expanded at 37°C and 5% CO2 with culture medium changes every 7 days. When cultures reached 90% confluence, cells were transferred to new culture flasks. Cells were phenotypically characterized by their ability to differentiate into chondrogenic, osteogenic, and adipogenic lineages.

[0057] ASCs are cultured in a suitable tissue culture vessel comprising a surface suitable for the adhesion of ASCs, e.g., plastic. Non-adherent cells are removed, e.g., by washing in a suitable buffer, to obtain an isolated population of adherent stromal cells (e.g., ASCs). The cells thus isolated are seeded (preferably at 2-3×10 cells / ml) into tissue culture flasks. 4 pieces / cm 2 ) and can be expanded at 37 °C and 5% CO2, changing the culture medium every 3-4 days. When the cultures reach approximately 90% confluence, the cells are preferably detached from the adhesive surface (e.g., by trypsin) and transferred to new culture flasks (1,000 cells / cm). 2 ) ("passage").

[0058] The ASCs may be cultured for at least about 15 days, at least about 20 days, at least about 25 days, or at least about 30 days. Generally, expansion of cells in culture improves the homogeneity of cell phenotype in the population, such that a substantially pure population is obtained.

[0059] In some embodiments, the ASCs are expanded in culture for at least three culture passages, or "passaged at least three times." In other embodiments, the cells are passaged at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times. It is preferred that the cells are passaged more than three times to improve the homogeneity of the cell phenotype in the cell population. Indeed, the cells can be expanded in culture indefinitely, so long as the homogeneity of the cell phenotype is improved and differentiation potential is maintained.

[0060] In some embodiments, the ASCs are grown in culture for at least 3 population doublings, e.g., the cells are expanded in culture for at least 4, 5, 6, 7, 8, 9, 10, 15, or 20 population doublings. In some embodiments, the cells are expanded in culture for less than 7, 8, 9, 10, 15, or 20 population doublings. In certain embodiments, the cells are expanded in culture for about 5-10 population doublings. In certain embodiments, the cells are expanded in culture for about 10-15 population doublings. In certain embodiments, the cells are expanded in culture for about 15-20 population doublings, e.g., about 16 population doublings. Isolation of ASCs is preferably performed under sterile or GMP conditions.

[0061] "Allogeneic therapy" as used herein refers to cell-based therapy in which the donor is different from the recipient of cells.Preferably, allogeneic therapy does not involve HLA matching between donor and recipient.In the manufacture of pharmaceutical products, allogeneic methods are promising because HLA-mismatched allogeneic therapy can form the basis of "off the shelf" products.

[0062] "Presensitization" as used herein means that a patient exhibits pre-existing immunity to a particular SC population before the actual administration of the SC population. Pre-existing immunity may be associated with the presence of donor-specific antibodies (DSA), in particular DSA that are specific for HLA-class I molecules. This can be determined by methods known in the art, such as spectrophotometric flow cytometry crossmatch (FCXM), fluorescent or luminescent assays (e.g., ELISA (enzyme-linked immunosorbent assay)), where HLA-class I molecules bind to the plate. Pre-existing immunity may be due, for example, to early blood transfusion or, in women, to early pregnancy.

[0063] "Retreatment" as used herein means that after administration of a first dose of a first SC population derived from a first donor to a patient, the patient receives either another dose of the first SC population derived from the first donor or a dose of a second SC population derived from a second donor. Retreatment may be necessary to increase therapeutic efficacy compared to after administration of a single dose.

[0064] "Culturing a sample of a SC population" as used herein means that the SC population is contained in a suitable cell culture medium to maintain the viability of the SC population. A suitable cell culture medium may contain nutrients, such as amino acids, carbohydrates, vitamins and / or salts, essential for the SC population. The pH is adapted to suitable conditions by using a buffer. Preferably, the cell culture medium is selected from RPMI (available from Gibco) or DMEM (e.g., available from Sigma-Aldrich).

[0065] "IFN-γ concentration capable of inducing maximum HLA-class I expression in the SC population" refers herein to the IFN-γ concentration capable of providing the maximum amount of HLA-class I molecules on the surface of the SC population. The amount of HLA-class I molecules on the surface of the SC population can be determined by fluorescence activated cell sorting (FACS) or in an ELISA assay format, such as a sandwich assay format, using an HLA-class I antibody that is directly bound to a chromophore or fluorophore or indirectly bound to a labeled secondary antibody.

[0066] Maximal HLA-class I expression is induced when cells are treated with higher IFN-γ concentrations, without further increase in HLA-class I expression.

[0067] IFN-γ is known to increase the expression and presentation of HLA-class I molecules. According to a preferred embodiment, the IFN-γ concentration capable of inducing maximum HLA-class I expression in the SC population is about 0.5 to about 30 ng / ml, preferably about 1 to about 15 ng / ml, more preferably about 2 to about 4 ng / ml, and most preferably 3 ng / ml. Preferably, IFN-γ is incubated with the SC population for a period of 12 to 72 hours, preferably a period of 24 to 60 hours, more preferably a period of 30 to 54 hours, and most preferably a period of 48 hours. Most preferably, the IFN-γ concentration capable of inducing maximum HLA-class I expression in the SC population is 3 ng IFN-γ / ml, and IFN-γ is incubated with the SC population for a period of 48 hours.

[0068] By "HLA-class I antibody" is meant herein an antibody capable of specifically binding to HLA-A, HLA-B and / or HLA-C. "Specifically" in this context is meant to mean that the binding is not non-specific. The term "specifically" includes binding to antigens that share an epitope that also allows cross-reactivity of the antibody to related antigens. The antibody may be of any subtype, including IgG and IgM. The antibody may be from any source, preferably the antibody is a mouse, rabbit, sheep or goat antibody, preferably a mouse antibody. The antibody preferably comprises a constant region (Fc region) capable of interacting with proteins of the complement system.

[0069] In a preferred embodiment, the HLA-class I antibody specifically binds to HLA-A, HLA-B and HLA-C. It is therefore preferred that the antibody is a pan-HLA-class I antibody that recognizes epitopes conserved in HLA-A, HLA-B and HLA-C heavy chains.

[0070] In a further preferred embodiment, said HLA-class I antibody has essentially the same binding affinity to HLA-A, preferably HLA-A2, most preferably HLA-A*0201, as the antibody produced by hybridoma clone w6 / 32 available from ATCC (designation number: HB-95) or ECACC (number: 84112003). Preferably, the binding affinity is determined by using an ELISA method. "Essentially the same binding affinity" means that the binding affinity of the HLA-class I antibody differs from the binding affinity of the antibody produced by hybridoma clone w6 / 32 by less than 10%, preferably less than 8%, more preferably less than 5%.

[0071] In an even more preferred embodiment, the antibody is produced by the hybridoma clone w6 / 32, which is available from the ATCC (designation number: HB-95) or the ECACC (number: 84112003).

[0072] "Different concentration ranges of HLA-class I antibodies" as used herein means that test and control samples are treated with different concentrations of HLA-class I antibodies to obtain different CDC values ​​determined by cell lysis, which can be shown in a test curve depending on the concentration of HLA-class I antibodies.

