Pre-transplant conditioning regimen

Abstract

The present invention relates to conditioning regimens for transplanting cells, tissues, or organs, optionally hematopoietic stem / progenitor cells, into a subject. The present invention also relates to methods for inducing hematopoietic chimerism in a subject. The present invention also relates to methods for preventing or treating a disease or condition in a subject, inducing hematopoietic chimerism to enhance the subject's benefit from a subsequent therapy. The subsequent therapy may be a cell, tissue, or organ transplant, or may be gene therapy performed using genetically modified hematopoietic stem / progenitor cells.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to pre-treatment regimens for transplantation of cells, tissues, or organs, optionally hematopoietic stem cells / progenitor cells (HSPCs). The present invention also relates to methods for inducing hematopoietic chimerism in a subject. The present invention also relates to methods for the prevention or treatment of a disease or condition in a subject, wherein the method involves inducing hematopoietic chimerism to enhance the benefit of the subject from subsequent therapy. The subsequent therapy may be cell, tissue, or organ transplantation, or gene therapy performed using genetically modified HSPCs.

[0002] Background of the Invention Tissue / organ transplants can be compromised by acute and / or chronic rejection, which can lead to graft failure. Both acute and chronic rejection are typically treated with immunosuppressants, which increase the risk of infection, increase the risk of cancer, and can also cause organ failure (such as graft failure). A technique that can reduce the need for immunosuppression (by establishing immune tolerance to the transplant) is the induction of hematopoietic chimerism, typically by transplanting hematopoietic stem cells and progenitor cells from the same donor in a bone marrow transplant (BMT) before transplanting cells, tissues, or organs. Induced chimerism results in a chimeric immune system that does not attack the graft, which would otherwise have the same immunological profile as the donor's hematopoietic system, while preserving the recipient's normal immune function to react to unrelated antigens.

[0003] Unfortunately, the complex immunology involved in HSPC transplantation can be problematic, especially when there has been prior sensitization to donor antigens. The presence of the donor and recipient immune systems can lead to acute and chronic rejection reactions with both human and cellular components. Both robust host-versus-graft (HVG) and graft-versus-host disease (GVHD) are observed. Often, the transplanted cells do not engraft well in the recipient. Current methods attempt to address these problems through pre- and post-transplant immunosuppression. The procedures performed before transplantation, called conditioning regimens, may include treatments other than immunosuppression. For example, radiation may be used to eliminate some or all of the recipient's existing bone marrow cells to create space for the transplanted cells to engraft. However, engraftment often does not occur successfully. An improved conditioning regimen is needed for HSPC transplantation.

Summary of the Invention

[0004] Summary of the Invention Conditioning regimens for the transplantation of hematopoietic stem and progenitor cells (HSPCs) typically include treatments to deplete T lymphocytes and / or reduce donor-specific antibodies (DSAs) either directly (e.g., by administration of plasmapheresis or mismatched platelet transfusions that adsorb DSAs) or indirectly by inhibiting antibody production (e.g., using rituximab or bortezomib). However, existing conditioning regimens are often ineffective and engraftment often does not occur successfully. This may be because the high expression of MHC in bone marrow-derived cells increases their sensitivity to residual functional DSAs.

[0005] The inventors have surprisingly shown that conditioning regimens such as enzymatic inactivation of the subject's serum IgG are more likely to result in hematopoietic chimerism in the subject, as they significantly improve the engraftment rate (in contrast to the antibody removal techniques previously used).

[0006] The present invention provides a pretreatment regimen for transplanting HSPCs into a subject, which comprises administering to the subject an enzyme that inactivates the serum IgG molecules of the subject. The amount of the enzyme administered is preferably an amount sufficient to inactivate all or substantially all of the IgG molecules present in the serum of the subject.

[0007] The pretreatment regimen may further comprise one or more of the following: (a) Administration to the subject of a non-lethal dose of irradiation and / or any other agent that removes the subject's HSPCs (b) Administration of an agent that reduces the number of lymphocytes and / or down-regulates the activity of lymphocytes in the subject, wherein the lymphocytes include: i. T cells; and / or ii. B cells (optionally including antibody-producing cells); (c) Administration of any other agent or regimen that reduces the activity of the immune system, such as an inhibitor of complement, an inhibitor of cytokines, an inhibitor of innate immune cells, an inducer of tolerance.

[0008] The pretreatment regimen preferably includes at least (a), and most preferably includes at least (a) and (b).

[0009] The present invention also provides a method of inducing hematopoietic chimerism in a subject, the method comprising performing the pretreatment regimen of the present invention and then administering HSPCs to the subject in an amount sufficient to induce hematopoietic chimerism in the subject and under suitable conditions. The HSPCs may be autologous (using the subject's own cells) or allogeneic (the cells are derived from another donor). The HSPCs may be genetically modified, and in that case, it is desirable that they be autologous. The genetic modification may be one that expresses any gene, but typically it is a gene that is therapeutically beneficial to the recipient, and in that case, the HSPCs may be said to be expressing gene therapy. The HSPCs are preferably allogeneic cells or genetically modified autologous cells. Most preferably, the HSPCs are allogeneic.

[0010] The present invention also provides a method for the prevention or treatment of a disease or condition of interest, and a method for inducing hematopoietic chimerism in a subject according to the method of the present invention in order to enhance the subject's benefit of the therapy for the disease or condition. The therapy may be a transplant of cells, tissues, or organs, typically from the same donor as the HSPC. The transplanted cells, tissues, or organs may be of any type, such as kidneys, livers, hearts, pancreas, lungs, small intestines, skin, blood vessels / vascular tissue, face, arms, trachea, part of an eye, pancreatic islets, substantia nigra, bone marrow, or stem cells. The transplanted cells may be of any type, such as the same HSPC used in the method itself, so as to avoid the need for additional therapy.

[0011] In other words, the present invention also provides a method for preventing or treating immune rejection of cell, tissue, or organ transplants, the method comprising inducing hematopoietic chimerism in a subject according to the method of the present invention, and applying a cell, tissue, or organ transplant to the subject, optionally, said cells, tissue, or organ from the same donor as the HSPC. The cells, tissue, or organ is typically administered after hematopoietic chimerism has been induced in the subject. The transplant of cells, tissue, or organ may be of any type, such as kidney, liver, heart, pancreas, lung, small intestine, skin, blood vessels / vascular tissue, face, arm, trachea, part of the eye, pancreatic islets, substantia nigra, bone marrow, or stem cells. The transplanted cells may be of any type, such as the same HSPC used to induce hematopoietic chimerism, so as to avoid the need for additional transplants.

[0012] Expressed in other words, the present invention provides a method for preventing or treating immune rejection of cell, tissue, or organ transplants, and includes: (i) Perform the pretreatment regimen of the present invention; (ii) Administering HSPC to the subject in an amount and under appropriate conditions sufficient to induce hematopoietic chimerism in the subject; and (iii) Applies to cell, tissue, or organ transplants from the same donor as the HSPC, optionally, the transplant is the administration of the HSPC in step (ii).

[0013] If the cells, tissues, or organs of step (iii) are the HSPCs of step (ii), the method of the present invention may be a method for treating a disease or condition treated by transplantation of the HSPCs. If the HSPCs are genetically modified for the purpose of administering gene therapy, the method of the present invention may be for the prevention or treatment of a disease or condition targeted by the gene therapy. [Brief explanation of the drawing]

[0014] [Figure 1] Figure 1. EndoS inhibits monoclonal DSA-mediated death of donor bone marrow cells. Naive NOD (Panel AB) or B6.H-2g7 (Panel CD) were infused with a mixture of CFSE-labeled NOD / CTV-labeled B6 bone marrow cells (BMC; Panel AB) or CFSE-labeled B6.H-2g7 / CTV-labeled NOD.H-2bBMC (Panel CD) four hours prior to intravenous administration of 30 μg of EndoS and / or anti-H-2Kb mAb (10 μg or 100 μg). Experimental protocols for NOD (Panel A) and B6.H-2g7 (Panel C) are shown. The ratios of dye-labeled B6 (Panel B) to NOD cells, or NOD.H-2b (Panel D) to B6.H-2g7 cells, are shown in blood collected 1 to 3 hours post-bone marrow transplantation (BMT) (left panel), host spleen (center panel), and bone marrow (BM, right panel) collected 4 hours post-BMT. Mean ± SEM values ​​are shown. Data were pooled from 5 (Panel B) and 4 (Panel D) independent experiments. The Mann-Whitney U test (center and right panels) was used for the shown comparisons; *p<0.05 and **p<0.01. [Figure 2]Figure 2. EndoS-immunifidase reduces DSA-mediated death of donor BMCs in sensitized recipients. (AC) Naive NOD mice were immunized with FVB spleen cells 4 weeks prior to EndoS-immunifidase administration. Serum was collected pre-immunization, pre-enzyme treatment, and 4 hours after enzyme treatment. The representative histograms on the left are for DSA-IgG Fc (Panel A), DSA-IgG1 Fc (Panel B), DSA-IgG3 Fc (Panel C), and DSA-IgG3 heavy chain (Panel D) with serum at 1:25 dilutions. The mean fluorescence intensity (MFI) of DSA in titrated serum is shown on the right. Mean ± SEM is shown. Ratio-paired t-tests were used to compare the MFI of DSA before and after enzyme treatment in each serum dilution, with *p<0.05 and **p<0.01. (D) Schematic of the experiment shown in EF. Naive NOD mice were immunized with B6.CD45.1 spleen cells 4 weeks prior to injection of a T-cell depletion mAb. EndoS-immunifidase was administered 2 days after T-cell depletion. Four hours after enzyme treatment, 80 million B6.CD45.2 bone marrow cells were intravenously injected into the NOD mice. Spleen and bone marrow cells were analyzed for MHC-I H-2Kb and CD45.2 expression. (EF) Representative pointillisms of the four different treatment groups (left) and donor cell percentages (right, mean ± SEM) are shown. Values ​​between the three sensitization groups were compared using Holm-Sidak one-way ANOVA with multiple comparisons, with *p<0.05. [Figure 3]Figure 3. Bortezomib / cyclophosphamide before BMT reduces bone marrow B cells in sensitized recipients. (A) Schematic of the experiment shown in BE. NOD mice were intravenously treated with cyclophosphamide and bortezomib (CyBor) four weeks after immunization with FVB spleen cells. Four days after CyBor treatment, bone marrow transplantation with 20 million FVB BMCs was performed. Spleen and bone marrow cells were collected five days after BMT for analysis. Serum was collected before CyBor treatment and five days after BMT. Cell counts of B cells and plasma cells in the bone marrow (panel B) and spleen (panel C) of mice given CyBor or vehicle are shown. (D) Serum was collected before immunization and five days after BMT, i.e., nine days after CyBor treatment. MFI of DSA-IgG Fc in titrated serum from individual controls (left) or CyBor-treated mice (right) is shown. (E) The percentile change in MFI at 1:25 dilution of DSA on day 9 is shown compared to before treatment. Filled and empty symbols represent data collected in two separate experiments. [Figure 4] Figure 4. EndoS-immunifidase enables hematopoietic chimerism in pre-sensitized recipients. (A) Outline of the chimerism induction protocol; naive B6.H-2g7 or NOD mice were immunized with FVB spleen cells 4 to 6 weeks prior to chimerism induction. CyBor was administered on day -4 relative to the day of BMT for chimerism induction. T cell depletion (TCD) antibody was administered intravenously on days -2, 2, 6, 11 and 16. Recipients sensitized to FVB spleen cells were treated intravenously with EndoS-immunifidase on day -6 and with repeated doses on day 0, 4 hours prior to BMT. 6 Gy of whole-body irradiation was administered 4 hours prior to BMT on day 0. FVB BMC (80 × 10⁶) was administered on day 0. (B) Percentage of donor cells in the lymphocyte gate in peripheral blood over time. (C) Shows the proportion of different lineages of donor cells in the lymphocyte gate in peripheral blood from naive NOD chimeras (n=4, left, mean ± SEM) and primed NOD chimeras (n=2, right). Data were pooled from six independent experiments. [Figure 5]Figure 5. Mean immunofidase concentration versus nominal time since administration (N=15). Data BLQ is included in the mean as BLQ / 2. SD is shown as a bar. [Figure 6] Figure 6. Time course of in vitro cleavage of rATG by immunofidase. The column shows the number of subjects with visible intact rATG on the Western blot after immunofidase (N=11). [Modes for carrying out the invention]

