Desensitization with clazakizumab and improvement of renal transplantation in HLA-sensitized patients

Abstract

The present invention provides methods for desensitizing patients in need of organ transplantation. Patients sensitized to human leukocyte antigens awaiting incompatible kidney transplantation have been treated with clazakizumab to show reduced or eliminated donor-specific antibody levels and improved transplantation rates. The present invention provides clazakizumab and variants for use in various embodiments of the method. In some embodiments, clazakizumab or its variants are administered simultaneously or sequentially with intravenous immunoglobulin.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 757,676 filed on November 8, 2018 and U.S. Provisional Patent Application No. 62 / 855,988 filed on June 1, 2019, the entire contents of which are incorporated herein by reference. Field of the Invention

[0003] The present invention relates to therapies and treatment methods for desensitizing and improving organ transplantation in sensitized patients.

[0004] background

[0005] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. No admission is made that any information provided herein is prior art or relevant to the invention claimed herein, or that any publication explicitly or implicitly referenced is prior art.

[0006] The causes of accelerated decline in renal allografts are multifactorial. Recent data suggest a reversal of the long-standing belief that calcineurin inhibitor (CNI) toxicity is the primary cause of most chronic allograft failures. It is now recognized that alloimmune responses are responsible for the majority of renal allograft failures, which total 5,000 per year in the United States. The costs associated with allograft failures represent a considerable financial burden on the healthcare system while reducing the length and quality of life of those affected. Patients returning to the transplant list after allograft failure now represent the fourth largest category of new patient lists in the United States. These patients represent a major problem for transplant centers because they are highly human leukocyte antigen (HLA) sensitized and are therefore unlikely to receive another transplant without significant desensitization. Currently, there are no U.S. FDA-approved drugs in this category. The development of new therapies that reduce allosensitization and improve transplant rates is of great importance. Today, this represents one of the most important goals of transplant medicine.

[0007] HLA molecules are polymorphic. Each HLA molecule expresses one or more polymorphic private epitopes as well as a number of public determinants representing epitopes shared by more than one HLA molecule. Immunization following a previous transplant, transfusion, or pregnancy can result in the development of HLA-specific antibodies. An important responsibility of the immunogenetics laboratory is the identification and analysis of HLA-specific antibodies present in the patient's serum before or after transplantation. Knowledge of alloantibody specificity can help predict the likelihood of finding a cross-match compatible donor, to avoid transplantation with a donor carrying the HLA antigens used to sensitize the patient, to select the best cross-matching method, and / or to avoid false-positive cross-matches with donors by excluding clinically irrelevant antibodies.

[0008] Antibodies against HLA antigens have a strong influence on the mediation of allograft damage and loss, and remain a persistent and often insurmountable barrier to successful transplantation for the tens of thousands of patients on the kidney transplant list worldwide. Preformed or de novo synthesized donor-specific antibodies (DSA) activate complement, induce endothelial cell proliferation and mediate antibody-dependent cellular cytotoxicity (ADCC), which renders the recipient highly HLA-sensitized, subject to a sustained immune attack on the allograft, and lead to the progression of interstitial fibrosis, tubular atrophy (IF / TA), and allograft dysfunction and loss. Patients who resume dialysis have little hope of receiving a subsequent transplant and often face a higher risk of death while on dialysis. DSA is also known to accelerate atherosclerosis in the allograft, thereby accelerating vascular death of the kidney.

[0009] In order to increase the rate of renal transplantation in sensitized patients, new protocols for HLA desensitization have emerged. These methods require the use of intravenous immunoglobulin (IVIG), rituximab, and plasma exchange (plasmapheresis, PLEX). There is increasing interest in developing new, less expensive and more convenient immunomodulatory drugs for improving antibody reduction in transplantation.

[0010] There are two main desensitization regimens for existing IVIG-related therapies, namely low-dose intravenous immunoglobulin with plasma exchange (IVIG / PLEX) and high-dose IVIG (HD-IVIG). IVIG / PLEX has been successfully used for ABO-incompatible and positively cross-matched (+CMX) living donor kidney transplants, while HD-IVIG has been used to desensitize living donors +CMX and highly HLA-sensitized deceased donor (HS-DD) recipients on the waiting list. HD-IVIG (2g / kg) in a multiple-dose regimen is considered a reasonable approach for desensitization. The B cell depleting agent rituximab is often used in combination with HD-IVIG and IVIG / PLEX regimens. Rituximab in the IVIG / rituximab regimen has been shown to modify alloreactive B cells and prevent DSA rebound.

[0011] The major problem with existing desensitization protocols is the interpretation of CMX and DSA results when IVIG and rituximab are present in the test serum. IVIG given at a dose of 2 gm / kg (maximum 140 grams) may interfere with the LUMINEX test for DSA, giving false-positive results. In theory, this can be avoided by waiting at least 1 month after IVIG administration to perform the LUMINEX single antigen bead (LSA) test, as IVIG has a half-life of 30-40 days. Rituximab does not interfere with the LSA test platform, but does give "false-positive" CDC+ and flow cytometry crossmatch (FCMX)-positive B cell crossmatches, which may be incorrectly interpreted as being due to DSA. Pronase treatment of B cells prior to FCMX and CDC testing generally reduces the effect of rituximab, but this is not always reliable.

[0012] Allogeneic antibodies are the main obstacle to obtaining and successfully performing life-saving organ transplants. Despite advances in desensitization, efficient and effective methods designed to remove pathogenic anti-HLA antibodies remain a major medical challenge. Existing desensitization regimens have many obvious defects. For example, existing therapies fail to substantially completely remove the risk of acute rejection of DSA before transplantation. There is also a risk of post-transplant rebound DSA formation accompanied by acute and chronic damage to the allogeneic graft. Moreover, some existing regimens (especially those that use complement inhibitors to prevent chronic antibody-mediated rejection (cABMR)) still fail to achieve the desired results. Therefore, there is an unmet medical need to improve the ability to reduce or eliminate pre-existing HLA antibodies to a level that allows patients to receive life-saving organ transplants.

[0013] Therefore, it is an object of the present invention to provide a composition for use in combination with, to improve, or to replace existing standard treatments for desensitization to increase the rate of solid organ transplantation in HLA-sensitized subjects. SUMMARY OF THE INVENTION

[0015] The present invention describes and illustrates the following embodiments and aspects thereof in conjunction with compositions and methods which are intended to be exemplary and illustrative rather than limiting in scope.

[0016] The present invention provides a method for reducing donor-specific antibodies and / or desensitizing in human subjects sensitized to human leukocyte antigens (HLA). The method comprises administering to the subject an effective amount of clazakizumab; an IL-6 binding fragment of clazakizumab; or a V having a CDR1, CDR2, and CDR3 polypeptide contained in SEQ ID NO: 1, 2, or 3 and 4, respectively. H Polypeptides and V sequences comprising the CDR1, CDR2 and CDR3 polypeptides respectively contained in SEQ ID NOs: 5, 6 and 7 L wherein the subject requires or has undergone solid organ transplantation. Various embodiments provide that the subject is a human.

[0017] In one embodiment, clazakizumab, an IL-6 binding fragment of clazakizumab, or a polypeptide disclosed herein is administered prior to transplantation. In another embodiment, clazakizumab, an IL-6 binding fragment of clazakizumab, or a polypeptide disclosed herein is administered after transplantation. Another embodiment provides administration of clazakizumab, an IL-6 binding fragment of clazakizumab, or a polypeptide disclosed herein before and after transplantation.

[0018] Some embodiments of the disclosed methods provide that in addition to the administration of clazakizumab, the IL-6 binding fragment of clazakizumab, or the polypeptide disclosed herein, a standard of care treatment is administered, including intravenous immunoglobulin (IVIG) administration, rituximab administration, plasmapheresis, or a combination thereof. In one aspect, the standard of care treatment is administered before clazakizumab, the IL-6 binding fragment of clazakizumab, or the polypeptide of the present disclosure. In another aspect, the standard of care treatment is administered simultaneously with or after clazakizumab, the IL-6 binding fragment of clazakizumab, or the polypeptide of the present disclosure.

[0019] One embodiment provides a method for desensitizing an HLA-sensitized human patient awaiting a kidney transplant, wherein the method comprises administering an effective amount of clazakizumab, an IL-6 binding fragment of clazakizumab, or a polypeptide disclosed herein. Another embodiment provides a method for desensitizing an HLA-sensitized human patient awaiting a kidney transplant, comprising administering an effective amount of (1) clazakizumab, an IL-6 binding fragment of clazakizumab, or a polypeptide disclosed herein, (2) standard of care treatment, such as IVIG, plasmapheresis, rituximab, or a combination thereof, and optionally (3) an anti-infective agent.

[0020] Other embodiments provide one or more methods for desensitizing HLA-sensitized human patients for other solid organ transplants (including heart, liver, lung, pancreas, or intestine).

[0021] In one embodiment, clazakizumab, an IL-6 binding fragment of clazakizumab, or a polypeptide of the present disclosure is administered subcutaneously 1, 2, 3, 4, 5, or 6 times before transplantation and 4, 5, 6, 7, 8, 9, 10, 11, or 12 times after transplantation at an average dose of about 1-5, 5-10, 10-20, or 20-30 mg / time, wherein the subject has a reduced amount of donor-specific antibodies after treatment compared to before treatment. Various aspects provide for administration of clazakizumab, an antigen-binding fragment thereof, or a polypeptide disclosed herein at approximately monthly intervals after transplantation. One embodiment provides for administration of one dose of clazakizumab, IVIG, and plasmapheresis to a subject before transplantation, followed by administration of six doses or 12 doses of clazakizumab after transplantation. Various embodiments provide disclosed methods comprising administering clazakizumab, an antigen-binding fragment thereof, or a polypeptide disclosed herein to a human subject who is HLA-sensitized and in need of or has undergone a kidney transplant, wherein the subject's creatinine level is reduced after treatment compared to before treatment, there is no or no detectable presence of donor-specific antibodies, and / or the subject does not develop detectable symptoms or evidence of antibody-mediated rejection (e.g., no deterioration of allograft function as measured by serum creatinine and estimated glomerular filtration rate; no detectable evidence of capillaritis, inflammation, or complement (C4d) deposition). Another aspect of this embodiment provides that, concurrently or after administration of clazakizumab, an antigen-binding fragment thereof, or a polypeptide disclosed herein, the subject's creatinine level is reduced and maintained at a reduced level for 1, 2, 3, 4, 5 months or longer.

[0022] The present invention also provides a pharmaceutical composition for administration to an HLA-sensitized subject to desensitize the subject and improve the transplantation rate. The pharmaceutical composition comprises clazakizumab, an IL-6 binding fragment of clazakizumab, or a polypeptide disclosed herein, and a pharmaceutically acceptable excipient, such as an amino acid, sorbitol, and a diluent.

[0023] Various embodiments provide for the use of clazakizumab in patients who are HLA-sensitized and awaiting an incompatible kidney transplant, wherein DSA, complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC) are reduced or eliminated (e.g., DSA is reduced or eliminated from the serum). In some embodiments, patients treated with clazakizumab are suitable for transplantation with a lesser likelihood of an antibody response.

[0024] Some aspects provide that one or more of the disclosed methods further include selecting a mammalian (eg, human) patient who is HLA-sensitized and awaiting an incompatible deceased donor (DD) or living donor (LD) kidney transplant.

[0025] Other features and advantages of the invention will become apparent from the following detailed description taken in conjunction with the accompanying drawings which illustrate, by way of example, various features of embodiments of the invention. Sequence Listing <110> Cedars-Sinai Medical Center <120> Desensitization with clazakizumab and improvement of renal transplantation in HLA-sensitized patients <130> 065472-000762WO00 <150> 62 / 757,676 <151> 2018-11-08 <150> 62 / 855,988 <151> 2019-06-01 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> VH CDR1 <400> 1 Asn Tyr Tyr Val Thr 1 5 <210> 2 <211> 16 <212> PRT <213> Artificial sequence <220> <223> VH CDR2 <400> 2 Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Trp Ala Ile Gly 1 5 10 15 <210> 3 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Another VH CDR2 <400> 3 Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Ser Ala Ile Gly 1 5 10 15 <210> 4 <211> 12 <212> PRT <213> Artificial sequence <220> <223> VH CDR3 <400> 4 Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn Leu 1 5 10 <210> 5 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VL CDR1 <400> 5 Gln Ala Ser Gln Ser Ile Asn Asn Glu Leu Ser 1 5 10 <210> 6 <211> 7 <212> PRT <213> Artificial sequence <220> <223> VL CDR2 <400> 6 Arg Ala Ser Thr Leu Ala Ser 1 5 <210> 7 <211> 12 <212> PRT <213> Artificial sequence <220> <223> VL CDR3 <400> 7 Gln Gln Gly Tyr Ser Leu Arg Asn Ile Asp Asn Ala 1 5 10 <210> 8 <211> 166 <212> PRT <213> Artificial sequence <220> <223> VH <400> 8 Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser 35 40 45 Asn Tyr Tyr Val Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Trp 65 70 75 80 Ala Ile Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn Leu Trp Gly Gln 115 120 125 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys 165 <210> 9 <211> 109 <212> PRT <213> Synthetic Sequence <220> <223> Another VH <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Asn Tyr 20 25 30 Tyr Val Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Trp Ala Ile 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn Leu 100 105 <210> 10 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Another VH <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Asn Tyr 20 25 30 Tyr Val Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Ser Ala Ile 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn Leu 100 105 <210> 11 <211> 163 <212> PRT <213> artificial sequence <220> <223> VL <400> 11 Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser 20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 35 40 45 Gln Ser Ile Asn Asn Glu Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60 Arg Pro Lys Leu Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val 65 70 75 80 Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 Gly Tyr Ser Leu Arg Asn Ile Asp Asn Ala Phe Gly Gly Gly Thr Glu 115 120 125 Val Val Val Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn <210> 12 <211> 99 <212> PRT <213> artificial sequence <220> <223> another VL <400> 12 Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp 1 5 10 15 Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Asn Asn Glu Leu 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Leu Arg Asn Ile 85 90 95 Asp Asn Ala

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Exemplary embodiments are shown in the accompanying drawings. It is intended that the embodiments and drawings disclosed herein be considered illustrative rather than restrictive.

