WO2025226628A1

WO2025226628A1 - Use of il-6 inhibitors for preventing or inhibiting lung transplantation rejection in a subject

Info

Publication number: WO2025226628A1
Application number: PCT/US2025/025686
Authority: WO
Inventors: Derek E. BYERS; Ramsey HACHEM
Original assignee: Recordati Rare Diseases Inc
Current assignee: Recordati Rare Diseases Inc
Priority date: 2024-04-22
Filing date: 2025-04-22
Publication date: 2025-10-30
Anticipated expiration: 2026-10-22

Abstract

This invention relates to the use of IL-6 inhibitors for preventing or inhibiting lung transplantation rejection in a subject. In particular, this invention relates to methods of treatment of antibody mediated rejection (AMR) of a lung transplant in a subject in need thereof comprising a combination therapy of (i) administration to the subject of an antibody or fragment which is capable of inhibiting human IL-6; and (ii) administration to the subject of an immunosuppressive therapy, wherein the immunosuppressive therapy does not comprise any antibody or fragment thereof which is capable of inhibiting human IL-6.

Description

USE OF IL-6 INHIBITORS FOR PREVENTING OR INHIBITING LUNG TRANSPLANTATION REJECTION IN A SUBJECT

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/637,004, filed on April 22, 2024, which is hereby incorporated by reference in its entirety.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on April 16, 2025, is named P93947PC_sequence_listing_2025- 04-16 and is 8,692 bytes in size.

FIELD

BACKGROUND

Lung transplantation (LT) is the ultimate treatment option for individuals with advanced lung disease, and approximately 2,500 people undergo lung transplantation annually in the US (1). LT improves quality of life and survival; however, long-term outcomes remain disappointing, and the median survival after LT is 6.7 years (2). Chronic lung allograft dysfunction (CLAD) is the leading cause of death beyond the first year after LT (2,3). Two phenotypes of CLAD are recognized; bronchiolitis obliterans syndrome (BOS) is characterized by an obstructive ventilatory defect without radiographic opacities, and restrictive allograft syndrome (RAS) is characterized by a restrictive ventilatory defect and upper-lobe predominant fibrotic opacities on imaging studies (4,5). A mixed phenotype with features of both BOS and RAS is also recognized. CLAD typically follows a progressive clinical course that culminates in respiratory failure and death or retransplantation.

Indeed, the median survival after the diagnosis of CLAD is approximately 2.5 years (6). Although all phenotypes are generally progressive, RAS and the mixed phenotype are typically more aggressive and result in significantly worse survival than BOS (7-9). CLAD is widely viewed as the end-result of innate and alloimmune insults that damage the allograft (10,11). These include gastroesophageal reflux disease (GERD), primary graft dysfunction (PGD), acute cellular rejection (ACR), lymphocytic bronchiolitis, donor-specific antibodies (DSA) to mismatched human leukocyte antigens (HLA), antibody-mediated rejection (AMR), and community-acquired respiratory viral (CARV) infections (12-22).

AMR is an increasingly recognized form of lung rejection, in large part because of improved awareness and the development of a standardized definition that facilitates research and communication between centres.

According to the International Society for Heart and Lung Transplantation (ISHLT) definition of AMR, the diagnosis is based on 5 criteria: DSA, abnormal lung histology, allograft dysfunction, complement component 4d (C4d) deposition, and the exclusion of other potential causes of allograft dysfunction (23). Diagnostic certainty depends on the number of criteria present; for example, a diagnosis of definite AMR is made if all 5 criteria are present, and a diagnosis of probable AMR is made if 4 of the 5 criteria are present.

The incidence of AMR has varied in previous studies depending on study design and diagnostic certainty used to identify cases. In several older single-centre retrospective cohort studies the incidence of AMR varied between 4-25% (20, 21, 24-26). In more recent multicentre prospective studies that used donor-derived cell-free DNA (cfDNA) as part of clinical monitoring, the incidence of AMR was 27% (27,28). A variety of immunosuppressive treatments focused on antibody-depletion have been used, and generally, the standard of care for AMR patients, is a multi-drug regimen including various combinations of high-dose corticosteroids, intravenous immune globulin (IVIG), Rituximab, Bortezomib or Carfilzomib, anti-thymocyte globulin (ATG), and plasma exchange (PLEX) (20, 21, 24-28). However, there have been no randomized controlled trials (RCT) and no head-to-head comparisons of different regimens to guide management. Moreover, outcomes after the diagnosis of AMR are dismal. Approximately 20% of patients die within 30 days, 50% die within 1 year, and 80% die within 2 years of the diagnosis of AMR (20, 21, 27). The majority of survivors develop CLAD early after the diagnosis of AMR. In a multicentre study, 73% developed severe CLAD, defined as >50% loss of lung function compared to baseline, within 1 year of the diagnosis of AMR (20, 21, 24, 25). Indeed, the leading causes of death after AMR are progressive CLAD and refractory AMR (20, 21, 24-27). Clearly, identifying better treatment regimens for the management of AMR is a critical unmet need for LT recipients.

All documents referred to herein are incorporated by reference in their entirety.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

SUMMARY OF INVENTION

An object of the invention is to provide a treatment for antibody mediated rejection (AMR) in lung transplant recipients.

In a first aspect, the present invention provides a method of treatment of antibody mediated rejection (AMR) of a lung transplant, the method comprising a combination therapy of: (i) administration to the subject of an antibody or fragment which is capable of inhibiting human IL-6; and (ii) administration to the subject of an immunosuppressive therapy, wherein the immunosuppressive therapy does not comprise any antibody or fragment thereof which is capable of inhibiting human IL-6.

In a second aspect, the present invention provides an anti-IL-6 antibody or fragment for use in treatment of an antibody mediated rejection (AMR) of a lung transplant, wherein the treatment comprises a combination therapy of administration of the antibody or fragment and administration of an immunosuppressive therapy, wherein the immunosuppressive therapy does not comprise any antibody or fragment thereof which is capable of inhibiting human IL-6.

In a third aspect, the present invention provides a use of an anti-IL-6 antibody or fragment in treatment of antibody mediated rejection (AMR) of a lung transplant, wherein the treatment comprises a combination therapy of administration of the antibody or fragment and administration of an immunosuppressive therapy, wherein the immunosuppressive therapy does not comprise any antibody or fragment thereof which is capable of inhibiting human IL-6.

In a fourth aspect, the present invention provides a use of an anti-IL-6 antibody or fragment for the manufacture of a medicament for treatment of antibody mediated rejection (AMR) of a lung transplant, wherein the medicament is to be administered in a combination therapy with administration of an immunosuppressive therapy, wherein the immunosuppressive therapy does not comprise any antibody or fragment thereof which is capable of inhibiting human IL-6.

DETAILED DESCRIPTION

IL-6 is a pleiotropic cytokine that can be secreted by many cell types upon appropriate stimulation and promotes inflammatory, immune, and fibrotic responses. Different modes of IL-6 signalling exist.

In classic signalling, IL-6 binds to its receptor (IL-6R), which is devoid of signalling capacity, and the complex of IL-6 and IL6R binds to glycoprotein 130 (gpl30) which dimerizes and initiates intracellular signalling (29).

In trans-signalling, the soluble form of IL-6R (sIL-6R) binds to IL-6, and the complex binds to gpl30 inducing dimerization and intracellular signalling (29). This enables the transmission of IL-6R-initiated signals to cells that do not express the receptor thereby expanding the activation capacities of IL-6.

Both classic and trans-signalling activate signal transduction pathways leading to janus kinase (JAK)-dependent signal transducer and activator of transcription (STAT) 3 signalling as well as PI3K/AKT and RAS/MAPK pathways (30).

Trans-presentation is a third mode of IL-6 signalling where IL-6 binds to IL-6R in the cytoplasm of dendritic cells and the complex is transported to the plasma membrane where it binds to gpl30 expressed by CD4⁺ T-cells (31). Trans-presentation was recently discovered in mice but has not been confirmed in humans.

IL-6 expands effector and memory CD8⁺ T-cells by augmenting the expression of IL-2 and receptor CD122/CD25 and is essential for the differentiation of naive CD4⁺ T-cells towards the Thl7 phenotype (32-34). IL-17 produced by Thl7 cells promotes neutrophil proliferation and migration, endothelial cell activation, and fibroblast activation and proliferation which result in cytopathic responses implicated in acute and chronic rejection (33, 34). IL-6 was initially identified as B-cell stimulating factor 2 (BSF-2) because of its role in promoting antibody synthesis by B-cells (35-37).

Furthermore, IL-6 in conjunction with other cytokines is responsible for normal antibody production and is critical for the induction of follicular helper T-cells (Tfh) as well as the production of IL-21 which regulates immunoglobulin synthesis (38, 39). IL- 6 is also crucial for B-cell differentiation into plasmablasts and for enhancing plasmablast survival (40, 41).

Blocking IL-6 signalling effectively reduces B-cell activation, plasmablast differentiation, and antibody production (42-44). IL-6 also promotes innate immune responses. NK cells express IL-6R and are activated by IL-6 to induce endothelial cell cytotoxicity (45). IL-6 promotes fibrosis by inducing the differentiation of fibroblasts into myofibroblasts and participating in vascular smooth muscle cell and endothelial cell proliferation and activation (46). These are all important components of the pathogenesis of AMR. High concentrations of IL-6 in bronchoalveolar lavage (BAL) fluid and peripheral blood are seen with severe PGD after LT and are associated with prolonged length of stay in the intensive care unit and the hospital. However, IL-6 levels have not been consistently associated with acute cellular rejection and there have been no published results of IL-6 levels in AMR after LT. The inventors have surprisingly found that blocking IL-6 signalling may be a potential therapeutic approach for treatment of AMR in LT patients, especially when delivered with the standard of care for AMR in LT patients.

IL-6 signalling is a therapeutic target in solid organ transplantation. In a non-human primate (NHP) model of lung transplantation, all cynomolgus monkeys rejected their lung allografts in spite of conventional immunosuppression with tacrolimus, mycophenolate mofetil (MMF), and steroids, and all had DSA at the time of rejection (47). Tocilizumab (IL6R inhibitor) has also been studied for kidney (44, 49-54) and cardiac (48) AMR.

Clazakizumab (IL6 inhibitor) has been studied for AMR in kidney transplants (55, 56). In a pilot randomised controlled trial (RCT), clazakizumab attenuated the decline in GFR and depleted DSA; however, 25% of treated patients developed serious infections and 10% developed complicated diverticulitis (56). A Phase 3 RCT examining the efficacy and safety of clazakizumab for the treatment of chronic AMR after kidney transplantation is currently enrolling patients (NCT03744910).

High concentrations of IL-6 in bronchoalveolar lavage (BAL) fluid and peripheral blood are seen with severe PGD after LT and are associated with prolonged length of stay in the intensive care unit and the hospital (57-59). In contrast, IL-6 levels have not been consistently associated with ACR (60, 61). Of note, there is no association between serum and BAL fluid concentrations of IL-6 as serum concentrations do not reflect intrapulmonary production (57, 58, 60).

There have been no published results of IL-6 levels in AMR after LT. There have been no published studies of using IL-6 inhibitors, such as siltuximab, to treat AMR after LT.

The inventors have surprisingly found that from immunofluorescence staining for IL-6 in lung biopsies of patients with AMR, there is evidence of intrapulmonary IL-6 expression. These data illustrate increased IL-6 levels in serum and increased expression in lung allografts with AMR.

There are several FDA approved IL-6 inhibitors available today for treatment of rheumatoid arthritis, giant cell arteritis, systemic sclerosis-associated interstitial lung disease, cytokine release syndrome, idiopathic multicentric Castleman's disease (MCD), and neuromyelitis optica.

One example of a known IL-6 inhibitor is Siltuximab which is the only monoclonal antibody to IL-6 that is FDA-approved. In previous studies, Siltuximab has been used in patients with hematologic diseases/malignancies, solid tumors and MCD (Multicentric Castleman's Disease).

Siltuximab is a chimeric (human-murine) immunoglobulin G monoclonal antibody that binds IL-6 and is approved for the treatment of idiopathic MCD. Siltuximab prevents IL-6 from binding to both soluble and membrane bound IL-6R with high affinity and specificity resulting in deactivation of IL-6. However, there have been no published reports describing the use of Siltuximab after LT or for the treatment of AMR after any solid organ transplant. Further, there have been no published reports of use of Siltuximab in combination with routine treatment for AMR.

As discussed above, there is an unmet need for improved treatments of AMR in lung transplant patients. The present invention relies on the use of IL-6 inhibitors, such as but not limited to Siltuximab, to prevent, treat, inhibit, or block AMR or the recurrence thereof in lung transplant patients. These IL-6 inhibitors, such as Siltuximab, are to be used in combination with the routine immunosuppressive therapy (standard of care) for AMR in lung transplant recipients.

These and other aspects of the disclosure are described in detail below.

Unless indicated otherwise, all technical and scientific terms used herein will have their common meaning as understood by one of ordinary skill in the art to which this invention pertains.

It is contemplated that any method, use or treatment described herein can be implemented with respect to any other method, use or treatment described herein. All features disclosed herein in connection with any particular aspect are applicable to each of the other aspects, mutatis mutandis. In particular, all features disclosed herein in connection with the first aspect are applicable to each of the second to fourth aspects, mutatis mutandis. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

The term "comprises" or "comprising" will take its usual meaning in the art, namely indicating that the component includes but is not limited to the relevant features (i.e. including, among other things). As such, the term "comprises" will include references to the component consisting essentially of (such as consisting of) the relevant features. The term "consists of" or "consisting of" will take its usual meaning in the art, namely indicating that the component includes and is limited to the relevant features.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The word "about" means plus or minus 5% of the stated number.

Where a numerical range is provided herein for any parameter, it is understood that all numerical subsets of that numerical range, and all the individual integer values contained therein, are provided as part of the invention. As used herein, the term "optionally" means that the subsequently described event(s) may or may not occur, and includes both event(s) which occur, and events that do not occur.

Methods of Treatment

The first aspect of the invention is a method of treatment of antibody mediated rejection (AMR) of a lung transplant in a subject in need thereof, the method comprising a combination therapy of:

(i) administration to the subject of an antibody or fragment which is capable of inhibiting human IL-6; and

(ii) administration to the subject of an immunosuppressive therapy, wherein the immunosuppressive therapy does not comprise any antibody or fragment thereof which is capable of inhibiting human IL-6.