[0015] A brief explanation of arrays Sequence ID 1 is the complete sequence of IdeS, including the N-terminal methionine and signal sequence. It is also available as NCBI reference sequence ID WP_010922160.1. Sequence ID 2 is the mature sequence of IdeS lacking the N-terminal methionine and signal sequence. It is also available as Genbank acceptance number ADF13949.1. Sequence ID 3 is the complete sequence of IdeS, including the N-terminal methionine and signal sequence. It is also available as NCBI reference sequence ID WP_014622780.1. Sequence ID 4 is the mature IdeZ sequence, lacking the N-terminal methionine and signal sequence. Sequence ID 5 is the sequence for a hybrid IdeS / Z. The N-terminus is based on IdeZ lacking an N-terminal methionine and signal sequence. Sequence IDs 6 to 25 are exemplary protease sequences for use in the method of the present invention. Sequence ID 26 is the sequence of the IdeS polypeptide. It includes the sequence of Sequence ID 2 (internal standard pCART124) with additional N-terminal methionine and histidine tags. Sequence ID 27 is the sequence of the IdeZ polypeptide. It includes the sequence of Sequence ID 4 (internal standard pCART144) with additional N-terminal methionine and histidine tags. Sequence ID 28 is the sequence of the IdeS / Z polypeptide. It includes the sequence of Sequence ID 5 (internal standard pCART145) with additional N-terminal methionine and histidine tags. Sequence ID 29 is the continuous sequence PLTPEQFRYNN, which corresponds to positions 63-73 of sequence ID 3. Sequence ID 30 is the continuous sequence PPANFTQG, corresponding to positions 58-65 of sequence ID 1. Sequence ID 31 is the sequential sequence DDYQRNATEAYAKEVPHQIT, which corresponds to positions 35-54 of Sequence ID 3. Sequence ID 32 is the continuous sequence DSFSANQEIRYSEVTPYHVT, which corresponds to positions 30-49 of sequence ID 1. Sequence IDs 33 to 55 are nucleotide sequences that encode the proteases defined above. Sequence IDs 56 to 69 are exemplary protease sequences for use in the method of the present invention. Sequence ID 70 is a continuous sequence NQTN corresponding to positions 336-339 of sequence ID 1. Sequence ID 71 is the continuous sequence DSFSANQEIR YSEVTPYHVT, which corresponds to positions 30-49 of sequence ID 1. Sequence IDs 72 to 86 are nucleotide sequences encoding polypeptides disclosed herein. Sequence ID 87 is the sequence SFSANQEIRY SEVTPYHVT, which corresponds to positions 31-49 of sequence ID 1. Sequence ID 88 is the sequence DYQRNATEAY AKEVPHQIT, which corresponds to positions 36-54 of the IdeZ polypeptide NCBI reference sequence ID WP_014622780.1. Sequence ID 89 is the sequence DDYQRNATEA YAKEVPHQIT, which may be present at the N-terminus of the polypeptide of the present invention. Sequence ID 90 is the mature sequence of EndoS (endoglycosidase of S. pyogenes).

[0016] Detailed description of the invention General matters It should be understood that the different applications of the disclosed products and methods may be adapted to the specific needs of the art. It should also be understood that the terms used herein are solely for the purpose of describing specific aspects of the invention and are not intended to limit them.

[0017] Furthermore, as used herein and in the appended claims, the singular forms "a," "an," and "the" include multiple references unless the context explicitly indicates otherwise. Thus, for example, a reference to "a polypeptide" includes "polypeptides."

[0018] In this specification, the term "polypeptide" is used in its broadest sense to refer to compounds of two or more subunits of amino acids, amino acid analogs, or other peptide mimetic compounds. Thus, the term "polypeptide" includes short peptide sequences, as well as longer polypeptides and proteins. As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acids, including both D and L optical isomers, as well as amino acid analogs and peptide mimetic compounds.

[0019] The terms "patient" and "subject" are used interchangeably and typically refer to humans. References to IgG typically refer to human IgG unless otherwise specified.

[0020] Amino acid identity can be calculated using any suitable algorithm. For example, the PILEUP and BLAST algorithms can be used (typically in their default settings) to calculate homology or to line up sequences (identify identical or corresponding sequences, etc.), as described, e.g., Altschul SF (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information. This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that, when aligned with words of identical length in a database sequence, either match or satisfy a certain positive-valued threshold score T. T is called the neighbor word score threshold (Altschul et al, above). These first neighbor word hits serve as seeds to initiate a search for HSPs containing them. The word hit is extended in both directions along each sequence as long as the cumulative alignment score can increase. The extension of the word hit in each direction stops when: the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score becomes zero or less due to the accumulation of residue alignments with a negative score of 1 or more; or when it reaches the end of either sequence. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment.The BLAST program uses the following initial settings: word length (W) 11, BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919), alignment (B) 50, expected value (E) 10, M=5, N=4, and comparison of both strands.

[0021] The BLAST algorithm performs statistical analysis of the similarity between two sequences (e.g., see Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two polynucleotide or amino acid sequences occurs by chance. A sequence is considered similar to another sequence if, for example, the smallest sum probability in a comparison between the first and second sequences is less than approximately 1, preferably less than approximately 0.1, more preferably less than approximately 0.01, and most preferably less than approximately 0.001. Alternatively, the UWGCG package provides the BESTFIT program (e.g., used in its initial settings) which can be used to calculate identity (Devereux et al (1984) Nucleic Acids Research 12:387-395).

[0022] All publications, patents, and patent applications cited herein, whether above or below, are incorporated herein by reference in their entirety.

[0023] Pre-treatment regimen The present invention provides a pre-treatment regimen for transplanting cells, tissues, or organs into a subject, comprising administering an enzyme to the subject that inactivates target serum IgG molecules. The amount of the enzyme administered is preferably sufficient to inactivate all or substantially all IgG molecules present in the subject's serum. If necessary, two or more IgG inactivating enzymes can be combined and administered in any order, such as simultaneously or sequentially.

[0024] The term “serum IgG molecule” or “IgG molecule present in serum” refers to any gamma immunoglobulin (IgG1, IgG2, IgG3, and IgG4) molecule present in human tissue or circulation before the method of the present invention is carried out. Such IgG molecules may be endogenously produced from individual B cells or may be exogenous gamma immunoglobulins administered to the subject before the method of the present invention is carried out, such as any therapeutic IgG molecule of any origin. Inactivation of serum IgG typically means a decrease in Fc receptor interaction of the IgG molecule. The term “Fc receptor” refers to the Fc gamma immunoglobulin receptor (FcγR) present on cells. In humans, FcγR refers to one, part, or all of the family of receptors, including FcγRI(CD64), FcγRIIA(CD32A), FcγRIIB(CD32B), FcγRIIC(CD32C), FcγRIIIA(CD16a), and FcγRIIIB(CD16b). As used herein, the term FcγR includes the spontaneous polymorphisms FcγRI(CD64), FcγRIIA(CD32A), FcγRIIB(CD32B), FcγRIC(CD32C), FcγRIIIA(CD16a), and FcγRIIIB(CD16b).

[0025] The enzyme used in the method of the present invention may be any enzyme that inactivates serum IgG, but is typically an IgG cysteine ​​protease that cleaves IgG such that the antigen-binding domain and the Fc-interaction domain are separated from each other. In such a case, the amount of intact IgG molecules in the serum is reduced, thus reducing the Fc receptor interaction of serum IgG molecules. As another example, the enzyme may be an IgG endoglycosidase that cleaves the glycan structure on the Fc-interaction domain of IgG, particularly the N-linked nianthentria glycan at position Asn-297 (Kabat numbering). This glycan structure plays a crucial role in Fc receptor binding and complement activation. Therefore, if this glycan is removed entirely or partially by the protein, it leads to a decrease in Fc receptor binding or complement activation by other intact IgG molecules. Suitable enzymes for use in pretreatment regimens are described in more detail in the following sections.

[0026] The enzyme is preferably administered by intravenous infusion, but may be administered by any suitable route, such as intradermal, subcutaneous, transdermal, intramuscular, intra-arterial, intraperitoneal, intra-articular, intraosseous, or other appropriate route of administration. The amount of enzyme administered may be between 0.01 mg / kgBW and 2 mg / kgBW, between 0.05 and 1.5 mg / kgBW, between 0.1 mg / kgBW and 1 mg / kgBW, preferably between 0.15 mg / kg and 0.7 mg / kgBW, most preferably between 0.2 mg / kg and 0.3 mg / kgBW, and particularly 0.25 mg / kgBW. The enzyme may be administered multiple times to the same subject, provided that the amount of anti-drug antibodies (ADAs) in the serum of the subject capable of binding to the enzyme does not exceed a threshold determined by the clinician. The amount of ADAs in the serum of the subject capable of binding to the protease may be determined by any suitable method, such as a drug-specific CAP FEIA (ImmunoCAP) test or titration (titre) assay. If the target ADA exceeds the aforementioned threshold, the pretreatment regimen may include the administration of an alternative enzyme.