[0028] Figure 1 Depicted is the DSA profile of the subject "ClazaDES01" in the study, a 50-year-old African American female with a history of end-stage renal disease (ESRD) secondary to biopsy-proven focal segmental glomerulosclerosis (FSGS) and has been on dialysis since November 2008 (i.e., approximately 10 years of waiting time for a B+ blood type), with a calculated population reactive antibodies (cPRA) of 58%. The patient's sensitization events included pregnancy x 4 and blood transfusions.

[0029] Figure 2 Depicted are the DSA spectra of subject "ClazaDES05" before and after transplantation. (Median fluorescence intensity, MFI). Subject "ClazaDES05" is a 36-year-old female with a history of ESRD secondary to IgA nephropathy, and she has been on dialysis since June 2008 (i.e., a waiting time of about 10 years for A+ blood type), with 100% cPRA. The patient's sensitization events included previous transplants and blood transfusions. Subject "ClazaDES05" received a deceased donor kidney transplant after 4 doses of ClazadES05. The patient underwent 2 DSAs (Class I and Class II) before and after transplantation. The DSA intensity of Class I decreased from MFI>12,500MFI at the time of transplantation to MFI=0 10 days after transplantation, and the DSA intensity of Class II decreased from MFI>17,500 at the time of transplantation to MFI>3250 10 days after transplantation. According to the study protocol, patients received clazakizumab monthly for 6 months after transplantation.

[0030] Figure 3ATotal C-reactive protein levels in the clazakizumab desensitization study are depicted. Overall, C-reactive protein (CRP) decreased from baseline to almost zero by month 2. The number of subjects included in the analysis at each time point is indicated in parentheses.

[0031] Figure 3B Individual C-reactive protein amounts from baseline to month 7 in the clazakizumab desensitization study are depicted.

[0032] Figure 4 The total MFI over time from before plasmapheresis (PLEX) (pre-PLEX) to the 5th dose of clazazumab (N=9) is depicted. Typically, MFI tends to rebound approximately 1-3 months after completion of PLEX / IVIG. Here, with monthly clazazumab injections, the total MFI remained reduced over time compared to pre-PLEX. To date, 3 patients have undergone transplantation. Patients ClazaDES01 and ClazaDES03 underwent transplantation after the 1st dose of clazazumab. Patient ClazaDES05 underwent transplantation after the 4th dose of clazazumab injection.

[0033] Figure 5 It is a schematic diagram of an exemplary method for desensitizing patients with HLA sensitization before renal transplantation. At the selected time point during the study, the patient will receive up to 6 dosages of clazakizumab, while monitoring anti-HLA antibodies (DSA levels), Treg cells and plasmablasts. For example, DSA levels are collected at all time points (including the 0th day, except the 7th day). The amount of C-reactive protein (CRP) and quantitative immunoglobulin (QIG) is collected at all time points (including baseline (-15 days), except the 0th day). In addition, the following is collected at baseline (-15 days) and the 180th day: CD4+ / CD25+ / Fox P3+ / CD127 low cell count (Treg); Th17+ cell count; and CD19+ / CD38+ / CD27+ / IL-6+ (plasmablasts). For the subject who was not transplanted before 180 days before transplantation, a special test will be carried out. For the subject who received transplantation before 180 days before transplantation, a special test will be carried out at the 0th day of transplantation. Maintenance of the standard regimen included tacrolimus, mycophenolate mofetil, and steroids.

[0034] Figure 6 Schematic diagram of an exemplary method for post-transplant prevention and / or treatment to reduce donor-specific antibodies. Figure 5 In one aspect of the pre-transplant treatment shown, IVIG and clazakizumab were administered post-transplant (on the day of transplantation). Figure 6In the figure, DSA levels were monitored at 0, 90 and 180 days; and were also monitored at 270 days in patients receiving the second round of administration. CRP and QIG levels were collected at 0, 30, 60, 90, 120, 150 and 180 days; and were also collected at 240 and 300 days in patients receiving the second round of administration. On the 180th day after transplantation (about 6 months), the following levels were collected: CD4+ / CD25+ / Fox P3+ / CD127 low cell count (Tregs); Th17+ cell count; and CD19+ / CD38+ / CD27+ / IL-6+ (plasmablasts). Viral PCT tests (for cytomegalovirus, Epstein-Barr virus, polyomavirus, BK virus, JC virus and parvovirus B19) were performed at 30, 90 and 180 days; and were also tested at 270 and 330 days if the patient received the second round of administration. Maintenance of the standard regimen included tacrolimus, mycophenolate mofetil, and steroids. The "add-on schedule" consisted of patients continuing to receive an additional 6 doses of clazakizumab per month (approximately on days 180, 210, 240, 270, 300, and 330) if they demonstrated stability or improvement in a) the 6M protocol biopsy Banff 2015 reading; b) glomerular filtration rate (GFR); c) DSA.

[0035] Figure 7 Depicted is the treatment timeline of patient "ClazaDES03" studied in the Examples and his creatinine levels (mg / dL) from pre-treatment to post-treatment.

[0036] Figure 8 Shown is a kidney transplant biopsy of patient "ClazaDES03" approximately 2 months after transplantation (including tubular damage, arteriosclerosis, and very focal tubulitis).

[0037] Fig. 9 Shown is a kidney transplant biopsy of patient "ClazaDES03" approximately 6 months after transplantation (including acute tubular necrosis, rare isometric alveoli, and mild tubulointerstitial inflammation).

[0038] Fig. 10A and 10B Flow-PRA class I / II antibodies before desensitization and after transplantation (post-Tx) are depicted, respectively.

[0039] Fig.11 Depicted are HLA class I and class II antibodies and total amounts of various markers before desensitization and at last follow-up (F / U) approximately 6-12 months after transplantation for subject DES03 who received clazakizumab. DSA was not detected at transplantation or after transplantation.

[0040] Figures 12A-12C HLA class I and class II antibodies and total amounts of various markers are depicted before desensitization and after transplantation in subject DES05 who had received clazakizumab.

[0041] Figures 13A-13C HLA class I and class II antibodies and total amounts of various markers are depicted before desensitization and after transplantation in subject DES07 who had received clazakizumab.

[0042] Fig.14 HLA class I and class II antibodies and total amounts of various markers are depicted for subject DES02 (untransplanted) before desensitization and after clazakizumab.

[0043] Fig.15 HLA class I and class II antibodies and total amounts of various markers are depicted for subject DES09 (untransplanted) before desensitization and after clazakizumab.

[0044] Fig.16 HLA class I and class II antibodies before and after clazakizumab are depicted for all study patients (N=10).

[0045] Figures 17A-17C Figure 3 HLA class I antibody ( Fig.17A ), Class II antibodies ( Fig. 17B )( Fig. 17C (combined comparison).

[0046] Fig.18 Depicted are DSA for individual patients of category I and II: pre-desensitization, at transplantation, and post-transplantation (N=8).

[0047] Fig.19 Depicted are the mean DSA MFI for categories I and II: before desensitization, at transplantation, and after transplantation. SUMMARY OF THE INVENTION

[0049] All references cited herein are incorporated herein by reference in their entirety as if fully set forth. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3 rded., Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7 th ed., J. Wiley & Sons (New York, NY2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4 th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012) provides those skilled in the art with a general guide to many of the terms used in this application. For reference on how to prepare antibodies, see D. Lane, Antibodies: A Laboratory Manual 2 nd ed. (Cold Spring Harbor Press, Cold Spring Harbor NY, 2013); Kohler and Milstein, (1976) Eur. J. Immunol. 6:511; Queen et al. US Patent No. 5,585,089; and Riechmann et al., Nature 332:323 (1988); US Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Ward et al., Nature 334:544-54 (1989); Tomlinson I. and Holliger P. (2000) Methods Enzymol, 326, 461-479; Holliger P. (2005) Nat. Biotechnol. Sep; 23(9):1126-36).

[0050] Those skilled in the art will recognize that many methods and materials similar or equivalent to the methods or materials described in the present invention can be used in the practice of the present invention. In fact, the present invention is by no means limited to the methods and materials described. For purposes of the present invention, the following terms are defined as follows.

[0051] The term "transplant rate" generally refers to the number of patients who receive a transplant in every 100 patients on the waiting list in a year. In some respects, it is a measure of the frequency with which a patient on the waiting list is scheduled to receive a transplant. In order to more easily compare figures, in some respects, the ratio is given as "per 100 patient-years," which means that the ratio is standardized to 100 patients on the list in a year. For example, a transplant rate of 5 per 100 patient-years means that 5 per 100 patients on the list undergo a transplant in a year. Because this is a standardized ratio, this number may contain decimals, such as 5.1 per 100 patient-years. This means that slightly more than 5 patients per 100 patients on the list undergo a transplant in a year.

[0052] A positive crossmatch (+CMX) indicates the presence of donor-specific alloantibodies (DSA) in the serum of the potential recipient and is often associated with a graft loss rate of more than 80%.

[0053] "HLA sensitized (HS) patients" in the study of the examples refer to patients waiting for a kidney transplant on the United Network for Organ Sharing (UNOS) waiting list, whose calculated population reactive antibodies (cPRA) or the percentage of possible cross-matched incompatible donors is ≥50%, and who in various embodiments also have a history of DSA and sensitization events (e.g., previous transplants, transfusions, and / or pregnancies) that can be confirmed using LUMINEX bead technology. The presence of HLA-specific antibodies can be determined by testing the patient's serum for cells from a panel of HLA-typed donors or soluble HLA antigens attached to a solid support. Typically, HLA-sensitized patients are patients with a cPRA of not less than 10%, 20%, 30%, 40%, or 50%.

[0054] "Subject" refers to a person or an animal. Typically, an animal is a vertebrate, such as a primate, a rodent, a domestic animal, or a wild animal. Primates include chimpanzees, crab-eating monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Domestic animals and wild animals include cattle, horses, pigs, deer, bison, buffalo, felines (e.g., house cats), and canine species (e.g., dogs, foxes, wolves). The terms "patient," "individual," and "subject" are used interchangeably herein. In one embodiment, a subject is a mammal. The mammal may be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples. In addition, the methods described herein may be used to treat domestic animals and / or pets.

[0055] The term "treatment", "treatment", "treatment" or "relief" refers to therapeutic treatment, wherein the purpose is to reverse, alleviate, improve, inhibit, slow down or stop the progression or severity of the condition associated with the disease or disorder. The term "treatment" includes alleviating or relieving at least one adverse effect or symptom of the condition, disease or disorder, such as weight loss or muscle loss caused by cancer cachexia. If one or more symptoms or clinical signs are alleviated, the treatment is usually "effective". Alternatively, if the progression of the disease is reduced or stopped, the treatment is "effective". That is, "treatment" includes not only the improvement of symptoms or signs, but also the cessation or at least slowing down of the progression or deterioration of symptoms (which would be expected to occur in the absence of treatment). Beneficial or ideal clinical results include, but are not limited to, alleviating one or more symptoms, alleviating the extent of the disease, stabilizing the disease state (i.e., not worsening), delaying or slowing the progression of the disease, improving or alleviating the disease state and alleviating (whether in part or in whole), whether detectable or undetectable. The term "treatment" of the disease also includes providing relief of the symptoms or side effects of the disease (including palliative care).

[0056] The term "antibody" refers to a monoclonal or polyclonal antigen-binding fragment (referred to herein as "Fc fragment" or "Fc domain") of a complete immunoglobulin or an FcRn binding fragment having an Fc (crystallizable fragment) region or an Fc region. Antigen-binding fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of a complete antibody. Antigen-binding fragments include, in particular, Fab, Fab', F(ab')2, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (scFv), single-domain antibodies, chimeric antibodies, double antibodies, and polypeptides containing at least a portion of an immunoglobulin sufficient to bind the polypeptide to a specific antigen. The Fc domain includes portions of two heavy chains that contribute to two or three classes of antibodies. The Fc domain can be produced by recombinant DNA technology or by enzymatic (e.g., papain cleavage) or by chemical cleavage of a complete antibody. The antibody can be a chimeric antibody, a humanized antibody, or a human antibody. The antibody can be an IgG1, IgG2, IgG3, or IgG4 antibody. In some aspects, the antibodies herein have an Fc region that has been modified to alter at least one of effector function, half-life, proteolysis, or glycosylation.