[0073] In one embodiment, the different concentrations of HLA-class I antibodies are in the range of about 1 to about 50 ng / ml. In one embodiment, two or three different concentrations of HLA-class I antibodies in the range of about 1 to about 50 ng / ml are used. In one embodiment, four or five different concentrations of HLA-class I antibodies in the range of about 1 to about 50 ng / ml are used. In one embodiment, six or seven different concentrations of HLA-class I antibodies in the range of about 1 to about 50 ng / ml are used. In one embodiment, eight or nine different concentrations of HLA-class I antibodies in the range of about 1 to about 50 ng / ml are used.

[0074] In one embodiment, the different concentrations of HLA-class I antibodies produced by hybridoma clone w6 / 32 are in the range of about 1 to about 50 ng / ml. In one embodiment, two or three different concentrations of HLA-class I antibodies produced by hybridoma clone w6 / 32 in the range of about 1 to about 50 ng / ml are used. In one embodiment, four or five different concentrations of HLA-class I antibodies produced by hybridoma clone w6 / 32 in the range of about 1 to about 50 ng / ml are used. In one embodiment, six or seven different concentrations of HLA-class I antibodies produced by hybridoma clone w6 / 32 in the range of about 1 to about 50 ng / ml are used. In one embodiment, eight or nine different concentrations of HLA-class I antibodies produced by hybridoma clone w6 / 32 in the range of about 1 to about 50 ng / ml are used.

[0075] In a further preferred embodiment, the range of different concentrations of the HLA-class I antibody is at least two or three, preferably four or five, more preferably six or seven, and most preferably seven or eight concentrations selected from 1 ng / ml, 3 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml and 50 ng / ml.

[0076] "Under conditions such that HLA-class I antibodies bind to HLA-class I expressed in the test sample and the control sample" means herein that the SC populations of the test sample and the control sample are contained in a suitable medium that allows binding of the antibodies to the stem cells. The medium therefore has a pH and salt concentration that does not denature the antibodies and / or the cells. Preferably, the medium contains a suitable buffer system to provide a pH similar to the physiological pH in human blood (pH 7.35-7.45). More preferably, the medium is a phosphate buffered saline (PBS) solution having a pH of 7.4 and containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4.

[0077] "Complement" as used herein means complement derived from any source of blood, including plasma or serum. Complement may also be used as a mixture of purified or synthetic complement proteins. The complement system consists of a number of small proteins that are synthesized by the liver and found in circulating blood as inactive precursors. When stimulated by one of several triggers, proteases in the system cleave specific proteins, releasing cytokines and initiating an amplifying cascade of further cleavages. The end result of this complement activation or complement binding cascade is the stimulation of phagocytes to remove foreign and damaging substances, inflammation to attract additional phagocytes, and activation of the membrane attack complex (MAC) to kill cells resulting in cytolysis of the attacked cells. During allorecognition, complement-dependent cytotoxicity (CDC) is initiated when C1q, the initiating mediator of the classical pathway, binds to the Fc portion of HLA class-I antigen-binding antibodies, resulting in activation of C1q and subsequent complement cascade signaling.

[0078] "Saturated with complement" as used herein means that the amount of complement added in the method of the present invention is in molar excess relative to the amount of HLA-class I antibodies bound to the surface of the IFN-γ-induced SC population (test sample). Saturation with complement can be determined experimentally by performing assays using different concentrations of complement. Saturation is achieved once CDC activity has reached its maximum and can no longer be increased by the addition of higher concentrations of complement.

[0079] In a preferred embodiment, the complement used in step (c) of claim 1 is derived from serum. Preferably, the serum is not heat treated. Heat inactivation can denature complement proteins, thereby rendering them unable to form MAC complexes. Also, preferably, the serum is not fetal bovine serum. More preferably, the serum is derived from rabbit, goat or sheep. Most preferably, the serum is derived from rabbit. Such serum is available from One Lambda.

[0080] In a preferred embodiment, the serum concentration resulting in saturation of complement-bound HLA-I antibodies is 50 to 83.33% (v / v), preferably, the serum concentration resulting in saturation of complement-bound HLA-I antibodies is 66.66 to 83.33% (v / v), more preferably, the serum concentration resulting in saturation of complement-bound HLA-I antibodies is 75 to 83.33% (v / v), and most preferably, the serum concentration resulting in saturation of complement-bound HLA-I antibodies is 83.33% (v / v).

[0081] "Complement-dependent cytotoxicity (CDC)" is an effector function of IgG and IgM antibodies. Their binding to surface antigens triggers the classical complement pathway, leading to the formation of the membrane attack complex (MAC) and cytolysis of the target. In the context of the present invention, CDC is determined by measuring the cytolysis induced in the SC population by the binding of HLA-class I antibodies to HLA class I molecules expressed on the SC population.

[0082] In a preferred embodiment, the cell lysis is determined by measuring chemiluminescent or fluorescent dyes that are selective for live or lysed cells or radioactive material released by lysed cells. Live cells with intact cell membranes can be distinguished from lysed cells by methods known in the art using different types of agents.

[0083] Chromium release assays have been established as a radioactive method to distinguish live from dead cells. As an alternative to radioactive assays, fluorescent dyes have been developed. For example, non-fixable viability dyes for DNA binding such as propidium iodide, DAPI, 7-aminoactinomycin D (7-AAD), TO-PRO-3, etc., can be used. If sample fixation is required, Invitrogen's amine-reactive dyes or eBioscience's Fixable Viability Dyes (eFluor450, eFluor660, eFluor780) or Becton Dickinson's Violet fixable dye (BD Horizon VD450) can be added to the sample before fixation to distinguish live from dead cells. Further dyes suitable for the assessment of cell viability by cell functionality are available, such as dyes that require esterase activity (e.g. Calcein AM from eBioscience / Invitrogen) or dyes that accumulate in mitochondria (JC-1, Rhodamine 123).

[0084] As further alternatives, chemiluminescence- or bioluminescence-based assays have been developed (e.g., menadione-catalyzed H2O2 production), all of which are contemplated by the present invention to determine CDC by measuring cell lysis. In a preferred embodiment, CDC activity is determined by using 7-aminoactinomycin D as a dye in FACS analysis, as further described in the Examples.

[0085] "EC" as defined in the claims 50 The "EC value" is the 50% of the maximum CDC in the test and control samples. 50 EC is the concentration of HLA class I antibody that induces the EC value. 50"Determining values" may be performed visually using the determined CDC values ​​for different HLA-class I antibody concentrations, or using commercially available software for biological data analysis. Software capable of linear regression analysis may be used. A suitable software may be FCS Express Version 5 (DeNovo Software).

[0086] In a preferred embodiment, the SC population for allogeneic therapy is derived from the EC 50 EC of control samples versus values 50 When the ratio of the values ​​is about 0.1 to about 1.25, preferably about 0.1 to about 1.0, more preferably about 0.1 to about 0.5, and particularly preferably about 0.1 to about 0.25, it is selected according to step f1).

[0087] In a further preferred embodiment, the SC population for allogeneic therapy is 50 If the value is between about 3.5 ng / ml and about 30 ng / ml, preferably between about 9 ng / ml and about 25 ng / ml, more preferably between about 10 ng / ml and about 20 ng / ml of HLA-class I antibodies, then selection is made according to step f2).