The term "treatment", "treat" or "treating" as used herein, refers to therapeutic (curative) treatment including amelioration. Treatment also includes stopping the disease from developing or slowing further progression of the disease. For example, treatment may include preventing symptoms from worsening. The terms "prevention", "prevent" and "preventing" as used herein, refers to prophylaxis treatment i.e., action taken to prevent disease.

The terms "patient", "recipient" and "subject" are used interchangeably. By "a patient", "a subject" or "a recipient" we intend a human patient, subject or recipient. Typically the patient is an adult i.e. > 18 years old at the commencement of the treatment. Alternatively the patient may be a paediatric patient i.e. < 18 years old at the commencement of the treatment.

In a preferred embodiment of any of the aspects of the invention, the subject is a human.

In the context of this invention, "combination therapy" means that both (i) an antibody or fragment which is capable of inhibiting human IL-6; and (ii) an immunosuppressive therapy, wherein the immunosuppressive therapy does not comprise any antibody or fragment thereof which is capable of inhibiting human IL-6, are administered in combination as a treatment to the patient. By "in combination" we do not necessarily mean that the antibody or fragment which is capable of inhibiting human IL-6 is administered at the same time as the immunosuppressive therapy.

Antibody Mediated Rejection (AMR)

"AMR" and "antibody mediated rejection" are used interchangeably throughout this document. Antibody-mediated rejection (AMR) is a recognized cause of allograft dysfunction in lung transplant recipients. Diagnostic criteria and a working consensus definition are established by the International Society for Heart and Lung Transplantation (ISHLT) (23). AMR is a complex pathologic, serologic and clinical process.

A process of immune activation, whereby allospecific B-cells and plasma cells produce antibodies directed against donor lung antigens, is central to the concept of pulmonary AMR. The antigen-antibody complex may result in an amplified immune response, via both complement-dependent and independent pathways, which may result in lung tissue pathology and graft dysfunction to a variable degree. Complement is a multifunctional system of receptors, regulators and effector molecules that may amplify both innate and adaptive immunity contributing to the pathogenesis of AMR.

Key diagnostic criteria for AMR in lung transplant recipients may include the presence of antibodies directed toward donor human leukocyte antigens and characteristic lung histology with or without evidence of complement 4d within the graft. Exclusion of other causes of allograft dysfunction increases confidence in the diagnosis but is not essential. Pulmonary AMR may be clinical (allograft dysfunction which can be asymptomatic) or sub-clinical (normal allograft function). Both clinical and sub-clinical AMR were further sub-categorized into 3 mutually exclusive possibilities (definite, probable, and possible) by the ISHLT. These categories are based on the degree of certainty related to the presence or absence of a number of pathologic, serologic, clinical and immunologic criteria and are summarised in Table 1 (clinical) and Table 2 (subclinical).

Table 1 : Definition and Diagnostic Certainty of Clinical Pulmonary Antibody-mediated Rejection (23); + means item present; - means item absent or missing.

Table 2: Definition and Diagnostic Certainty of Sub-clinical Pulmonary Antibody- mediated Rejection (23); + means item present; - means item absent or missing.

For avoidance of doubt, there are five criteria that may be met to diagnose AMR in a lung transplant recipient. These five criteria that may be present to diagnose AMR are:

• Allograft dysfunction;

• Other potential causes of allograft dysfunction excluded;

• Abnormal lung histology;

• Lung Biopsy complement component (C4d) deposition; and

• Donor-specific antibodies (DSA).

Diagnostic certainty depends on the number of criteria present. Specifically, a diagnosis of definite clinical pulmonary AMR may be made if all five criteria are present, and a diagnosis of probable clinical pulmonary AMR may be made if any four of the five criteria are present. Specifically, a diagnosis of definite sub-clinical pulmonary AMR may be made if all three criteria (abnormal lung histology, lung biopsy C4d, and donorspecific antibodies) are present, and a diagnosis of probable sub-clinical pulmonary AMR may be made if any two of the three criteria (abnormal lung histology, lung biopsy C4d, and donor-specific antibodies) are present. Measurement and determination of each of these criteria can be achieved by any appropriate method known in the art.

In a particular embodiment of any aspect of the invention, the AMR is probable AMR or definite AMR.

AMR is increasingly recognized as a potential contributing factor to acute lung allograft dysfunction and the development of CLAD in pediatric lung transplant recipients (> 18 years of age). Although the frequency is unknown in children, AMR has clearly been documented across all pediatric age groups from infancy to early adulthood. Adult diagnostic criteria for lung allograft rejection are often applied to pediatric patients, with recent confirmation that these criteria are consistent in children.

After treatment according to the present invention, pulmonary AMR may stabilize, and/or improve. Improvement may be partial or complete. A complete response may be a return to baseline graft function if applicable, abolition of DSA titers and reversal of pathologic changes. A partial response may be an improvement in graft function if applicable, but not all parameters return to baseline. Stabilization may be defined as no further clinical deterioration.

Donor-specific anti-human leukocyte antigen (HLA) antibodies, are known to contribute to antibody-mediated rejection (AMR) in solid-organ transplantation. The best-characterized donor antigens are HLA, which are further divided, based on their structure and function, into HLA Class I and Class II. The presence of DSA at the time of transplant or detected de novo post-transplant is well described in renal transplantation, where it has been associated with compromised renal allograft survival. De novo DSA and an increase in DSA titers, perhaps via an anamnestic response, have also been associated with lung allograft dysfunction, occasionally in asymptomatic patients.

In the context of this invention, "donor-specific antibodies" is intended to mean antibodies to donor human leukocyte antigens (HLA) with a Mean Fluorescence Intensity > 1000. These can be either pre-existing or de novo. These antibodies can be measured by any appropriate method known in the art.

In a particular embodiment of any aspect of the invention, the AMR comprises the presence of donor-specific antibodies to human leukocyte antigens in the subject and/or wherein the AMR comprises allograft dysfunction. In the context of the present invention, allograft dysfunction may, for example, be considered as chronic lung allograft dysfunction.

In the context of the present invention, allograft survival may be defined as freedom of the transplant recipient from death or retransplantation.

In a particular embodiment of any aspect of the invention, the administration of the combination therapy described herein reduces one or more of the severity of allograft dysfunction, the severity of abnormal lung histology, and the amount of donor-specific antibodies in the subject. Suitably, the administration of the combination therapy described herein reduces the severity of allograft dysfunction and the amount of donorspecific antibodies in the subject, and optionally reduces the severity of abnormal lung histology. For example, the administration of the combination therapy described herein may reduce or eliminate one, two, three, four or all five criteria for AMR, such that the subject would no longer have definite AMR or probable AMR.

The skilled person would be able to determine an "abnormal lung histology" by techniques known in the art.

Lung Transplant

By "lung transplant" we mean lung transplantation, or pulmonary transplantation, which is a surgical procedure in which one (single lung transplant) or both (bilateral lung transplant) lungs are replaced by lungs from a donor. Donor lungs can be retrieved from a living or deceased donor, such as a donor that has been declared brain dead. A living donor can only donate one lung lobe.

Another type of lung transplant that is relevant to the present invention is a lobe transplant, which is a surgery in which part of a living or deceased donor's lung is removed and used to replace the recipient's diseased lung. In living donation, this procedure requires the donation of lobes from two different people, replacing a lung on each side of the recipient. Donors who have been properly screened should be able to maintain a normal quality of life despite the reduction in lung volume. In deceased lobar transplantation, one donor can provide both lobes.

A donor-recipient matching system may be used to determine an appropriate donor for a patient. For example the system administered by the United Network for Organ Sharing (UNOS) finds an appropriate match based on specific criteria, including blood type, size of organ compared with chest cavity, geographic distance between donor organ and transplant recipient, severity of the recipient's lung disease, recipient's overall health, and likelihood that the transplant will be successful.

In an embodiment of the invention, the method is initiated after the transplant of a lung from a donor. In an embodiment of the invention, the lung transplant may be a single or bilateral lung transplant. A bilateral lung transplant (BLT) is sometimes referred to as a double lung transplant or a sequential single lung transplant.

Lung transplantation may be used as a therapeutic measure of last resort for patients with end-stage lung disease who have exhausted all other available treatments without improvement. A variety of conditions may make such surgery necessary. For example, chronic obstructive pulmonary disease (COPD), including emphysema; idiopathic pulmonary fibrosis; cystic fibrosis; idiopathic (formerly known as "primary") pulmonary hypertension; alpha 1-antitrypsin deficiency; replacing previously transplanted lungs that have since failed; and other causes, including bronchiectasis and sarcoidosis. A double lung transplant may be required for example in patients with cystic fibrosis.

Some patients may require a combined heart and lung transplantation, where the patient receives a heart and two lungs. This type of transplant may be required for example where a patient has a congenital condition(s) or patients in whom a lung condition has caused significant heart disease.

IL-6 inhibitors

By "IL-6" we include any natural or synthetic protein with structural and/or functional identity to the human IL-6 protein, such as defined in UniProt Accession No. P05231, or natural variants thereof. IL-6 gene and/or amino acid sequences are disclosed in references 84-86.

By "capable of inhibiting human IL-6" we intend that any antibody or fragment that can or may be able to bind to, inhibit, block or reduce human IL-6. IL-6 exerts its biological functions via two major pathways: classic signaling and trans-signaling pathways (87). In the classic signaling pathway, IL-6 binds to the IL-6 receptor (IL- 6R) on hepatocytes and some leukocytes. The IL-6 IL-6R complex further recruits the ubiquitously expressed membrane-bound or soluble gpl30 (sgpl30), triggering the dimerization of gpl30 and intracellular signaling. In the trans-signaling pathway IL-6 interacts with soluble IL-6R (sIL-6R) to form the IL-6 sIL-6R complex, which can bind to gpl30 on any cell and initiate intracellular signaling without a requirement for the stimulated cell to express IL-6R. An antibody which is capable of inhibiting human IL- 6 must be capable of specifically binding to human IL-6, and of inhibiting its interaction with sIL-6R or IL-6R, or otherwise preventing gpl30 activation. By "capable of specifically binding", we include the ability of the antibody or antigen-binding fragment to bind at least 10-fold more strongly to the relevant polypeptide, e.g. IL-6, than to any other polypeptide; preferably at least 50-fold more strongly and more preferably at least 100-fold more strongly. Inhibitory antibodies to IL-6 can typically be divided into two groups; and the putative epitopes on the IL-6 molecule designated Site I and Site II. Site I binders prevent binding to the IL-6R or sIL-6R and thereby prevent gpl30 activation. The Site I epitope was further characterized as comprising regions of both amino terminal and carboxy terminal portions of the IL-6 molecule. Site II- binders prevent gpl30 activation and therefore may recognize a conformational epitope involved in signalling. Binding of the antibody may be measured by surface plasmon resonance, for example, by immobilizing the antibody on a chip and using recombinant human IL-6 as analyte, as described in WO 2004/039826A1. Suitable antibodies may bind IL-6 with an affinity (Kd) of at least IO^-9 M, preferably at least 10’

¹⁰ M, preferably at least 10 ¹¹ or 5 x 10 ¹¹ M. Epitope mapping to identify Site I or Site

11 binders may be performed by binding to human IL-6-mutant proteins (88). Inhibition of IL-6 activity may be measured by assaying proliferation of the murine B myeloma cell line, 7TD1, in response to IL-6, as described in WO 2004/039826A1. Suitable antibodies may inhibit >50%, such as >90%, such as substantially 100% of 7TD1 cell proliferation in response to IL-6.

By "antibody" we include substantially intact antibody molecules, as well as chimeric antibodies, humanised antibodies, human antibodies (wherein at least one amino acid is mutated relative to the naturally occurring human antibodies), single chain antibodies, bi-specific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and/or light chains, and antigen binding fragments and derivatives of the same. The term also includes antibody-like molecules which may be produced using phage-display techniques or other random selection techniques for molecules. The term also includes all classes of antibodies, including IgG, IgA, IgM, IgD, and IgE. Also included for use in the invention are antibody fragments such as Fab, F(ab')2, Fv, Fab', scFv (single-chain variable fragment), or di- scFv and other fragments thereof that retain the antigen-binding site. Similarly, the term "antibody" includes genetically engineered derivatives of antibodies such as single-chain Fv molecules (scFv) and single-domain antibodies (dAbs).

Preferred antibodies are chimeric, such as mouse-human chimeric antibodies, CDR- grafted antibodies, humanised antibodies, or human antibodies. Although the antibody may be a polyclonal antibody, it is preferred if it is a monoclonal antibody, or that the antigen-binding fragment is derived from a monoclonal antibody. Suitable monoclonal antibodies may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies; A manual of techniques" (89) and in "Monoclonal Hybridoma Antibodies: Techniques and Application" (90). The antibodies may be human antibodies in the sense that they have the amino acid sequence of human antibodies with specificity for the IL-6; however, it will be appreciated that they may be prepared using methods known in the art that do not require immunisation of humans. Suitable antibodies may be prepared from transgenic mice which contain human immunoglobulin loci, as described in "Complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery" (91).

Suitably prepared non-human antibodies can be "humanised" in known ways, for example, by inserting the CDR regions of mouse antibodies into the framework of human antibodies. Chimeric antibodies are discussed in Neuberger et al (92).

It will be appreciated by persons skilled in the art that the binding specificity of an antibody or antigen-binding fragment thereof is conferred by the presence of complementarity determining regions (CDRs) within the variable regions of the constituent heavy and light chains. As discussed below, in a particularly preferred embodiment of the antibodies and antigen-binding fragments, binding specificity for IL-6 is conferred by the presence of one or more and typically all six of the CDR amino acid sequences defined herein.