[0057] The term "antibody fragment" refers to a protein fragment that contains only a portion of an intact antibody, generally containing the antigen binding site of the intact antibody and thus retaining the ability to bind antigen. Examples of antibody fragments encompassed by this definition include: (i) L ,CL,V H and CH1 domain; (ii) Fab' fragment, which is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) having V Hand an Fd fragment of the CH1 domain; (iv) a fragment having V H and a CH1 domain and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) a V having a single arm of the antibody L and V H Fv fragment of the structural domain; (vi) V H (vii) isolated CDR regions; (viii) F(ab')2 fragments, which are bivalent fragments comprising two Fab' fragments linked by a disulfide bond in the hinge region; (ix) single-chain antibody molecules (e.g., single-chain Fv; scFv); (x) "diabodies" with two antigen-binding sites, which comprise a light chain variable domain (V L ) connected to the heavy chain variable domain (V H ); (xi) "linear antibodies" comprising a pair of tandem Fd segments (V H -CH1-V H -CH1), which together with the complementary light chain polypeptide form a pair of antigen binding regions. The antibody or antibody fragment can be scFv, camel antibody, nanobody, IgNAR (single chain antibody derived from shark) and Fab, Fab' or F(ab')2 fragment.

[0058] "Selective binding" or "specific binding" refers to the antibodies or antibody fragments described herein that bind to D 10 -5 M (10000nM) or less, for example, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 The ability of a polypeptide agent to selectively bind to a target (e.g., a molecule present on a cell surface) at a concentration of 1 M or less. Specific binding can be affected by, for example, the affinity and avidity of the polypeptide agent and the concentration of the polypeptide agent. One of ordinary skill in the art can use any suitable method, such as titration of the polypeptide agent in a suitable cell binding assay, to determine suitable conditions for the polypeptide agent described herein to selectively bind to a target.

[0059] An "ineffective" treatment means that when the treatment is administered to a subject, the symptom improvement is less than 5%. If specifically provided for the claims, an ineffective treatment may mean that the symptom improvement is less than 1%, 2%, 3%, 4%, 6%, 7%, 8%, 9% or 10%.

[0060] "Adverse Events" are any unfavorable and unintended signs, symptoms, or illnesses temporally associated with the use of an investigational medicinal product (IMP) or other protocol-imposed interventions, regardless of attribution. An adverse event can be any unfavorable and unintended sign (including abnormal laboratory findings), symptom, or illness temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product. Surgical procedures are not adverse events; they are treatments for conditions requiring surgery. However, conditions requiring surgery are adverse events if they occur or are detected in the studies of the embodiments. Planned surgical procedures and the conditions leading to these procedures are not adverse events if the condition is known prior to the start of study treatment. In the latter case, the condition should be reported as medical history.

[0061] A pre-existing condition is one that was present at the start of the study. A pre-existing condition that worsens during the study is considered an adverse event. A pre-existing condition should be recorded as an adverse event if the frequency, intensity, or character of the condition worsens during the study.

[0062] “Abnormal test findings”, which meet any of the following criteria, should be considered an adverse event:

[0063] Test results are related to accompanying symptoms;

[0064] Test results require additional diagnostic testing or medical / surgical intervention;

[0065] Test results that lead to changes in study treatment dosing (e.g., dose adjustment, interruption, or permanent discontinuation) or changes in concomitant medications (e.g., addition, interruption, or discontinuation) or any other changes in concomitant medications or therapy;

[0066] The test result results in any outcome included in the definition of a critical adverse event (note: this will be reported as a critical adverse event);

[0067] The test results were considered adverse events by the investigators.

[0068] Laboratory results that fall outside the reference range and do not meet one of the above criteria should not be reported as adverse events. Repeating an abnormal test without the above conditions does not constitute an adverse event. Any abnormal test result that is determined to be an error does not need to be reported as an adverse event.

[0069] "Serious Adverse Event" A serious adverse event (SAE) is any untoward medical occurrence that, at any dose:

[0070] is fatal (causes death);

[0071] Is life-threatening: At the time of the adverse event, the patient is in immediate danger of death as a result of the adverse event. This does not include events that occurred in a more severe form or were allowed to continue and could have resulted in death;

[0072] requiring an inpatient hospital stay or prolonging an existing hospital stay;

[0073] Causes persistent or severe disability / incapacity;

[0074] are congenital anomalies / birth defects (in children of patients receiving study treatment);

[0075] Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered SAEs when, based on appropriate medical judgment, the event may endanger the subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. Examples of such events are intensive treatment in the emergency room or at home for allergic bronchospasm, blood cachexia, or seizures that do not result in hospitalization or the development of drug addiction or abuse.

[0076] Adverse events that resulted in hospitalization were considered critical. Any adverse event that resulted in initial admission to a healthcare facility or transfer within the hospital to an emergency / intensive care unit was considered critical.

[0077] Hospitalization or prolonged hospital stay in the absence of a precipitating clinical adverse event is not, in itself, a critical adverse event. Examples of events that are not considered critical adverse events include: (1) hospitalization for a pre-existing condition that is not associated with the development of a new adverse event or the exacerbation of a pre-existing condition, (2) social or administrative hospitalization, (3) elective hospitalization that is not associated with a precipitating clinical adverse event, and (4) pre-planned treatment or surgical procedures that should be noted in the baseline documentation.

[0078] All adverse events require an investigator's assessment of severity. The following criteria are used to assess severity:

[0079] Mild: Discomfort is noticed but normal daily activities are not disrupted;

[0080] Moderate: Discomfort sufficient to reduce or interfere with daily activities; and

[0081] Severe: Unable to work or carry out daily activities.

[0082] To clarify the distinction between the terms “critical” and “serious” (they are not synonyms), note that the term “serious” is often used to describe the intensity (severity) of a specific event, such as a mild, moderate, or severe myocardial infarction. However, the event itself may be of relatively minor medical significance, such as a severe headache. This is different from “critical,” which is based on patient / event outcomes or action criteria that are generally associated with an event that threatens a patient’s life or functioning. Seriousness (not severity) is used as a guide for defining regulatory reporting obligations.

[0083] The causality assessment is a determination of whether there is a reasonable possibility that the study treatment caused or contributed to the adverse event. “Unrelated” describes a temporal relationship to study treatment administration that is lost or implausible, or there is evidence of another cause. “Unlikely Related” describes a temporal relationship to study treatment administration that makes a causal relationship unlikely; and other drugs, chemicals, or underlying illness provide a reasonable explanation. “Possibly Related” describes a reasonable temporal sequence to study treatment administration, but the event could also be explained by concurrent illness with other drugs or chemicals. Information regarding discontinuation may be lacking or unclear. “Definitely Related” describes a reasonable temporal relationship to study treatment administration; the event cannot be explained by concurrent illness or other drugs or chemicals. The response to discontinuation (dechallenge) should be clinically reasonable. The event must be definitive pharmacologically or phenomenologically, using satisfactory rechallenge procedures if necessary.

[0084] Interleukin-6 is an important mediator of inflammation and the development, maturation and activation of T cells, B cells and plasma cells. Excessive IL-6 production is associated with a number of human diseases characterized by excessive and uncontrolled antibody production and autoimmunity.

[0085] The disclosed method for desensitizing HLA-sensitized subjects, reducing the amount of donor-specific antibodies and / or improving organ transplantation rates or transplant survival comprises administering to the subject an effective amount of clazakizumab or an antibody or antigen-binding fragment thereof having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology (identity) with clazakizumab or the complementary determining region (CDR) of clazakizumab. One or more methods provided in some embodiments further comprise administering an effective amount of IVIG or plasmapheresis.

[0086] Clazakizumab is a glycosylated humanized (from rabbit parent antibody) monoclonal antibody targeting interleukin 6. The peptide sequence and structural information of Clazakizumab can be obtained from IMGT / mAb-db record number #414. BLAST peptide sequence analysis revealed identical matches to peptides protected in U.S. Patent No. 8,062,864, which is incorporated herein by reference in its entirety. Further description of Clazakizumab and its variants is shown in U.S. Patent No. 7,935,340 (which is incorporated herein by reference in its entirety), and its antibodies or antibody fragments are suitable for the methods disclosed herein to reduce or eliminate donor-specific antibodies in subjects in need or who have undergone allogeneic transplantation. For example, the antibody has a peptide sequence comprising SEQ ID NO: 1 (for V H CDR1), 2 or 3 (for V H CDR2) and 4 (for V H CDR1, CDR2 and CDR3 H Polypeptides and V sequences comprising the CDR1, CDR2 and CDR3 polypeptides respectively contained in SEQ ID NOs: 5, 6 and 7 L The anti-human IL-6 antibody comprises the variable heavy chain contained in SEQ ID NO: 8, 9 or 10, and the variable light chain contained in SEQ ID NO: 11 or 12.

[0087] Asn-Tyr-Tyr-Val-Thr (SEQ ID NO:1)

[0088] Ile-Ile-Tyr-Gly-Ser-Asp-Glu-Thr-Ala-Tyr-Ala-Thr-Trp-Ala-Ile-Gly (SEQ ID NO: 2)

[0089] Ile-Ile-Tyr-Gly-Ser-Asp-Glu-Thr-Ala-Tyr-Ala-Thr-Ser-Ala-Ile-Gly (SEQ ID NO: 3)

[0090] Asp-Asp-Ser-Ser-Asp-Trp-Asp-Ala-Lys-Phe-Asn-Leu (SEQ ID NO: 4)

[0091] Gln-Ala-Ser-Gln-Ser-Ile-Asn-Asn-Glu-Leu-Ser (SEQ ID NO: 5)

[0092] Arg-Ala-Ser-Thr-Leu-Ala-Ser (SEQ ID NO: 6)

[0093] Gln-Gln-Gly-Tyr-Ser-Leu-Arg-Asn-Ile-Asp-Asn-Ala (SEQ ID NO: 7).

[0094] The variable heavy chain sequence is set forth in SEQ ID NO: 8 - METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTASGFSLSNYYVTWVRQAPGKGLEWIGIIYGSDETAYATWAIGRFTISKTSTTVDLKMTSLTAADTATYFCARDDSSDWDAKFNLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK.

[0095] The substituted variable heavy chain sequence is set forth in SEQ ID NO: 9 -

[0096] EVQLVESGGGLVQPGGSLRLSCAASGFSLSNYYVTWVRQ APGKGLEWVGIIYGSDETAYATWAIGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCARDDSSDWDAKFNL.

[0097] Another substituted variable heavy chain sequence is set forth in SEQ ID NO: 10 -

[0098] EVQLVESGGGLVQPGGSLRLSCAASGFSLSNYYVTWVRQ APGKGLEWVGIIYGSDETAYATSAIGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCARDDSSDWDAKFNL.

[0099] The variable light chain sequence is set forth in SEQ ID NO: 11 -

[0100] MDTRAPTQLLGLLLLWLPGARCAYDMTQTPASVSAAVGGTVTIKCQASQSINNELSWYQQKPGQRPKLLIYRASTLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQQGYSLRNIDNAFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN.

[0101] The variable light chain sequence to be replaced is set forth in SEQ ID NO: 12 -

[0102] IQMTQSPSSSLSASVGDRVTITCQASQSINNELSWYQQKPGK APKLLIYRASTLASGVPSRFSGSGSGTDFTLTISSLQPDDFATYY CQQGYSLRNIDNA.

[0103] Clazazumab is a genetically engineered humanized immunoglobulin G1 (IgG1) antibody that binds to human IL-6 with an affinity of about 4pM. By using a combination of IL-6 (to measure classical signaling) and IL-6 and sIL-6R (to measure trans-signaling) for responding to a single signal transduction and a variety of cell function assays, it is demonstrated that Clazazumab is an effective and complete antagonist of IL-6-induced signal transduction (as measured by phosphorylation of signal transducer and activator of transcription 3 (STAT3)) and cell functions such as cell proliferation, differentiation, activation, B cell production of immunoglobulins and acute phase proteins (C-reactive protein [CRP] and fibrinogen) produced by hepatocytes. In addition, it is shown that Clazazumab is a competitive antagonist of IL-6-induced cell proliferation.

[0104] Clazakizumab exhibits broad immunomodulatory effects that can resolve destructive allogeneic antibody responses to allogeneic transplants and can be used as a desensitizing agent to improve renal transplant rates in highly HLA-sensitized patients. Clazakizumab has been extensively evaluated in patients with rheumatoid arthritis but has not yet been approved by the U.S. FDA for any condition. Since the introduction of IL-6 / IL-6R blocking drugs, reports have suggested that inhibition of the IL-6 / IL-6R pathway may have significant benefits in systemic lupus erythematosus (SLE) and other vascular diseases and reduce antibody-producing cells in treated patients. There is currently no information on clazakizumab in highly sensitized patients awaiting incompatible (HLAi) transplants or for the treatment of antibody-mediated rejection.

[0105] To date, although clazakizumab has been studied in healthy subjects and subjects with rheumatoid arthritis (RA), psoriatic arthritis (PsA), Crohn's disease, graft-versus-host disease (GVHD), and tumors, no studies using clazakizumab have been conducted in subjects with highly sensitized patients undergoing renal transplantation.