[0088] "CD46 expression level" can be determined on nucleic acid level by standard techniques in the art, such as Northern blot and RT-qPCR. CD46 mRNA sequence is available from public database (NCBI reference sequence: NM_002389.4). Suitable primers can be prepared by methods known in the art. CD46 expression level can also be determined on protein level. Suitable methods include Western blot and FACS analysis using labeled HLA-class I antibody molecules.

[0089] According to a further aspect, there is provided a SC population suitable for allotherapy, in particular for the treatment of presensitized patients or for retreatment with allotherapy, said SC population having the following characteristics: i) EC of the test sample 50EC of control samples versus values 50 The ratio of the EC values ​​is less than 1.25, preferably less than 1.0, more preferably less than 0.5, and particularly preferably less than 0.25, wherein the EC 50 The value is determined according to the present invention; ii) EC of the test sample 50 a value of at least 3.5 ng / ml HLA-class I antibodies, preferably at least 9 ng / ml, more preferably at least 15 ng / ml, particularly preferably at least 20 ng / ml HLA-class I antibodies, wherein said EC 50 The value is determined in accordance with the present invention; or iii) the ratio of CD46 expression in the test sample to the CD46 expression in the control sample is greater than 2.0, preferably greater than 2.5, particularly preferably greater than 3.0, wherein said CD46 expression is determined according to the invention; The present invention provides a population of SCs having any of the following:

[0090] In a preferred embodiment, said SC population is selected by the method according to the invention. Also preferably, said SC population is a mesenchymal stem cell (MSC) population, preferably a BM-MSC population or an ASC population, preferably human BM-MSC or human ASC.

[0091] According to a further aspect, there is provided a pharmaceutical composition comprising the SC population according to the invention and optionally a pharma- ceutically acceptable carrier.

[0092] The term "pharmaceutical acceptable carrier" as used herein means any pharmaceutical acceptable carrier known in the art that does not adversely affect the viability or efficacy of the SC cell population. The pharmaceutical acceptable carrier may be a cell culture medium containing amino acids, salts and vitamins. Preferably, the cell culture medium is RPMI or DMEM, more preferably, the cell culture medium is DMEM. The cell culture medium may be supplemented with human serum albumin (HSA) at a concentration of about 5% (v / v) to about 30% (v / v), more preferably, the HSA concentration may be 20% (v / v). In one embodiment, the pharmaceutical acceptable carrier is DMEM containing 20% ​​(v / v) HSA.

[0093] The pharmaceutical composition may be in the form of an injectable suspension. The concentration of the SC population in the suspension is 1×10 5 ~8×10 6 / ml, preferably 1 x 10 6 ~6×10 6 Most preferably, the concentration is in the range of 5×10 6 The pharmaceutical composition may be administered by injection.

[0094] According to a further aspect, there is provided a method for preparing a pharmaceutical composition comprising the steps of: a) carrying out the method of the present invention; and b) formulating the selected SC population with at least one pharma- ceutically acceptable carrier; A method is provided comprising:

[0095] In a further aspect, the SC population according to the present invention is provided for use in allogeneic stem cell therapy in a patient in need thereof, preferably in a pre-sensitized or re-treated patient. In a further preferred embodiment, said patient in need thereof suffers from a disease selected from fistula, leukemia, lymphoma, neurodegenerative disease, brain and spinal cord injury, heart disease, blindness and vision impairment, pancreatic beta cell loss of function, cartilage repair, osteoarthritis, musculoskeletal disease, wounds, infertility, autoimmune disease and inflammatory disease, such as inflammatory bowel disease, preferably said disease is fistula, more preferably said disease is complex perianal fistula.

[0096] According to a further aspect, there is provided a method of allogeneic stem cell therapy comprising administering the SC population of the present invention to a patient in need thereof, preferably a presensitized or re-treated patient, said patient may be suffering from any of the diseases mentioned above.

[0097] In one embodiment, the present invention relates to an in vitro method for selecting a stem cell (SC) population suitable for allogeneic therapy, in particular for the treatment of a presensitized patient or for the re-treatment of a patient with allogeneic therapy, comprising the following steps: a) culturing a sample of an SC population in the presence of an IFN-γ concentration capable of inducing maximum HLA-class I expression in said SC population (test sample) and separately culturing a sample of an SC population in the absence of IFN-γ (control sample), wherein said SC population is an ASC population and said IFN-γ concentration is 3 ng / ml applied over a period of 48 hours; b) under conditions such that HLA-class I antibodies bind to HLA-class I expressed in the test sample and the control sample. , different The range of concentrations HLA-class I antibodies Test sample and Control sample Tocontacting, wherein said HLA-class I antibody is w6 / 32, and wherein the antibody concentrations are 1 ng / ml, 3 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml and 50 ng / ml; c) adding complement to the test and control samples so as to saturate the bound HLA-class I antibodies with complement and induce complement-dependent cytotoxicity (CDC), said complement being added in the form of rabbit serum; d) determining CDC for a range of different concentrations of HLA-class I antibodies by measuring induced cell lysis in test and control samples; e) 50% of the maximum CDC in the test and control samples (EC 50 determining the concentration of HLA-class I antibodies that induces a .alpha.-amino acid value; and f1) EC of test sample 50 EC of control samples versus values 50 Selecting the SC population for allogeneic therapy if the ratio of values ​​is less than 1.25, preferably less than 1.0, more preferably less than 0.5; particularly preferably less than 0.25; or f2) EC of test sample 50 Selecting the SC population for allogeneic therapy if the value is at least 3.5 ng / ml, preferably at least 9 ng / ml, more preferably at least 15 ng / ml, particularly preferably at least 20 ng / ml of HLA-class I antibodies. The present invention relates to a method comprising the steps of:

[0098] In one embodiment, the present invention relates to an in vitro method for selecting a stem cell (SC) population suitable for allogeneic therapy, in particular for the treatment of a presensitized patient or for the re-treatment of a patient with allogeneic therapy, comprising the following steps: a) culturing a sample of an SC population in the presence of an IFN-γ concentration capable of inducing maximum HLA-class I expression in said SC population (test sample) and separately culturing a sample of an SC population in the absence of IFN-γ (control sample), wherein said SC population is an ASC population and said IFN-γ concentration is 3 ng / ml applied over a period of 48 hours; b) under conditions such that HLA-class I antibodies bind to HLA-class I expressed in the test sample and the control sample. , different The range of concentrations HLA-class I antibodies Test sample and Control sample To contacting, wherein said HLA-class I antibody is w6 / 32, and wherein the antibody concentrations are 1 ng / ml, 3 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml and 50 ng / ml; c) adding complement to the test and control samples so as to saturate the bound HLA-class I antibodies with complement and induce complement-dependent cytotoxicity (CDC), said complement being added in the form of rabbit serum; d) determining CDC for a range of different concentrations of HLA-class I antibodies by measuring induced cell lysis in test and control samples; e) 50% of the maximum CDC in the test and control samples (EC 50 determining the concentration of HLA-class I antibodies that induces a .alpha.-amino acid value; and f1) EC of test sample 50 EC of control samples versus values 50 selecting the SC population for allogeneic therapy when the ratio of values ​​is about 0.1 to about 1.25, preferably about 0.1 to about 1.0, more preferably about 0.1 to about 0.5, and particularly preferably about 0.1 to about 0.25; or f2) EC of test sample 50If the value is between about 3.5 ng / ml and about 30 ng / ml, preferably between about 9 ng / ml and about 25 ng / ml, more preferably between about 10 ng / ml and about 20 ng / ml of HLA-class I antibodies, the SC population for allogeneic therapy is selected. The present invention relates to a method comprising the steps of:

[0099] Some embodiments of the present invention relate to: 1. An in vitro method for selecting a stem cell (SC) population suitable for allogeneic therapy, in particular for the treatment of a presensitized patient or for re-treatment of a patient with allogeneic therapy, comprising the following steps: a) culturing a sample of the SC population in the presence of a concentration of IFN-γ capable of inducing maximal HLA-class I expression in said SC population (test sample) and separately culturing a sample of the SC population in the absence of IFN-γ (control sample); b) under conditions such that HLA-class I antibodies bind to HLA-class I expressed in the test sample and the control sample. , different The range of concentrations HLA-class I antibodies Test sample and Control sample To To make contact; c) adding complement to the test and control samples so that bound HLA-class I antibodies are saturated with complement and complement-dependent cytotoxicity (CDC) is induced; d) determining CDC for a range of different concentrations of HLA-class I antibodies by measuring induced cell lysis in test and control samples; e) 50% of the maximum CDC in the test and control samples (EC 50 determining the concentration of HLA-class I antibodies that induces a .alpha.-amino acid value; and f1) EC of test sample 50 EC of control samples versus values 50 Selecting the SC population for allogeneic therapy if the ratio of values ​​is less than 1.25, preferably less than 1.0, more preferably less than 0.5; particularly preferably less than 0.25; or f2) EC of test sample50 Selecting the SC population for allogeneic therapy if the value is at least 3.5 ng / ml, preferably at least 9 ng / ml, more preferably at least 15 ng / ml, particularly preferably at least 20 ng / ml of HLA-class I antibodies. The method comprising:

[0100] 2. The in vitro method according to item 1, wherein the method further comprises the steps of determining the CD46 expression level in the test sample and in the control sample; and selecting the SC population for allogeneic therapy if the ratio of CD46 expression in the test sample to the CD46 expression in the control sample is greater than 2.0, preferably greater than 2.5, particularly preferably greater than 3.0.

[0101] 3. The in vitro method according to item 1 or 2, wherein said SC population is a mesenchymal stem cell (MSC) population, preferably a human MSC population.

[0102] 4. The in vitro method of item 3, wherein the mesenchymal stem cell population is a bone marrow derived stem cell (BM-MSC) population.

[0103] 5. The in vitro method of item 3, wherein the mesenchymal stem cell population is an adipose tissue-derived stem cell (ASC) population.

[0104] 6. The in vitro method of item 5, wherein the ASC population expresses CD29, CD73, CD90 and / or CD105.

[0105] 7. The in vitro method according to any one of items 1 to 6, wherein the IFN-γ concentration capable of inducing maximum HLA-class I expression in the SC population is about 0.5 to about 30 ng / ml, preferably about 1 to about 15 ng / ml, and more preferably about 2 to about 4 ng / ml.

[0106] 8. The in vitro method according to item 7, wherein the IFN-γ concentration capable of inducing maximum HLA-class I expression in the SC population is 3 ng IFN-γ / ml, preferably applied for a period of 48 hours.

[0107] 9. The in vitro method according to any one of items 1 to 8, wherein the HLA class I antibody specifically binds to HLA-A, HLA-B and / or HLA-C.

[0108] 10. The in vitro method according to item 9, wherein the HLA class I antibody specifically binds to HLA-A, HLA-B and HLA-C, preferably the HLA class I antibody is a mouse monoclonal antibody.

[0109] 11. The in vitro method according to item 9 or 10, wherein the HLA-class I antibody has essentially the same binding affinity for HLA-A as the antibody produced by hybridoma clone w6 / 32 available from ATCC (designation number: HB-95) or ECACC (number: 84112003).

[0110] 12. The in vitro method according to any one of items 9 to 11, wherein the antibody is produced by the hybridoma clone w6 / 32 available from ATCC (designation number: HB-95) or ECACC (number: 84112003).

[0111] 13. The in vitro method according to any one of items 1 to 12, wherein two or three different concentrations of the HLA-class I antibody within the range of about 1 to about 50 ng / ml are used, preferably four or five different concentrations of the HLA-class I antibody within the range of about 1 to about 50 ng / ml are used, more preferably six or seven different concentrations of the HLA-class I antibody within the range of about 1 to about 50 ng / ml are used, even more preferably eight or nine different concentrations of the HLA-class I antibody within the range of about 1 to about 50 ng / ml are used.

[0112] 14. The in vitro method according to item 13, wherein the ranges of different concentrations of the HLA-class I antibody are 1 ng / ml, 3 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml and 50 ng / ml.

[0113] 15. The in vitro method according to any one of items 1 to 14, wherein the complement used in step (c) of claim 1 is derived from serum.

[0114] 16. The in vitro method of claim 15, wherein the serum is not heat treated.

[0115] 17. An in vitro method according to claim 15 or 16, wherein the serum is from rabbit, goat or sheep, preferably from rabbit.

[0116] 18. An in vitro method according to any one of claims 15 to 17, wherein the serum concentration is 50 to 83.33% (v / v), preferably the serum concentration is 66.66 to 83.33% (v / v).

[0117] 19. The in vitro method according to any one of items 1 to 18, wherein the cell lysis is determined by measuring a chemiluminescent or fluorescent dye that is selective for live or lysed cells or radioactive material released from lysed cells.

[0118] 20. A SC population suitable for allogeneic therapy, in particular for the treatment of presensitized patients or retreatment with allogeneic therapy, having the following characteristics: i) EC of the test sample 50 EC of control samples versus values 50 The ratio of the EC values ​​is less than 1.25, preferably less than 1.0, more preferably less than 0.5, and particularly preferably less than 0.25, wherein the EC 50 The value is determined as indicated in item 1; ii) EC of the test sample 50a value of at least 3.5 ng / ml HLA-class I antibodies, preferably at least 9 ng / ml, more preferably at least 15 ng / ml, particularly preferably at least 20 ng / ml HLA-class I antibodies, wherein said EC 50 The value is determined as indicated in item 1; and / or iii) the ratio of CD46 expression in the test sample to the CD46 expression in the control sample is greater than 2.0, preferably greater than 2.5, particularly preferably greater than 3.0, wherein said CD46 expression is determined as indicated in item 2; SC group, which has any of the following:

[0119] 21. The SC population according to item 20, wherein the SC population is selected by the method according to any one of items 1 to 19.

[0120] 22. The SC population according to item 21 or 22, wherein the SC population is a mesenchymal stem cell (MSC) population, preferably a BM-MSC population or an ASC population, preferably human BM-MSC or human ASC.

[0121] 23. A pharmaceutical composition comprising a population of SCs according to any one of items 20 to 22 and optionally a pharma- ceutically acceptable carrier.

[0122] 24. A method for preparing a pharmaceutical composition comprising the steps of: a) carrying out the method according to any one of items 1 to 19; and b) formulating the selected SC population with at least one pharma- ceutically acceptable carrier; The method comprising:

[0123] 25. The SC population according to any one of items 20 to 22 for use in allogeneic stem cell therapy in a patient in need thereof, preferably in a presensitized patient or a patient undergoing re-treatment.