Preferably, the antibody or antigen-binding fragment comprises an antibody Fc region. It will be appreciated by the skilled person that the Fc portion may be from an IgG antibody, or from a different class of antibody (such as IgM, IgA, IgD, or IgE). For example, the Fc region may be from an IgGl, IgG2, IgG3, or IgG4 antibody. Advantageously, however, the Fc region is from an IgGl antibody. It is preferred that the antibody or antigen-binding fragment is an IgG molecule, or is an antigen-binding fragment or variant of an IgG molecule. Suitable antibodies and fragments are described in WO 2004/039826A1. Suitably, the antibody or fragment which is capable of inhibiting human IL-6 is a chimeric, humanized or CDR grafted antibody or fragment thereof. Suitably, the antibody or fragment which is capable of inhibiting human IL-6 is a chimeric, humanized or CDR grafted antibody or fragment thereof comprising a heavy chain variable region in which CDR1, CDR2, and CDR3 comprise the amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and a light-chain variable region in which CDR1, CDR2, and CDR3 comprise the amino acid sequences SEQ ID NO: 4, SEQ ID NO:, 5 and SEQ ID NO: 6, respectively, and a constant region derived from a human IgG antibody.

VH CDR1 Ser Phe Ala Met Ser (SEQ ID NO. 1)

VH CDR2 Glu He Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr Vai Thr Gly (SEQ ID NO. 2)

VH CDR3 Gly Leu Trp Gly Tyr Tyr Ala Leu Asp Tyr (SEQ ID NO. 3) VL CDR1 Ser Ala Ser Ser Ser Vai Ser Tyr Met Tyr (SEQ ID NO. 4) VL CDR2 Asp Thr Ser Asn Leu Ala Ser (SEQ ID NO. 5) VL CDR3 Gin Gin Trp Ser Gly Tyr Pro Tyr Thr (SEQ ID NO. 6)

In a preferred embodiment the antibody is siltuximab, or an antigen-binding fragment thereof. Siltuximab, also known as CNTO328 and CLLB8, with the US FDA UNII Identifier T4H8FMA7IM and the WHO ATC code L04AC11 is a chimeric (human-murine) IgGlK monoclonal antibody that binds to human IL-6. The intact molecule contains 1324 amino acid residues and is composed of two identical heavy chains (approximately 50 kDa each) and two identical light chains (approximately 24 kDa each) linked by inter-chain disulfide bonds. Siltuximab contains the antigen-binding variable region of the murine antibody, CLB-IL-6-8, and the constant region of a human IgGlK immunoglobulin.

The complete amino acid sequences of the heavy and light chains of siltuximab are shown below.

SEQ ID NO. 7 Siltuximab heavy chain amino acid sequence

EVQLVESGGKLLKPGGSLKLSCAASGFTFSS FAMSWFRQS PEKRLEWVAE I SSGGSYTYY PDTVTGRFTI SRDNAKNTLYLEMSSLRSEDTAMYYCARGLWGYYALDYWGQGTSVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPRE PQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLS PGK

SEQ ID NO. 8 Siltuximab light chain amino acid sequence

QIVLIQS PAIMSAS PGEKVTMTCSASSSVSYMYWYQQKPGSS PRLLIYDTSNLASGVPVR FSGSGSGTSYSLTI SRMEAEDAATYYCQQWSGYPYTFGGGTKLE IKRTVAAPSVFI FPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSS PVTKS FNRGEC

Siltuximab and methods of preparing it, including by recombinant expression of encoding nucleic acid sequences, are described in WO 2004/039826A1.

Other suitable antibodies include olokizumab, which is a IgG4K antibody humanized from rat (93); elsilimomab (also known as B-E8), which is a mouse IgGlK monoclonal antibody (94); or the human monoclonal antibody clone 1339, which is a high-affinity fully humanized anti-IL-6 monoclonal antibody (IgGl) derived from elsilimomab (95). Further suitable antibodies include clazakizumab (formerly ALD518 and BMS-945429), which is an aglycosylated, humanized rabbit IgGl monoclonal antibody against interleukin-6 (96); sirukumab, which is a human monoclonal IgGl kappa antibody (97). Further suitable antibodies include: the MH166 antibody (98); the SK2 antibody (99); Levilimab, which is an anti IL-6 monoclonal antibody initially developed to treat rheumatoid arthritis (100); and ARGX-109, which is a preclinical stage human antibody candidate developed by arGEN-X from its SIMPLE Antibody™ platform and which is said to have outstanding neutralization potency for IL-6. Fragments of any of these antibodies may also be used.

The antibody or fragment should be prepared under sterile conditions. The appropriate volume of antibody or fragment should be withdrawn from the vials. It is recommended that the antibody solution is filtered (0.2 to 1.2 pm) before injection into the patient either by using an in-line filter during infusion or by filtering the solution with a particle filter (e.g., filter Nr. MF1830, Impromediform, Germany). The volume of the antibody is typically added to an infusion bag containing 5% dextrose. Siltuximab is available as a single-use vial containing 100 mg or 400 mg siltuximab powder for concentrate for solution for infusion, and should be stored at refrigeration temperature. The siltuximab powder is typically provided with one or more excipients, typically histidine, histidine hydrochloride monohydrate, polysorbate 80, and sucrose. After reconstitution with single-use sterile water for injection, the solution contains 20 mg siltuximab per mL. Antibodies or fragments may be formulated in other ways, as known in the art.

The dose of the antibody when administered by intravenous administration, such as by infusion, may be 11 ± 3 mg/kg patient body weight, optionally 11 mg/kg. A suitable dose of the fragment is a dose having an equivalent antagonistic effect on human IL- 6.

The combination therapy of the present invention partly comprises administration to the subject of an antibody or fragment which is capable of inhibiting human IL-6, not human IL-6R.

Without wishing to be bound by theory, the inventors consider that inhibitors of human IL6 will be advantageous over inhibitors of IL-6R for treating AMR in lung transplant recipients for many reasons.

For example, treatment with monoclonal antibodies to IL-6R may result in significant increases in IL-6 and IL-6R levels (44, 65). Although this may not cause deleterious effects while treatment is ongoing, there is a theoretical possibility that a rebound IL- 6 effect may be seen when treatment is discontinued. Indeed, the occurrence of allograft loss in 4 kidney recipients with chronic AMR who stopped Tocilizumab supports this possibility (50, 56). Furthermore, the inventors consider that considerably lower doses of monoclonal antibodies to IL-6 may be used compared to those to IL-6R because soluble IL-6R is present at high concentrations compared to IL-6, and all soluble IL-6R have to be saturated with a therapeutic antibody to exert its pharmacodynamic effect (66). In addition, IL-6R binds not only IL-6 but also IL-27p28, so the blockade of IL-6R may affect other signaling pathways (67). Turning to specific inhibitors, Siltuximab (IL6 inhibitor) has a higher binding affinity for IL-6 than Tocilizumab (IL-6R inhibitor) has for IL-6R (68).

In a preferred embodiment of any of the aspects of the invention, the antibody or fragment which is capable of inhibiting human IL-6 is selected from siltuximab, olokizumab, elsilimomab, mAb 1339, clazakizumab, sirukumab, levilimab and ARGX- 109.

Siltuximab In a particularly preferred embodiment of any of the aspects of the invention, the antibody or fragment which is capable of inhibiting human IL-6 is siltuximab.

In previous studies, Siltuximab has been used in patients with hematologic diseases/malignancies, solid tumors and MCD. In Phase 1 studies that included patients with advanced solid tumors, refractory multiple myeloma, or MCD, common grade > 3 adverse events (AE) included leukopenia, neutropenia, thrombocytopenia, hypercholesterolemia, anemia, fatigue, infections, and abnormal liver enzymes (62, 69-71). In Phase 2 studies in MCD, Siltuximab has generally been well-tolerated during long-term follow-up, with a similar pattern of grade > 3 AEs as in Phase 1 studies (72- 74). Siltuximab's safety data are consistent with those of other IL-6/IL-6R monoclonal antibodies (64). Serious infections are the most common serious adverse events (SAE) in clinical trials and post-marketing surveillance studies; other SAE include GI tract perforation, hepatitis, and pancreatitis (64).

Siltuximab has not previously been used in combination with routine treatment for AMR.

Siltuximab dosing studies for indications other than AMR have examined different doses ranging between 3 mg/kg to 15 mg/kg with most doses given every 2 or 3 weeks in patients with hematologic diseases/malignancies, solid tumors, or Multicentric Castleman's Disease (62, 70, 71, 75,76). Measuring IL-6 concentrations accurately during treatment with Siltuximab is not feasible because Siltuximab-neutralized IL-6 complexes distort quantification methods (76). Thus, levels of C-reactive protein (CRP) are used as a surrogate for IL-6 activity because IL-6 is the primary factor that drives CRP production by hepatocytes (76, 77). In these studies, doses of 11 mg/kg or 15 mg/kg every 3 weeks resulted in the most pronounced suppression of CRP, and the half-life of Siltuximab was approximately 21 days (62, 70, 71, 76). Consequently, the currently approved dose of Siltuximab for MCD is 11 mg/kg every 3 weeks. However, because suppression of CRP to levels below 1 mg/L may not be seen until after the second dose, weekly dosing for 4 weeks is recommended for patients with severe MCD (78). No apparent differences in pharmacokinetics were observed across different disease states, and serum concentrations of Siltuximab reached steady state by the sixth infusion. The mean terminal half-life of the 11 mg/kg dose is 20.6 days (range: 14.2-29.7 days).

Immunosuppressive Therapy By "immunosuppressive therapy" we include the standard, routine, common immunosuppressive therapies which are given to transplant recipients before, after or during the transplant procedure to suppress their immune system and reduce the risk of the recipient's body rejecting the donor lung(s) (transplant). The purpose is to stop an unwanted immune response that would damage survival of the transplanted tissue and/or of the subject. Some transplant recipients may be on a life-long immunosuppressive treatment regimen after the transplant. Some patients may be able to reduce or stop taking certain immunosuppressive therapies (e.g., corticosteroids) after the lung transplant, which may reduce the risk of the side effects and complications associated with the immunosuppressive therapies. This is particularly because continuation of immunosuppressive therapies may result in immunodeficiency, by which the subject may be susceptible to opportunistic infection and/or increase the likelihood of cancer due to reduced surveillance of damaged cells by the immune system.

Some common immunosuppressive therapy options administered to patients after a lung transplant include glucocorticoids (corticosteroids), monoclonal antibodies (mAbs), nucleotide blocking agents, calcineurin inhibitors, and mTOR inhibitors. Although monoclonal antibodies are known immunosuppressants, antibodies or fragments which are capable of inhibiting human IL-6 are not known for use in treatment of AMR in lung transplant patients. Suitable immunosuppressive therapy options are known to the person skilled in the art.

The immunosuppressive therapy may comprise a multi-drug regimen. For example, the combination therapy may comprise two or more of the immunosuppressive therapy treatments described herein.

In particular, the immunosuppressive therapy may comprise administering one or more of a baseline immunosuppression maintenance therapy, and/or a standard of care treatment for AMR.

Immunosuppressive therapies may be categorised as induction agents, maintenance therapy, or rescue therapy (i.e. to treat rejection when observed). Induction agents are administered at the time of transplant and are generally more powerful immunosuppressive agents; for this reason they are given for a short acute period. Maintenance therapy comprises medications used over a longer period. Rescue therapy is given when clinical signs of rejection require additional action, such as when the transplant rejection becomes refractory to other medications. In the context of this invention, "baseline immunosuppression maintenance therapy" is defined as immunosuppressive therapy that is categorised as maintenance therapy and used as a form of therapy to be administered chronically to ensure and improve tolerance and continued acceptance of the recipient organ or graft. It typically is a combination of agents and includes one or more of calcineurin inhibitors (cyclosporine and tacrolimus), mammalian target of rapamycin (mTOR) inhibitors (sirolimus and everolimus), antiproliferative agents (azathioprine and mycophenolic acid), costimulatory blockers (belatacept), and corticosteroids.

In general, lung transplant recipients may be treated with a combination of Tacrolimus, Mycophenolate Mofetil (MMF), and Prednisone (or methylprednisolone) for maintenance immunosuppression. In some cases, Cyclosporine A is substituted for Tacrolimus or Azathioprine is substituted for MMF because of toxicity. In accordance with the present invention, patients may be administered a routine maintenance immunosuppression therapy as described herein.

In particular, the baseline immunosuppression maintenance therapy may comprise one or more of tacrolimus, cyclosporine A, Mycophenolate Mofetil (MMF), azathioprine, and prednisone. Suitably, the baseline immunosuppression maintenance therapy may comprise:

(a) tacrolimus, or cyclosporine A;

(b) Mycophenolate Mofetil (MMF), or azathioprine; and

(c) optionally prednisone.

In the context of the present invention, the standard of care treatment for AMR is defined as a routine or standard treatment for pulmonary AMR that is commonly or generally administered to a patient before, during or after receiving a lung transplant. For example, this may include preventative immunosuppressive treatment administered before, during or after transplantation of one or more lung(s) or lobe(s) thereof, to prevent AMR developing, and/or routine immunosuppressive treatment administered before, during or after transplantation to ameliorate, stabilise, improve, or (partially) treat AMR in a lung transplant recipient.

The standard of care treatment for AMR may comprise one or more of a corticosteroid, intravenous immune globulin (IVIG), Rituximab, Bortezomib, Carfilzomib, antithymocyte globulin (ATG), plasma exchange (PLEX), tacrolimus, cyclosporine A, Mycophenolate Mofetil (MMF), azathioprine, prednisone, and methylprednisolone, and optionally acetaminophen (paracetamol) and/or diphenhydramine.

The corticosteroid may be selected from one or more of prednisolone, prednisone, methylprednisolone, cortisone, dexamethasone, betamethasone and hydrocortisone. These are all commercially available corticosteroids.

In particular, the immunosuppressive therapy (standard of care for AMR) may comprise acetaminophen, diphenhydramine, and methylprednisolone. In such cases, the acetaminophen, diphenhydramine, and methylprednisolone may be administered 30- 60 minutes prior to an or each administration to the subject of the antibody or fragment which is capable of inhibiting human IL-6. For example, the antibody or fragment which is capable of inhibiting human IL-6 may be siltuximab.