[0106] Various embodiments provide one or more methods for reducing donor-specific antibodies in an HLA-sensitized subject characterized by having a calculated population reactive antibody (cPRA) of at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% or a percentage of donors that are likely to be cross-matched incompatible, wherein the method comprises administering to the subject an effective amount of clazakizumab; an antigen-binding fragment thereof; or a V-binding fragment thereof comprising the CDR1, CDR2, and CDR3 polypeptides contained in SEQ ID NOs: 1, 2, or 3, and 4, respectively, prior to kidney transplantation, after kidney transplantation, or both. H Polypeptides and V sequences comprising the CDR1, CDR2 and CDR3 polypeptides respectively contained in SEQ ID NOs: 5, 6 and 7 L A polypeptide of a polypeptide. Various embodiments of the method provide administering an effective amount of an anti-human IL-6 antibody or antibody fragment comprising a variable heavy chain in SEQ ID NO: 8, 9 or 10 and a variable light chain in SEQ ID NO: 11 or 12 to a subject with HLA sensitization before or after the subject receives an allogeneic transplant to reduce or eliminate donor-specific antibodies in the subject after the allogeneic transplant. Some embodiments of the method further include selecting a subject having a calculated population reactive antibody (cPRA) of at least 10%, 20%, 30%, 40%, 50%, 60% or 70% or a percentage of donors that may be cross-matched incompatible. Some embodiments of the method further include performing a kidney transplant in the subject. Other embodiments of the method are characterized in that donor-specific antibodies are reduced after kidney transplantation due to the administration of clazakizumab or an antigen-binding fragment thereof; or characterized in that there is no detectable amount of donor-specific antibodies starting from about one month, two months, three months, four months, five months or six months after kidney transplantation due to the administration of clazakizumab or an antigen-binding fragment thereof.

[0107] Various embodiments provide one or more methods for reducing donor-specific antibodies in an HLA-sensitized subject, wherein the method comprises administering an effective amount of (1) IVIG and (2) an effective amount of clazakizumab; an IL-6 binding fragment of clazakizumab; or a V polypeptide comprising a CDR1 comprised in SEQ ID NO: 1, a CDR2 comprised in SEQ ID NO: 2 or 3, and a CDR3 comprised in SEQ ID NO: 4. H A polypeptide and a V sequence of a polypeptide comprising the CDR1 contained in SEQ ID NO: 5, the CDR2 contained in SEQ ID NO: 6 and the CDR3 contained in SEQ ID NO: 7 LIn one embodiment, the IVIG is clazakizumab; an IL-6 binding fragment of clazakizumab; or a V polypeptide having a CDR1, CDR2, and CDR3 polypeptide contained in SEQ ID NO: 1, 2 or 3 and 4, respectively. H Polypeptides and V sequences comprising CDR1, CDR2 and CDR3 polypeptides respectively contained in SEQ ID NOs: 5, 6 and 7 L The polypeptide is administered before or simultaneously with the polypeptide of the present invention. In some aspects, the HLA-sensitized subject will receive a solid organ transplant - on the one hand, the solid organ is from a cross-matched donor, i.e., the HLA-sensitized subject comprises pre-transplant antibodies against the HLA of the organ from the donor; on the other hand, the solid organ is not from a positive cross-matched donor. In other aspects, the one or more methods are in addition to administering an effective amount of (1) IVIG and (2) an effective amount of clazakizumab; an IL-6 binding fragment of clazakizumab; or a V polypeptide comprising the CDR1, CDR2, and CDR3 polypeptides contained in SEQ ID NOs: 1, 2, or 3 and 4, respectively. H Polypeptides and V sequences comprising the CDR1, CDR2 and CDR3 polypeptides respectively contained in SEQ ID NOs: 5, 6 and 7 L In addition to polypeptides, it also includes solid organ transplantation.

[0108] Various embodiments of the method for reducing donor-specific antibodies in an HLA-sensitized subject comprise administering (1) an effective amount of IVIG in combination with clazakizumab; (2) an effective amount of IVIG in combination with an IL-6 binding fragment of clazakizumab; or (3) an effective amount of IVIG in combination with a V polypeptide comprising the CDR1, CDR2, and CDR3 polypeptides contained in SEQ ID NOs: 1, 2, or 3, and 4, respectively. H Polypeptides and V sequences comprising the CDR1, CDR2 and CDR3 polypeptides respectively contained in SEQ ID NOs: 5, 6 and 7 L In one embodiment, the IVIG is clazakizumab; an IL-6 binding fragment of clazakizumab; or a V polypeptide having a CDR1, CDR2, and CDR3 polypeptide contained in SEQ ID NO: 1, 2 or 3 and 4, respectively. H Polypeptides and V sequences comprising the CDR1, CDR2 and CDR3 polypeptides respectively contained in SEQ ID NOs: 5, 6 and 7 L In another embodiment, IVIG is further administered to the subject before, during or immediately after solid organ transplantation.

[0109] Still further embodiments provide methods for reducing donor-specific antibodies in an HLA-sensitized subject, wherein the method comprises administering (1) an effective amount of a combination of IVIG, plasmapheresis, and clazakizumab; (2) an effective amount of a combination of IVIG, plasmapheresis, and an IL-6 binding fragment of clazakizumab; or (3) an effective amount of IVIG, plasmapheresis, and a V6 binding fragment having a polypeptide comprising the CDR1, CDR2, and CDR3 polypeptides contained in SEQ ID NOs: 1, 2, or 3, and 4, respectively. H Polypeptides and V sequences comprising the CDR1, CDR2 and CDR3 polypeptides respectively contained in SEQ ID NOs: 5, 6 and 7 L In one embodiment, IVIG and plasmapheresis are performed in the presence of clazakizumab; an IL-6 binding fragment of clazakizumab; or a V polypeptide comprising the CDR1, CDR2, and CDR3 polypeptides contained in SEQ ID NOs: 1, 2, or 3, and 4, respectively. H Polypeptides and V sequences comprising the CDR1, CDR2 and CDR3 polypeptides respectively contained in SEQ ID NOs: 5, 6 and 7 L The polypeptide is administered before or simultaneously with the polypeptide.

[0110] In various embodiments, desensitization due to clazakizumab resulted in (1) transplantation in 8 of 10 patients in the study of the Examples; (2) a significant reduction in HLA specificity despite no significant change in cPRA; and (3) a reduction in DSA at the time of transplantation and after transplantation with continued administration of effective amounts of clazakizumab.

[0111] In a further embodiment, anti-IL-6 treatment (e.g., clazakizumab administration) has a significant effect on reducing the MFI level of class II / class II HLA antibodies; thereby increasing the transplant rate of HLA-sensitized patients or the probability or percentage of transplant survival of individual HLA-sensitized patients. Anti-IL-6 mediates this effect by reducing plasma cells (anti-HLA) that produce IL-6. After treatment, anti-IL-6 therapy reduces or eliminates DSA levels and prevents de novo DSA production. A further aspect provides that patients receiving clazakizumab and solid organ transplants do not develop antibody-mediated graft rejection.

[0112] The disclosed methods provided by each embodiment include identifying HLA sensitized patients who need solid organ transplantation, administering PLEX and IVIG and monthly doses of clazakizumab, performing solid organ transplantation (e.g., kidney transplantation), administering induction therapy, such as alemtuzumab and / or THYMOGLOBULIN (anti-thymocyte globulin), and administering immunosuppressive therapy, such as tacrolimus, CELLCEPT (mycophenolate mofetil) and gradually reducing prednisone. On the one hand, the transplantation rate of HLA sensitized subjects after desensitization with clazakizumab and PLEX / IVIG is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. On the other hand, for HLA sensitized subjects, the time to start transplantation after completing pre-transplant desensitization with clazakizumab and PLEX / IVIG is 0 days to 1 week, 1 week to 1 month, 1 month to 3 months, 3 months to six months, six months to one year, one year to two years or longer. On the other hand, a previously HLA-sensitized subject who has received desensitization treatment with an effective amount of clazakizumab (optionally in combination with PLEX / IVIG) and received an allogeneic kidney transplant is at least 95%, 90%, 85%, 80%, 75% or 70% free of signs or symptoms of antibody-mediated transplant rejection. Other aspects of the method include that the subject does not have rheumatoid arthritis (RA), psoriatic arthritis (PsA), Crohn's disease, graft-versus-host disease (GVHD), cancer or a combination thereof. Other aspects of the method also include selecting a subject who does not have or has not had rheumatoid arthritis (RA), psoriatic arthritis (PsA), Crohn's disease, graft-versus-host disease (GVHD) or cancer and is HLA-sensitized and needs or has undergone a solid organ (e.g., kidney) transplant for a method of reducing and / or eliminating donor-specific antibodies.

[0113] One or more of the disclosed methods provided by various embodiments further include performing one or more assays for the presence or absence of an infection associated with cytomegalovirus, Epstein-Barr virus, polyomavirus, BK virus, JC virus, parvovirus B19, or a combination thereof, in a subject prior to and / or after an allogeneic transplant. In other embodiments, one or more of the disclosed methods are characterized in that the subject has no detectable amount of an infection associated with cytomegalovirus, Epstein-Barr virus, polyomavirus, BK virus, JC virus, parvovirus B19, or a combination thereof, prior to and / or before the allogeneic transplant. Further embodiments provide that the subject in one or more of the disclosed post-transplant clazakizumab methods does not have chronic antibody-mediated rejection of a solid organ transplant or has been tested for lack of evidence of chronic antibody-mediated rejection.

[0114] Various embodiments of the method for reducing or eliminating donor-specific antibodies in an HLA-sensitized subject requiring or having undergone an allogeneic transplant and / or desensitizing the subject comprise administering an effective amount of clazakizumab, an IL-6 binding fragment of clazakizumab, a polypeptide comprising a variable heavy chain of SEQ ID NO: 8, 9, or 10 and a variable light chain of SEQ ID NO: 12 or 12, or a polypeptide comprising a variable heavy chain having CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2 or 3, and CDR3 of SEQ ID NO: 4, and a polypeptide comprising a variable heavy chain having CDR1 of SEQ ID NO: 5, CDR2 of SEQ ID NO: 6, and CDR3 of SEQ ID NO: 7 in one or more doses over time. NO:7, wherein (1) the subject has or had preformed donor-specific antibodies (DSA) prior to allogeneic transplantation, and / or develops DSA after allogeneic transplantation, (2) the subject has a calculated population reactive antibody of 50% or more, (3) the subject has high intensity donor-specific antibodies, such as determined by a single antigen LUMINEX bead assay and expressed as a mean fluorescence intensity (MFI) of greater than 9,000, 10,000, 11,000, 12,000 or more for class I or class II, (4) or the subject has had one or more pregnancies, transfusions, and / or previous transplants. In one aspect, the subject has one of the above characteristics. In another aspect, the subject has two of the mentioned characteristics. In another aspect, the subject has three of the mentioned characteristics. In another aspect, the subject has four of the mentioned characteristics.

[0115] Additional embodiments of the methods disclosed herein include one or more steps of immune monitoring prior to and / or following allogeneic transplantation.In various aspects, methods for reducing or eliminating donor-specific antibodies in subjects with pre-formed DSA, 50% or greater cPRA, or high-intensity DSA prior to allogeneic transplantation and in subjects who subsequently underwent allogeneic transplantation (e.g., the allogeneic transplant is HLA-incompatible with the subject) include (i) administering an effective amount of clazakizumab, an IL-6 binding fragment of clazakizumab, or a polypeptide disclosed above in one or more doses; (ii) performing (a) immune monitoring of the subject, such as assaying a blood sample from the subject to quantify Treg, Tfh, Th17, B cells, IL-6, CRP, plasma cells, plasmablast IgG levels, or a combination thereof, b) biopsy assessment of the transplant, (c) measuring glomerular filtration rate, and / or (d) measuring the amount of DSA in the subject, each performed once or more, for example, each time over a period of time (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months , 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 months or longer) after one or more doses of clazakizumab, an IL-6 binding fragment of clazakizumab, or a polypeptide of the disclosure within 12 months; and optionally (iii) when (a) immune monitoring indicates improvement in immune responsiveness, such as as characterized by decreased levels of CRP, Treg, Tfh, Th17, B cells, IL-6, plasma cells, or plasmablast IgG compared to previous immune monitoring or baseline measurements at or before the time of allogeneic transplantation, or levels of CRP, Treg, Tfh, Th17, B cells, IL-6, plasma cells, or plasmablast IgG relative to comparable levels in healthy subjects or desensitized subjects, when (b) biopsy evaluation of the transplant indicates absence of cell-mediated and antibody-mediated rejection, absence of or reduced evidence of allograft dysfunction (e.g., using Banff assay). 2015 criteria as determined by C4d staining and transplant glomerular lesions), and / or improvement according to the Banff 2015 criteria, (c) when the glomerular filtration rate is stable, such as a level similar to or decreased compared to the last measurement or before transplantation, or (d) when the amount of DSA is stable, such as a level similar to or decreased compared to the last measurement or before transplantation, then interrupt or limit further administration of clazakizumab, the IL-6 binding fragment of clazakizumab, or the polypeptide of the present disclosure to no more than an additional six months; when immune monitoring indicates that the immune reactivity (such as described above) has not improved or the glomerular filtration rate or the amount of DSA has not stabilized, then continue to administer clazakizumab, the IL-6 binding fragment of clazakizumab, or the polypeptide of the present disclosure or administer it at an adjusted dose; and when a biopsy evaluation of the transplant indicates the presence of cell-mediated and / or antibody-mediated rejection, then administer standard of care therapy to treat the rejection, such as IVIG, plasmapheresis, or both.In some cases, steps (ii) and (iii) are repeated one, two, three, four, five, six, seven, eight, nine, or ten times, or continued as needed, or until improvement, stabilization, or even cure is observed.