[0027] The pretreatment regimen may further include one or more of the following: (a) Non-lethal dose of irradiation and / or administration of any agent to the subject that removes the target HSPC (b) Administration of a drug that reduces the number and / or downregulates the activity of lymphocytes in a subject, wherein the lymphocytes include: i T cells; and / or ii. B cells (including optionally antibody-producing cells); (c) Administration of any other drugs or regimens that control (e.g., reduce) the activity of the immune system, such as complement inhibitors, cytokine inhibitors, innate immune cell inhibitors, or tolerance inducers.

[0028] Step (a) typically involves administering a dose of radiation sufficient to partially or completely eradicate (or excise) the bone marrow of the subject. Partial eradication is preferred because the side effects are typically less severe and it is desirable to retain some of the recipient's bone marrow. Excision of the recipient's bone marrow creates space for donor HSPCs to engraft within the bone marrow, but removes the subject's lymphocytes and therefore also reduces the activity of the immune system in a similar manner to step (b). Thus, the preconditioning regimen preferably includes at least (a), but most preferably at least (a) and (b). Alternatively, in step (a), it may be preferable to use an approach that does not involve irradiation for the removal of the subject's HSPCs, such as the administration of anti-CD117 and / or anti-CD47. This creates space for donor HSPC engraftment but avoids some of the undesirable side effects of irradiation. In addition to HSPC removal, the subject may optionally receive an infusion of donor CD8-alpha cells, which may increase the frequency of stable chimerism in sensitized recipients. Injecting donor T cells may promote the engraftment of donor HSPCs by reducing the survival of host T cells.

[0029] Step (b) can be carried out using any appropriate method and any appropriate drug. The same drug or combination of drugs may be effective in reducing the number and / or downregulating the activity of two or more types of lymphocytes. For example, preclinical studies in non-human primate models of transplantation into pre-sensitized recipients suggest that co-stimulus deprivation with beratacept combined with plasma cell depletion therapy with bortezomib can sustainably suppress DSA and reduce the risk of antibody-mediated rejection.

[0030] Examples of drugs suitable for T cell removal are known in the art and include anti-thymocyte globulins (ATG, rabbit or equine ATG, etc.); or panels of antibodies such as anti-CD4, anti-CD8, and anti-CD90; anti-CD52 antibodies (such as alemtuzumab); anti-CD117 antibodies; anti-CD45 antibodies; busulfan; cyclophosphamide; fludarabine; treosulfan; cyclosporine; tacrolimus; or immunotoxins that target T cells.

[0031] Examples of drugs suitable for the removal of B cells (including plasma cells optionally) are known in the art and include anti-CD20 antibodies (such as rituximab); anti-CD19 antibodies; bortezomib; fludarabine; cyclophosphamide; or B cell-targeting immunotoxins such as anti-CD20 immunotoxins (e.g., MT-3724).

[0032] An exemplary regimen comprising steps (a) and (b) is shown in the Examples, which includes, in addition to the administration of a non-lethal dose of radiation, the administration of a panel of antibodies such as anti-CD4, anti-CD8, and anti-CD90 to eliminate T cells, and the administration of bortezomib and cyclophosphamide to eliminate B cells (such as antibody-producing cells).

[0033] Steps (a) and (b) are typically separated from each other, and, if necessary, separated from the administration of the enzyme that inactivates the target serum IgG molecule, for a time interval suitable for administration to achieve the desired effect. For example, if steps (a) and / or (b) include an antibody-based drug, it would be preferable that these steps be performed at a sufficient time interval after the administration of the enzyme so that the enzyme does not inactivate the antibody-based drug in step (a) or (b). Exemplary time intervals are illustrated in Example 2. Administration of rATG (or other antibody-based therapy) may be started as early as 4 days after the administration of immunofidase. Alternatively, the enzyme may be added at an appropriate interval after the antibody-based drug, such that the antibody-based drug has already exerted its effect.

[0034] The administration of the enzyme that inactivates serum IgG molecules, and steps (a) and (b), may be performed at different times relative to the transplantation of cells, tissues, or organs to the subject. For example, the administration of the enzyme, and steps (a) and (b), may all be performed before cell, tissue, or organ transplantation. Alternatively, the administration of the enzyme, and steps (a) and (b), may all be performed after cell, tissue, or organ transplantation. Alternatively, the administration of the enzyme may be performed before cell, tissue, or organ transplantation, and steps (a) and (b) may be performed thereafter. Alternatively, the administration of the enzyme and (if any) step (a) may be performed before cell, tissue, or organ transplantation, and step (b) may be performed thereafter. A typical method may include the administration of the enzyme, followed by the application of cell, tissue, or organ transplantation (such as a kidney transplant), followed by the administration of ATG at appropriate intervals after the enzyme. In the case of HPSC transplantation (e.g., bone marrow transplantation), the order of the procedures may typically be: step (b) antibody-based drug, then step (a) removal of the recipient HPSC, then an enzyme to inactivate the target serum IgG molecule, and then transplantation.

[0035] Methods for inducing hematopoietic chimerism The present invention provides a method for inducing hematopoietic chimerism in a subject, comprising performing a pre-treatment regimen of the present invention, and then administering HSPCs to the subject in an amount and under appropriate conditions sufficient to induce hematopoietic chimerism. The method may alternatively be described as a method for stable transplantation of HSPCs. HSPCs may be autologous (cells from the patient's own body are used), syngeneic (cells are obtained from genetically identical twins), or allogeneic (cells are obtained from another non-identical donor).

[0036] Immune complications that reduce the likelihood of successful engraftment of HSPCs in the recipient are most pronounced in allogeneic cells, and therefore the method of the present invention is most beneficial in such cells. However, even in autologous cells, immune complications can occur if products that the recipient has not been previously exposed to are expressed. If autologous cells are genetically modified to express gene therapy, the cells may be sufficiently altered to trigger an immune response. For example, an immune response may occur to the expressed gene therapy product. The same is true if the HSPCs are genetically modified to express a different HLA type that does not match the recipient's HLA. Therefore, it is preferable that the HSPCs are allogeneic cells, or genetically modified autologous cells or syngeneic cells. Most preferably, the HSPCs are allogeneic cells. In a particularly preferred embodiment, the HSPCs are donor-derived, meaning the donor is also the donor of another organ or tissue to be transplanted into the recipient. That is, the same donor provides both the HSPCs and another cell, organ, or tissue.

[0037] HSPCs are found in adult bone marrow, particularly in the pelvis, femur, and sternum. They are also found in umbilical cord blood and, to a lesser extent, in peripheral blood. HSPCs may be collected from these locations using any appropriate techniques established in the art.

[0038] For example, HSPCs may be harvested from human bone marrow by directly aspirating from the center of the donor's bone with a large needle. The posterior iliac crest is the usual harvesting site. This technique is called bone marrow harvesting and can be performed under local or general anesthesia. When the HSPCs to be administered are derived from the donor's bone marrow, the administration of HSPCs is sometimes referred to as bone marrow transplantation (BMT).

[0039] HSPCs can be collected from umbilical cord blood immediately after the birth of an infant. The umbilical cord is secured with a double clamp from the navel, and the procedure is performed between the clamps. The umbilical vein is aseptically punctured, and the blood flows freely by gravity into an anticoagulant-treated sterile closed collection system, from which the HSPCs can be isolated.

[0040] Highly vascularized progenitor cells (HSPCs) can typically be collected from peripheral blood by apheresis. However, since the number of HSPCs in peripheral blood is usually small, it is first necessary to mobilize them from the bone marrow. In healthy donors, this can be achieved by administering granulocyte colony-stimulating factor (G-CSF). If the donor is not healthy, another method may be required. This may be a common case when the planned HSPC transplant is an autologous transplant.

[0041] HSPCs are preferably used as soon as possible after collection (i.e., fresh), but may be cryopreserved for storage and thawed for use in the method of the present invention. Cryopreservation typically involves volume reduction due to the removal of red blood cells and plasma. The amount of stem cells in the collection may be quantified, for example, by flow cytometry analysis of the sample to determine the percentage of cells positive for CD34 (a marker for stem cells).

[0042] HSPC may be administered to the subject by any appropriate method. The preferred method is infusion, typically through a central line. To reduce the risk of infection, the patient may be kept in a highly clean or sterile environment, such as a room with a high-efficiency particulate air (HEPA) filter under positive pressure, before, during, and after infusion.

[0043] This method may be used to monitor whether HSPC transplantation has successfully resulted in hematopoietic chimerism. This is achieved by determining the percentage of donor-derived hematopoietic cells present in a blood sample taken from the subject after a specific time interval, typically 28 days after HSPC administration. For example, hematopoietic chimerism can be defined as achieved if at least 5% of the lymphocytes and / or myelocytes in the sample are found to be donor-derived, preferably at least 5% of the lymphocytes in the sample are found to be donor-derived. Chimerism is described as mixed if 90% or less of the lymphocytes and / or myelocytes in the sample are found to be donor-derived (i.e., at least 10% are still recipient-derived), preferably if 90% or less of the lymphocytes in the sample are found to be donor-derived (i.e., at least 10% of the lymphocytes are still recipient-derived). Chimerism can be described as complete if 98% or more of the lymphocytes and / or myelocytes in the sample are found to be donor-derived. Mixed chimerism is typically preferred for the method of the present invention because it results in the recipient having a higher level of immune function. However, in the treatment of cancer such as leukemia, the goal is to eliminate host cells that may cause cancer recurrence and replace them with transplanted HSPCs, so in some situations, flu chimerism may be effective.

[0044] The proportion of donor-derived cells and recipient-derived cells in the sample can be determined by any suitable method in the art, such as flow cytometry analysis as described in the examples. Real-time PCR may also be used. Other methods are described in Agrawal et al. Bone Marrow Transplantation 2004 (34) p-12.

[0045] Methods of treating or preventing a disease or condition The present invention provides a method for preventing or treating a disease or condition of interest. The method comprises inducing hematopoietic chimerism in a subject according to the method described above, thereby treating or preventing the disease or condition, in order to enhance the subject's benefit to the therapy for the disease or condition. The therapy may be a transplant of cells, tissues, or organs, typically from the same donor as the HSPC. The transplanted cells, tissues, or organs may be of any kind, such as kidneys, livers, hearts, pancreases, lungs, small intestines, skin, blood vessels / vascular tissue, face, arms, trachea, part of an eye, pancreatic islets, substantia nigra, bone marrow, etc. The transplanted cells may be of any kind, such as the same HSPC used in the method itself, so as not to require additional therapy. The therapy may be a gene therapy performed using genetically modified HPSCs.