In particular, the administration of the immunosuppressive therapy may comprise the administration of acetaminophen 650 or 1000 mg by mouth (oral administration e.g, by tablet or capsule), diphenhydramine 25 or 50 mg by mouth (oral administration e.g, by tablet or capsule), and methylprednisolone 60 mg intravenously. In this case, antibody or fragment which is capable of inhibiting human IL-6 is preferably Siltuximab. Further, the acetaminophen, diphenhydramine, and methylprednisolone may be administered 30-60 minutes prior to an or each administration of the antibody of fragment (e.g., Siltuximab). The acetaminophen, diphenhydramine, and methylprednisolone do not necessarily need to be administered with every dose of the antibody or fragment but they may be administered with each dose of antibody or fragment (e.g., Siltuximab) if required.

The skilled person would be able to design a suitable treatment plan of the immunosuppressive therapy according to the needs of the patient. The treatment plan may be revised during the treatment (e.g. between doses of the antibody or fragment) as the patient requires (for example in reaction to the patient's toleration of a particular dose or type of antibody or fragment, or to take into account the perceived efficacy of treating AMR in a particular patient a particular dose or type of antibody or fragment).

Administration of the Combination Therapy

The antibody or fragment which is capable of inhibiting human IL-6 may be administered via the same or a different administration route as the immunosuppressive therapy. In one embodiment, the antibody or fragment which is capable of inhibiting human IL-6 may be prepared e.g. for parenteral administration e.g., subcutaneous, intramuscular, intravenous, intra-dermal, intra-articular or periarticular administration, particularly in the form of liquid solutions or suspensions; or for inhalation to the lungs e.g. pulmonary administration, particularly in the form of solutions, suspensions including nanosuspensions for nebulisation, or suspension or solution pressurised or non-pressurised aerosols. In such embodiments, the immunosuppressive therapy may be prepared for parenteral administration e.g., subcutaneous, intramuscular, intravenous, intra-dermal, intra-articular or peri-articular administration, particularly in the form of liquid solutions or suspensions; for oral administration, particularly in the form of tablets, capsules, powder, granules, solid dispersions or in the form of liquid solutions or suspensions including nanosuspensions; for inhalation to the lungs or nose e.g. pulmonary or intranasal administration, particularly in the form of dry powders, solutions, suspensions including nanosuspensions for nebulisation, nasal sprays or drops comprising solutions or suspensions or suspension or solution pressurised or non-pressurised aerosols; for topical or transdermal administration e.g. as creams, sprays, foams, gels, ointments, liquids, patches; for mucosal administration e.g. to buccal, sublingual or vaginal mucosa, and for rectal administration e.g. in the form of a foam or suppository.

The antibody or fragment which is capable of inhibiting human IL-6, and/or the immunosuppressive therapy may be administered by inhalation. An advantage of inhaled medications is their direct delivery to the area of rich blood supply in comparison to many medications taken by oral route. Thus, the absorption is very rapid as the alveoli have an enormous surface area and rich blood supply and first pass metabolism is bypassed.

The antibody or fragment which is capable of inhibiting human IL-6, and/or the immunosuppressive therapy may conveniently be administered in unit dosage form and may be prepared by any of the methods well-known in the pharmaceutical art. The antibody or fragment which is capable of inhibiting human IL-6, and/or the immunosuppressive therapy may also conveniently be administered in multiple unit dosage form.

The present invention also provides an inhalation device containing the antibody or fragment which is capable of inhibiting human IL-6, and/or the immunosuppressive therapy of the present invention. Typically said device is a metered dose inhaler (MDI), which contains a pharmaceutically acceptable chemical propellant to push the medication out of the inhaler. The immunosuppressive therapy may be administered by intranasal administration. The nasal cavity's highly permeable tissue is very receptive to medication and absorbs it quickly and efficiently. Nasal drug delivery is less painful and invasive than injections, generating less anxiety among patients. By this method absorption is very rapid and first pass metabolism is usually bypassed, thus reducing inter-patient variability.

The immunosuppressive therapy may be administered by transdermal administration. For topical delivery, transdermal and transmucosal patches, creams, ointments, jellies, solutions or suspensions may be employed.

The immunosuppressive therapy may be administered by sublingual administration.

In a preferred embodiment of any of the aspects of the invention, the administration to the subject of the antibody or fragment which is capable of inhibiting human IL-6 is by intravenous administration, optionally by infusion, optionally wherein the infusion is over the course of one hour.

The present disclosure provides pharmaceutical compositions comprising IL-6 inhibitors and other pharmaceutical compositions for administering immunosuppressive therapy. Such compositions may comprise a prophylactically or therapeutically effective amount of the active drug (the IL-6 inhibitor and/or a drug required by the immunosuppressive therapy), and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The various compositions in the context of the therapies and administrations described herein, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in "Remington's Pharmaceutical Sciences." Such compositions will contain a prophylactically or therapeutically effective amount of the agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, intra-rectal, vaginal, topical or delivered by mechanical ventilation.

Pharmaceutically acceptable salts include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Generally, the ingredients of compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

As the immunosuppressive therapy is intended to be a routine (standard) immunosuppressive therapy generally administered to lung transplant recipients, a person skilled in the art would be aware of the appropriate administration routes for different immunosuppressive therapies. For example, the administration route may vary inter alia according to patient characteristics, dosage amount(s) and/or frequency, and/or the type of immunosuppressive therapy. Further, when the immunosuppressive therapy is a multi-drug regimen, the administration route (technique) may be different across different immunosuppressive therapies within the multi-drug regimen.

The antibody or fragment which is capable of inhibiting human IL-6 may be administered according to a dosage regimen. For instance, the administration may include one or more (including two or more, three or more, four or more) doses of the antibody or fragment. For example, the administration may comprise one, two, three, four, five, six, seven, eight, nine or more doses to a subject of the antibody or fragment which is capable of inhibiting human IL-6. Preferably, the antibody or fragment which is capable of inhibiting human IL-6 is administered as 2 or more doses. For example, the antibody or fragment which is capable of inhibiting human IL-6 may be administered as 2 or 3 doses. For example, the antibody or fragment which is capable of inhibiting human IL-6 may be administered as 2 doses. For example, the antibody or fragment which is capable of inhibiting human IL-6 may be administered as 3 doses.

The dose of the antibody or fragment is determined according to the weight in kg of the patient. Where a dose is given in the units of "mg/kg", this refers to mg of antibody or fragment or other drug per kg of body weight of the patient. An antibody fragment is to be administered at an equivalent fragment dose having an equivalent antagonistic effect on human IL-6 to the whole antibody from which the fragment is derived. The equivalent fragment dose may be calculated according to the fragment molecular weight compared to the molecular weight of the whole antibody, also referred to as parent antibody. For example, if a given antibody has a molecular weight of 150 kD, and a Fab fragment has a molecular weight of 50 kD, then a fragment dose that is one third of the antibody dose should provide an equivalent antagonistic effect on human IL-6. Thus, if the antibody dose was 12 mg/kg, then the equivalent fragment dose for the Fab fragment would be 4 mg/kg. The equivalent antagonistic effect on human IL-

6 may also be determined according to the amount of human IL-6 that the fragment can specifically bind to, compared to the amount of human IL-6 that the parent antibody can specifically bind to. These amounts may be determined by various assays, including ELISA.

In a particular embodiment of any of the aspects of the invention, the administration to the subject of the antibody or fragment which is capable of inhibiting human IL-6 is in a dose of 1 mg/kg to 15 mg/kg, such as 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg,

7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9 mg/kg, 9.5 mg/kg, 10 mg/kg, 10.5 mg/kg, 11 mg/kg, 11.5 mg/kg, 12 mg/kg, 12.5 mg/kg, 13 mg/kg, 13.5 mg/kg, 14 mg/kg, 14.5 mg/kg or 15 mg/kg. For example, the dose may be 5.5 mg/kg or 11 mg/kg. Preferably, the antibody or fragment which is capable of inhibiting human IL-6 is siltuximab. More preferably, the antibody or fragment which is capable of inhibiting human IL-6 is siltuximab, and is administered in a dose of 5.5 mg/kg or 11 mg/kg.

Particular dosage regimens of the antibody or fragment which is capable of inhibiting human IL-6 include:

• administering to a subject on day 1 of treatment a first dose of the antibody or fragment (preferably siltuximab) (i.e., a one dose treatment); or

• administering to a subject on day 1 of treatment a first dose of the antibody or fragment (preferably siltuximab) and administering to a subject on day 8 a second dose of the antibody or fragment (preferably siltuximab) (i.e., a two dose treatment);

• administering to a subject on day 1 of treatment a first dose of the antibody or fragment (preferably siltuximab) and administering to a subject on day 22±3 (particularly day 22) a second dose of the antibody or fragment (preferably siltuximab) (i.e., a two dose treatment); and

• administering to a subject on day 1 of treatment a first dose of the antibody or fragment (preferably siltuximab), administering to a subject on day 8 a second dose of the antibody or fragment (preferably siltuximab), and administering to a subject on day 22±3 (particularly day 22) a third dose of the antibody or fragment (preferably siltuximab) (i.e., a three dose treatment).

In any of the above dosage regimens, the administration to the subject of the antibody or fragment which is capable of inhibiting human IL-6 is in each dose may be from 1 mg/kg to 15 mg/kg. Suitably, each dose may be 5.5 mg/kg or 11 mg/kg.

Each dose administered in the same dosage regimen does not have to be of the same dosage amount. For example, a subject may be administered a first dose of 11 mg/kg of the antibody or fragment (preferably siltuximab) on day 1 of treatment, and then administered a second dose of 5.5 mg/kg of the antibody or fragment (preferably siltuximab) on day 8 of treatment.

In a particular embodiment of any aspect of the invention, a first dose of siltuximab is administered at 11 mg/kg on day 1 of treatment, and a second dose of siltuximab is administered at 11 mg/kg on day 8 of treatment. In a particular embodiment of any aspect of the invention, a first dose of siltuximab is administered at 11 mg/kg on day 1 of treatment, and a second dose of siltuximab is administered at 11 mg/kg on day 22±3 of treatment. In a particular embodiment of any aspect of the invention, a first dose of siltuximab is administered at 11 mg/kg on day 1 of treatment, and a second dose of siltuximab is administered at 11 mg/kg on day 22 of treatment.

In a particular embodiment of any aspect of the invention, a first dose of siltuximab is administered at 11 mg/kg on day 1 of treatment, a second dose of siltuximab is administered at 11 mg/kg on day 8 of treatment, and a third dose of siltuximab is administered at 11 mg/kg on day 22±3 of treatment. In a particular embodiment of any aspect of the invention, a first dose of siltuximab is administered at 11 mg/kg on day 1 of treatment, a second dose of siltuximab is administered at 11 mg/kg on day 8 of treatment, and a third dose of siltuximab is administered at 11 mg/kg on day 22 of treatment.

In a particular embodiment of any aspect of the invention, a first dose of siltuximab is administered at 5.5 mg/kg on day 1 of treatment, and a second dose of siltuximab is administered at 5.5 mg/kg on day 8 of treatment. In a particular embodiment of any aspect of the invention, a first dose of siltuximab is administered at 5.5 mg/kg on day 1 of treatment, and a second dose of siltuximab is administered at 5.5 mg/kg on day 22±3 of treatment. In a particular embodiment of any aspect of the invention, a first dose of siltuximab is administered at 5.5 mg/kg on day 1 of treatment, and a second dose of siltuximab is administered at 5.5 mg/kg on day 22 of treatment. In a particular embodiment of any aspect of the invention, a first dose of siltuximab is administered at 5.5 mg/kg on day 1 of treatment, and a second dose of siltuximab is administered at 5.5 mg/kg on day 8 of treatment and a third dose of siltuximab is administered at 5.5 mg/kg on day 22±3 of treatment. In a particular embodiment of any aspect of the invention, a first dose of siltuximab is administered at 5.5 mg/kg on day 1 of treatment, and a second dose of siltuximab is administered at 5.5 mg/kg on day 8 of treatment and a third dose of siltuximab is administered at 5.5 mg/kg on day 22 of treatment.

In a particular embodiment of any aspect of the invention, a first dose of siltuximab is administered at 11 mg/kg on day 1 of treatment, and a second dose of siltuximab is administered at 5.5 mg/kg on day 8 of treatment. In a particular embodiment of any aspect of the invention, a first dose of siltuximab is administered at 11 mg/kg on day 1 of treatment, and a second dose of siltuximab is administered at 5.5 mg/kg on day 22±3 of treatment. In a particular embodiment of any aspect of the invention, a first dose of siltuximab is administered at 11 mg/kg on day 1 of treatment, and a second dose of siltuximab is administered at 5.5 mg/kg on day 22 of treatment. In a particular embodiment of any aspect of the invention, a first dose of siltuximab is administered at 11 mg/kg on day 1 of treatment, and a second dose of siltuximab is administered at 5.5 mg/kg on day 8 of treatment and a third dose of siltuximab is administered at 5.5 mg/kg on day 22±3 of treatment. In a particular embodiment of any aspect of the invention, a first dose of siltuximab is administered at 11 mg/kg on day 1 of treatment, and a second dose of siltuximab is administered at 5.5 mg/kg on day 8 of treatment and a third dose of siltuximab is administered at 5.5 mg/kg on day 22 of treatment.

Further, the subject may be administered a different antibody or fragment which is capable of inhibiting human IL-6 as part of the same dosing regime. For example, the first dose may comprise siltuximab, and the second dose may comprise a different antibody or fragment which is capable of inhibiting human IL-6, such as one or more IL-6 inhibitors selected from olokizumab, elsilimomab, mAb 1339, clazakizumab, sirukumab, levilimab, SK2, MH166, and ARGX-109. A third dose may comprise the same antibody or fragment as the first and/or second dose, or a different antibody or fragment as the first and/or second dose.

Alternatively, the same antibody or fragment which is capable of inhibiting human IL- 6 may be used throughout the treatment (e.g., for each dose). Preferably, each dose comprises or consists of siltuximab.

The treatment of the present invention is a combination therapy of (i) an antibody or fragment which is capable of inhibiting human IL-6; and (ii) an immunosuppressive therapy, wherein the immunosuppressive therapy does not comprise any antibody or fragment thereof which is capable of inhibiting human IL-6. In particular, the immunosuppressive therapy does not comprise any of siltuximab, olokizumab, elsilimomab, mAb 1339, clazakizumab, sirukumab, levilimab, SK2, MH166, and ARGX- 109. The immunosuppressive therapy may comprise IL6R inhibitors such as Tocilizumab. However, in most embodiments it is envisioned that the only antibody inhibitor of IL-6 signalling will be the antibody or fragment which is capable of inhibiting human IL-6. Hence, in such embodiments, the immunosuppressive therapy would not comprise any antibody or fragment thereof which is capable of inhibiting human IL-6 or human IL-6R. Moreover, it will be appreciated that in most embodiments the only inhibitor of IL-6 signaling will be the antibody or fragment which is capable of inhibiting human IL-6. Hence, in such embodiments, the immunosuppressive therapy would not comprise any drug which is capable of inhibiting human IL-6 or human IL-6R.