[0116] In some aspects, if evidence of antibody-mediated rejection is observed, the subject is directed to a treatment for antibody-mediated rejection. In some aspects, after steps (i) and (ii), no further doses of clazakizumab, IL-6 binding fragments of clazakizumab, or polypeptides of the disclosure are administered for a period of time (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, and 3 months), and after the "rest," immunoreactivity is monitored or a biopsy of the allograft is assessed, and depending on the results, such as the characterization in step (iii), one of skill in the art will discontinue or continue administration of clazakizumab, IL-6 binding fragments of clazakizumab, or polypeptides of the disclosure to further reduce or eliminate DSA in the subject.

[0117] dose

[0118] In one embodiment, the method for reducing donor-specific antibodies and HLA desensitization in a subject (e.g., a human subject) comprises administering an effective amount of clazakizumab or an antibody binding fragment of clazakizumab before transplantation (e.g., about 25 mg / dose subcutaneously, once every 4 weeks, up to 6 doses). In one embodiment, the method for reducing donor-specific antibodies and HLA desensitization in a subject (e.g., a human subject) comprises administering five, six or seven sessions of plasma exchange (or plasmapheresis) before transplantation, followed by administration of an effective amount of IVIG (e.g., about 2g / kg subject, up to 140g), and administration of an effective amount of clazakizumab (e.g., about 25 mg subcutaneously, once every 4 weeks, up to 6 doses). In another embodiment, the method comprises transplanting an allogeneic transplant into a subject. On the other hand, the transplantation of the allogeneic transplant is between the last dose of clazakizumab and about 270 days after IVIG administration. This is Figure 5 In one aspect, for 4, 5, 6 or more doses, the effective amount of clazakizumab for reducing DSA levels in HLA-sensitized subjects is about 12.5 mg / dose. In one aspect, for 4, 5, 6 or more doses, the effective amount of clazakizumab for reducing DSA levels in HLA-sensitized subjects is about 25 mg / dose. In one aspect, the effective amount of clazakizumab for reducing DSA levels in HLA-sensitized subjects is not 25 mg / dose at a monthly dose of 4, 5 or 6 doses.

[0119] In another embodiment, the method for reducing donor-specific antibodies and HLA desensitization in a subject (e.g., a human subject) comprises administering an effective amount of clazakizumab or an antigen-binding fragment thereof after transplantation (e.g., about 25 mg subcutaneously, once every 4 weeks starting about 5 to 7 days after transplantation, up to 6 doses). In another embodiment, the method comprises administering an additional effective amount of clazakizumab (e.g., an additional 6 doses until day 330 after transplantation). This Figure 6 In one aspect, for 1, 2, 3, 4, 5 or more doses, the effective amount of clazakizumab for reducing DSA levels after solid organ transplantation in HLA-sensitized subjects is about 12.5 mg / dose. In one aspect, for 1, 2, 3, 4, 5 or more doses, the effective amount of clazakizumab for reducing DSA levels after solid organ transplantation in HLA-sensitized subjects is about 25 mg / dose. In one aspect, the effective amount of clazakizumab for reducing DSA levels after solid organ transplantation in HLA-sensitized subjects is not 25 mg / dose administered every 4 weeks.

[0120] In another embodiment, a method for reducing donor-specific antibodies and HLA desensitization in a subject (e.g., a human subject) comprises administering an effective amount of clazakizumab or an antibody-binding fragment thereof prior to transplantation (e.g., about 25 mg / dose subcutaneously, once every 4 weeks, up to 6 doses), and administering an effective amount of clazakizumab or an antigen-binding fragment thereof after transplantation (e.g., about 25 mg subcutaneously, once every 4 weeks starting about 5 to 7 days after transplantation, up to 6 doses).

[0121] Some embodiments of these methods provide for measuring biopsies from patients and confirming stable levels of glomerular filtration rate (GFR) over time (e.g., less than 10%, 20%, or 30% change between two, three, or four consecutive biopsies) and lower levels of DSA (e.g., less than 10%, 20%, or 30%) compared to before desensitization therapy or compared to earlier biopsies performed after transplantation. In some embodiments, when the level of GFR is unstable or the level of DSA is high, the method further comprises repeatedly administering an effective amount of IVIG and clazakizumab until the level of GFR is stable and the level of DSA is low.

[0122] Yet another embodiment provides a method for reducing donor-specific antibodies and HLA desensitization in a highly HLA-sensitized subject (e.g., a human subject), the method comprising administering plasma exchange (or plasmapheresis) prior to transplantation; administering an effective amount of IVIG before, during, or after transplantation; before, after, or before and after administering an effective amount of clazakizumab to the subject, wherein the subject has a level of glomerular filtration rate (GFR) that is stable over time (e.g., less than 10%, 20%, or 30% change between two, three, or four consecutive biopsies) and a lower level (e.g., less than 10%, 20%, or 30%) of DSA compared to before desensitization treatment.

[0123] After administration of clazakizumab as a 1-hour IV infusion, the pharmacokinetics of clazakizumab were linear in the dose range of 30 mg to 640 mg in healthy subjects and in the dose range of 80 mg to 320 mg in subjects with rheumatoid arthritis (RA), as indicated by consistent clearance at these dose levels. The half-life (T-half) of clazakizumab at all doses was very similar in healthy male subjects and subjects with RA and was consistent with that expected for a humanized IgG1 antibody. At all doses studied, the mean half-life of clazakizumab was 19.5 to 31.0 days in healthy male subjects and 26.4 to 30.9 days in subjects with RA. In healthy male subjects, the half-life of clazakizumab after SC administration was similar to IV administration. In a Phase 1 study comparing IV and SC dosing in healthy male subjects, the mean half-life of clazakizumab after a single IV dose was 30.7 days, compared with 31.1 to 33.6 days after SC administration. The bioavailability of clazakizumab after SC administration was 60% of that of the IV formulation. As expected, Cmax was lower and Tmax was longer with SC administration relative to IV administration.

[0124] Population PK analysis of data from clinical studies in RA, PsA, and healthy subjects showed that body weight affects the PK of clazakinumab, such that clearance and central volume of distribution increase with increasing body weight. Therefore, subjects with a heavier body weight will have lower drug exposure compared to subjects with a lighter body weight.

[0125] The effective amount of clazakizumab for a subject can be studied or limited based on safety assessments. Safety assessments include medical interviews, adverse event records, physical examinations, blood pressure, and laboratory measurements. Subjects are typically assessed for adverse events (all grades), critical adverse events, and adverse events that require interruption or discontinuation of study drug at each study visit during their participation in the study.

[0126] In some embodiments, clazakizumab, an IL-6 binding fragment of clazakizumab, or a V sequence having a CDR1, CDR2, and CDR3 polypeptide comprising SEQ ID NO: 1, 2 or 3, and 4, respectively, suitable for administration in the disclosed methods is H Polypeptides and V sequences comprising the CDR1, CDR2 and CDR3 polypeptides respectively contained in SEQ ID NOs: 5, 6 and 7 L An effective amount of a polypeptide can be about 10-50 μg / dose, 50-100 μg / dose, 100-150 μg / dose, 150-200 μg / dose, 100-200 μg / dose, 200-300 μg / dose, 300-400 μg / dose, 400-500 μg / dose, 500-600 μg / dose, 600-700 μg / dose, 700- 800μg / dose, 800-900μg / dose, 900-1000μg / dose, 1000-1100μg / dose, 1100-1200μg / dose, 1200-1300μg / dose, 1300-1400μg / dose, 1400-1500μg / dose, 1500-1600μg / dose, 1600-1700μg / dose, 1700 -1800μg / dose, 1800-1900μg / dose, 1900-2000μg / dose, 2000-2100μg / dose, 2100-2200μg / dose, 2200-2300μg / dose, 2300-2400μg / dose, 2400-2500μg / dose, 2500-2600μg / dose, 2600-2700μg / dose , 2700-2800 μg / dose, 2800-2900 μg / dose or 2900-3000 μg / dose, for a total of one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15 or more doses, or as needed to continue to reduce the amount of DSA in the subject, and administered at a frequency of daily, weekly, biweekly, monthly or bimonthly, or a combination thereof.

[0127] In some embodiments, clazakizumab, an IL-6 binding fragment of clazakizumab, or a V sequence having a CDR1, CDR2, and CDR3 polypeptide comprising SEQ ID NO: 1, 2 or 3, and 4, respectively, suitable for administration in the disclosed methods is H Polypeptides and V sequences comprising the CDR1, CDR2 and CDR3 polypeptides respectively contained in SEQ ID NOs: 5, 6 and 7 LThe effective amount of the polypeptide per unit weight of the subject in the above method includes 10-100 μg, 100-200 μg, 200-300 μg, 300-400 μg, 400-500 μg, 500-600 μg, 600-700 μg, 700-800 μg, 800-900 μg, 1-5 mg, 5-10 mg, 10-20 mg, 20-30 mg, 30-40 mg, 40-50 mg, 50-60 mg, 60-70 mg, 70-80 mg, 80-90 mg, 90-100 mg, 100-200 mg, 200-300 mg, 300-400 mg, 400 mg-500 mg, 500 mg-1 g or 1 g-10 g. The unit weight of the subject can be per kg body weight or per subject. In one embodiment, the effective amount of clazakizumab for reducing DSA levels and desensitizing previously HLA-sensitized human subjects who need or have received an allogeneic kidney transplant is about 25 mg / month. In one embodiment, the effective amount of clazakizumab for reducing DSA levels and desensitizing previously HLA-sensitized human subjects who need or have received an allogeneic kidney transplant is not 25 mg / month.

[0128] In further embodiments, clazakizumab, an IL-6 binding fragment of clazakizumab, or a V sequence comprising a CDR1, CDR2, and CDR3 polypeptide contained in SEQ ID NO: 1, 2 or 3, and 4, respectively, suitable for administration in the disclosed methods. H Polypeptides and V sequences comprising the CDR1, CDR2 and CDR3 polypeptides respectively contained in SEQ ID NOs: 5, 6 and 7 LThe effective amount of the polypeptide of the polypeptide can be in the range of 0.01-0.05 mg / kg, 0.05-0.1 mg / kg, 0.1-1 mg / kg, 1-5 mg / kg, 5-10 mg / kg, 10-50 mg / kg, 50-100 mg / kg. In other embodiments, the effective amount of clazakizumab, the antigen-binding fragment of clazakizumab, or the disclosed polypeptide is about 1-2 mg / kg, 2-3 mg / kg, 3-4 mg / kg, 4-5 mg / kg, 5-6 mg / kg, 6-7 mg / kg, 7-8 mg / kg, 8-9 mg / kg, 9-10 mg / kg, 10-11 mg / kg, 11-12 mg / kg, 12-13 mg / kg, 13-15 mg, 15-20 mg / kg, or 20-25 mg / kg. In additional embodiments, the effective amount of clazakizumab, an antigen-binding fragment of clazakizumab, or a disclosed polypeptide is about 100-125 mg, 125-150 mg, 150-175 mg, 160-170 mg, 175-200 mg, 155-165 mg, 160-165 mg, 165-170 mg, 155-170 mg, or a combination thereof, which can be administered for more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 doses, some of which are before transplantation and others are after transplantation.

[0129] In various embodiments, clazakizumab, an IL-6 binding fragment of clazakizumab, or a V sequence having a CDR1, CDR2, and CDR3 polypeptide contained in SEQ ID NO: 1, 2, or 3 and 4, respectively, suitable for administration in the disclosed methods. H Polypeptides and V sequences comprising the CDR1, CDR2 and CDR3 polypeptides respectively contained in SEQ ID NOs: 5, 6 and 7 L The polypeptides of the polypeptides are administered at least once a week 1-7 times, 1-7 times a month, or 1-12 times a year, or one or more times as needed for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 14 months, 16 months, 18 months, about 24 months, about 30 months, about 36 months, or a combination thereof, at any one or more doses described herein.

[0130] Pharmaceutical composition

[0131] In various embodiments, the present invention provides a pharmaceutical composition. The pharmaceutical composition includes (1) clazakizumab, an IL-6 binding fragment of clazakizumab, or a V polypeptide having a CDR1, CDR2, and CDR3 polypeptide contained in SEQ ID NO: 1, 2 or 3 and 4, respectively. HPolypeptides and V sequences comprising the CDR1, CDR2 and CDR3 polypeptides respectively contained in SEQ ID NOs: 5, 6 and 7 L The invention relates to a polypeptide comprising: a polypeptide, and (2) a pharmaceutically acceptable excipient.