[0124] 26. The SC population for use according to item 25, wherein said patient in need thereof is suffering from a disease selected from fistula, leukemia, lymphoma, neurodegenerative diseases, brain and spinal cord injuries, heart diseases, blindness and visual impairment, pancreatic beta cell function loss, cartilage repair, osteoarthritis, musculoskeletal diseases, wounds, infertility, autoimmune diseases and inflammatory diseases such as inflammatory bowel diseases, preferably said disease is fistula, more preferably said disease is complex perianal fistula.

[0125] 27. A method of allogeneic stem cell therapy comprising administering to a patient in need thereof, preferably a presensitized patient or a patient undergoing re-treatment, the SC population according to any one of items 20 to 22.

[0126] 28. The method of allogeneic stem cell therapy according to item 27, wherein said patient in need thereof is suffering from a disease selected from leukemia, lymphoma, neurodegenerative diseases, brain and spinal cord injuries, heart diseases, blindness and vision impairment, pancreatic beta cell function loss, cartilage repair, osteoarthritis, musculoskeletal diseases, wounds, infertility, autoimmune diseases and inflammatory diseases such as inflammatory bowel diseases, preferably said disease is a fistula, more preferably said disease is a complex perianal fistula.

[0127] The present invention is further described in the following examples, which are merely for the purpose of illustrating certain embodiments of the invention and are not to be construed as limiting the scope of the invention in any way. EXAMPLES

[0128] Materials and Methods Monitoring DSA production in ADMIRE CD1 patient A subgroup of 123 patients from the randomized, double-blind, parallel-group, placebo-controlled trial ADMIRE CD1 (Panes et al. (2016), Lancet 388, 1281-1290). Briefly, all patients were adult patients (≥18 years) with CD and treatment-resistant complex draining perianal fistulas and were selected to receive a single intralesional injection of 120 million ASCs or 24 mL saline (placebo). A total of 60 and 63 patients received placebo or ASC injections, respectively, of which 105 (58 ASCs, 47 placebo) were successfully followed up to 52 weeks after treatment.

[0129] ASC donors Human lipoaspirate tissue from healthy donors was processed as described elsewhere (Lopez-Santalla et al., 2015, cited above). ASCs from seven different donors were used in this study, with DonA being the donor used in the ADMIRE CD1 clinical trial. DonA and DonB were from the ADMIRE CD2 clinical trial (NCT03279081), which used two different donors. Additionally, ASCs from donors DonC, DonD, DonE, DonF and DonG were analyzed. All ASC donors met the identity and purity standards set by the International Federation for Adipose Therapeutics and the International Society for Cellular Therapy (Bourin et al., (2013), Cytotherapy (2013) 15(6): 641-648). Culture of ASCs has been described elsewhere (DelaRosa et al., 2009, cited above).

[0130] Anti-HLA detection Plasma samples were obtained by centrifugation of peripheral blood tubes containing ethylenediaminetetraacetic acid (Vacutainer® spray-coated K2EDTA tubes, BD) taken from all patients at baseline and at 12 and 52 weeks after administration of placebo or ASC. Anti-HLA antibodies were detected on the Luminex platform using the LabscreenMixed™ kit (One Lambda Inc.®, Canoga Park, CA, USA) according to the manufacturer's instructions. All samples with a signal above 800 units of median fluorescence intensity (MFI) were considered positive, and the specificity of HLA antibodies was determined using the Labscreen Single Antigen™ kit (One Lambda Inc.®, Canoga Park, CA, USA). All signals were normalized according to Quantiplex™ bead fluorescence, and a standard fluorescence intensity unit above 20,000 was considered relevant. Qualitatively, the HLA antibody titer was defined as the resulting sum of MFI of all determinant beads from the HLA class I molecules contained in the Labscreen Mixed kit, allowing comparison of the resulting level of humoral responses independent of the presented anti-HLA specificity.

[0131] HLA typing The assignment of the expressed HLA alleles in each patient was determined from DNA samples obtained from peripheral blood samples using chemagic DNA Blood250 KIT (PerkinElmer). After checking the purity by examining the A260 / 280 absorbance ratio, all samples with a DNA concentration of 20 ng / μL or more were tested using the LABType® SSO assay (One Lambda, Canoga Park, CA) specific for HLA loci A, B and C according to the manufacturer's instructions. Characterization of mismatches between patient ASC and donor ASC was defined as non-shared unique chains of polymorphic residues (hereafter referred to as eplets) using the algorithm HLA Matchmaker (Duquesnoy (2002), Hum Immunol 63, 339-352).

[0132] Standardization of flow cytometry crossmatch (FCXM) binding with recombinant anti-HLA antibodies (rHLA) standard curve Levels of class I and class II HLA expression were determined in ASCs from DonA and DonB (class I) and DonA (class II) under basal conditions and preactivated with interferon IFNγ (3 ng / mL for 48 h). 50,000 ASCs were stained with increasing concentrations (0–15 ng / mL for clone W6 / 32 and 0–3 ng / mL for clone L243) of PE (phycoerythrin)-labeled anti-human class I HLA Ab (clone W6 / 32) and PerCP (peridinin-chlorophyll-protein)-labeled anti-human class II HLA Ab (clone L243) (Becton Dickinson, Franklin Lakes, NJ, USA) and incubated for 30 min at room temperature in the dark.

[0133] FCXM binding strength in plasma samples Plasma samples were tested 12 weeks (W12) and 52 weeks (W52) before treatment from all patients who received ASCs previously inactivated at 56°C for 30 min and washed once with magnetic activated cell sorting (MACS) buffer. 50 μL of inactivated plasma was incubated with 50,000 ASCs (final volume 100 μL) for 30 min at room temperature. HLA-class I and HLA-class II MFIs were determined using a FACS-Fortessa X20 cell analyzer by acquiring 10,000 events in the P1 gate (total population of ASCs) per sample. Analysis was performed using BD FacsDiva™ software (BD).

[0134] CDC assay for plasma samples For the measurement of cytotoxicity, 250 μL of rabbit serum as a source of complement anti-human class I HLA (CABC-1D, One Lambda Inc®, Canoga Park, CA, USA) was added to the cells for 1 h. The cells were then washed twice and incubated with 20 μL FITC anti-human IgG within 20 min. Finally, after washing, cytotoxicity was determined after the addition of 5 μL of 7-aminoactinomycin D (7-AAD) viability dye with acquisition on an LSR Fortessa flow cytometer (BD).

[0135] mCRP quantification by FACS ASCs were grown for 48 hours in either normal or 3ng / mL IFNγ conditions. ASCs were then trypsinized and counted. A total of 50,000 cells were resuspended in 100μL MACS buffer. For staining, we used CD46 (cat. no. 564253, BD), CD55 (MCA1614PE, Serotec) and CD59 (BRA-10G, Novus Biologicals) Abs, and their respective isotypes as controls (IgG2a-APC, IgG1-PE and IgG2b-PE, BD). After 20 minutes of incubation on ice, ASCs were washed with MACS and centrifuged at 1,500 rpm for 4 minutes. Finally, ASCs were resuspended in 100 μL MACS, transferred to cytometry tubes, acquired on an LSR Fortessa flow cytometer (BD) and analyzed using BD FacsDiva™ (BD).