In an embodiment, the combination therapy may comprise (i) an antibody or fragment which is capable of inhibiting human IL-6; and (ii) an immunosuppressive therapy, wherein the immunosuppressive therapy does not comprise the antibody or fragment which is capable of inhibiting human IL-6 from (i). For example, if the antibody or fragment which is capable of inhibiting human IL-6 of (i) is Siltuximab, the immunosuppressive therapy of (ii) does comprise not Siltuximab.

The administration of (i) an antibody or fragment which is capable of inhibiting human IL-6; may occur before, after, at substantially the same time as, or during the administration of (ii) an immunosuppressive therapy, wherein the immunosuppressive therapy does not comprise any antibody or fragment thereof which is capable of inhibiting human IL-6. In the combination therapy of the invention, each or a dose of (i) an antibody or fragment which is capable of inhibiting human IL-6 may be administered with or without a dose of (ii) immunosuppressive therapy, as long as a dose of (ii) immunosuppressive therapy is administered at least once during the treatment.

In a particular embodiment of any aspect of the invention, the administration to the subject of the antibody or fragment which is capable of inhibiting human IL-6 is given in a first treatment dose initiated within the period 24 hours before or after initiation of the immunosuppressive therapy. The administration of the first treatment dose of the antibody of fragment may be initiated within the period 2 hours before or after initiation of the immunosuppressive therapy. The administration of the first treatment dose of the antibody of fragment may be initiated within the period one hour before or after initiation of the immunosuppressive therapy. The administration of the first treatment dose of the antibody of fragment may be initiated substantially at the same time as initiation of the immunosuppressive therapy.

In a preferred embodiment, the immunosuppressive therapy is administered 30 to 60 minutes prior to an or each administration to the subject of the antibody or fragment which is capable of inhibiting human IL-6. Preferably, the antibody or fragment which is capable of inhibiting human IL-6 is siltuximab.

This means that, in such an embodiment, the immunosuppressive therapy is administered 30 to 60 minutes prior to one or more doses (particularly the first dose, or the only dose if there is only one dose administered) of the antibody or fragment (preferably Siltuximab). If the treatment comprises more than one dose of the antibody or fragment which is capable of inhibiting human IL6, the immunosuppressive therapy for example may be administered 30 to 60 minutes prior to every dose, only the first dose, only the last dose, only the second dose, only one dose, only one or two doses out of a three dose treatment, every other dose, every second dose, or every third dose and so on, of the antibody or fragment (for example, Siltuximab).

The administration of the combination therapy may further comprise administration to the subject of a prophylactic medication, wherein the prophylactic medication is an antiviral and/or antimicrobial medication. The prophylactic medication may be one or more of an antiviral medication, or an antimicrobial medication. The person skilled in the art will appreciate that any suitable antiviral or antimicrobial medication may be administered prophylactically, i.e. to prevent possible viral or microbial infection. By microbial infection we include bacterial, mycobacterial and fungal infections, such that an antimicrobial medication may be an antibiotic agent against bacteria or mycobacteria, or an antifungal agent against fungi (particularly yeasts or molds). In particular, the prophylactic medication may comprise one or more of Valganciclovir, Trimethoprim-Sulfamethoxazole, Voriconazole, Posaconazole, and Isavuconazonium.

Such prophylactic medications are considered standard treatments for lung transplant recipients (i.e., these medications are part of the standard of care), and the skilled person would be able to determine the appropriate dosage, medication, and prophylactic treatment regimen. These prophylactic medications are known and commercially available.

Participants at risk for cytomegalovirus (i.e., seronegative recipients of seropositive donors or seropositive recipients) may be treated with Valganciclovir prophylactically after treatment for AMR. Similarly, all lung transplant recipients may be maintained on prophylaxis for Pneumocystis jirovecii with either Trimethoprim-Sulfamethoxazole or an alternative drug if allergic to sulfa.

All patients being evaluated for AMR may undergo bronchoscopy with lung biopsies, bronchial washings, and/or bronchoalveolar lavage as part of the routine clinical evaluation. Bronchoscopy specimens may be routinely tested for mycobacterial, fungal, and bacterial organisms as well as polymerase chain reaction (PCR) testing for CARV. Although positive mycobacterial, fungal, or bacterial cultures do not necessarily represent invasive disease or infection, patients may be routinely started on appropriate antimicrobial therapy before initiating treatment for AMR to minimize the risk of invasive disease and infection after treatment for AMR.

Patients who have positive cultures for a mold from bronchoscopy specimens may be routinely treated with Voriconazole, Posaconazole, or Isavuconazonium. Patients who have a positive bacterial culture may be treated with appropriate antimicrobials. For patients who have a positive mycobacterial culture, anti-mycobacterial therapy may be initiated depending on the specific organism.

DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

Figure 1 shows (A) serum IL-6 concentrations in a cohort of lung transplant patients with AMR compared to healthy non-transplant individuals, and (B) immunofluorescence staining for IL-6 in lung biopsies of patients with AMR.

Figure 2 shows the traditional 3+3 Phase 1 clinical trial design used in Example 2.

Figure 3 shows the dosing and follow-up timelines for Cohorts A-D in Example 2.

EXAMPLES

Example 1: Measurements of IL-6 in patients with AMR

Using an ELISA method, serum IL- 6 concentrations were measured in a cohort of LT recipients with AMR (n = 15), which revealed a median concentration of 1.94 (range: 0.91-4.26) pg/mL compared to healthy non-transplant individuals who had a median concentration of 0.56 (range: 0.06-1.83) pg/mL (Figure 1A, p < 0.001).

Studies in other disease states (e.g., multiple myeloma, non-Hodgkin's lymphoma) have shown wide interpatient variability in baseline serum IL-6 concentrations, and baseline serum concentrations were not predictive of clinical response to IL-6 blockade because serum concentrations may not reflect local production or concentrations (62).

Hence, localised detection is important. Immunofluorescence staining for IL-6 in lung biopsies of patients with AMR demonstrated evidence of intrapulmonary IL-6 expression (Figure IB).

These preliminary data illustrate increased IL-6 levels in serum and increased expression in lung allografts with AMR. Example 2: A Phase 1 Clinical Trial of Siltuximab for the Treatment of Antibody-Mediated Rejection after Lung Transplantation

This is a Phase 1, prospective, multicenter, open-label clinical trial in which 18 lung transplant recipients will be treated with escalating doses of Siltuximab in addition to standard of care therapy for antibody-mediated rejection after lung transplantation.

1. Protocol Synopsis

1.1. Primary Objective

The primary objective of the study is to assess the safety and tolerability of Siltuximab added to routine immunosuppressive treatment for antibody-mediated rejection after lung transplantation.

1.2. Secondary Objectives

The secondary objectives are:

1. Assess pharmacodynamics,

2. Determine the optimal dose for a subsequent trial, and

3. Assess functional biological measurements and clinical outcomes feasible for use in a subsequent trial that examines efficacy.

1.3. Primary Endpoint

The primary endpoint is safety and tolerability which will be assessed based on the development of dose limiting toxicities (DLT), adverse events (AE), and serious adverse events (SAE).

1.4. Secondary Endpoints

The following secondary endpoints will be assessed between enrolment and day 90:

1. Undetectable serum C-reactive protein (CRP) (< 10 mg/L),

2. Clearance of donor-specific antibodies (DSA),

3. Hyperuricemia (> 7 mg/dL),

4. Hyperlipidemia requiring the initiation or dose increase of medical therapy,

5. Confirmed bacterial, cytomegalovirus (CMV), mold, mycobacterial, or community-acquired respiratory viral infection (each assessed independently),

6. Development of probable chronic lung allograft dysfunction (CLAD) according to the 2019 ISHLT criteria (4, 5),

7. Allograft survival defined as death or undergoing re-transplantation.

1.5. Exploratory Endpoints The following exploratory endpoints will be assessed between enrolment and day 90:

1. Donor-derived cell-free-DNA < 1%,

2. Serum IL-6 levels at enrolment only (before administration of the first dose of Siltuximab),

3. Exhaled breath condensate IL-6 levels at enrolment,

4. Serial measurements of IL-6 bioavailability by reporter gene assay (RGA).

1.6. Inclusion Criteria

Individuals who meet all the following criteria are eligible for enrolment as study participants:

1. 18 years of age or older,

2. Single or bilateral lung transplant recipient,

3. New diagnosis of definite or probable antibody-mediated rejection according to the 2016 International Society for Heart and Lung Transplantation (ISHLT) definition (23),

4. Donor-specific antibodies (DSA) to human leukocyte antigens (HLA),

5. Able to understand the purpose of the study and willing to participate and sign informed consent.

1.7. Exclusion Criteria

Individuals who meet any of these criteria are not eligible for enrolment as study participants:

1. Pregnant or breast feeding,

2. History of lymphoma or hematologic malignancy,

3. Treatment with IL-6 signaling blockade with 6 months of enrolment,

4. Cancer other than non-melanoma skin cancer with disease-free period < 3 years,

5. Positive respiratory virus PCR detected within 7 days of enrolment,

6. Active cytomegalovirus infection within 7 days of enrolment,

7. Positive respiratory culture for Mycobacterium tuberculosis, Mycobacterium abscessus, Mycobacterium chelonae, or Mycobacterium avium complex within 4 weeks of enrolment,

8. Absolute neutrophil count (ANC) < 1,000 cells/mm3 at enrolment,

9. Platelet count < 75,000 cells/mm3 at enrolment,

10. Hemoglobin > 17 g/dL at enrolment,

11. ALT or AST > 2.5 times upper limit of normal at enrolment,

12. Total bilirubin > 2.5 times upper limit of normal at enrolment,

13. History of gastrointestinal tract perforation, 14. Diagnosis of diverticulitis within 4 weeks of enrolment,

15. Inability or unwillingness to give written informed consent or comply with the study protocol,

16. Any condition that in the opinion of the site investigator introduces undue risk by participating in this study or impacts the quality or interpretation of the study results.

1.8. Study Stopping Rules

The principal investigator team, the Data and Safety Monitoring Board (DSMB), and NHLBI will review safety data on an ongoing basis. If a safety concern arises, enrollment and randomization of participants in the trial will be suspended pending DSMB review. Subjects already enrolled will continue to be treated per protocol.

The criteria described below provide additional guidance for suspending the trial pending DSMB review based on the occurrence of selected adverse events. Selected adverse events of concern and their thresholds in this trial are:

1. Any occurrence of gastrointestinal tract perforation,

2. Death in greater than 20% of enrolled subjects,

3. Opportunistic infections in greater than 25% of enrolled subjects.

2. Study Definitions

Allergic reaction: An allergic reaction is defined as a disorder characterized by an adverse local or general response from exposure to an allergen. Grade 3 allergic reaction is one that results in bronchospasm, requires hospitalization, or intravenous intervention.

Allograft survival: Freedom from death or re-transplantation.

Antibody-Mediated Rejection: Antibody-mediated rejection will be defined based on the

2016 ISHLT definition. Only cases of definite or probable AMR with donor-specific antibodies (DSA) to human leukocyte antigens (HLA) will be considered as AMR in the study.

Bronchoalveolar lavage (BAL): Bronchoalveolar lavage is medical procedure performed during bronchoscopy in which a small scope is inserted into a patient's nose or mouth and passed into the lungs. Once in the lungs, sterile saline is instilled, coating the lining the lungs, and then quickly aspirated out of the lungs.

Clearance of donor-specific antibodies (DSA): Clearance of donor-specific antibodies (DSA) is defined as all DSA being below the level of detection (Mean Fluorescence Intensity < 1000). Death as a DLT: Death is defined as a Dose Limiting Toxicity if the CEAC adjudicates that death was due to infection.

Donor-specific antibodies (DSA): Donor-specific antibodies are antibodies to donor human leukocyte antigens (HLA) with a Mean Fluorescence Intensity > 1000. These can be either pre-existing or de novo.

Hepatitis as a DLT: Hepatitis is defined as a Dose Limiting Toxicity if the AST or ALT > 5 times the upper limit of normal or if the total bilirubin > 3 times the upper limit of normal.

Hyperlipidemia as a DLT: Hyperlipidemia is defined as a Dose Limiting Toxicity if it results in pancreatitis, requires hospitalization, or prolongs a hospitalization.

Hypersensitivity reaction: A hypersensitivity reaction is defined as a Dose Limiting Toxicity if this meets the definition of a Grade > 3 allergic reaction.

Infection as a DLT: Infections including CMV, fungemia, and respiratory tract infections are defined as Dose Limiting Toxicities if they require hospitalization or prolong a hospitalization.

Neutropenia as a DLT: Neutropenia is defined as a Dose Limiting Toxicity if the absolute neutrophil count < 500 cells/mm³ for at least 3 days.

Opportunistic infection: Opportunistic infections are infections that occur more often or are more severe in immunocompromised patients. These include tissue invasive cytomegalovirus, varicella zoster, tuberculosis, Pneumocystis jirovecii, nocardia, endemic mycoses, and toxoplasmosis.

Probable CLAD: Probable CLAD is defined as a >20% decline in FEVi compared to the baseline value on 2 measurements at least 3 weeks apart and after exclusion or adequate treatment of potential secondary causes of allograft dysfunction (e.g., respiratory infection, acute cellular rejection).

Pruritis as a DLT: Pruritis is defined as a Dose Limiting Toxicity if it is widespread and constant, limits self-care, or requires systemic steroids.

Rash as a DLT: Rash is defined as a Dose Limiting Toxicity if it covers > 30% of the body surface area with moderate or severe symptoms limiting self-care.

Thrombocytopenia as a DLT: Thrombocytopenia is defined as a Dose Limiting Toxicity if the platelet count < 50,000 cells/mm3 for at least 3 days.