[0132] Pharmaceutical compositions according to the present invention may include any pharmaceutically acceptable excipient." pharmaceutically acceptable excipient " refers to a generally safe, nontoxic and desirable excipient that can be used to prepare a pharmaceutical composition, and includes veterinary use and acceptable excipients for human pharmacy. Such excipients may be solid, liquid, semisolid, or may be gas in the case of aerosol compositions. Examples of excipients include, but are not limited to, amino acids, starches, sugars, microcrystalline cellulose, diluents, granulators, lubricants, adhesives, disintegrants, wetting agents, emulsifiers, colorants, release agents, coating agents, sweeteners, flavoring agents, aromatics, preservatives, antioxidants, plasticizers, gelling agents, thickeners, hardeners, curing agents, suspending agents, surfactants, wetting agents, carriers, stabilizers and combinations thereof.

[0133] In one embodiment, the disclosed method comprises administering a pharmaceutical composition comprising L-histidine, L-histidine monohydrochloride, sorbitol, polysorbate 80, and water for injection, and clazakizumab, an IL-6 binding fragment of clazakizumab, or a V polypeptide comprising the CDR1, CDR2, and CDR3 polypeptides contained in SEQ ID NOs: 1, 2, or 3, and 4, respectively. H Polypeptides and V sequences comprising the CDR1, CDR2 and CDR3 polypeptides respectively contained in SEQ ID NOs: 5, 6 and 7 L Peptides of polypeptides.

[0134] In various embodiments, the pharmaceutical composition according to the present invention can be prepared for delivery by any route of administration. In one embodiment, the pharmaceutical composition is administered intravenously or subcutaneously to the subject." route of administration " can refer to any route of administration known in the art, including but not limited to aerosol, nasal cavity, oral, transmucosal, percutaneous, parenteral or enteral." parenteral " refers to the route of administration usually associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subarachnoid, subcapsular, subcutaneous, transmucosal or transtracheal. Via parenteral route, the composition can be the form of a solution or suspension for infusion or injection, or the form of a lyophilized powder. Via parenteral route, the composition can be the form of a solution or suspension for infusion or injection. Via the enteral route, the pharmaceutical composition can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles that allow controlled release. Typically, the composition is administered by injection. These methods of administration are known to those skilled in the art.

[0135] The pharmaceutical composition according to the present invention may include any pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle that participates in carrying or transporting a compound of interest from one tissue, organ or part of the body to another tissue, organ or body part. For example, the carrier can be a liquid or solid filler, diluent, excipient, solvent or encapsulating material, or a combination thereof. Each component of the carrier must be "pharmaceutically acceptable" because it must be compatible with the other ingredients of the preparation. It must also be suitable for contact with any tissue or organ that it may contact, which means that it must not carry the risk of toxicity, irritation, allergic reaction, immunogenicity or any other complication that overly exceeds its therapeutic benefit.

[0136] The pharmaceutical composition according to the present invention can also be encapsulated, tableted or prepared in an emulsion. Pharmaceutically acceptable solid or liquid carriers can be added to enhance or stabilize the composition, to facilitate the preparation of the composition, or to provide a sustained release or controlled release (or increase the half-life) of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerol, saline, alcohol and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, gypsum powder, magnesium stearate or stearic acid, talc, pectin, gum arabic, agar or gelatin. Emulsion carriers include liposomes or controlled release polymer nanoparticles known in the art. Methods for preparing liposome delivery systems are discussed in Gabizon et al., Cancer Research (1982) 42: 4734; Cafiso, Biochem Biophys Acta (1981) 649: 129; and Szoka, Ann Rev Biophys Eng (1980) 9: 467. Other drug delivery systems are known in the art and are described, for example, in Poznansky et al., DRUG DELIVERY SYSTEMS (RL Juliano, ed., Oxford, NY 1980), pp. 253-315; ML Poznansky, Pharm Revs (1984) 36: 277. The carrier may also contain a sustained release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

[0137] The pharmaceutical preparations are prepared according to conventional techniques of pharmacy, including grinding, mixing, granulating and compressing (if necessary for tablet form); or grinding, mixing and filling for hard gelatin capsule form. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or aqueous or non-aqueous suspension. Such liquid preparations can be administered directly or filled into soft gelatin capsules.

[0138] The pharmaceutical composition according to the present invention can be delivered in a therapeutically effective amount. An accurate therapeutically effective amount is the amount of the composition that will produce the most effective results in a given subject in terms of therapeutic efficacy. This amount will vary depending on a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics and bioavailability), the subject's physiological condition (including age, sex, disease type and stage, general physical condition, responsiveness to a given dose and drug type), the properties of one or more pharmaceutically acceptable carriers in the preparation, and the route of administration. The technician in the clinical and pharmacological fields will be able to determine the therapeutically effective amount by routine experiments, for example, by monitoring the subject's reaction to the compound administration and adjusting the dosage accordingly. For other guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

[0139] Prior to administration to a patient, the agent may be added to clazakizumab, an IL-6 binding fragment of clazakizumab, or a V vector having a polypeptide comprising the CDR1, CDR2, and CDR3 polypeptides contained in SEQ ID NOs: 1, 2, or 3 and 4, respectively. H Polypeptides and V sequences comprising the CDR1, CDR2 and CDR3 polypeptides respectively contained in SEQ ID NOs: 5, 6 and 7 L Polypeptides of polypeptides. Liquid preparations may be preferred. For example, these preparations may include oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, fillers or combinations thereof.

[0140] Carbohydrate preparations include sugar or sugar alcohol, such as monosaccharide, disaccharide or polysaccharide, or water-soluble glucan. Sugar or glucan can include fructose, dextrose, lactose, glucose, mannose, sorbitol, xylose, maltose, sucrose, dextran, pullulan, dextrin, α and β cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethyl cellulose or its mixture." sugar alcohol " is defined as the C4-C8 hydrocarbon with -OH group, and includes galactitol, inositol, mannitol, xylitol, sorbitol, glycerol and arabitol. These sugars or sugar alcohols mentioned above can be used alone or in combination. As long as sugar or sugar alcohol is soluble in aqueous preparation, there is no fixed restriction on the amount used. In one embodiment, sugar or sugar alcohol concentration is 1.0w / v% to 7.0w / v%, more preferably 2.0 to 6.0w / v%.

[0141] The amino acid formulation includes the levorotatory (L) forms of carnitine, arginine, and betaine; however, other amino acids may be added.

[0142] In some embodiments, the polymer as a formulation includes polyvinyl pyrrolidone (PVP) having an average molecular weight of 2,000 to 3,000 or polyethylene glycol (PEG) having an average molecular weight of 3,000 to 5,000.

[0143] It is also preferred to use a buffer in the composition to minimize pH changes in the solution before or after lyophilization or reconstitution. Most physiological buffers can be used, including but not limited to citrate, phosphate, succinate and glutamate buffers or mixtures thereof. In some embodiments, the concentration is 0.01 to 0.3 molar. Surfactants that can be added to the formulation are shown in EP 270,799 and 268,110.

[0144] After the liquid pharmaceutical composition is prepared, it can be lyophilized to prevent degradation and maintain sterility. Methods for lyophilizing liquid compositions are known to those of ordinary skill in the art. Just before use, the composition can be reconstituted with a sterile diluent (e.g., Ringer's solution, distilled water, or sterile saline) that can include other ingredients. After reconstruction, the composition is administered to a subject using those methods known to those skilled in the art.

[0145] Anti-infective agents

[0146] Various embodiments provide that the method for desensitization further comprises administering one or more anti-infective agents (preferably after transplantation) as a prophylactic or therapeutic agent against bacterial, viral or fungal infection.

[0147] Exemplary anti-infective agents suitable for use in the disclosed methods include antibiotics such as aminoglycosides (e.g., amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, paromomycin), ansamycins (e.g., geldanamycin, herbicide), carbacephems (e.g., loracarbef), carbapenems (e.g., ertapenem, doripenem, imipenem, cilastatin, meropenem), cephalosporins (e.g., first First generation: cefadroxil, cefazolin, cefalotin or cephalothin, cefalexin; second generation: cefaclor, cefadroxil, cefoxitin, cefprozil, cefuroxime; third generation: cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone; fourth generation: cefepime; fifth generation: ceftobiprole), glycopeptides (e.g. teicoplanin, vancomycin), macrolides (e.g. such as azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, tiliromycin, spectinomycin), monolactone rings (such as aztreonam), penicillins (such as amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin, piperacillin, ticarcillin), antibiotic polypeptides (such as bacitracin, colistin, polymyxin, b), quinolones (e.g., ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin), rifamycins (e.g., rifampicin or rifampin, rifabutin, rifapentine, rifaximin), sulfonamides (e.g., mafenide, prontosil, sulfacetamide, sulfamethoxazole, sulfabenzamide, sulfasalazine, sulfonylsalicylic acid), Azole, trimethoprim, trimethoprim-sulfamethoxazole Sulfadiazine (sulfamethoxazole azole, "tmp-smx") and tetracyclines (e.g., demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline), as well as arsphenamine, chloramphenicol, clindamycin, lincomycin, ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin, plate mycin, pyrazinamide, quinupristin / dalfopristin combination and tinidazole. In some embodiments, the method of reducing donor-specific antibodies before and / or after allogeneic transplantation in HLA-sensitized subjects comprises administering to the subject an effective amount of clazakizumab or an IL-6 binding antibody fragment of clazakizumab, and administering to the subject an effective amount of ganciclovir, valganciclovir, fluconazole, trimethoprim, sulfamethoxazole, or the like. azole or a combination thereof.

[0148] Reagent test kit

[0149] In various embodiments, the present invention provides a kit for desensitization of organ transplant recipients. The kit is a collection of materials or components, including clazakizumab, an IL-6 binding fragment of clazakizumab, or a V having a polypeptide comprising CDR1, CDR2, and CDR3 contained in SEQ ID NO: 1, 2 or 3 and 4, respectively. H Polypeptides and V sequences comprising the CDR1, CDR2 and CDR3 polypeptides respectively contained in SEQ ID NOs: 5, 6 and 7 L A polypeptide of a polypeptide; instructions or manual for desensitization administration before and after organ transplantation; one or more vessels as containers; and optionally one or more diluents.

[0150] The exact nature of the components configured in the kit of the present invention depends on its intended purpose. In one embodiment, the kit is particularly configured for human subjects. In a further embodiment, the kit is configured for veterinary applications, to treat subjects such as but not limited to farm animals, livestock and experimental animals.

[0151] Instructions for use may be included in the kit. "Instructions for use" generally include a tangible expression that describes the techniques employed in using the components of the kit to achieve a desired result (e.g., to treat or inhibit cancer cachexia in a subject). Optionally, the kit also includes other useful components, e.g., measuring tools, diluents, buffers, pharmaceutically acceptable carriers, syringes, or other useful appliances as will be readily recognized by those skilled in the art.

[0152] The materials or components assembled in the kit can be stored and provided to the practitioner in any convenient and appropriate manner that maintains their operability and practicality. For example, the components can be in dissolved, dehydrated or lyophilized form; they can be provided at room temperature, refrigerated or frozen temperature. The components are generally contained in suitable packaging materials. As used herein, the phrase "packaging material" refers to one or more physical structures used to contain the contents of the kit (e.g., the composition of the present invention, etc.). The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. As used herein, the term "packaging" refers to a suitable solid matrix or material capable of containing the various kit components, such as glass, plastic, paper, foil, etc. Thus, for example, a package can be a bottle for containing an appropriate amount of a composition of the present invention, comprising clazakizumab, an IL-6 binding fragment of clazakizumab, or a V having a CDR1, CDR2 and CDR3 polypeptide comprising SEQ ID NO: 1, 2 or 3 and 4, respectively. H Polypeptides and V sequences comprising the CDR1, CDR2 and CDR3 polypeptides respectively contained in SEQ ID NOs: 5, 6 and 7 LThe packaging material typically has an external label that indicates the contents and / or purpose of the kit and / or its components. Example

[0153] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that specific materials are mentioned, they are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art may develop equivalent means or reactants without exerting creative ability and without departing from the scope of the invention.

[0154] Study: Phase I / II trial evaluates clazakizumab in patients with highly HLA-sensitized (HS) awaiting kidney transplantation The safety and tolerability of this approach is to eliminate donor-specific HLA antibodies in patients with leukemia and improve the transplantation rate.

[0155] This study was designed as an open-label study to evaluate the safety and efficacy of clazakizumab in eliminating DSA and inducing Treg subtypes in HS patients awaiting HLA-incompatible (HLAi) kidney transplantation. Figure 5 and 6 Safety assays were designed to assess any side effects associated with clazakizumab administration and the risk of infectious complications associated with clazakizumab therapy for desensitization of HS patients awaiting renal HLAi transplantation. Limited efficacy assays included assessment of reductions in DSA levels that allowed for increased transplantation rates and subsequent prevention of ABMR (eGFR, SCr) following clazakizumab treatment.