[0136] Generation of CD46 knockout cell lines Guide RNA was designed to target CD46 exon 3 using the following public genomic tools: www.genome.ucsc.edu and www.www.ncbi.nlm.nih.gov / gene. For CRISPR RNA (crRNA) delivery, the Alt-R® CRISPR-Cas9 System (IDT Integrated DNA Technologies) was used according to the manufacturer's instructions. Briefly, ASCs were thawed and left overnight. After this, a ribonucleoprotein complex mixture was prepared and delivered using Lipofectamine™ RNAiMAX (Thermo-Fisher). crRNA and transactivating crRNA (tracrRNA) were mixed in equimolar concentrations in a sterile microcentrifuge tube with a final oligoduplex working concentration of 1 μM. The mixture was incubated at room temperature for 20 minutes. After this, the transfection complex was added to the culture plate, followed by the ASC suspension. After 24 hours, the ASC medium was replaced and fluorescent tracrRNA-ATTO was added. 550 The efficacy of lipofection was confirmed under a microscope using a .

[0137] result Presence of long-term DSA in ADMIRE CD1-treated patients Blood samples were collected from 123 patients in the ADMIRE CD1 study reported in Panes, J. et al. (2016), Lancet 388, 1281-1290, of whom 63 were ASC and 60 were placebo, at baseline and 12 weeks after treatment administration. At 52 weeks after treatment administration, 105 patients (58 ASC and 47 placebo) provided blood samples (Figure 1A). Analysis by solid-phase assay using Luminex technology revealed that 23 patients produced DSA at 12 weeks after treatment. As expected, no patients who received placebo produced samples containing DSA (Figure 1A, right chart). Furthermore, the results showed that 16% (10 / 63) of ASC patients and 15% (9 / 60) of placebo patients were presensitized at baseline. Of the 53 untreated patients, 17 produced ASC DSA at W12, and 6 of the 10 presensitized patients produced ASC DSA at W12 (Figure 1A, left chart). In all cases, DSA specificity was detected only for HLA class I molecules, not for HLA class II molecules. Long-term follow-up revealed that no further production of DSA was detected at W52, and that 30% of patients (7 / 23 cases) had cleared DSA at this time point. Interestingly, the group of untreated patients who produced DSA at W12 showed a clearance rate of 35% (6 / 17 cases), whereas the presensitized patients who produced DSA at W12 showed a clearance rate of 17% (1 / 6 cases) at W52. Interestingly, presensitized patients tended to show sustained humoral responses at W52. In contrast, untreated patients who developed DSA showed a tendency to return to their basal DSA levels. In summary, the above observations suggest that the response to ASC in these ADMIRE CD1 patients follows primary immune response kinetics.

[0138] As mentioned in the methods (section on anti-HLA detection), the level of DSA positivity for a given sample was selected using the most restrictive threshold of single antigen results, i.e. according to categorical values ​​(above or below a certain cutoff). However, the amount of bound antibody compared to the total antigen present on purified HLA-coated beads (microspheres used in the Luminex® technology) can also be quantified as the sum of MFI HLA class I LSM (least squares mean) microspheres. A time course curve measuring plasma DSA titers throughout the period (pre-treatment and 12-52 weeks post-treatment) was calculated to show the response kinetics and determine the likelihood of decreasing DSA levels (Figure 1B). Patients were clustered into the following groups based on the presence of DSA: untreated patients who did not produce DSA (baseline levels from these patients were used for comparison); untreated patients who produced DSA after allo-ASC administration; pre-sensitized patients with specificity of the administered donor ASC; and pre-sensitized patients without specificity for the donor cells used. As expected, untreated patients who did not develop DSA did not show HLA class I antibodies throughout the course of the study (Figure 1B, top left panel). In contrast, untreated patients who developed DSA showed increased antibody titers. Furthermore, the change in MFI from baseline to W12 was not similar between all patients, and indeed, in certain patients, the response seemed to be more potent than others, with patient 92 (Pat92) being the most alloreactive (Figure 1B, circle). However, at W52, MFI was found to have decreased in all cases, which is consistent with the antibody profile of a classical primary response, with a reduced increase in antibody titers after the booster effect. In the group of presensitized patients sharing specificity with ASC, a profile consistent with a secondary or memory response was observed, with antibody titers above baseline levels at both W12 and W52 (Figure 1B, bottom left panel). These patients have a profile of a booster response that in this case tends to be limited in duration and intensity. Interestingly, similar kinetics were observed in the untreated patient population and in presensitized patients who did not show ASC specificity at baseline signifying a non-responsive profile (FIG. 1B, bottom right panel).

[0139] We investigated the possible association between donor-patient HLA match grade and the probability of producing DSA. We aimed to identify precursors of allorecognition by identifying polymorphic residues present in the HLA type (eplets) of the ASC donor that were missing in the patient. The eplets of each patient were aligned with the HLA alleles of the ASC donor for mismatch quantification. In this study, allosensitization occurred mainly against HLA class I, and therefore we focused on characterizing loci A and B of HLA class I. The total number of eplets correlated with the susceptibility of patients to produce DSA (Figure 1C). Linear regression analysis showed no significant correlation between eplets mismatch and the patient's DSA production at 12 weeks after administration.

[0140] In vitro binding of anti-HLA antibodies to ASC MSCs found in different tissues share common features, including immunomodulatory properties or identity markers, but different immunogenic responses have been reported in in vivo models. Using FACS analysis to characterize the immunogenic response of ASCs, we aimed to quantify the expression of HLA-class I and HLA-class II molecules on ASCs, as well as their ability to bind to patients' anti-HLA Abs (Figure 2A). ASCs were incubated with increasing concentrations of fluorescently labeled HLA-class I (6 and 32 weeks) or HLA-class II (L243) recombinant Abs, with or without pre-stimulation with IFNγ, and the MFI after staining was quantified. As expected, ASCs expressed HLA-class I at basal levels and showed strong overexpression after IFNγ stimulation (Figure 2A). Conversely, HLA-class II levels were negative at baseline and moderately increased after IFNγ stimulation.

[0141] The plasma sample, Pat92 (Figure 1B, circles), had the highest DSA titer after treatment. In basal conditions, no significant increase was detected in the binding intensity of DSA, or in the complement-dependent toxicity assay. However, when ASCs were stimulated with IFNγ, a significant upregulation of ASC binding intensity was observed in both the positive control (pool of hyperimmune samples, HI pool) and the Pat92 sample (Figure 2B, bottom left panel). The increased binding was accompanied by a high percentage of cytotoxic death (34%) in Pat92 (Figure 2B, light gray). This percentage was significantly higher than the percentage of death quantified in the other 22 patients tested (range 3.3-9.3%), confirming that Pat92 was the most alloreactive sample. Interestingly, Pat92 was the one sample that showed the highest level of mismatch with the administered ASCs, yet showed antibody clearance in W52. Understanding the safety and immunogenic toxicity of allogeneic ASC therapy will help assess the feasibility of retreatment and, furthermore, it will help determine their potential impact in presensitized patients and / or those with exacerbated de novo DSA production (e.g., Pat92).