[0046] Expressed in other words, the present invention also provides a method for the prevention or treatment of immune rejection of cell, tissue, or organ transplants, the method comprising inducing hematopoietic chimerism in a subject according to the method of the present invention, and applying a cell, tissue, or organ transplant to the subject, optionally, said cells, tissue, or organ from the same donor as the HSPC. The cells, tissue, or organ are typically administered after hematopoietic chimerism has been induced in the subject, but may be administered before. For example, if an organ is taken from a deceased donor, it may be preferable to perform the organ transplant first, and then induce hematopoietic chimerism using an HSPC taken from the same deceased donor or a close donor. The transplant of cells, tissue, or organ may be of any kind, such as kidney, liver, heart, pancreas, lung, small intestine, skin, blood vessels / vascular tissue, face, arm, trachea, part of the eye, pancreatic islets, substantia nigra, bone marrow, etc. The transplanted cells may be of any kind, such as the same HSPC used to induce hematopoietic chimerism, so as not to require additional transplantation.

[0047] The transplanted cells, tissues, or organs may originate from a different species than the recipient, i.e., xenotransplantation. Suitable species for xenotransplantation into human recipients include pigs and non-human primates. In such cases, the HSPC may be genetically modified to aid in transplant tolerance. The xenotransplanted cells, tissues, or organs may also be genetically modified.

[0048] The subject being treated is preferably sensitized or highly sensitized. “Sensitized” means that the subject expresses antibodies against human major histocompatibility (MHC) antigens (also known as human leukocyte antigens (HLA)). Anti-HLA antibodies originate from allogeneically sensitized B cells and are typically present in patients who have been previously sensitized due to blood transfusion, previous transplantation, or pregnancy. Achieving hematopoietic chimerism in sensitized patients may reverse allogeneic sensitization by reducing donor-specific immune responses such as DSA through specific tolerance to T cells and B cells.

[0049] Whether a candidate transplant recipient is sensitized can be determined by any appropriate method. For example, a panel-reactive antibody (PRA) test may be used to determine whether a recipient is sensitized. A PRA score > 30% is typically understood to mean that the patient is at “high immunological risk” or “sensitized.” Alternatively, a cross-matching test may be performed, in which a blood sample from a candidate transplant donor is mixed with a sample from the intended recipient. A positive cross-match means that the recipient has antibodies that react to the donor sample, indicating that the recipient is sensitized and should not be transplanted. Cross-matching tests are typically performed as a final check immediately before transplantation.

[0050] Methods for preventing or treating immune rejection of cell, tissue, or organ transplants include: (i) Perform the pretreatment regimen of the present invention; (ii) Administer HSPC to the subject in an amount and under appropriate conditions sufficient to induce hematopoietic chimerism in the subject.

[0051] The method may optionally include (iii) applying a cell, tissue, or organ transplant to the subject, which typically originates from the same donor as the HSPC. The HSPC administered in step (ii) may itself be the transplant, in which case the additional step (iii) is not required. The method can be considered a method for treating a disease or condition treated by the transplantation of cells, tissue, or organs. For example, if the HSPC is itself the transplant, the method may be a method for the prevention or treatment of any disease or condition treated by the transplantation of an HSPC.

[0052] Diseases or conditions typically treated by HSPC transplantation can be acquired or congenital. Acquired diseases or conditions that can be treated by HSPC transplantation include: - Hematological malignancies, e.g., leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)); lymphoma (e.g., Hodgkin's disease, non-Hodgkin lymphoma) and myeloma (e.g., multiple myeloma (Kahler's disease)), -Solid tumors, such as neuroblastoma, fibroplastic round cell tumor, Ewing's sarcoma, choriocarcinoma, - Hematological disorders, e.g., phagocyte disorders (e.g., myelodysplasia); anemia (e.g., paroxysmal nocturnal hemoglobinuria (PNH; severe dysplasia), aplastic anemia, acquired pure red blood cell dysplasia); myeloproliferative disorders (e.g., polycythemia vera, essential thrombocythopathy, myelofibrosis), - Metabolic disorders, e.g., amyloidosis (e.g., amyloid light chain (AL) amyloidosis), - Environmentally induced diseases, for example, radiation damage, - Viral diseases, such as human T-lymphotropic virus (HTLV), human immunodeficiency virus (HIV), - Autoimmune diseases, such as multiple sclerosis.

[0053] The following are examples of congenital disorders or conditions that can be treated with HSPC transplantation: - Lysosome storage disorders, e.g., lipidosis; lipid storage disorders (e.g., neuronal ceroid lipofuscinosis, infant neuronal ceroid lipofuscinosis (INCL, Santavuori disease), Jansky-Bierszowski disease (late stage of infant neuronal ceroid lipofuscinosis)); sphingoglycolipidosis (e.g., Niemann-Pick disease, Gaucher disease); leukodystrophy (e.g., adrenaline leukodystrophy, metachromatic leukodystrophy, Krabbe disease (globoid cell leukodystrophy)); mucopolysaccharidosis (e.g., Hurler syndrome (MPS IH, α-L-idronidase deficiency), Schayle syndrome (MPS IS), Hurler-Schey syndrome (MPS I HS), Hunter syndrome (MPS II, idronidase sulfate deficiency), Sanfilippo syndrome (MPS III), Mochio syndrome (MPS IV), Marotex-Lamy syndrome (MPS VI), Sly syndrome (MPS VII); Glycoproteinosis (e.g., mucolipidosis II (I-cell disease), fucosidosis, aspartylglucosamineuria, alpha-mannosidosis); or others (e.g., Wolmann disease (acid lipase deficiency), - Immunodeficiency disorders, e.g., T-cell deficiencies (e.g., ataxia-telangiectasia, DiGeorge syndrome); combined T-cell and B-cell deficiencies (e.g., severe combined immunodeficiency (SCID), all types); Wiscott-Aldrich syndrome; phagocytic disorders (e.g., Kostman syndrome, Schwakman-Diamond syndrome); immune dysregulation disorders (e.g., Glycerin syndrome, type II); innate immunodeficiency (e.g., NF-kappa-B essential modulator (NEMO) deficiency) - Blood disorders, such as hemoglobin disorders (e.g., sickle cell anemia, severe β-thalassemia (Cooley's anemia)); anemia (e.g., aplastic anemia, Diamond-Blackfan anemia, Fanconi anemia); cytopenia (e.g., anegemakaryocytic thrombocytopenia); and hemophagocytic syndromes (e.g., hemophagocytic lymphohistiocytosis (HLH)).

[0054] When HSPCs are genetically modified for the application of gene therapy, the method of the present invention may be for the prevention or treatment of the disease or condition targeted by the gene therapy.

[0055] The present invention also provides an enzyme for use in a method for preventing or treating a disease or condition, wherein the method is as described above.

[0056] The present invention also provides the use of an enzyme in the manufacture of a pharmaceutical product that inactivates serum IgG molecules in a target, wherein the pharmaceutical product is for the prevention or treatment of a disease or condition in the manner described above.

[0057] enzyme IgG cysteine ​​protease The IgG cysteine ​​protease used in the present invention is IgG-specific. In a preferred embodiment, the protease used in the method of the present invention is IdeS( I mmunoglobulin G-degrading e nzyme of SIdeS is an immunoglobulin G-degrading enzyme of S. pyogenes, also known as immunofidase. IdeS is an extracellular cysteine ​​protease produced by the human pathogen S. pyogenes. IdeS was originally isolated from serotype M1 of group A Streptococcus strain, but the ideS gene has now been identified in all group A Streptococcus strains tested. IdeS has very high substrate specificity, and its sole substrate has been identified as IgG. IdeS catalyzes single proteolytic cleavage in the lower hinge region of the heavy chains of all subclasses of human IgG. IdeS also catalyzes equivalent cleavage of the heavy chains of several subclasses of IgG from various animals. IdeS efficiently cleaves IgG into Fc and F(ab')2 fragments in a two-step mechanism. In the first step, one (primary) heavy chain of IgG is cleaved, producing a single cleaved IgG (scIgG) molecule with a non-covalently bonded Fc molecule. The scIgG molecule is essentially an intermediate product, retaining the remaining (secondary) heavy chain of the original IgG molecule. In the second step of the mechanism, this primary double chain is cleaved by IdeS, releasing an F(ab')2 fragment and a homodimer Fc fragment. These are products commonly observed under physiological conditions. Under reducing conditions, the F(ab')2 fragment may dissociate into two Fab fragments, and the homodimer Fc may dissociate into its constituent monomers. IdeS has been shown to be particularly effective in cleaving IgG in humans. Within minutes of IdeS administration, the entire IgG pool in the plasma is cleaved, and blood IgG concentrations remain low for over a week until newly synthesized IgG appears in the plasma. This indicates that not only the plasma pool (i.e., serum IgG molecules) but the entire extracellular IgG pool is cleaved by IdeS (Winstedt et al; PloS One 2015; 10(7): e0132011).

[0058] Sequence ID 1 is the complete sequence of IdeS, including the N-terminal methionine and signal sequence. It can also be used as NCBI reference sequence ID WP_010922160.1. Sequence ID 2 is the mature sequence of IdeS, lacking the N-terminal methionine and signal sequence. It can also be used as Genbank acceptance number ADF13949.1.

[0059] In an alternative embodiment, the protease for use in the method of the present invention is IdeZ, an IgG cysteine ​​protease produced by Streptococcus equi ssp. Zooepidemicus, a bacterium mainly found in horses. Sequence ID 3 is the complete sequence of IdeZ, including the N-terminal methionine and signal sequence. This can also be used as NCB reference sequence number WP_014622780.1. Sequence ID 4 is the mature sequence of IdeZ, lacking the N-terminal methionine and signal sequence.

[0060] In an alternative embodiment, the protease for use in the method of the present invention is a hybrid IdeS / Z, such as the sequence of SEQ ID NO: 5. The N-terminus is based on IdeZ lacking an N-terminal methionine and a signal sequence.

[0061] In a preferred embodiment, the protease for use in the present invention may include or be derived from SEQ ID NOs: 2, 4, or 5. The protease for use in the present invention may include an additional methionine (M) residue at the N-terminus and / or a tag at the C-terminus to facilitate expression and isolation therefrom in a standard bacterial expression system. Suitable tags include histidine tags, which may be directly bound to the C-terminus of the polypeptide or indirectly bound by any suitable linker sequence, such as 3, 4, or 5 glycine residues. The histidine tag typically consists of 6 histidine residues, but can be longer, typically up to 7, 8, 9, 10, or 20 amino acids, or shorter, for example, 5, 4, 3, 2, or 1 amino acid.