3. Study Hypothesis

We hypothesize that the addition of Siltuximab to standard of care immunosuppressive therapy for AMR is sufficiently safe and clinically tolerable to proceed with a subsequent Phase 2 clinical trial that assesses efficacy. To test this hypothesis, we will conduct a Phase 1 clinical trial of 18 adult lung transplant recipients with AMR. 4. Study Design

4.1. Description of study design

This is a two-center, prospective, open-label, Phase 1 clinical trial in which LT recipients diagnosed with definite or probable AMR according to the 2016 ISHLT definition (23) (n = 18) will be treated with escalating doses of Siltuximab plus standard of care (SOC) immunosuppressive treatment for AMR and baseline maintenance immunosuppression. We will use a traditional 3 + 3 Phase 1 clinical trial design (Figure 2). This safe and easy to implement design proceeds with cohorts of 3 participants:

Cohort A

In Cohort A, 3 participants will be treated with Siltuximab 5.5 mg/kg for 2 doses on days 1 and 8 (Figure 3). If none of the 3 participants in Cohort A experiences a DLT (Table 3), the next 3 participants will be enrolled in Cohort B (see below). However, if 1 participant in Cohort A experiences a DLT, 3 more participants will be added to Cohort A and treated with the same dose (Figure 2). If no additional participants experience a DLT in Cohort A, the study will progress to Cohort B. If 2 or more of the 6 participants or 2 of the initial 3 participants in Cohort A experience a DLT, the next 3 participants will be enrolled in Cohort A2.

Cohort A2

In Cohort A2, 3 participants will be treated with 5.5 mg/kg for 2 doses on days 1 and 22. If none of the 3 participants in Cohort A2 experiences a DLT, the study will progress to Cohort B. If 1 participant in Cohort A2 experiences a DLT, 3 more participants will be added to Cohort A2 and treated with the same dose.

Cohort B

In Cohort B, 3 participants will be treated with Siltuximab 11 mg/kg for 2 doses on days 1 and 22 (Figure 3). If none of the 3 participants in Cohort B experiences a DLT, the next 3 participants will be enrolled in Cohort C. If 1 participant in Cohort B experiences a DLT, 3 more participants will be added to Cohort B and treated with the same dose. If no additional participants experience a DLT in Cohort B, the study will progress to Cohort C. If 2 or more of the 6 participants or 2 of the initial 3 participants in Cohort B experience a DLT, the maximum tolerated dose (MTD) will be designated as that used in Cohort A (or Cohort A2 if that was the previous Cohort before Cohort B).

Cohort C In Cohort C, 3 subjects will be treated with Siltuximab 11 mg/kg for 2 doses on days 1 and 8 (Figure 3). If none of the 3 participants in Cohort C experiences a DLT, the next 3 participants will be enrolled in Cohort D. If 1 participant in Cohort C experiences a DLT, 3 more participants will be added to Cohort C and treated with the same dose. If no additional participants experience a DLT in Cohort C, the study will progress to Cohort D. If 2 or more of the 6 participants or 2 of the initial 3 participants in Cohort C experience a DLT, the MTD will be designated as that used in Cohort B.

Cohort D

In Cohort D, 3 subjects will be treated with Siltuximab 11 mg/kg on days 1, 8, and 22 for a total of 3 doses (Figure 3). If none of the 3 participants in Cohort D experiences a DLT, we will designate this as the MTD. If 1 participant in Cohort D experiences a DLT, 3 more participants will be added to Cohort D and treated with the same dose. If no additional participants experience a DLT, we will consider this the MTD. If 2 or more of the 6 participants or 2 of the 3 initial participants in Cohort D experience a DLT, the MTD will be designated as that used in Cohort C.

The MTD

The MTD is the highest dosing schedule where < 33% of patients experience a DLT. Once the MTD is identified, we will enrol additional patients to reach a total number of 18 (in all cohorts) and treat them with this dosing schedule to confirm MTD and develop preliminary data to inform the design of a future Phase 2 clinical trial. The approved dose of Siltuximab for MCD is 11 mg/kg intravenously every 3 weeks. However, the optimal dose and dosing schedule for the treatment of AMR after LT are not known. The role of IL-6 and its concentrations in serum and local tissues is different in different disease states. Indeed, IL-6 concentrations in serum are generally higher in MCD than in AMR. In one study, patients with MCD had median serum IL-6 concentrations of 24.5 (range: 6.5-93) pg/mL whereas patients with AMR have median serum IL-6 concentrations of 2.20 (range: 1.58-5.06) pg/mL (78). Likewise, IL-6 concentrations in hyperplastic lymph nodes in MCD are likely different than in the lung allograft in AMR. Therefore, it is possible that lower doses of Siltuximab are needed for the treatment of AMR. We selected the starting dose of 5.5 mg/kg for 2 doses 1 week apart (Cohort A) although this dose did not suppress CRP to undetectable levels in MCD because it is possible that it may be sufficient in AMR (62, 74). In Cohort B, we will examine the safety and pharmacodynamics of the dose approved in MCD (11 mg/kg every 3 weeks for 2 doses). In Cohort C, we will examine an expedited dosing schedule (11 mg/kg on days 1 and 8 for 2 doses) to assess if this more rapid suppression of IL-6 activity is safe. Finally, we will assess the safety of 3 doses in Cohort D. 4.2. Primary endpoint

The primary endpoint is safety and tolerability which will be assessed based on the development of dose limiting toxicities (DLT), adverse events (AE), and serious adverse events (SAE). Table 3 below details and defines the different DLT.

Table 3 - Dose limiting toxicities

The development of DLT will be assessed during the DLT follow-up period which is defined as 30 days after the last dose of Siltuximab (Figure 3). All DLT, AE, and SAE will be reviewed by the CEAC to adjudicate if a DLT has occurred. Where applicable, the study will use the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. The study defines a hypersensitivity reaction as a DLT if this meets CTCAE Grade > 3 allergic reaction. An allergic reaction is defined as a disorder characterized by an adverse local or general response from exposure to an allergen. Grade 3 allergic reaction is one that results in bronchospasm, requires hospitalization, or intravenous intervention. Death is defined as a DLT if the CEAC adjudicates that death was due to infection because approximately 20% of LT recipients with AMR die within 30 days of the diagnosis without treatment with IL-6 signaling blockade (20). Grade 3 thrombocytopenia (platelet count < 50,000 cells/mm3) and Grade 4 neutropenia (absolute neutrophil count < 500 cells/mm3) lasting at least 3 days are defined as DLT. Grade 3 rash is defined as a DLT if it covers > 30% of the body surface area (BSA) with moderate or severe symptoms limiting self-care. Grade 3 pruritis is defined as a DLT if it is widespread and constant, limits self-care, or requires systemic steroids. Hyperlipidemia is defined as a DLT if it results in pancreatitis or requires hospitalization or prolongs a hospitalization. Infections are defined as DLT if they require hospitalization or prolong a hospitalization. Typically, requirement for intravenous antibiotics is the threshold for Grade 3 infections in the CTCAE. However, LT recipients are frequently treated with intravenous antibiotics for non-serious upper or lower respiratory tract infections with antibiotic-resistant organisms. Yet, the use of intravenous antibiotics in this setting is not an indication of the clinical severity of infection. Thus, we have modified the definition of infection as a DLT to be more applicable to LT recipients.

4.3. Secondary Endpoints

See section 1.4 above.

4.4. Exploratory Endpoints

See section 1.5 above.

5. Selection of Participants and Sites

5.1. Lung transplant recipients with AMR

The study will enrol adult lung transplant recipients who develop probable or definite AMR according to the 2016 ISHLT definition (23). The study will not enrol children as the lung transplant programs enrolling participants do not provide care for children. Lung transplant recipients who develop AMR have a poor prognosis. IL-6 plays an important role in mediating allograft injury, and IL-6 blockade might mitigate this. The eligibility criteria for enrolment have been determined based on known adverse events associated with IL-6 blockade and Siltuximab, the package insert, and the risk of infection associated with augmented immunosuppression.

For inclusion and exclusion criteria, see sections 1.6 and 1.7 above.

6. Investigational Agent

6.1. Siltuximab (Sylvant®)

Siltuximab is the investigational agent in this study. Details describing the study related product, labeling, supply, storage, preparation, dispensing, monitoring and accountability are described below. Siltuximab is manufactured by EUSA Pharma and is approved by the FDA as detailed below. Siltuximab is a first-in-class chimeric (humanmouse) immunoglobulin G1K (IgGlK) monoclonal antibody against human IL-6 in a Chinese hamster ovary cell line.

6.2. Dosage and administration

The Siltuximab dose is given as an intravenous infusion over 60 minutes. This Phase 1 study will assess the safety and tolerability of 4 dosing schedules to determine the MTD (see Section 4.1 above and Figure 3). Complete blood count should be checked before each dose of Siltuximab. Before the first administration, the absolute neutrophil count must be > 1,000 cells/mm³, the platelet count must be > 75,000 cells/mm³, and the hemoglobin must be < 17 g/dL. Before subsequent doses, the absolute neutrophil count must be > 1,000 cells/mm³, the platelet count must be > 50,000 cells/mm³, and the hemoglobin must be < 17 g/dL. If these criteria are not me, the dose may be delayed but the dose should not be reduced. Participants will be premedicated 30-60 minutes prior to the start of the Siltuximab infusion using:

1. Acetaminophen 650 or 1000 mg by mouth,

2. Diphenhydramine 25 or 50 mg by mouth, and

3. Methylprednisolone 60 mg intravenously.

6.3. Instructions to followed in the study for preparation and administration Use aseptic technique for reconstitution and preparation of dosing solution. Parenteral drug products should be inspected visually for particulate matter and discoloration prior to administration, whenever solution and container permit.

1. Calculate the dose (mg), total volume (mL) of reconstituted Siltuximab solution required and the number of vials needed. A 21-gauge 1.5-inch needle is recommended for preparation. Infusion bags (250 mL) must contain Dextrose 5% in water and must be made of polyvinyl chloride (PVC), polyolefin (PO), polypropylene (PP), or polyethylene (PE).

2. Allow the vial(s) of Siltuximab to come to room temperature over approximately 30 minutes. Siltuximab should remain at room temperature for the duration of the preparation.

3. Aseptically reconstitute each Siltuximab vial as below: powder. Do not shake or swirl vigorously. Do not remove the contents until all the solids have been completely dissolved. The lyophilized powder should dissolve in less than 60 minutes. Once reconstituted, and prior to further dilution, inspect the vials for particulates and discoloration. Do not use if particles or solution discoloration are present or if visibly opaque. The reconstituted product should be kept for no more than 2 hours prior to addition into the infusion bag.

4. Dilute the reconstituted Siltuximab solution dose to 250 mL with sterile Dextrose 5% in water by withdrawing a volume equal to the total calculated volume of reconstituted Siltuximab from the Dextrose 5% in water, 250 mL bag. Slowly add the total calculated volume (mL) of reconstituted Siltuximab solution to the Dextrose 5% in water infusion bag. Gently invert the bag to mix the solution.

5. Administer the diluted Siltuximab solution in 5% Dextrose in water 250 mL by intravenous infusion over a period of 1 hour using administration sets lined with PVC, or polyurethane (PU), or PE, containing a 0.2-micron inline polyethersulfone (PES) filter. The infusion should be completed within 4 hours of the dilution of the reconstituted solution to the infusion bag.

6. Do not infuse Siltuximab concomitantly in the same intravenous line with other agents.

7. Siltuximab does not contain preservatives. Do not store any unused portion of the reconstituted product or of the infusion solution. Waste material should be disposed of in accordance with local requirements.

6.4. Infusion supervision

Siltuximab will be administered in a hospital or outpatient infusion center where full resuscitation facilities are immediately available and under close supervision of the investigator or designated safety accessor. The intravenous infusion will be supervised by the clinical staff (e.g., clinical nurse or physician) at the participating institutions. A history of each infusion and any adverse side effects will be recorded and reported using appropriated case report forms (CRF). Prior to each infusion, clinical labs (ANC, platelets, bilirubin, ALT, and AST) will be completed. Results must be available prior to initiating the infusion. Vital signs (temperature, blood pressure, pulse, and respiratory rate) will be obtained prior to the start of each infusion and every 15 minutes until the end of the infusion, and every hour thereafter for a total of 2 hours after the first infusion, and 1 hour after subsequent infusions. After the infusion, the intravenous line should remain in the participant for at least 1 hour to enable the administration of drugs if necessary. Additional vital signs may be obtained as clinically indicated. 6.5. Instructions regarding siltuximab dosing delays and discontinuation criteria

6.5.1. Serious infections

Closely monitor patients for the development of signs and symptoms of infection during and after treatment with Siltuximab, as signs and symptoms of acute inflammation may be lessened due to suppression of the acute phase reactants. Hold Siltuximab if a patient develops a serious infection, an opportunistic infection, or sepsis. A patient who develops a new infection during treatment with Siltuximab should undergo a prompt and complete diagnostic workup appropriate for an immunocompromised patient, initiate appropriate antimicrobial therapy, and closely monitor the patient. Siltuximab may be resumed at the next scheduled dose timepoint if the infection has resolved.

6.5.2. Neutropenia

Treatment with Siltuximab was associated with a higher incidence of neutropenia. Infections have been uncommonly reported in association with treatment-related neutropenia in studies and post marketing clinical experience. Per package insert, it is not recommended to initiate Siltuximab in patients with an ANC < 1000 cells/mm³. If the ANC is < 1000 cells/mm³, treatment with Siltuximab may be delayed, but the dose should not be reduced. For this study, if the ANC is < 1000 cells/mm³, the scheduled dose of Siltuximab may be delayed up to 3 days. If during this time the ANC is > 1000 cells/mm³, the dose may be given. However, if the ANC remains < 1000 cells/mm³, the dose will not be administered.

6.5.3. Thrombocytopenia

Treatment with Siltuximab was associated with a reduction in platelet count. Treatment-related reduction in platelets was not associated with serious bleeding events in clinical trials. Per the package insert, it is not recommended to initiate Siltuximab in patients with a platelet count < 75,000 cells/mm³, and it is not recommended to receive subsequent doses of Siltuximab in patients with a platelet count < 50,000 cells/mm³. For this study, if the platelet count is < 50,000 cells/mm³, the scheduled dose of Siltuximab may be delayed up to 3 days. If during this time the platelet count is > 50,000 cells/mm³, the dose may be given. However, if the platelet count remains < 50,000 cells/mm³, the dose will not be administered.