[0156] The tested agent, clazakizumab, has an active ingredient of a genetically engineered humanized anti-IL-6 monoclonal antibody. Produced by Ajinomoto Althea (San Diego CA), it has a strength of 25 mg / mL. It contains excipients including L-histidine, L-histidine monohydrochloride, sorbitol, polysorbate 80, and water for injection. In a single-dose vial of 25 mg / mL for undiluted injection. Clazakizumab vials should be stored at ≤-20°C (-4°F) away from light. The prepared syringe can be stored in a refrigerator at 2°C-8°C (36°F-46°F) ≤-20°C (-4°F) for up to 24 hours, and up to 4 hours of the 24 hours can be stored at room temperature 15°C-25°C (59°F-77°F). The prepared syringe should be protected from light. Before administration, clazakizumab should be brought to room temperature by storage without refrigeration for 30 to 60 minutes before use.

[0157] program

[0158] This is a Phase I / II clinical study of clazakizumab in highly HLA-sensitized patients awaiting a kidney transplant. The study is intended to last 3 years. HLA antibodies are detected using a solid phase assay system currently used at the Cedars-Sinai Medical Center HLA Laboratory.

[0159] Typically, patients entering the study initially receive PLEX (5-7 treatments) + IVIG one week after IVIG, and receive 25mg clazakizumab subcutaneously (SC). If no safety / tolerance / efficacy issues are observed after the initial dose, these patients receive 5 additional injections every four weeks (Q4W). If the patient receives an HLAi transplant, clazakizumab is continued for 6 months (starting from the 5th day after transplantation) with 6 doses of 25mg SC Q4W after transplantation. A protocol biopsy is performed 6 months after transplantation to assess the ABMR evidence of the allograft, including C4d staining and TG performed using the Banff 2015 criteria. If improvement is seen after the 6th dose of clazakizumab, the patient will continue to receive another 6 doses within 6 months. Patients who show evidence of persistent allograft dysfunction may therefore have a non-protocol biopsy. Patients who receive 12 doses of clazakizumab after transplantation will receive a 12M protocol biopsy. In the event that the patient did not show improvement after receiving 6 doses of clazakizumab, no further treatment was given and the patient returned on Day 365 for a final study visit.

[0160] Specifically, at the time of transplantation (day 0), a single dose of IVIG was administered (IVIG #1). A second dose of IVIG was administered on day 1. The treatment period then began. Clazakizumab was administered 6 times, on days 5 ± 2d, 30 ± 7d, 60 ± 7d, 90 ± 14d, 120 ± 14d, and 150 ± 14d, respectively.

[0161] Figure 7Depicted is the study timeline of patient ClazaDes03 and his creatinine levels over time. Patient "ClazaDES03" is a 32-year-old male with a history of end-stage renal disease secondary to unknown etiology. His status was alive after an unrelated kidney transplant in 2012 but failed in 2016. For patient "ClazaDES03", the transplant was performed after the first dose of clazazumab (25 mg subcutaneously) and before the second dose of clazazumab. The patient received PLEX (5-7 times) on day -15; 2g / kg IVIG on day 0; pre-transplant #1 clazakizumab (25mg SQ) on day 7 (April 5, 2018); received a kidney transplant from a deceased donor on day 21 (April 29, 2018); post-transplant #1-#6 clazakizumab (25mg SQ) at approximately monthly intervals; induced with alemtuzumab (CAMPATH 1H) and maintained on tacrolimus, mycophenolate mofetil (MMF), and prednisone. When the renal allograft biopsy was performed on June 28, 2018 ( Figure 8 ) was examined and showed suboptimal creatinine levels, monthly dosing of Belatacept was started on August 14, 2018. On October 23, 2018, the 6M biopsy ( Fig. 9 ). Starting October 31, 2018, the second round (#7-#12) of clazakizumab administration (#7 post-transplant clazakizumab) was started. For this patient, no donor-specific antibodies are present since transplantation.

[0162] Anti-HLA antibodies can occur naturally or as a result of a previous pregnancy, blood transfusion, or previous transplant. Patients treated with 6 doses of clazakizumab for desensitization underwent blood sampling for HLA antibodies, as well as other monitoring blood samples and immunological studies. If patients received an HLAi transplant during the study, they will receive a standard post-transplant immunosuppression regimen and 6 doses of 25 mg clazakizumab subcutaneously every 4 weeks with immune monitoring. Immune monitoring in blood samples included monitoring for Treg, Tfh, Th17, and B cell subsets, as well as IL-6 and CRP, which was performed at the Cedars-Sinai Transplant Immunology Laboratory.

[0163] result

[0164] Figure 1Shown is the DSA profile of the subject "ClazaDES01" in the study, a 50-year-old African American female with a history of end-stage renal disease (ESRD) secondary to biopsy-proven focal segmental glomerulosclerosis (FSGS) and who has been on dialysis since November 2008 (i.e., a waiting time of approximately 10 years for a B+ blood type), with a calculated population reactive antibodies (cPRA) of 58%. The patient's sensitization events included pregnancy x 4 and blood transfusions.

[0165] Figure 2 The DSA spectra of subject "ClazaDES05" before and after transplantation are shown. (Median fluorescence intensity, MFI). Subject "ClazaDES05" is a 36-year-old female with a history of ESRD secondary to IgA nephropathy, and she has been receiving dialysis since June 2008 (i.e., a waiting time of about 10 years for A+ blood type), with 100% cPRA. The patient's sensitization events included previous transplants and blood transfusions. Subject "ClazaDES05" received a deceased donor kidney transplant after 4 doses of ClazadES05. The patient underwent 2 DSAs (Class I and Class II) before and after transplantation. The DSA intensity of Class I decreased from MFI>12,500MFI at the time of transplantation to MFI=0 10 days after transplantation, and the DSA intensity of Class II decreased from MFI>17,500 at the time of transplantation to MFI>3250 10 days after transplantation. According to the study protocol, patients received clazakizumab monthly for 6 months after transplantation.

[0166] Figure 3A Total C-reactive protein levels in the clazakizumab desensitization study are shown. Overall, C-reactive protein (CRP) decreased from baseline to almost zero by month 2. The number of subjects included in the analysis at each time point is indicated in parentheses. Figure 3B Individual C-reactive protein amounts from baseline to month 7 in the clazakizumab desensitization study are shown.

[0167] Figure 4 The total MFI over time from before plasmapheresis (PLEX) (pre-PLEX) to the 5th dose of clazazumab (N=9) is shown. Typically, MFI tends to rebound approximately 1-3 months after completion of PLEX / IVIG. Here, with monthly clazazumab injections, the total MFI remained reduced over time compared to pre-PLEX. To date, 3 patients have undergone transplantation. Patients ClazaDES01 and ClazaDES03 underwent transplantation after the 1st dose of clazazumab. Patient ClazaDES05 underwent transplantation after the 4th dose of clazazumab injection.

[0168] For patient "ClazaDES03", Figure 7 His creatinine (mg / dL) levels over time are shown, comparing before desensitization therapy and after desensitization therapy and kidney transplantation. Post-transplantation, lower levels of creatinine were maintained with two rounds of post-transplant clazakizumab administration. Figure 8 A 2 month renal transplant biopsy of patient "ClazaDES03" is shown. In this biopsy, mild acute tubular injury was present; mild to moderate atherosclerosis and mild arteriolar sclerosis, consistent with donor disease; no diagnostic evidence of acute rejection (borderline for cell-mediated rejection at best according to the Banff criteria); mesangial IgA deposition without associated glomerular proliferative changes. As there was no IgA staining on the biopsy from this patient's previous renal transplant in 2012, it was considered that the IgA present on this biopsy may be donor related. Fig. 9 A 6-month renal allograft biopsy of patient "ClazaDES03" is shown. In this biopsy, there is acute tubular necrosis with rare isometric epithelial blebs; mild tubulointerstitial inflammation (most borderline change for cell-mediated rejection); arteriosclerosis; and minimal interstitial fibrosis / tubular atrophy. Rare isometric epithelial blebs were detected and may be related to acute calcineurin inhibitor toxicity from recent IVIG treatment. There are no diagnostic features of antibody-mediated rejection or polyomavirus nephropathy.

[0169] Overall, desensitization therapy with clazakizumab reduced HLA antibodies and allowed transplantation in at least 40% of the patients in the study.

[0170] From March to November 2018, a total of ten patients were enrolled. Nine patients were allowed to receive a transplant: eight patients were transplanted during the study, and the ninth patient was transplanted two months after the study was completed. Four patients have reached the 12-month study period. All infusions received were well tolerated. No graft loss or patient death was observed, and there were no major infections attributed to clazakizumab or requiring discontinuation of clazakizumab. Renal function was stable at six months. Demographic information and immunological / transplant characteristics are summarized in Tables 1 and 2.

[0171] Table 1. Demographic information and baseline characteristics of the 9 subjects who underwent transplantation.

[0172]

[0173] Table 2. Immunological and transplant characteristics of the 9 subjects who received transplants.

[0174]

[0175] ^: 1 patient received 0MM

[0176] All patients except the ninth underwent a 6-month protocol biopsy. Two patients (25%) had biopsy-confirmed rejection: 1 patient had chronic active cell-mediated rejection, Banff grade 1A; 1 patient had chronic active antibody-mediated rejection and cell-mediated rejection, Banff grade 1B. Both patients responded to treatment according to the study center's standard of care protocol.

[0177] 7 of 9 transplant patients (78%) had DSA before desensitization and at the time of transplantation. Only 3 patients (33%) had DSA detected at one month; 2 patients (22%) had DSA detected at 3 months; and no patient had DSA detected at six months (6M), nine months (9M) and 12 months (12M) (including patients with DSA detected at one month and three months). The DSA MFI values ​​(mean ± SD) before desensitization, at the time of transplantation, at 1 month and 3 months were as follows: 11060 ± 6990, 7980 ± 6260, 1923 ± 3973, 1040 ± 2650 and 0 ± 0 for 6M, 9M and 12M, respectively. Mean DSA MFI was significantly decreased when comparing pre-desensitization vs. 1M (p=0.0004) and 3M (p=0.0001) and at transplant vs. 1M (p=0.007) and 3M (p=0.001).

[0178] There were seven SAEs, but all were considered unrelated to clazakizumab. These included wound dehiscence requiring wound reclosure (1 SAE), hematuria and UTI (1 SAE), and bacteremia (1 SAE) before receiving the first dose of study drug, right external iliac artery thrombosis with graft loss (1 SAE), persistent surgical site pain requiring CT-guided drainage of perigraft fluid with MSSA (1 SAE), biopsy-proven chronic active ABMR and chronic thrombocytopenia due to delay in clazakizumab administration d / t infection (1 SAE), and perinephric fluid collections requiring CT-guided drainage (1 SAE).

[0179] Primary and secondary goals

[0180] To determine whether clazakizumab treatment can significantly reduce or eliminate ABMR episodes and C4d deposition in incompatible allografts transplanted into highly HLA-sensitized patients after clazakizumab desensitization. Allograft function will be assessed for up to 6-12 months (6-12M) after transplantation, and renal function will be determined using serum creatinine (SCr), Modification of Diet in Renal Disease (MDRD) calculated GFR (creatinine clearance (CrCl) will be estimated using the Schwartz equation for patients younger than 18 years), and DSA levels. A protocol biopsy will be performed 6M after clazakizumab treatment. In addition, several immunological assays of blood samples will be evaluated at time points before and after the start of clazakizumab treatment. These include:

[0181] Evaluate T reg Cells (CD4+, CD25+, FoxP3+CD127 dim )

[0182] Evaluate T fh Cells (CD4+, ICOS+, CXCR5+, IL-21+)

[0183] Assessment of circulating plasmablasts (CD19+, CD38+, CD27+, IL-6+)

[0184] Assessment of CRP and IL-6 levels

[0185] These secondary endpoints will help us understand the biology of the allogeneic immune response to the allograft and define the magnitude and mechanism of the beneficial effects of clazakizumab. Monitor viral PCR as per standard of care.

[0186] Inclusion Criteria

[0187] Age 15-75 years at screening. HS patients (cPRA ≥ 50%) on the UNOS list awaiting DD or LD kidney transplant. Previous history of pregnancy, blood transfusion and / or kidney transplant. Subject / parent / guardian must be willing to fully participate in the study requirements. Subject / parent / guardian must be able to understand and provide informed consent. Pneumococcal vaccination. Negative tuberculin (ppd) placement result or negative Quantiferon TB Gold result. These individuals must also have a sufficient waiting time on the UNOS list to allow for frequent provision of a history of a positive crossmatch (DD) or incompatibility (LD) with a positive flow cytometry (FCMX) and a negative complement dependent cytotoxicity (CDC+) crossmatch. Patients undergoing HLAi transplantation after desensitization will have a negative CDC CMX at a 1:2 dilution, FCMX <225 channel shift, and a DSA at an acceptable MFI as previously defined.

[0188] Exclusion criteria

[0189] Multi-organ transplantation (eg, kidney and pancreas).

[0190] Intolerance to treatment with clazakizumab or other IL-6 inhibitors.

[0191] Women who are breastfeeding or pregnant.

[0192] Women of childbearing potential and male partners of women of childbearing potential who are unwilling or unable to practice FDA-approved forms of contraception during the study period and for 5 months after the last dose.

[0193] HIV positive subjects.

[0194] Subjects who tested positive for HBV by HBVeAg / DNA or for HCV infection [positive anti-HCV (EIA) and confirmed HCV RIBA].