[0142] Plasma DSA binds to ASC and induces moderate killing in vitro In the results above, it was shown that, of the cohort of 63 ADMIRE CD1 patients, 10 had pre-existing HLA-class I Abs and 17 produced de novo DSAs. ASCs were also demonstrated to express HLA-class I antigens and bind rHLA-class I Abs, however, it is unclear whether the patient's DSAs have the capacity to bind and subsequently induce cytotoxic death of ASCs. To test this, we aimed to quantify the different affinities of the presensitized and de novo DSA-positive (DSA+) groups for the binding of HLA-class I antigens to ASCs in vitro. In addition to the first donor DonA (administered in the ADMIRE CD1 study), an additional donor DonB was included to serve as a control for DSA specificity, since it was not administered to the patient (Figure 3A). Samples at baseline and W12 were measured by FCXM for HLA-I binding strength to ASCs cultured under basal conditions or prestimulated with IFNγ (Figure 3A, upper panel). No high binding capacity was observed under basal conditions in samples derived from either presensitized or de novo DSA+ patients in donor ASCs. When ASCs were prestimulated with IFNγ, it was observed that presensitized patients had high and comparable binding affinity at both week 0 (W0) and week 12 (W12) visits with DonA only (Figure 3A, upper panel). The basal condition and therefore the low HLA-I antigen expression in the membrane of ASC donors correlated with low binding affinity in both DonA and DonB (Figure 3A, lower panel). Of note, a significant increase in binding affinity was observed in the comparison of patient samples derived from DonA between W12 and baseline in de novo DSA+ samples (Figure 3A, lower panel). The above data suggest that the concentration of HLA in basal ASCs is not sufficient to show significant binding of DSA in vitro. Moreover, the concentration of DSA in most patients does not seem to be sufficient to show significant binding strength in both non-activated or activated ASCs.These results are consistent with the solid organ transplant flow cytometry cross-matching observations from organ transplantation studies, in which only patients with high DSA levels that also shared immune specificity with the donor yielded significant HLA-class I binding.

[0143] We next aimed to understand whether pre-existing HLA-I antibodies and DSA produced after allogeneic administration were able to fix complement and thus initiate in vitro CDC. We optimized a CDC assay by flow cytometric analysis (FCtox) and established 7-AAD incorporation as a readout for cell death. Plasma samples from presensitized and new DSA+ patients (baseline to W12) containing DonA and DonB ASCs were incubated in the presence of rabbit C3 complement either under basal conditions or before IFNγ stimulation. In the presensitized patient cohort, W12 samples induced moderate cytotoxic death (0-5% 7-ADD+ cells) in DonB, both in basal and IFNγ conditions. As observed in DonA ASCs, incubation of the same samples induced higher cytotoxic death in basal (2 patients) and IFNγ (3 patients) conditions (Figure 3B). The novel DSA+W12 samples were also able to fix complement and induce cytotoxic death under basal conditions. As expected, a higher percentage of cell death was obtained only in DonA ASCs when stimulated with IFNγ (Figure 3B). These data suggest that pre-existing HLA-class I Abs and DSA can bind to ASCs and induce moderate CDC specifically in DonA ASCs. Interestingly, DonB did not induce a significant percentage of cytotoxicity in either the presensitized or novel DSA+ cohorts, although there was a trend toward increased cell death in W12 compared to baseline. One possible explanation for this could be the presence of shared HLA polymorphic alleles between the two donors used in this study. HLA typing was performed and DonB was found to share a common allele with DonA.

[0144] DNA from DonA and DonB was purified and tested by the LABType® SSO assay for HLA allele characterization. In bold are shown the HLA-A alleles shared by DonA and DonB (see Table 1).

[0145] [Table 1]

[0146] This shared HLA allele may be responsible for the tendency towards increased levels of cytotoxicity in W12 samples in DonB.

[0147] High expression of mCRP in ASCs To understand the moderate death of ASCs affected by DSA, we aimed to identify complement inhibition strategies that may enable ASCs to cope with and / or avoid cytotoxic death. One classical mechanism for complement signaling inhibition is the induction of mCRP CD46, CD55, and CD59 (Gancz and Fishelson, 2009;Ricklin et al., 2010;Tegla et al., 2011). Some authors have shown that MSCs express low levels of CD46 and CD55, and high levels of CD59, while others suggest that MSCs express moderate levels of total mCRP. To address this controversy, the expression levels of CD46, CD55, and CD59 were analyzed in a panel of ASCs stimulated or not stimulated with IFNγ, and the expression levels were compared with commercially available BM-MSCs (Figure 4A). It was observed that the basal levels of CD46, CD55, and CD59 were higher in ASCs compared to BM-MSCs. To recapitulate this physiologically relevant scenario, mCRP levels were tested in the presence of IFNγ, a key mediator of ASC immunoregulatory responses (proinflammatory environment). No significant regulation of mCRP in BM-MSCs was observed, but ASCs appeared to strongly induce mCRP after IFNγ stimulation. CD46 induction was particularly prominent in ASCs, approximately 2.14-fold compared to BM-MSCs. The above results suggest that ASCs strongly express mCRP under basal conditions, and expression is further enhanced in the presence of IFNγ. This suggests an important role for mCRP in the negative regulation of CDC and may hint at a cytoprotective mechanism in ASCs to cope with DSA-induced cytotoxicity. This may explain the moderate level of death imposed by DSA.

[0148] Although the expression patterns of mCRP in the different ASC donors were comparable, some donors showed specific expression of certain mCRPs (Figure 4B). Specifically, DonC showed higher CD46 and CD55 levels compared to the other donors, while DonE and DonF preferentially overexpressed CD55. We further investigated whether differential expression of mCRPs could affect the differential susceptibility to CDC. To answer this, we investigated the kinetics of HLA-I antigen expression and its binding affinity among the ASC donors of this panel. To test this, ASC donors were subjected to increasing concentrations of rHLA-class I W6 / 32. Their binding affinity was then measured by FACS (Figure 4C). Differential binding affinities between donors under basal conditions (i.e., a 12-fold difference when comparing DonC with DonG at 10 ng / mL W6 / 32) were observed. After IFNγ stimulation, HLA-I antigens were induced in ASC donors as shown by increased W6 / 32 binding (Figure 4C). Also, different binding affinities between donors (6.25-fold difference at 10 ng / mL when comparing DonB with DonG) were observed. In parallel, the sensitivity of different ASC donors to the CDC assay was tested. Under basal conditions, DonE showed about 25% cell death at the highest W6 / 32 concentration, whereas in the remaining donors this was approximately 15%, with the exception of DonD and DonG, which showed lower percentages (Figure 4C). As expected, after IFNγ stimulation, the CDC sensitivity level increased dramatically in all ASC donors, but to a lesser extent in DonB and DonC, which remained relatively resistant to CDC-mediated cell death (Figure 4C).