[0062] In a further preferred embodiment, the protease for use in the present invention may substantially consist of, or be composed of, any one of the sequences of SEQ ID NOs: 6 to 25. These sequences represent IdeS and IdeZ polypeptides with increased protease activity and / or decreased immunogenicity. Each of SEQ ID NOs: 6 to 25 may optionally contain an additional methionine at the N-terminus and / or a histidine tag at the C-terminus. The histidine tag preferably consists of six histidine residues. The histidine tag is preferably linked to the C-terminus by a linker of 3x glycine residues or 5x glycine residues.

[0063] In a further preferred embodiment, the protease for use in the present invention may substantially consist of, or be composed of, any one of the sequences of SEQ ID NOs. 56 to 69. These sequences represent an IdeS polypeptide with increased protease activity and / or decreased immunogenicity. Each of SEQ ID NOs. 56 to 69 may optionally contain an additional methionine at the N-terminus and / or a histidine tag at the C-terminus. The histidine tag preferably consists of six histidine residues. The histidine tag is preferably linked to the C-terminus by a linker of 3x glycine or 5x glycine residues.

[0064] In a further preferred embodiment, the protease for use in the present invention substantially comprises, or may comprise, one of the sequences of SEQ ID NOs: 6 to 25, which may have up to three (e.g., one, two, or three) amino acid substitutions. Each of SEQ ID NOs: 6 to 25 and its variants may optionally contain an additional methionine tag at the N-terminus and / or a histidine tag at the C-terminus.

[0065] In a further preferred embodiment, the protease for use in the present invention substantially consists of, or may consist of, one of the sequences of SEQ ID NOs. 56 to 69, which may have up to three (e.g., one, two, or three) amino acid substitutions. Each of SEQ ID NOs. 56 to 69 and its variants may optionally contain an additional methionine tag at the N-terminus and / or a histidine tag at the C-terminus.

[0066] The polypeptides of the present invention are typically of a length of at least 100, 150, 200, 250, 260, 270, 280, 290, 300, or 310 amino acids. The polypeptides of the present invention are typically of a length of 400, 350, 340, 330, 320, or 315 amino acids or less. It will be understood that any of the lower limits listed above can be combined with any of the upper limits listed above to provide a range for the length of the polypeptides of the present invention. For example, the polypeptide may be of a length of 100 to 400 amino acids, or of a length of 250 to 350 amino acids. The polypeptide is preferably of a length of 290 to 320 amino acids, most preferably of 300 to 315 amino acids.

[0067] The primary structure (amino acid sequence) of the protease of the present invention is based on the primary structure of IdeS, IdeZ, or IdeS / Z, specifically on the amino acid sequence of SEQ ID NO: 2, 4, or 5, respectively. The sequence of the protease of the present invention may include variants of the amino acid sequence of SEQ ID NO: 2, 4, or 5 that are at least 80% identical to the amino acid sequence of SEQ ID NO: 2, 4, or 5. The variant sequence may be at least 80%, at least 85%, preferably at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 2, 4, or 5. The variant may be identical to the sequence of SEQ ID NO: 2, 4, or 5, except that it includes one or more specific modifications identified in WO2016 / 128558 or WO2016 / 128559. The identity of sequence SEQ ID NO: 2, 4, or 5 can be measured over a region of at least 50, at least 100, at least 200, at least 300 or more consecutive amino acids in the sequence shown in SEQ ID NO: 2, 4, or 5, or more preferably over the entire length of SEQ ID NO: 4 or 5.

[0068] The proteases for use in the present invention may be IdeS, IdeZ, or IdeS / Z polypeptides containing variants of the amino acid sequence of SEQ ID NOs. 2, 4, or 5, which are modified by adding, deleting, or substituting amino acids into the sequence of SEQ ID NOs. 2, 4, or 5. Such modifications are preferably conservative amino acid substitutions. Conservative substitutions involve substituting an amino acid with another amino acid having a similar chemical structure, similar chemical properties, or similar side chain volume. The introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acid they substitute for. Alternatively, a conservative substitution may involve introducing another aromatic or aliphatic amino acid in place of an existing aromatic or aliphatic amino acid. Conservative amino acid modifications are well known in the art.

[0069] IgG cysteine ​​protease activity may be evaluated by any suitable method, for example, by incubating a polypeptide with an IgG-containing sample and determining the presence of an IgG cleavage product. Suitable methods are described in publication WO2016 / 128559. Suitable assays include ELISA-based assays, such as those described in WO2016 / 128559. In such assays, the wells of the assay plate are typically coated with an antibody target, such as bovine serum albumin (BSA). Next, a sample of the polypeptide to be tested is added to the well, followed by a sample of a target-specific antibody, which in this example is an antibody specific to BSA. The polypeptide and antibody are allowed to interact under conditions suitable for IgG cysteine ​​protease activity. After a suitable time, the assay plate is washed, and a detection antibody that specifically binds to the target-specific antibody is added under conditions suitable for binding to the target-specific antibody. The detection antibody binds to any intact target-specific antibody bound to the target in each well. After washing, the amount of detection antibody present in a well is proportional to the amount of target-specific antibody bound to that well. The detection antibody can be directly or indirectly labeled or conjugated to other reporter systems (such as enzymes), allowing the amount of detection antibody remaining in each well to be measured. The higher the potency of the test polypeptide in the well, the less intact target-specific antibody remains, and therefore the less detection antibody there is. Typically, at least one well on a given assay plate contains IdeS instead of the polypeptide being tested, allowing for a direct comparison of the potency of the test polypeptide to that of IdeS. IdeZ or IdeS / Z may also be included for comparison.

[0070] Other assays can determine the potency of a test polypeptide by directly visualizing and / or quantifying IgG fragments resulting from cleavage of IgG by the test polypeptide. This type of assay is also described in WO2016 / 128559. Such assays typically involve incubating an IgG sample with different concentrations of the test polypeptide (or one or more of IdeS, IdeZ, and IdeS / Z as controls) in a titration system. The products resulting from incubation at each concentration are then separated using gel electrophoresis, e.g., SDS-PAGE. The total IgG and fragments resulting from IgG cleavage are then identified by size and quantified by the intensity of staining with an appropriate dye. A larger amount of cleavage fragments indicates greater potency of the test polypeptide at a given concentration. The polypeptide of the present invention will typically produce detectable amounts of cleavage fragments at lower concentrations (lower points in the titration system) than IdeZ and / or IdeS. This type of assay can also determine the amount of different fragments resulting from each cleavage event, thus enabling the identification of test polypeptides that are more effective at cleaving the primary or secondary heavy chain of the IgG molecule. The polypeptides of the present invention may be particularly effective at cleaving the primary chain of the IgG molecule more than the secondary chain when IgG is of the IgG2 isotype. The polypeptides of the present invention may be more effective at cleaving IgG1 than IgG2.

[0071] IgG endoglycosidase The enzyme may preferably have IgG endoglycosidase activity that cleaves the sugar chain portion at Asn-297 (Kabat numbering) in the Fc region of IgG. An example of such a protein is EndoS( Endo glycosidase of SAn example is *S. pyogenes* (endoglycosidase). EndoS hydrolyzes the β-1,4-di-N-acetylchitobiose core of the asparagine-linked glycans of normally glycosylated IgG. The mature sequence of EndoS is provided as SEQ ID NO: 90. The drug may be a protein containing or consisting of the amino acid sequence of SEQ ID NO: 90, or its homologue from alternative bacteria such as *Streptococcus equi* or *Streptococcus zooepidemicus*, or *Corynebacterium pseudotuberculosis*, *Enterococcus faecalis*, or *Elizabethkingia meningoseptica*. The drug may be CP40, EndoE, or EndoF2.

[0072] Alternatively, the drug may be a variant of the EndoS protein having at least 80%, 85%, 90%, or 95% identity with SEQ ID NO: 90 and containing or comprising any amino acid sequence having IgG endoglycosidase activity. If the variant has IgG endoglycosidase activity, the EndoS protein variant may contain or comprise an amino acid sequence in which up to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more amino acid substitutions, insertions, or deletions have been made to the amino acid sequence of SEQ ID NO: 90. The amino acid substitutions are preferably conserved. Conservative substitutions are as defined in the preceding paragraph.

[0073] Alternatively, the drug may be a protein containing or comprising the fragment of SEQ ID NO: 90 and having IgG enodoglycosidase activity, preferably the fragment being 400 to 950, 500 to 950, 600 to 950, 700 to 950, or 800 to 950 amino acids in length. A preferred fragment consists of amino acids 1 to 409 of SEQ ID NO: 90 and corresponds to the enzymatically active α-domain of EndoS produced by cleavage by streptococcal cysteine ​​proteinase SpeB. The fragment may be prepared by deleting one or more amino acid residues in the amino acid sequence of SEQ ID NO: 90. Up to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 550 residues may be deleted, or more. The deleted residues may be contiguous with other residues.

[0074] Any fragment or variant of SEQ ID NO: 90 preferably includes residues 191 to 199 of SEQ ID NO: 90, namely Leu-191, Asp-192, Gly-193, Leu-194, Asp-195, Val-196, Asp-197, Val-198, and Glu-199. These amino acids constitute the active site of the complete chitinase family 18, which ends in glutamic acid. Glutamic acid in the chitinase active site is essential for enzyme activity. Most preferably, and therefore, variants of SEQ ID NO: 90 include Glu-199 of SEQ ID NO: 90. However, if the variant includes Glu-199 of SEQ ID NO: 90, the variant of SEQ ID NO: 90 may also include residues 191 to 199 of SEQ ID NO: 90 having one or more conservative substitutions.

[0075] Polypeptide production The enzyme used in the method of the present invention is a polypeptide, which may be produced by any suitable means. For example, the polypeptide may be synthesized directly using standard techniques known in the art, such as Fmoc solid-phase chemistry, Boc solid-phase chemistry, or solution-phase peptide synthesis. Alternatively, the polypeptide may be produced by transforming cells, typically bacterial cells, with a nucleic acid molecule or vector encoding the polypeptide. The production of enzyme polypeptides by expression in bacterial host cells is described and illustrated in WO2016 / 128558 and WO2016 / 128559.

[0076] Polypeptide-containing compositions and formulations The present invention also provides compositions comprising enzymes for use in the methods of the present invention. For example, the present invention provides compositions comprising one or more polypeptides and at least one pharmaceutically acceptable carrier or diluent. The carrier must be “acceptable” in the sense that it is compatible with the other components of the composition and is not harmful to the subject to which the composition is administered. Typically, the carrier and the final composition are sterile and pyrogen-free.