6.5.4. Polycythemia

Per the package insert, Siltuximab may increase hemoglobin levels in patients with MCD. Treatment with Siltuximab is not recommended in patients with a hemoglobin > 17 g/dL. For this study, if the hemoglobin is > 17 g/dL, the scheduled dose of Siltuximab may be delayed up to 3 days. If during this time the hemoglobin is < 17 g/dL, the dose may be given. However, if the hemoglobin remains > 17 g/dL, the dose will not be administered.

6.5.5. Siltuximab discontinuation

Siltuximab will be discontinued in the following circumstances:

1. Participant's decision,

2. Anaphylaxis or other hypersensitivity reaction,

3. GI tract perforation,

4. Pregnancy during the study treatment period,

5. The site PI believes that study therapy is no longer in the best interest of the participant (e.g., due to medical decision, pneumonia, serious infection, opportunistic infection).

In the event that treatment with Siltuximab is discontinued, participants will be followed for the duration of the study period (90 days after enrollment), and data from all safety assessments will be collected.

7. Other Medications

7.1. Maintenance immunosuppression

In general, lung transplant recipients are treated with a combination of Tacrolimus, Mycophenolate Mofetil (MMF), and Prednisone for maintenance immunosuppression. In some cases, Cyclosporine A is substituted for Tacrolimus or Azathioprine is substituted for MMF because of toxicity. During this study, participants will continue their maintenance immunosuppressive regimen, but Prednisone will be held as they receive intravenous Methylprednisolone.

7.2. Standard of care treatment for AMR

Lung transplant recipients with AMR are routinely hospitalized because of allograft dysfunction and to initiate intensive immunosuppressive treatment. Depending on the clinical course and response to treatment, some patients may be discharged from the hospital to complete treatment in the outpatient infusion center. Standard of care (SOC) treatment for AMR varies based on patient-specific factors including overall performance status, previous rejection treatments, lung biopsy findings at the time of AMR diagnosis, and history of infection. The most common treatment combination consists of Carfilzomib, rabbit anti-thymocyte globulin (ATG), high-dose corticosteroids, and IVIG. Carfilzomib 20 mg/m² is given on days 1, 2, 8, 9, 15, and 16, ATG (1-1.5 mg/kg) is given on days 1-5 targeting a cumulative dose of 5-7.5 mg/kg, methylprednisolone 1 mg/kg is given daily for 7-10 days with a tapering corticosteroid schedule based on clinical response, and IVIG 500-1000 mg/kg is first given on day 10 and continued monthly thereafter. PLEX is not routinely used in the management of AMR because of lack of efficacy and confounding the dosing of antibody treatments including ATG and IVIG. In some cases, a single dose of Rituximab 375 mg/m² may be administered instead of ATG or for refractory disease at least 72 hours apart from the first dose of IVIG. After enrolling in the study, subjects will receive the first dose of Siltuximab intravenously on day 1. Siltuximab will be given on the same day as the first dose of Carfilzomib and rabbit ATG. These would all be administered in the hospital. Subsequent doses may be given either during the hospitalization or in an outpatient infusion center depending on the patient's clinical status.

7.3. Prophylactic medications

Participants at risk for CMV (i.e., seronegative recipients of seropositive donors or seropositive recipients) will be treated with Valganciclovir prophylactically after treatment for AMR. Similarly, all lung transplant recipients are maintained on prophylaxis for Pneumocystis jirovecii with either Trimethoprim-Sulfamethoxazole or an alternative drug if allergic to sulfa. All patients being evaluated for AMR undergo bronchoscopy with lung biopsies, bronchial washings, and/or bronchoalveolar lavage as part of the routine clinical evaluation. Bronchoscopy specimens are routinely tested for mycobacterial, fungal, and bacterial organisms as well as polymerase chain reaction (PCR) testing for CARV. Although positive mycobacterial, fungal, or bacterial cultures do not necessarily represent invasive disease or infection, patients are routinely started on appropriate antimicrobial therapy before initiating treatment for AMR to minimize the risk of invasive disease and infection after treatment for AMR. Patients who have positive cultures for a mold from bronchoscopy specimens are routinely treated with Voriconazole, Posaconazole, or Isavuconazonium. Likewise, patients who have a positive bacterial culture are treated with appropriate antimicrobials. For patients who have a positive mycobacterial culture, anti-mycobacterial therapy is initiated depending on the specific organism. However, patients who have a positive mycobacterial culture with a potentially pathogenic organism (e.g., Mycobacterium abscessus, Mycobacterium avium complex) will not be eligible for enrolment.

7.4. Rescue treatments

Participants may require rescue treatments for progressive or refractory allograft dysfunction due to AMR. Rescue treatments for AMR may include plasma exchange and Eculizumab, a monoclonal antibody to complement component (C5). The decision to add one of these treatments will be made by the treating lung transplant clinician. If during the course of therapy with Siltuximab a participant requires rescue treatment, subsequent doses of Siltuximab will not be given, and the patient will continue study follow-up per protocol. The treating lung transplant clinician may decide to use rescue therapy with Tocilizumab for progressive or refractory AMR. There are limited data in the literature about the use of rescue Tocilizumab after treatment with Siltuximab. The data are in the setting of cytokine release syndrome (CRS) or immune effector cell associated neurotoxicity syndrome (ICANS) after chimeric antigen receptor (CAR) T-cell therapy (79). In this setting, rescue Tocilizumab has been given for persistent grade 1 CRS at 7 days after the first dose of Siltuximab or at 72 hours for grade 2 CRS. In this study, rescue Tocilizumab may be given 4 half-lives (64 days) after the last administered dose of Siltuximab.

8. Study Procedures

8.1. Informed consent

Sites will identify potential study participants from their cohorts of lung transplant recipients. The research study will be explained in lay terms to each potential participant, and the potential participant will sign an informed consent form before undergoing any study procedures. The investigator or study research coordinator will meet with the study candidate to review all the required elements of informed consent. After the informed consent has been signed, and the participant meets eligibility criteria, the participant will be enrolled in the study.

8.2. Study follow-up and testing

All study participants will be followed for 90 days after enrolment. Participants will be followed more closely for 30 days after the last dose of Siltuximab to assess for DLT. Participants in Cohorts A and C receive the last dose of Siltuximab on day 8 and will be followed for DLT through day 38. Participants in Cohorts B and D receive the last dose of Siltuximab on day 22 and will be followed for DLT through day 52. If Cohort A2 is necessary, participants in this cohort receive the last dose of Siltuximab on day 22 and will be followed for DLT through day 52. The schedule of events for each cohort is shown in Appendix 1.

During the hospitalization, participants will be assessed on a daily basis. This includes a comprehensive History and Physical Exam and review of daily lab work results to assess for treatment-related AE and DLT. On days where testing and Siltuximab dosing are both scheduled, testing will be performed first. Accurate measurements of serum IL-6 concentrations by ELISA during treatment with Siltuximab is not feasible. Thus, CRP will be a surrogate measurement of IL-6 activity (76, 77). IL-6 bioavailability will be assessed by reporter gene assay (RGA) which employs an IL-6-responsive reporter cell line (DS-l/SIE-luciferase) that has been shown to have high dynamic range and specificity for IL-6 blockade in the setting of treatment with anti-IL-6 or anti-IL-6R monoclonal antibodies (80). Additional study-related testing includes baseline serum and exhaled breath condensate (EBC) IL-6 concentrations, and serial measurements of donor-derived cfDNA, DSA, and spirometry as functional biologic assessments of treatment outcome. EBC IL-6 concentrations will be assessed as an exploratory metric at baseline to assess intra-pulmonary levels as serum levels may not reflect intrapulmonary production (57, 58, 60). EBC IL-6 concentrations are significantly higher among cigarette smokers and patients with chronic obstructive pulmonary disease (COPD) than healthy controls but have not been measured in LT recipients (81, 82). Fasting lipids and uric acid levels may be affected by IL-6 blockade and will be measured serially.

9. Criteria for Participant Completion, Withdrawal, and Replacement

9.1. Participant completion

Participants have completed the study when they have completed the final visit on day 90.

9.2. Participant withdrawal

Participants may be withdrawn from the study for the following reasons:

1. The participant elects to withdraw consent from all future study activities including follow-up.

2. The participant is lost to follow-up (i.e., no further follow-up is possible because attempts to reestablish contact with the participant have failed).

3. The participant dies.

9.3. Participant replacement

In the event that a subject is withdrawn for a reason other than a DLT or death (of any cause), another subject will be enrolled into that cohort. Advancement to a successive dosing cohort will not proceed until at least 3 subjects have completed 30 days of follow-up after the last dose of Siltuximab.

10. Safety Monitoring and Reporting

10.1. Safety Definitions

10.1.1. Adverse event (AE)

Any untoward or unfavorable medical occurrence associated with the subject's participation in the research, whether or not considered related to the subject's participation in the research (modified from the definition of adverse events in the 1996 International Conference on Harmonization E-6 Guidelines for Good Clinical Practice) (from CHRP "Guidance on Reviewing and Reporting Unanticipated Problems Involving Risks to Subjects or Others and Adverse Events" http://www.hhs.gOv/ohrp/policy/advevntguid.html#Q2 ).

10.1.2. Suspected adverse reaction (SAR)

An SAR is any adverse event for which there is a reasonable possibility that the study drug caused the adverse event. For the purposes of safety reporting, "reasonable possibility" means there is evidence to suggest a causal relationship between the drug and the adverse event. An SAR implies a lesser degree of certainty about causality than adverse reaction, which means any adverse event caused by a drug.

10.1.3. Unexpected adverse event

An adverse event or suspected adverse reaction is considered "unexpected" if it is not listed in the most recent version of the Siltuximab Investigator Brochure (IB) or is not listed at the specificity, severity or rate of occurrence that has been observed as described in the IB; or is not consistent with the risk information described in the general investigational plan or elsewhere in the IND. "Unexpected" also refers to adverse events or suspected adverse reactions that are mentioned in the IB as occurring with a class of drugs or as anticipated from the pharmacological properties of the drug but are not specifically mentioned in the IB as occurring with the particular drug under investigation.

10.1.4. Serious adverse event

An adverse event or suspected adverse reaction is considered "serious if, in the view of the investigator, it results in any of the following outcomes:

1. Death,

2. A life-threatening event: an AE or SAR is considered "life-threatening" if, in the view of the investigator, its occurrence places the subject at immediate risk of death. It does not include an AE or SAR that, had it occurred in a more severe form, might have caused death,

3. Inpatient hospitalization or prolongation of existing hospitalization,

4. Persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions,

5. Congenital anomaly or birth defect,

6. Important medical events that may not result in death, be life threatening, or require hospitalization may be considered serious when, based upon appropriate medical judgment, they may jeopardize the subject and may require medical or surgical intervention to prevent one of the outcomes listed above.

Elective hospitalizations or hospital admissions for the purpose of conduct of protocol mandated procedures are not to be reported as an SAE unless hospitalization is prolonged due to complications.

10.2. Grading of adverse events

The study site will grade the severity of adverse events experienced by participants according to the criteria set forth in NCI-CTCAE, version 5.0. This document provides a common language to describe levels of severity, to analyze and interpret data, and to articulate the clinical significance of all adverse events. Adverse events will be graded on a scale from 1 to 5 according to the following standards in the NCI-CTCAE manual:

Table 4 - Grading of adverse events

Events grade > 3 will be recorded on the appropriate AE case report form for this study. For grading an abnormal value or result of a clinical or laboratory evaluation, a treatment-emergent adverse event is defined as an increase in grade from baseline or from the last post-baseline value that doesn’ t meet grading criteria. Changes in grade from screening to baseline will also be recorded as adverse events but are not treatment emergent. If a specific event or result from a given clinical or laboratory evaluation is not included in the NCI-CTCAE manual, then an abnormal result would be considered an adverse event if changes in therapy or monitoring are implemented because of the event/result. Example 3: A Phase 1 Clinical Trial of Siltuximab for the Treatment of Antibody-Mediated Rejection after Lung Transplantation (Updated)

The following Phase 1 Clinical Trial protocol is based on the protocol set out in Example 2 but has been updated after consultation with regulatory agencies.

This is a Phase 1, two-center, double-blind, randomized placebo-controlled, clinical trial in which 30 lung transplant recipients with antibody-mediated rejection will be randomized 1: 1 : 1 to low dose Siltuximab, full dose Siltuximab, or Placebo intravenously on days 1 and 22 in addition to routine clinical treatment for antibody- mediated rejection.

Study Arm 1 : low dose Siltuximab (n = 10)

• Siltuximab 5.5 mg/kg intravenously on days 1 and 22 Study Arm 2: full dose Siltuximab (n = 10)

• Siltuximab 11 mg/kg intravenously on days 1 and 22 Study Arm 3: Placebo (n = 10)

• Placebo intravenously on days 1 and 22

1. Protocol synopsis

1.1. Primary Objectives

See Example 2, Section 1.1.

1.2. Secondary Objectives

The secondary objectives are:

1. Assess the pharmacokinetics of Siltuximab after dose 1 and dose 2 of treatment,

2. Assess the pharmacodynamics of Siltuximab after dose 1 and dose 2 of treatment,

3. Determine the optimal dose for future trials,

4. Assess functional biological measurements and clinical outcomes that may be used to assess efficacy in future trials.

1.3. Primary Endpoint

The primary endpoint is the incidence of CTCAE > grade 3 during a period of 90 days after randomization.