[0149] We next aimed to determine whether high W6 / 32 binding affinity correlated with enhanced CDC sensitivity. To perform this correlation analysis, the W6 / 32 concentration was set at 10 ng / mL, since this was the Ab amount that induced the transition phase of the curve (post-exponential and pre-plateau). No significant correlation was observed in either basal or IFNγ stimulation scenarios (Figure 6A). Of note, we identified a group of ASC donors that expressed relatively low W6 / 32 Ab MFI levels (low binding) yet showed high sensitivity to CDC. This suggests that there is no absolute correlation between W6 / 32 Ab binding and sensitivity to CDC. To determine which of the three mCRPs was a contributing factor to CDC inhibition of ASCs, we correlated the cell death levels achieved with 10 ng / mL W6 / 32 with MFI expression levels in both basal and IFNγ conditions (Figure 6B). No positive correlation was observed under basal conditions for any of the mCRP molecules tested. However, after IFNγ stimulation, lower CD46 and CD55 levels were found to be significantly correlated with higher cell death levels. Finally, the significance of the slope for CD46 was slightly higher compared to CD55.

[0150] CD46 depletion increases CDC susceptibility of ASCs in vitro Prompted by the robust induction of CD46 after IFNγ stimulation compared to CD55 or CD59, and the high significance of cytotoxicity correlation along with reduced donor-to-donor variability of CD46 compared to CD55, we performed a thorough analysis of CD46 and its potential impact on CDC susceptibility in ASCs. Using public genome browsers, we identified top gRNA sequences (ncbi.nlm.nih.gov / gene and crispr.mit.edu) for knockdown of CD46. Optimal guide RNA (gRNA) sequences were selected based on two parameters: high specificity and low off-target score. Two optimal crRNAs (crRNA1 and crRNA2) targeting exon 3 were selected for efficacy screening. crRNA: tracrRNA-ATTO550 : The delivery of the Cas9 complex was examined under a microscope. 24 hours after lipotransfection, it was found that the majority of ASCs had incorporated the ribonucleoprotein complex, which correlated with high Cas9-mediated double-strand break events (Figure 6C). To confirm the efficacy of CRISPR-mediated knockdown, ASCs were cultured in the presence or absence of IFNγ and analyzed for CD46 expression by FACS. The efficacy of crRNA1 was comparable to that of crRNA2 under both normal and IFNγ conditions, and therefore, we concluded that the ASCs-CD46 knockdown was significantly improved by the use of crRNA1. KO crRNA1 was selected for the generation of clones (Figure 6D).

[0151] After confirming knockdown specificity, one donor with low CDC susceptibility was selected to test whether selective depletion of CD46 could sensitize it to cytotoxic death. DonB was selected for W6 / 32-mediated cytotoxicity assays in basal and IFNγ conditions (Figure 5A). CD46 knockdown induced a moderate increase in cytotoxic death under basal conditions (Figure 5A, left graph), likely due to low HLA-I binding and subsequent complement fixation. After IFNγ stimulation, a significant increase in the percentage of cytotoxic death in parental ASCs was observed, which was consistent with the CD46 knockdown. KO This was further enhanced in ASCs (Figure 5A, right graph). At a physiological dose of 10 ng / mL W6 / 32, approximately 50% of 7-AAD-positive cells were observed in parental ASCs, and CD46 knockdown increased the percentage of death up to approximately 95%, suggesting that CD46 plays an important role in preventing cytotoxic death, at least in DonB.

[0152] To test whether the CD46 cytotoxicity inhibitory function is effective in other ASC donors, cytotoxicity assays were performed in a panel of seven donors and mean curves were plotted from basal conditions (Figure 5B, left graph) to ASC before IFNγ stimulation (Figure 5B, right graph). Under both test conditions, CD46 KOASC donors showed enhanced susceptibility to CDC compared to parental controls. 50 ) A shift in the curve to the left means that a decrease in the concentration of W6 / 32 Ab is required to induce CDC, which correlates with increased susceptibility to CDC. Next, to quantify the shift in the curve, we measured the EC 50 was calculated (Figure 5C).

[0153] Table 2 below shows the results of 7 parental ASCs and their corresponding CD46 KO The half effective concentration of W6 / 32 Ab (ng / mL) in ASCs is shown. In columns 4 and 7, the difference in fold change (CD46 KO EC 50 / Parent EC 50 ) was calculated.

[0154] [Table 2]

[0155] Under basal conditions, CD46 KO Donors had reduced EC compared to parent donors 50 The results showed that CD46 expression confers CDC resistance to ASCs in vitro, suggesting a higher sensitivity to W6 / 32 (Figure 5C, fourth row). A similar effect was observed under IFNγ conditions (Figure 5C, seventh row), confirming that CD46 expression confers CDC resistance to ASCs in vitro. The data confirm that CD46 mediates CDC and that its depletion in ASCs correlates with enhanced CDC sensitivity.

Claims

1. 1. An in vitro method for selecting a population of adipose tissue-derived stem cells (ASCs) suitable for allogeneic therapy, comprising: The following steps: a) culturing a sample of an ASC population in the presence of a concentration of IFN-γ capable of inducing maximal HLA-class I expression in said ASC population (test sample), and separately culturing a sample of an ASC population in the absence of IFN-γ (control sample); b) contacting the test and control samples with a range of different concentrations of HLA-class I antibodies under conditions such that the HLA-class I antibodies bind to HLA-class I expressed in the test and control samples; c) adding complement to the test and control samples such that bound HLA-class I antibodies are saturated with complement and complement dependent cytotoxicity (CDC) is induced; d) determining CDC for a range of different concentrations of HLA-class I antibodies by measuring induced cell lysis in test and control samples; e) 50% of the maximum CDC (EC ５０ determining the concentration of HLA-class I antibody that induces a β-blocking effect (β-blocking value); and f1) EC of the test sample ５０ EC of the control sample versus the value ５０ selecting the ASC population for allogeneic therapy if the ratio of values ​​is less than 1.25; or f2) EC of the test sample ５０ If the value is at least 3.5 ng / ml, selecting the ASC population for allogeneic therapy. The method comprising:

2. The in vitro method of claim 1, further comprising the steps of determining CD46 expression levels in a test sample and a control sample; and selecting an ASC population for allogeneic therapy if the ratio of CD46 expression in the test sample to the control sample is greater than 2.

0.

3. The in vitro method according to claim 1 or 2, wherein the IFN-γ concentration capable of inducing maximum HLA-class I expression in the ASC population is 0.5-30 ng / ml.

4. The in vitro method according to any one of claims 1 to 3, wherein the HLA-class I antibody specifically binds to HLA-A, HLA-B and / or HLA-C.

5. The in vitro method according to any one of claims 1 to 4, wherein two or three different concentrations of said HLA-class I antibodies in the range of 1 to 50 ng / ml are used.

6. The in vitro method according to any one of claims 1 to 5, wherein the complement used in step (c) of claim 1 is derived from serum.

7. The in vitro method according to any one of claims 1 to 6, wherein the complement is derived from serum and the serum concentration resulting in saturation of bound HLA-I antibodies with complement is between 50 and 83.33% (v / v).

8. 8. The in vitro method according to any one of claims 1 to 7, wherein the cell lysis is determined by measuring a chemiluminescent or fluorescent dye that is selective for live or lysed cells or radioactive material released from lysed cells.

9. 1. A method for preparing a pharmaceutical composition comprising the steps of: a) carrying out the method according to any one of claims 1 to 8; and b) formulating the selected population of ASCs with at least one pharma- ceutically acceptable carrier. The method comprising:

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