[0195] Subjects with latent or active TB. Subjects must have a negative Quantiferon TB Gold test result.

[0196] Recent recipient of any licensed or investigational live attenuated vaccine (including but not limited to any of the following: adenovirus [oral adenovirus type 7 vaccine], varicella [Varivax], hepatitis A [VAQTA], rotavirus [Rotashield], yellow fever [YF-Vax], measles and mumps [live measles and mumps virus vaccine], measles, mumps and rubella vaccine [MMR-II], Sabin oral polio vaccine, and rabies vaccine [IMOVAX Rabies ID, RabAvert]) within two months of the screening visit.

[0197] Significantly abnormal general serum screening laboratory results, defined as ANC < 2000 and platelet count < 100 × 10 3 / ml, SGOT or SGPT>1.5×upper limit of normal.

[0198] Individuals deemed unable to comply with the protocol.

[0199] Subjects with active CMV or EBV infection with or without incompatible disease as defined by CMV-specific serology (IgG or IgM) and confirmed by quantitative PCR (quantitative PCR cutoff defined as having >50 copies of CMV or EBV DNA / PCR) were allowed to use study agents within 4 weeks of participation.

[0200] History or active inflammatory bowel disease or diverticular disease or gastrointestinal perforation

[0201] Recent infection (within the past 6 weeks of screening) requiring any antibiotic use (oral, parenteral, or topical).

[0202] Current or previous (within 5 years) malignancy other than basal cell carcinoma, completely excised cutaneous squamous cell carcinoma, or non-recurrent (within 5 years) cervical carcinoma in situ.

[0203] Applicants also conducted a study that evaluated the cost / benefit analysis of desensitization compared to dialysis. The costs associated with transplantation after desensitization include all medications, organ acquisition, treatment of rejection episodes, and the cost of returning to dialysis for patients who lose their allografts (compared favorably to the cost of remaining on dialysis for the same period of time). Most important is the survival benefit of transplantation in this cohort. At 3 years, the mortality rate for patients who were desensitized and transplanted was 3.5%, while the mortality rate for patients who had been on dialysis was 22.8%.

[0204] If rejection occurs during the study (confirmed by biopsy), patients are treated with a “pulse” of methylprednisolone (10 mg / kg / day, maximum 1000 mg for >100 kg for 3 days) and antithymocyte globulin (1.5 mg / kg daily × 4) for episodes of cell-mediated rejection that do not respond to the loading steroid. Patients who experience a recurrent antibody-mediated rejection (ABMR) episode after study drug treatment will initially receive daily IV × 3 doses of loading methylprednisolone (10 mg / kg / day, maximum 1000 mg for >100 kg), followed by IV × 1 dose of IVIG 10% solution 2 gm / kg (maximum 140 g for >70 kg), depending on severity, followed by IV × 1 dose of rituximab (375 mg / m 2 In cases where rapid deterioration of allograft function is noted and / or thrombotic microangiopathy is diagnosed, the patient will receive 3-5 plasmapheresis sessions followed by weekly IV anti-C5 Continue for 4 weeks (1200 mg in week 1, followed by 900 mg / week for an additional 3 weeks). The efficacy of treatment will be evaluated by determining improvement in renal function, monitoring DSA response, and repeating allograft biopsy if necessary. For the purposes of this study, ABMR is defined as follows: worsening of allograft function (defined as a decrease of >30% from baseline) in high-risk graft recipients (i.e., sensitized patients with a history of DSA) as measured by serum Cr and eGFR; associated with the presence (usually increased intensity) of DSA measured by LUMINEX technology; biopsy evidence based on the BANFF 2015 grade, including: capillaritis, inflammation, and C4d deposition.

[0205] Adverse events (AEs) and critical AEs were monitored after treatment with clazakizumab. These included careful attention to infectious complications that may be associated with clazakizumab treatment. Our study group evaluated infectious complications associated with IVIG+rituximab desensitization and alemtuzumab induction therapy followed by maintenance therapy with tacrolimus, MMF, and prednisone. The use of this desensitization regimen followed by alemtuzumab induction did not increase the risk of common or critical infections after transplantation compared with patients in the low-risk group. Critical infections were defined as any viral infection and fungal or bacterial infection requiring iv antibiotics or hospitalization. Therefore, the risk of infection in the study group (clazakizumab) after ABMR treatment will likely be similar and comparable to that of non-sensitized patients. Vaccination against Streptococcus pneumoniae was required for all patients enrolled in this study.

[0206] In the study, all study patients, regardless of their cytomegalovirus (CMV) status, received IV ganciclovir for 6 months after kidney transplantation as an inpatient and valganciclovir as an outpatient, with dose adjustments for renal function used. Fungal prophylaxis was completed with 100 mg of fluconazole daily for 1 month after transplantation. 80 mg of trimethoprim and 400 mg of sulfamethoxazole daily were used for 12 months after transplantation. Pneumocystis jiroveci pneumonia and bacterial prophylaxis were completed with azoles. Study patients were tested for CMV, Epstein-Barr virus, parvovirus B-19, polyomavirus BK, and JC using viral polymerase chain reaction assays monthly for 6 months after transplantation.

[0207] Various embodiments of the present invention are described above in the detailed description. Although these descriptions directly describe the above-mentioned embodiments, it should be understood that modifications and / or changes to the specific embodiments shown and described herein may be conceived by those skilled in the art. Any such modifications or changes falling within the scope of this specification are also intended to be included therein. Unless otherwise specified, it is the intention of the inventor to give the words and phrases in the specification and claims the ordinary and customary meanings of those of ordinary skill in the applicable field.

[0208] The foregoing description of various embodiments of the present invention known to the applicant at the time of filing this application has been presented, and is intended for purposes of illustration and description. This specification is not intended to be exhaustive, nor is it intended to limit the invention to the precise form disclosed, and many modifications and variations are possible in light of the above teachings. The described embodiments serve to explain the principles of the present invention and its practical application, and to enable others skilled in the art to utilize the present invention in various embodiments with various modifications suitable for the particular use under consideration. Therefore, it is intended that the present invention is not limited to the specific embodiments disclosed for carrying out the present invention.

[0209] Although specific embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that, based on the teachings herein, changes and modifications may be made without departing from the present invention and its broader aspects and scope, and therefore, the appended claims are intended to include within their scope all such changes and modifications that are within the true spirit and scope of the present invention. It will be understood by those skilled in the art that, in general, the terms used herein are generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including but not limited to", the term "having" should be interpreted as "having at least", the term "comprising" should be interpreted as "including but not limited to", etc.).

[0210] As used herein, the terms "comprising" or "including" are used to refer to compositions, methods and corresponding components thereof that are useful for the embodiments, but open to including unspecified elements (whether useful or not). Those skilled in the art will understand that, in general, the terms used herein are generally intended to be "open" terms (e.g., the term "comprising" should be interpreted as "including but not limited to", the term "having" should be interpreted as "having at least", the term "including" should be interpreted as "including but not limited to", etc.). Although the open term "comprising" is used herein as a synonym for terms such as including, comprising or having to describe and claim the present invention, alternative terms such as "consisting of" or "consisting essentially of" may be used to describe.

[0211] Unless otherwise indicated, all numbers indicating quantities are to be understood in all cases as being modified by the term "about". The term "about" may refer to ±10% of the referenced value. If clearly defined and provided in the claims, the term "about" may refer to ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the referenced value; for example, a claim may state that a value is about X, where about ±6%.

[0212] In the case of providing a range of values, each numerical value between and including the upper and lower limits of the range is expected to be disclosed herein. It should be understood that any numerical range listed herein is intended to include all subranges contained therein. For example, the range "1 to 10" is intended to include all subranges between and including the listed minimum value 1 and the listed maximum value 10; that is, having a minimum value equal to or greater than 1 and a maximum value equal to or less than 10. Because the disclosed numerical ranges are continuous, they include every value between the minimum and maximum values.

Claims

1. Use of clazakizumab in the preparation of a medicament for use in a method for reducing and / or eliminating donor-specific antibodies (DSA) in a subject sensitized by human leukocyte antigen (HLA), wherein the medicament comprises clazakizumab or an interleukin-6 (IL-6) binding fragment of clazakizumab having a V sequence comprising a CDR1, CDR2 and CDR3 polypeptide as shown in SEQ ID NO: 1, 2 or 3 and 4, respectively. H Polypeptides and V sequences comprising CDR1, CDR2 and CDR3 polypeptides as shown in SEQ ID NOs: 5, 6 and 7, respectively. L polypeptide; and one or more pharmaceutically acceptable excipients, wherein the subject is in need of a solid organ transplant, and wherein the solid organ is a kidney.

2. The use of claim 1, wherein clazakizumab or the IL-6 binding fragment of clazakizumab is administered prior to solid organ transplantation.

3. The use of claim 1, wherein clazakizumab or the IL-6 binding fragment of clazakizumab is administered after and / or during solid organ transplantation.

4. The use of claim 1, wherein clazakizumab or the IL-6 binding fragment of clazakizumab is administered before and after solid organ transplantation.

5. The use of claim 1, wherein the subject is further administered standard of care treatment comprising intravenous immunoglobulin (IVIG) administration, rituximab administration, plasmapheresis, or a combination thereof.

6. The use of claim 5, wherein the standard of care therapy is administered prior to clazakizumab or the IL-6 binding fragment of clazakizumab.

7. The use of claim 1, wherein clazakizumab or the IL-6 binding fragment of clazakizumab is administered subcutaneously or intravenously.

8. The use of claim 1, wherein clazakizumab or the IL-6 binding fragment of clazakizumab is subcutaneously administered at an average dose of 0.1-1 mg / month, 1-5 mg / month, 5-10 mg / month, 10-20 mg / month, 20-30 mg / month or 30-40 mg / month for at least one month to up to 18 months.

9. The use of claim 1, wherein multiple doses of clazakizumab or an IL-6 binding fragment of clazakizumab are administered at monthly intervals for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months.

10. The use of claim 1, wherein clazakizumab or the IL-6 binding fragment of clazakizumab is subcutaneously administered 1, 2, 3, 4, 5 or 6 times before solid organ transplantation and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 times after solid organ transplantation at an average dose of 10-30 mg / time.

11. The use of claim 1, wherein the subject is a human.

12. The use of claim 1, wherein the subject is further administered one or more anti-infective agents.

13. The use of claim 12, wherein the one or more anti-infective agents are administered together with or after the solid organ transplant.

14. The method of claim 12, wherein the anti-infective agent comprises ganciclovir, valganciclovir, fluconazole, trimethoprim, sulfamethoxazole, azole or a combination thereof.

15. The use of claim 1, wherein the subject is further administered a standard of care treatment comprising intravenous immunoglobulin (IVIG) administration, rituximab administration, plasmapheresis, or a combination thereof; an anti-infective agent; or a combination of a standard of care treatment and an anti-infective agent.

16. The use of claim 1, wherein the reducing and / or eliminating further comprises selecting a human subject having a calculated population reactive antibody (cPRA) of 50% or more, or performing a population reactive antibody assay and identifying a human subject having a cPRA of 50% or more.

17. The use of claim 1, wherein following solid organ transplantation, the subject shows no detectable evidence of antibody-mediated rejection of the solid organ transplant, no detectable evidence of viral infection, or both.

18. The use of claim 1, wherein after solid organ transplantation, the subject is further administered antibody induction therapy comprising alemtuzumab, antithymocyte globulin, or both; administered immunosuppressive therapy comprising tacrolimus, mycophenolate mofetil, prednisone, or a combination thereof; or administered antibody induction therapy and immunosuppressive therapy.

19. The use of claim 1, wherein the subject in need of a solid organ transplant has undergone a solid organ transplant, and the reduction and / or elimination further comprises performing one or more immune monitoring of the subject, comprising measuring a blood sample of the subject to quantify the level of markers including CRP, Treg, Tfh, Th17, B cells, IL-6, plasma cells, plasmablast IgG, or a combination thereof.

20. The use of claim 19, wherein further administration of clazakizumab or an IL-6 binding fragment of clazakizumab is stopped or limited to no more than an additional 6 months when the immune monitoring indicates improvement based on one or more decreased levels of the marker compared to baseline measurements performed at or before the time of solid organ transplantation or based on one or more decreased levels compared to levels obtained from previous immune monitoring; and when the immune monitoring indicates poor performance based on comparable or increased levels of the marker compared to baseline measurements or to levels obtained from previous immune monitoring, administering one or more doses of clazakizumab or an IL-6 binding fragment of clazakizumab.

21. The use of claim 1, wherein the subject in need of a solid organ transplant has undergone a solid organ transplant, and the reducing and / or eliminating further comprises measuring the amount of glomerular filtration rate, DSA, or both after the solid organ transplant.

22. The use of claim 21, wherein further administration of clazakizumab or an IL-6 binding fragment of clazakizumab to the subject is stopped or limited to no more than an additional 6 months when the amount of glomerular filtration rate, DSA, or both is similar to or decreased compared to a baseline level measured at or before a solid organ transplant; and when the amount of glomerular filtration rate, DSA, or both is higher than the baseline level, the subject is administered one or more doses of clazakizumab or an IL-6 binding fragment of clazakizumab.

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