[0077] The formulation of suitable compositions can be carried out using standard pharmaceutical chemistry and methodologies, all of which are readily available to those skilled in the art. For example, an enzyme can be combined with one or more pharmaceutically acceptable excipients or vehicles. Auxiliary substances such as wetting agents or emulsifiers, pH buffers, and reducing agents may be present in the excipients or vehicles. Suitable reducing agents include cysteine, thioglycerol, thioredoxin, and glutathione. Excipients, vehicles, and auxiliary substances are generally pharmaceuticals that do not induce an immune response in the individual receiving the composition, and they can be administered without excessive toxicity. pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, thioglycerol, and ethanol. Pharmacologically acceptable salts, such as mineral salts like hydrochloride, hydrobromide, phosphate, and sulfate; and salts of organic acids such as acetate, propionate, malonate, and benzoate may also be included. A complete discussion of pharmaceutically acceptable excipients, vehicles, and auxiliary substances is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0078] Such compositions may be prepared, packaged, or sold in forms suitable for bolus or continuous administration. Injectable compositions may be prepared, packaged, or sold in unit dose forms, such as ampoules or multi-dose containers containing preservatives. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and embeddable sustained-release or biodegradable formulations. Such compositions may further contain, but are not limited to, one or more additional components, such as suspending agents, stabilizers, or dispersants. In one embodiment of a parenteral administration composition, the active ingredient is provided in a dry form (e.g., powder or granules) for reconstitution in a suitable vehicle (e.g., sterile pyrogen-free water), and the reconstituted composition is subsequently administered parenterally. Compositions may be prepared, packaged, or sold in the form of sterile, injectable aqueous or oily suspensions or solutions. These suspensions or solutions can be formulated according to known art and may contain, in addition to the active ingredient, additional components such as dispersants, wetting agents, or suspending agents as described herein. Such sterile injectable formulations may be prepared, for example, using water or a non-toxic, parenterally acceptable diluent or solvent such as 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixatives such as synthetic mono or diglycerides.

[0079] Other useful and parentally-administrable compositions include those containing the active ingredient in microcrystalline form, liposomal formulations, or as components of biodegradable polymer systems. Compositions for sustained release or implantation may contain pharmaceutically acceptable polymer materials or hydrophobic materials such as emulsions, ion exchange resins, poorly soluble polymers, or poorly soluble salts. Compositions may be suitable for administration by any suitable route, such as intradermal, subcutaneous, transdermal, intramuscular, intra-arterial, intraperitoneal, intra-articular, intraperiosteal, or other suitable routes of administration. Preferred compositions are suitable for administration by intravenous infusion.

[0080] kit The present invention also provides a kit for carrying out the methods described herein. The kit of the present invention may include an enzyme or an enzyme-containing composition, as described above. The kit may include means for administering the enzyme or composition to a target. The kit may include instructions for using various components in any method as described herein. [Examples]

[0081] Examples Unless otherwise noted, the methods used are standard biochemical and molecular biology techniques. Examples of appropriate methodological textbooks include Sambrook et al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley and Sons, Inc.

[0082] Example 1 Introduction Immurefidase cleaves all human IgG subclasses, but only mouse IgG2c and IgG3, while not cleaving mouse IgG1 and IgG2b. Interestingly, EndoS has been shown to reduce the complement and FcγR-mediated function of mouse IgG1 and IgG2b. However, EndoS-treated mouse IgG2a and IgG2c maintain cytolytic activity via FcγR, although IgG2c has also been shown to lose some binding affinity under certain conditions. Therefore, for the purposes of the animal model used in the following experiments, we are using a combination of immunorefidase and EndoS to maximize the effect on serum IgG in mouse subjects. It is expected that immunorefidase or EndoS (or other proteases or endoglycosidases with equivalent specificity / activity) alone would be sufficient to achieve equivalent effects in human subjects.

[0083] The following experiments use a stringent model of sensitized NOD recipients that are resistant to irradiation and tolerance induction. In this experiment, an approach that combines both Imurifidase and EndoS has demonstrated that it can generate mixed hematopoietic chimerism in these mice.

[0084] Materials and Methods Animals Adult NOD / ShiLtJ (H-2 g7 ; referred to as NOD), FVB / NJ (H-2 q ; referred to as FVB), C57BL / 6J (H-2 b ; referred to as B6.CD45.2), B6.SJL-Ptprc a Pepcb. / Boy (H-2 b , referred to as B6.CD45.1), B6.NOD-(D17Mit21-D17Mit10) (H-2 g7 ; referred to as B6.H-2 g7 ), NOD.B10Sn-H2 b / J (H-2 b ; referred to as NOD.H-2 b mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA), and were bred and housed in a specific pathogen-free facility at the University of Alberta. All animal care and handling were performed in accordance with the guidelines of the Canadian Council on Animal Care. All NOD mice used for chimerism induction were 8 to 10-week-old females.

[0085] Reagents for In Vivo Experiments Imurifidase and EndoS were provided by Hansa Biopharma AB (Lund, Sweden) and used under license. Anti-CD4 (clone Gk1.5, rat IgG 2b ), anti-CD90 (clone YTS154, rat IgG 2b ), anti-CD8α (clone YTS169.4, rat IgG 2b ) and anti-MHC-I H-2K b (clone B8.24.3, mouse IgG 2bThe mAbs used were those generated in-house. YTS 169.4 anti-mouse CD8α mAb-producing cells were developed by Prof. H Waldmann and Dr. SP Cobbold (Department of Pathology, University of Cambridge) and obtained via Cambridge Enterprise Limited (Hauser Forum, 3 Charles Babbage Road, Cambridge CB3 0GT). Cyclophosphamide (29875) and bortezomib (A2614) were purchased from Sigma (MO, USA) and ApexBio (TX, USA), respectively.

[0086] In vivo EndoS-mediated monoclonal DSA inhibition assay NOD or B6.H-2 g7 Mice were given a vehicle and anti-MHC-I H-2K as pretreatment. b (10 μg) alone, or EndoS (30 μg) with anti-MHC-I H-2K b A mixture of (10 μg or 100 μg) was administered intravenously. EndoS and anti-H-2Kb were mixed immediately before injection. Four hours after this pretreatment, 5 million cells of a 1:1 mixture of carboxyfluorescein succinimimidyl ester (CFSE)-labeled NOD and cell-trace violet (CTV)-labeled B6 bone marrow cells (BMC) were intravenously injected into pre-treated NOD mice. Similarly, CFSE-labeled B6.H-2 g7 and CTV marker NOD.H-2 b A 1:1 mixture of BMC was used to pre-treat B6.H-2 g7 The cells were injected into mice. Blood samples were collected 1, 2, and 3 hours after cell administration and analyzed by flow cytometry. Spleen cells and BMC were collected from one hind limb of each mouse and analyzed 4 hours after BMC injection.

[0087] Serum DSA detection assay NOD mice with 20 x 10 FVB spleen cells 6Individual cells were administered via ip to induce sensitization. Serum was collected before sensitization, 4 to 6 weeks after sensitization, and 4 hours after immunofidase and EndoS treatment. FVB spleen cells (2 × 10⁶) 5 (individual) FcR blockade (anti-mouse CD16 / CD32 rat IgG2 b Cells were treated with antibody (clone 2.4G2, BE0307, ​​Bio X cell) for 5 minutes, then incubated with a titration of serum in 100 μL for 30 minutes. Cells were washed twice and incubated with fluorescent dye-conjugated secondary antibody in 100 μL for 30 minutes. The following secondary antibodies were used: FITC-conjugated F(ab')2 fragment from rabbit anti-mouse IgG Fc antibody (1:200, 315-096-046, Jackson ImmunoResearch), APC-conjugated goat anti-mouse IgG1Fc antibody (1:100, 115-135-205, Jackson ImmunoResearch), and FITC-conjugated goat anti-mouse IgG3Fc antibody (1:100, 115-095-209, Jackson ImmunoResearch). Cells were washed twice and analyzed by flow cytometry. HBSS containing 2% FBS was used for cell washing and reconstitution.

[0088] BMT Protocol and Definition of Chimerism To determine the short-term survival of donor BMCs in sensitized recipients, NOD mice sensitized to B6.CD45.1 spleen cells were subjected to T cell depletion (anti-CD4 0.25 mg, anti-CD8 0.25 mg, anti-CD90 0.3 mg, ip) two days before BMT, followed by BMT (80 × 10⁻¹⁰). 6 EndoS and immunofidase were administered intravenously four hours prior to the intravenous injection of B6.CD45.2 BMC. Splenocytes and BMC were analyzed four hours after BMC injection.

[0089] For long-term chimerism induction, NOD mice sensitized to FVB spleen cells were treated IV with immunofidase and EndoS on day -6 relative to the BMT day. Cyclophosphamide (150 mg / kg, ip or iv) and bortezomib (1 mg / kg, iv) were administered on day -4. T-cell depletion antibody was administered ip on days -2, 2, 6, 11, and 16. Immurefidase and EndoS were repeatedly administered 4 hours before BMT on day 0, followed by 6 Gy of total body irradiation (TBI, Gammacell 1000 Elite). FVB bone marrow cells (80 × 10) were administered on day 0. 6 The drug was administered intravenously (iv) via the caudal vein. In experiments measuring the effects of cyclophosphamide and bortezomib on sensitized recipients before BMT, a low dose (20 × 10) was used to limit the adsorption potential of DSA to donor bone marrow cells. 6 Bone marrow cells were provided. Peripheral blood was collected at the specified time for flow cytometry analysis. For long-term chimerism, MHC-I cells were collected in the lymphocyte gate 28 days after BMT. + A recipient was considered a chimera if at least 5% of the cells were derived from the donor.

[0090] Antibodies and flow cytometry Mouse H-2K d (SF1-1.1.1), H-2K q (KH114), H-2K bFluorescently labeled antibodies against CD4 (AF6-88.5), CD45.2 (104), CD19 (6D5), CD138 (281-2), B220 (RA3-6B2), TCRβ (H57-597), CD4 (RM4-5 or RM4-4), CD8β (H35-17.2), CD11b (M1 / 70), CD11c (N418), CD49b (DX5), and CD122 (TM-β1) were purchased from BD Pharmingen (CA, USA), BioLegend (CA, USA), or Thermo Fisher Scientific (CA, USA). Data acquisition was performed using an LSR II (Becton Dickson, CA, USA) flow cytometer, and data analysis was performed using FlowJo (Treestar software, OR, USA).

[0091] statistical analysis The Mann-Whitney U test, ratio-paired t-test, one-way ANOVA with Holm-Sidak multiple comparison test, and Fisher's exact test were used as indicated. All statistical analyses were performed using Prism (GraphPad Software, CA, USA).

[0092] result EndoS inhibits monoclonal DSA-mediated death of donor BMCs. To evaluate the effect of EndoS on inhibiting antibody-mediated donor BMC death, passive DSA transplantation experiments were performed. Of all DSAs, anti-donor MHC antibodies or HLA antibodies are of the most clinical importance. Therefore, MHC-I K2 antibodies were used to transplant naive NOD mice. b Mouse IgG targeting expression cells 2b Bone marrow transplants were then performed from B6 mice, either by injecting antibodies and treating with EndoS or leaving them untreated.

[0093] As shown in Figure 1A-B, 10 μg of antipotassium bIn NOD recipients who received a single dose of mAb, the ratio of B6 to seronal NOD cells 1 hour post-BMT was significantly increased in EndoS-treated mice compared to untreated mice. This difference in the ratio of B6 to seronal NOD cells between the two groups remained stable at 2 and 3 hours post-BMT. Similarly, 100 μg of anti-potassium b Mice given mAb and EndoS were given 100 μg of anti-potassium. b Compared to treatment with mAb alone, it increased the ratio of B6 to NOD in the blood at 1 and 2 hours. However, this increase in the ratio did not last until 3 hours, suggesting that residual mAb effector function accumulates over time. Four hours after BMT, EndoS and 10 μg antipotassium were observed. b In mice treated with mAbs, 10 μg antipotassium was used. b Compared to mice that received mAb alone, a significant increase in the ratio of B6 to NOD cells was also observed in both the BM and spleen.

[0094] It is noteworthy that NOD mice lack hemolytic complement C5, which is essential for complement-dependent cell injury and is not genetically associated with MHC genes. Therefore, the effect of DSA may be reduced in NOD mice compared to complement-sufficient hosts. Thus, the role of EndoS in DSA in complement-sufficient hosts was also investigated. NOD MHC congenic B6.H-2 g7 Mice were used as recipients. EndoS was superior to the NOD host in B6.H-2 g7 The ratio of donor cells to recipient cells was increased to a similar extent in mice (Figure 1C-D).

[0095] In other words, EndoS improved donor cell survival in the presence of anti-MHC antibodies, regardless of whether the recipient was complement deficient, and suggested an effect against other removal mechanisms (e.g., FcgR-mediated) at least in this model system.

[0096] EndoS improves donor BMC survival in pre-sensitized recipients. Next, we investigated whether EndoS could improve donor BMC survival in allo-sensitized recipients, where the antibody repertoire against donor antigens is diversified. To verify this, EndoS was used in combination with an immunofidase. The immunofidase was mouse IgG 2c And IgG3 is cleaved, but mouse IgG1 and IgG 2bIt is not possible to cleave it. Therefore, EndoS was co-administered to reduce the effector function of mouse IgG isotypes that are not cleaved by immunofidase. As shown in Figure 2A, in NOD mice sensitized with FVB spleen cells, the combined use of immunofidase and EndoS significantly reduced DSA-IgG. The reduction in IgG targeting in donor cells is thought to be due to immunofidase, not EndoS, because the Fc-specific detection antibody can still bind after deglycosylation. The difference in sensitivity to mouse IgG isotypes is also shown by the fact that there was no change in the level of DSA-IgG1 (Figure 2B), while DSA-IgG3 (Figure 2C), a subclass cleaved by immunofidase, decreased by approximately 80%. While immunofidase-mediated degradation of IgG3 results in only a moderate reduction in intact IgG, EndoS may further contribute to the reduction of DSA-IgG effector function through deglycosylation of immunofidase-resistant IgG molecules. The combination of both enzymes allowed us to analyze donor cell survival in recipients sensitized with polyclonal DSA. In addition to DSA, prime donor antigen-specific cytotoxic T cells may contribute to the rapid death of donor BMCs. Therefore, CD45.1 NOD recipients sensitized with congenital B6.CD45.1 spleen cells underwent T cell depletion two days prior to immunofidase and EndoS treatment to avoid acute cytotoxicity mediated by sensitized T cells (Figure 2D). Two days after administration of the T cell-depleting mAb, over 95% of peripheral blood T cells were depleted in the recipients (data not shown). Here, the CD45.1 / 2 lineage was used to aid in the identification of viable donor BMCs, although its MHC staining may be interfered with by DSA. As shown in Figures 2E-F, B6.CD45.2 donor cells were almost completely eliminated 4 hours after BMT in sensitized NOD mice when given vehicle control (BM 0.22% and spleen 0.27%) or immunofidase only (BM 0.15% and spleen 0.46%).On the other hand, nearly 0.5% of BMCs and approximately 1.5% of spleen cells in sensitized NOD mice treated with EndoS and immunofidase were derived from B6.CD45.2 donors. Therefore, administration of immunofidase and EndoS 4 hours prior to BMT rescued a significant proportion of donor BMCs in allogeneic sensitized recipients compared to sensitized recipients treated with vehicle or immunofidase alone (Figure 2F). Interestingly, the majority of residual donor cells in recipients treated with immunofidase and EndoS had low MHC-I K levels. b The staining suggests that the donor's MHC epitopes were blocked by either deglycosylated DSA or DSA F(ab')2 (Figure 2E). Alternatively, the surviving donor cells may have been cells with low MHC class I expression.

[0097] In summary, these data indicate that the combination of immunofidase and EndoS improves the survival of donor BMS in allosensitized recipients. In other words, inactivating virtually all serum IgG improved the survival of donor BMS in allosensitized recipients.

[0098] Treatment with bortezomib and cyclophosphamide prior to BMT reduced B cells during BMT. Methods that reduce DSA-producing cells in addition to immunofidases and / or EndoS for BMT may provide a long window of low-DSA environment for continued survival and further development of donor cells after BMT. As an attempt to reduce existing plasma cells and B cells capable of differentiating into plasma cells after BMT, bortezomib was used to eliminate antibody-producing cells and cyclophosphamide to reduce B cells in sensitized mice before BMT (Figure 3A). The combination of bortezomib and cyclophosphamide (CyBor) is used in non-transplantable multiple myeloma patients and for graft-versus-host disease (GVHD) prevention after allogeneic BMT, but is rarely used for the purpose of DSA desensitization.

[0099] Five days after BMT, there was no difference in the cellularity of BMCs in BM mice between the groups. Interestingly, the total number of splenic cells increased in the CyBor-pretreated mouse group. However, compared to the vehicle group, the CD19 count of BM mice was different. + B cells, CD19 - CD138 + B220 + Plasmablasts and CD19 - CD138 + B220 - Plasma cells were significantly reduced in mice treated with CyBor (Figure 3B). In contrast to the reduction in B cells in the BM, CD19 in the spleen was reduced. + The reduction in B cells was not significant in the CyBor-treated group at the time of examination. Furthermore, in the spleen of CyBor-treated mice, CD19 - CD138 + B220 + Plasma blasts and CD19 - CD138 + B220 - A significant increase in plasma cells was observed (Figure 3C).

[0100] Next, we investigated whether CyBor treatment could inhibit the increase in DSA formation stimulated by BMC injection. As shown in Figure 3D, DSA levels increased significantly in 2 out of 5 mice in the control group and in 2 out of 5 mice in the CyBor-treated group, suggesting that CyBor cannot reduce DSA levels. However, when comparing the percentile changes in DSA levels 5 days after BMT (9 days after CyBor treatment), the increase in DSA tended to be smaller in CyBor-treated mice, suggesting that CyBor treatment before BMT may inhibit the increase in DSA stimulated by BMC injection (Figure 3E).

[0101] In summary, these data indicate that CyBor has a significant effect in reducing BM cell counts in BM, and that CyBor may suppress the increase in DSA caused by BMC injection.

[0102] Engraftment in pre-sensitized recipients can be achieved through a combination of immunofidase, EndoS, T cell removal, and CyBor. Based on the above data, it was hypothesized that combining non-lethal irradiation with a large dose of BMC, along with immunofidase and EndoS, and bone marrow plasma cell removal using a T cell removal antibody and CyBor, would enable engraftment of donor cells into pre-sensitized recipients. This protocol is applicable not only to NOD mice but also to B6.H-2 mice, which have a matching MHC profile with NOD mice but are not resistant to chimerism induction. g7 We also investigated whether chimerism could be induced in mice. Recipient mice were sensitized with FVB cells 4 weeks prior to chimerism induction. Naive and prime recipients were given the same pretreatment protocol as shown in the Methods section and Figure 4A.

[0103] As expected, all naive mice were almost completely chimeric with FVB cells at 4 weeks post-BMT, but prime mice not treated with immunofidase and EndoS rejected donor cells. As shown in Figure 4B, no donor cells were detected in sensitized NOD mice that were not treated with the enzymes, even at 2 days post-BMT. On the other hand, in 5 of the 7 sensitized NOD recipients treated with the enzymes, donor cells exceeded 5% at 4 or 9 days post-BMT. Furthermore, in the 4 sensitized NOD mice treated with the enzymes, the chimerism level steadily increased to over 50 percent at 16 days post-BMT. In the end, 8 NOD and B6.H-2 mice were treated. g7 Five of the pre-sensitized mice became chimeras with donor cells four weeks after BMT, and two prime NOD mice became stable mixed chimeras with multiple strains of donor cells in their periphery (Table 1 and Figure 4C). No signs of GVHD were observed in any of the chimeras. Attempts to simplify this protocol by removing either cyclophosphamide or bortezomib suggest that both are essential for the success of the current protocol in inducing chimerism in sensitized recipients (Table 1).

[0104] In summary, the combination of immunofidase and EndoS (i.e., inactivation of virtually all serum IgG), when combined with CyBor and standard pretreatment agents, enables donor BMC engraftment in pre-sensitized recipient mice.

[0105] [Table 1]

[0106] Consideration DSA is a major impairment of allogeneic BMT in sensitized recipients. Previous studies have shown in various models that immunofidases can be used to remove / reduce DSA and EndoS can suppress IgG-mediated cytotoxicity, but neither enzyme has been used in the context of HSPC transplantation / bone marrow transplantation, where high expression of MHC on bone marrow-derived cells may increase sensitivity to residual functional DSA.

[0107] Considering the results of recent clinical trials of kidney transplantation in sensitized recipients in conjunction with this experiment, immunofidases are shown to be usable as preconditioning for human patients undergoing HSPC / bone marrow transplantation. This study also shows that EndoS can be used in this context. EndoS alone was found to improve donor cell survival in the presence of DSA in vivo. Considering that EndoS-treated IgG reduces complement fixation capacity, as reported by Maria Allhorn and Mattias Collin, EndoS may be effective in B6.H-2 g7 Our findings that both NOD and B6.H-2 mice improved donor cell survival to a similar degree suggest that additional mechanisms, such as FcgR, are major mediators of DSA pathogenicity in this BMT model. g7 The differences in mice suggest that non-MHC genes may influence the effectiveness of EndoS on an individual basis. This NOD and B6.H-2 g7The difference is in mice with NOD and B6 backgrounds and IgG 2b This may be due to the differing binding aptitudes of various Fc receptors. FcR polymorphism may also be important. Furthermore, these results suggest that EndoS is more potent against low-titer DSA.

[0108] The combination of immunofidase and EndoS was found to improve donor BMC survival and enable donor chimerism in sensitized mice pre-treated with T cell depletion, CyBor, and sublethal irradiation. In the protocol tested, the effect of peripheral T cell depletion was not affected by EndoS. This suggests that, with proper timing design, enzymatic depletion of serum IgG can be used in conjunction with antibody-based products such as IVIG and B cell depletion antibodies such as rituximab. In other words, the enzyme can be used to inactivate DSA without adversely affecting the effector function of IgG-based biologics, provided that the timing of each administration is carefully selected.

[0109] Regarding the use of cyclophosphamide and bortezomib, both have immunomodulatory effects other than targeting B cells and plasma cells. For example, cyclophosphamide can promote chimerism induction in sensitized recipients by reducing memory T cells. With regard to bortezomib, published data and findings are consistent with the fact that splenic B cells increase compensatoryly after bortezomib treatment, resulting in humoral compensation. However, it remains unclear in this study whether the increase in splenic B cells after BMT is accompanied by a rebound in DSA. More importantly, the T cell depletion employed in this protocol may potentially inhibit the recovery and maturation of both naive and memory B cells, as well as the generation of de novo DSA.

[0110] Finally, the findings of this study must be considered in light of several limitations. Imrufidase cleaves all human IgG subclasses, but only two subclasses of mouse IgG, leaving IgM unaffected. Although IgM DSA levels were lower compared to IgG, this may have reduced the observed chimerism levels. Clinically, IgM DSA can be removed by plasmapheresis. To achieve the maximum effect against mouse DSA, it was necessary to combine EndoS with immunofidase. Imrufidase has been shown to transiently suppress memory B cell activation by cleaving membrane-bound BCRs in vitro, which may contribute to the success of chimerism. However, immunofidase does not cleave mouse IgG 2c Since it only cleaves IgG3, the effect of immunofidase on mouse IgG was not complete in this model (Figure 2A). A protocol using immunofidase as a desensitizer is more efficient in humans, where immunofidase completely removes / inactivates all extracellular IgG and completely inactivates the IgG DSA pool. Therefore, these findings may underestimate the potential of these enzymes in clinical settings.

[0111] The second limitation concerns the toxicity of the chimerism induction protocol. However, this study provides proof of a principled study showing that IgG Fc regulation may be strategically useful for BMT in sensitized recipients. Furthermore, EndoS or immunofidase can also be used in combination with other desensitization methods. Currently, it is unknown whether enzyme-mediated blockade of DSA prevents antibody rebound. Perhaps maintaining a certain level of DSA while blocking its function, i.e., deglycosylation of IgG Fc, may induce less rebound than complete removal of DSA. In these experiments, a short time frame (6-day interval between injections) was employed for repeated enzyme injections to avoid decreased activity as a result of host anti-enzyme antibody production. In humans, the higher efficacy of immunofidase may allow the enzyme to be administered separately (e.g., EndoS after immunofidase), which may mitigate any concerns that may arise with anti-enzyme antibodies.

[0112] Finally, we can conclude that the combination of immunofidase and EndoS (i.e., enzymatic inactivation of virtually all serum IgG) can be used in combination with other desensitization strategies to induce donor chimerism in allosensitized recipient mice.

[0113] Example 2: Optimal interval between immunofidase and antibody-based therapy background Immurefidase (conditionally approved in the EU for desensitization in kidney transplants) is a cysteine ​​protease that cleaves all subclasses of human and rabbit IgG into F(ab')2 and dimeric Fc fragments. Rabbit anti-thymocyte globulin (rATG) is a depletive antibody therapy approved for use in kidney transplants (it has a significant effect of reducing circulating T lymphocytes). Antibody-based therapies such as rATG may be inactivated when administered together with immunorefidase. The aim of this study was to determine the earliest possible timing to initiate rATG treatment while avoiding most of the residual immunorefidase cleavage activity.

[0114] method The cleavage pattern of rATG was examined in serum from healthy subjects (n=11) treated with 0.25 mg / kg immunofidase (EudraCT number: 2019-002770-31). Serum samples were incubated with clinically appropriate fixed concentrations of 50 μg / mL rATG (commonly observed after administration of 1.5 mg / kg) at 37°C for 2 hours. Serum samples were collected from before immunofidase administration to 14 days after immunofidase administration, and rATG cleavage was evaluated by analysis using SDS-PAGE and Western blotting developed with goat anti-rabbit IgG and F(ab')2 specific antibodies. Immurefidase concentrations were analyzed using validated electroluminescent immunoassays based on MSD technology.

[0115] result The serum immunofidase concentration in the subjects decreased rapidly, with an average concentration of 0.5 μg / mL at 96 hours, although there was considerable individual variation, ranging from <0.1 to 1.8 μg / mL (Figure 5). At this point, immunofidase activity levels had sufficiently decreased in 8 out of 11 subjects, avoiding complete cleavage of rATG (Figure 6).

[0116] conclusion rATG may be initiated as early as 4 days after immunofider, considering the possibility of partial interruption of the initial rATG dose in some patients. However, since rATG is administered in high doses and repeated over several days, this interruption at the start of therapy is not expected to negatively impact the overall effectiveness of rATG treatment.

[0117] Example 3 - Improved specificity and reduced toxicity of mixed chimerism protocols Stepwise modifications may be introduced to the mixed chimerism protocol shown in Example 1, with the aim of increasing specificity, reducing the potential toxicity of the approach, and achieving a higher probability of clinical translation. In particular: (i) Infusion of donor CD8-alpha cells may be administered to increase the frequency of stable chimerism in sensitized recipients. Infusion of donor T cells may promote BMT engraftment by reducing host T cell survival. (ii) Anti-CD117 / anti-CD47 can be administered along with the removal of DSAs by enzymes (IdeS and / or EndoS) and the maximal removal of T cells and NK cells. This may enable the first non-irradiated, non-myeloablative chimerism protocol for pre-sensitized recipients. Anti-CD117 / anti-CD47 antibodies help remove host HSCs.

Claims

1. A treatment agent for transplantation of hematopoietic stem and progenitor cells (HSPCs), comprising an enzyme that inactivates target serum IgG molecules, wherein the enzyme is selected from IgG cysteine ​​protease and IgG endoglycosidase.

2. The treatment agent according to claim 1, wherein the amount of enzyme administered is sufficient to inactivate all or substantially all IgG molecules present in the serum of the subject.

3. The treatment agent according to claim 1 or 2, wherein the enzymes are IgG cysteine ​​protease and IgG endoglycosidase.

4. (i) IgG cysteine ​​protease is derived from Streptococcus bacteria, and / or (ii) The IgG endoglycosidase is derived from Streptococcus bacteria, or from Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica. The treatment agent according to any one of claims 1 to 3.

5. (i) The IgG cysteine ​​protease is derived from Streptococcus pyogenes, and / or (ii) The IgG endoglycosidase is derived from Streptococcus pyogenes, Streptococcus equi, or Streptococcus zooepidemicus. The treatment agent according to claim 4.

6. (i) The IgG cysteine ​​protease is IdeS, IdeZ, or MAC2 polypeptide, and / or (ii) The IgG endoglycosidase is EndoS, CP40, EndoE, or EndoF2 polypeptide. The treatment agent according to claim 4 or 5.

7. - The IgG cysteine ​​protease is a polypeptide having a sequence that is at least 85%, 90%, 95%, or 99% identical to SEQ ID NOs: 2, 4, or 5, or the IgG cysteine ​​protease contains or consists of one of the sequences of SEQ ID NOs: 6 to 25 and 55 to 69; and / or - The IgG endoglycosidase is a polypeptide having a sequence that is at least 85%, 90%, 95%, or 99% identical to SEQ ID NO:

90. The treatment agent according to any one of claims 4 to 6.

8. The treatment agent according to claim 7, wherein the sequence comprises an additional methionine at the N-terminus and / or a histidine tag at the C-terminus.

9. A treatment agent according to any one of claims 1 to 8, wherein the enzyme is an immunofidase and / or EndoS.

10. A treatment agent according to any one of claims 1 to 9, to be used in combination with one or more of the following: (a) Non-lethal dose of irradiation and / or administration of a drug to remove the target HSPC, (b) Administration of a drug that reduces the number and / or downregulates the activity of lymphocytes in a subject, wherein the lymphocytes include: i. T cells; and / or ii. B cells, (c) Administration of an agent that reduces the activity of the immune system, selected from the group consisting of complement inhibitors, cytokine inhibitors, innate immune cell inhibitors, and tolerance inducers.

11. A treatment agent according to claim 10, comprising at least (a) and (b).

12. Furthermore, the treatment agent of claim 11, which is used in combination with the following: - (d) Infusion administration of donor CD8-alpha cells; and / or - (e) Administration of anti-CD117 antibody and / or anti-CD47 antibody; and / or - (f) Administration of anti-CD4, anti-CD8, and anti-CD90 antibodies, bortezomib, and cyclophosphamide, and / or administration of rATG.

13. An agent for inducing hematopoietic chimerism in a subject, comprising a treatment agent according to any one of claims 1 to 12 and a composition containing hematopoietic stems and progenitor cells (HSPCs), wherein the composition is administered to the subject in an amount and under conditions suitable for inducing hematopoietic chimerism after the treatment agent has been administered.

14. The agent according to claim 13, wherein the HSPC is of the same type, of the same type, or autologous to the target.

15. The agent according to claim 14, wherein HSPC is genetically modified.

16. An agent for preventing or treating immune rejection of cell, tissue, or organ transplants, comprising the agent according to any one of claims 13 to 15, wherein the transplanted cells, tissue, or organ are derived from the same donor as the HSPC.

17. The agent of claim 16, wherein the cells, tissues or organs to be transplanted are a kidney, liver, heart, pancreas, lung, small intestine, skin, blood vessels / vascular tissue, face, arm, trachea, part of an eye, islets, substantia nigra, bone marrow, stem cells, or HSPC, wherein the HSPC is contained in the agent of any one of claims 13 to 15 so that no additional transplantation is required.

18. A prophylactic or therapeutic agent for immune rejection of cell, tissue, or organ transplants, comprising an enzyme that inactivates serum IgG molecules, wherein the enzyme is selected from IgG cysteine ​​protease and IgG endoglycosidase, and is administered in combination with subsequent administration of hematopoietic stem and progenitor cells (HSPCs) and cell, tissue, or organ transplantation.

19. The agent according to claim 18, wherein the transplanted cells, tissues, or organs are derived from the same donor as the HSPC.

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