1.4. Secondary Endpoints

The following secondary endpoints will be assessed: 1. The incidence of CTCAE > grade 3 during a period of 180 days after randomization,

2. Undetectable serum high-sensitivity C-reactive protein (HS-CRP) (< 1 mg/L) 90 days after randomization,

3. Undetectable serum high-sensitivity C-reactive protein (HS-CRP) (< 1 mg/L) 180 days after randomization,

4. Siltuximab concentrations between randomization and day 90,

5. Clearance of donor-specific antibodies (DSA) between randomization and day 180,

6. Hyperuricemia (> 7 mg/dL) between randomization and day 180,

7. Hyperlipidemia requiring the initiation or dose increase of medical therapy between randomization and day 180,

8. Confirmed bacterial, cytomegalovirus (CMV) (defined as a positive blood viral load > 200 lU/mL), mold, mycobacterial, or community-acquired respiratory viral infection (each assessed independently) between randomization and day 180,

9. Opportunistic infection between randomization and day 180,

10. Change in Forced Vital Capacity (FVC) between randomization and day 180,

11. Change in Forced Expiratory Volume in One Second (FEV1) between randomization and day 180,

12. Development of probable chronic lung allograft dysfunction (CLAD) according to the 2019 ISHLT criteria between randomization and day 180,

13. Allograft survival defined as death or undergoing re-transplantation between randomization and day 180.

1.5. Exploratory Endpoints

The following exploratory endpoints will be assessed:

1. Donor-derived cell-free-DNA < 1%,

2. Change in circulating donor-derived cell-free-DNA after study drug treatment,

3. Change in HS-CRP between randomization and day 180,

4. Serum IL-6 levels at enrollment only (before administration of the first dose of Siltuximab),

5. Exhaled breath condensate IL-6 levels at enrollment,

6. Serial measurements of IL-6 bioavailability by reporter gene assay (RGA).

1.6. Inclusion criteria

Individuals who meet all the following criteria are eligible for enrolment as study participants: 1. 18 years of age or older,

2. Single or bilateral lung transplant recipient,

3. New diagnosis of clinical definite, probable, or possible antibody-mediated rejection according to the 2016 International Society for Heart and Lung Transplantation (ISHLT) definition with plans to be treated with Carfilzomib and/or anti-thymocyte globulin,

4. Admitted to the hospital for treatment of AMR,

5. Donor-specific antibodies (DSA) to human leukocyte antigens (HLA) with a Mean Fluorescence Intensity (MFI) > 1000,

6. Able to understand the purpose of the study and willing to participate and sign informed consent.

1.7. Exclusion Criteria

Individuals who meet any of these criteria are not eligible for enrolment as study participants.

1. Pregnant or breast feeding,

2. Airway anastomotic dehiscence on bronchoscopy,

3. Thoracotomy incision dehiscence,

4. Underwent lung transplantation less than 6 months before enrollment,

5. Underwent other invasive surgical procedure less than 6 weeks before enrollment,

6. History of lymphoma or hematologic malignancy,

7. Treatment with IL-6 signaling blockade with 6 months of enrollment,

8. Planned treatment with plasma exchange (PLEX) for AMR,

9. Cancer other than non-melanoma skin cancer with disease-free period <3 years,

10. Positive respiratory virus PCR detected within 7 days of enrollment,

11. Active cytomegalovirus infection within 7 days of enrollment,

12. Positive respiratory culture for Mycobacterium tuberculosis, Mycobacterium abscessus, Mycobacterium chelonae, or Mycobacterium avium complex within 4 weeks of enrollment,

13. Absolute neutrophil count (ANC) < 1,000 cells/mm³ at enrollment,

14. Platelet count < 75,000 cells/mm³ at enrollment,

15. Hemoglobin > 17 g/dL at enrollment,

16. ALT or AST > 2.5 times upper limit of normal at enrollment,

17. Total bilirubin > 2.5 times upper limit of normal at enrollment,

18. Uric acid > 7 mg/dL at enrollment.

19. History of gastrointestinal tract perforation,

20. History of diverticulitis (diverticulosis is not an exclusion), 21. Plan for surgical procedure (other than bronchoscopy) within 120 days of enrollment.

22. Inability or unwillingness to give written informed consent or comply with the study protocol,

23. Any condition that in the opinion of the site investigator introduces undue risk by participating in this study or impacts the quality or interpretation of the study results.

1.8. Study Stopping Rules

See Example 2, Section 1.8. Selected adverse events of concern and their thresholds in this trial are:

1. Any occurrence of gastrointestinal tract perforation,

2. Death in 20% or more of enrolled subjects,

3. Opportunistic infections in 25% or more of enrolled subjects.

2. Study Definitions

See the definitions in Example 2, Section 2, in addition to those below.

Serious infection: Infection requiring intravenous antimicrobial therapy and hospitalization.

Tissue invasive CMV: Tissue invasive CMV is defined as the presence of signs and symptoms of end-organ disease such as enteritis, colitis, pneumonitis, retinitis and CMV replication in blood.

In this study, Antibody-mediated rejection will be defined based on the 2016 ISHLT definition. Only cases of clinical definite, probable, or possible AMR with donor-specific antibodies (DSA) to human leukocyte antigens (HLA) will be considered as AMR in the study.

Moreover, in this study, further to the definition in Example 2, Section 2, the definition of opportunistic infection may include pathogenic non-tuberculous mycobacteria (NTM).

3. Study Hypothesis

We hypothesize that the addition of Siltuximab to routine clinical treatment for AMR is sufficiently safe and clinically tolerable to proceed with a subsequent Phase 2 clinical trial that assesses efficacy. To test this hypothesis, we will conduct a Phase 1 clinical trial of 30 adult lung transplant recipients with AMR. 4. Study Design

4.1. Description of study design

This is a Phase 1, two-center, double-blind, randomized, placebo-controlled clinical trial in which 30 LT recipients diagnosed with definite or probable AMR according to the 2016 ISHLT definition (23) will be randomized in a 1: 1 : 1 ratio to receive low dose Siltuximab, full dose Siltuximab, or Placebo (Dextrose 5% in sterile water) intravenously on days 1 and 22 in addition to routine clinical treatment for AMR and baseline maintenance immunosuppression. The placebo arm enhances the detection of safety signals by serving as a control group to compare AE rates in the Siltuximab group to.

Study Arm 1 : low dose Siltuximab (n = 10)

• Siltuximab 5.5 mg/kg intravenously on days 1 and 22 (two total doses)

• Routine clinical treatment for AMR

• Baseline maintenance immunosuppression

Study Arm 2: full dose Siltuximab (n = 10)

• Siltuximab 11 mg/kg intravenously on days 1 and 22 (two total doses)

• Routine clinical treatment for AMR

• Baseline maintenance immunosuppression Study Arm 3: Placebo (n = 10)

• Placebo (sterile D5W) intravenously on days 1 and 22 (two total doses)

• Routine clinical treatment for AMR

• Baseline maintenance immunosuppression

The study will use safety, pharmacokinetic, pharmacodynamic, functional biological measurements, and clinical outcomes data to determine an optimal dose for a future Phase 2 clinical trial that assesses the efficacy of Siltuximab in the treatment of AMR after LT. Although, the FDA-approved dose of Siltuximab for MCD is 11 mg/kg intravenously every 3 weeks, the optimal dose for the treatment of AMR after LT is not known. The role of IL-6 and its concentrations in serum and local tissues is different in different disease states. Indeed, IL-6 concentrations in serum are generally higher in MCD than in AMR. In one study, patients with MCD had median serum IL-6 concentrations of 24.5 (range: 6.5-93) pg/mL whereas patients with AMR have median serum IL-6 concentrations of 2.20 (range: 1.58-5.06) pg/mL (78). Likewise, IL-6 concentrations in hyperplastic lymph nodes in MCD are likely different than in the lung allograft in AMR. Therefore, it is possible that lower doses of Siltuximab are effective for the treatment of AMR. We selected the low dose of 5.5 mg/kg for 2 doses 21 days apart although this dose did not suppress CRP to undetectable levels in MCD because it is possible that it may be sufficient in AMR (62, 74). We will also examine the safety, pharmacokinetics, and pharmacodynamics of the dose approved in MCD (11 mg/kg every 21 days for 2 doses).

4.2. Primary Endpoint

See Section 1.3 above.

4.3. Secondary Endpoints

See Section 1.4 above.

4.4. Exploratory Endpoints

See Section 1.5 above.

4.5. Randomization and blinding

Eligible subjects will be randomized on the day of enrollment through a web-based randomization system with a 1: 1 : 1 ratio to low dose Siltuximab, full dose Siltuximab, or Placebo. The randomization list will be computer generated using a permuted block randomization and stratified by enrollment site. Treatment allocation will remain concealed except to each site's study pharmacist and unblinded statistician. The study staff at each site, the participants, and the clinical team will remain blinded to treatment assignment.

5. Selection of Participants and Sites

5.1. Lung transplant recipients with AMR

The study will enroll adult lung transplant recipients who develop clinical definite, probable, or possible AMR according to the 2016 ISHLT definition (23) and DSA to HLA. The study will not enroll children as the lung transplant programs enrolling participants do not provide care for children. Lung transplant recipients who develop AMR have a poor prognosis. IL-6 plays an important role in mediating allograft injury, and IL-6 blockade might mitigate this. The eligibility criteria for enrollment and randomization have been determined based on known adverse events associated with IL-6 blockade and Siltuximab, the package insert, and the risk of infection associated with augmented immunosuppression.

See Sections 1.6 and 1.7 for inclusion and exclusion criteria respectively.

6. Investigational Agent The investigational agents for this trial are Siltuximab (Sylvant®) and Placebo (Dextrose 5% in sterile water, D5W). Details describing the study related product labeling, supply, storage, preparation, dispensing, monitoring and accountability are described below.

6.1. Siltuximab (Sylvant®)

See Example 2, Section 6.1. In this study, Siltuximab is manufactured by Recordati Rare Diseases and is approved by the FDA as detailed below.

6.2. Dosage and administration

See Example 2, Section 6.2.

6.3. Instructions for preparation and administration

See Example 2, Section 6.3.

6.4. Placebo

Placebo, D5W, is considered an investigational agent in this study. Sites will use only sterile D5W that is produced by an FDA registered facility as designated by the presence of a National Drug Code number on product labeling. Product information including manufacturer, lot number, and expiration date will be captured in the pharmacy accountability log at each site. Placebo will be prepared with dose volume to match Siltuximab and will be stored using the standard procedures at each site's investigational pharmacy. Placebo should be prepared using appropriate aseptic techniques using the site's D5W stock. Placebo will be prepared and dispensed by an unblinded pharmacist at the site. Placebo will be labeled by the site's investigational pharmacy with the same infusion bag label used for active Siltuximab to maintain the blind.

6.5. Infusion supervision

See Example 2, Section 6.4. The investigational agents in this trial are Siltuximab and a placebo (Dextrose 5% in sterile water, D5W).

6.6. Instructions regarding investigational agent dosing delays and discontinuation

See Example 2, Section 6.5 and subsections. References in this section to "Siltuximab" in relation to actions in this study (i.e. other than references about known facts) should be read as "the investigational agent", in view of the blinding. 7. Other medications

7.1. Maintenance immunosuppression

See Example 2, Section 7.1.

7.2. Routine clinical treatment for AMR and investigational agent administration

Lung transplant recipients with AMR are routinely hospitalized because of allograft dysfunction and to initiate intensive immunosuppressive treatment. Depending on the clinical course and response to treatment, some patients may be discharged from the hospital to complete treatment in the outpatient infusion center. Routine clinical treatment for AMR varies based on patient-specific factors including overall performance status, previous rejection treatments, lung biopsy findings at the time of AMR diagnosis, and history of infection. The most commonly used clinical regimen at the two sites includes high-dose corticosteroids, IVIG, anti-thymocyte globulin (ATG), and Carfilzomib. ATG is not routinely used for induction immunosuppression in the immediate period after lung transplantation at the sites, but it is used to treat persistent ACR and CLAD. Patients who have been previously treated with ATG are eligible for enrollment in this study but will not be treated with an additional course of ATG for AMR; they will be treated with Carfilzomib. All other study participants will be treated with ATG and Carfilzomib. High-dose corticosteroids and IVIG are cornerstone treatments for AMR at the sites, and these will be given to all participants. Thus, study participants will be treated with:

• High-dose corticosteroids,

• IVIG,

• ATG, if it has not been administered previously, and

• Carfilzomib.

Carfilzomib 20 mg/m² is given on days 1, 2, 8, 9, 15, and 16, ATG (1-1.5 mg/kg) is given on days 1-5 targeting a cumulative dose of 5-7.5 mg/kg, methylprednisolone 1 mg/kg is given daily for 7-10 days with a tapering corticosteroid schedule based on clinical response, and IVIG 500-1000 mg/kg is first given on day 10 and continued monthly thereafter. PLEX is not part of the routine first-line treatment for AMR at the sites because of lack of effectiveness clearing DSA or improving the clinical course. Additionally, PLEX confounds the dosing of antibody treatments including ATG and IVIG and requires the placement of a central intravenous pheresis catheter. Known planned treatment with PLEX at the time of enrollment is an exclusion criterion for this study, but rescue treatment with PLEX for refractory AMR is allowed (see Section 7.4. Rescue treatments below). Subjects will be consented, enrolled, and randomized after admission to the hospital for management of AMR and will receive the first dose of the investigational agent intravenously within 72 hours of admission. Treatment for AMR will not be delayed for study-related reasons. The investigational agent may be given on the same day as the first dose of Carfilzomib and rabbit ATG. These would all be administered in the hospital. The subsequent dose of the investigational agent may be given either during the hospitalization or in an outpatient infusion center depending on the patient's clinical status.

7.3. Prophylactic medications

See Example 2, Section 7.3. In this study, participants at risk for CMB may be prophylactically treated with Letermovir as an alternative to valganciclovir, after treatment for AMR.

7.4. Rescue treatments

See Example 2, Section 7.4. In this study, rescue treatments for AMR may instead include PLEX, Rituximab, and Eculizumab. References in this section to "Siltuximab" should be read as "the investigational agent", in view of the blinding. Moreover, in this study, rescue Tocilizumab may be given after the end of the study (90 days) instead of 64 days after the last administered dose of Siltuximab as in Example 2.

8. Study Procedures

Study procedures including informed consent, History and Physical Exam, study drug dosing, and testing are detailed in the schedule of events for each dosing cohort (Appendix 2).

8.1. Informed consent

See Example 2, Section 8.1. In this study, Patients will be given ample time to decide if they would like to participate.

8.2. Randomization

Randomization will occur after the participant has signed the informed consent form.

8.3. Study follow-up and testing

All study participants will be followed for 180 days after enrollment and randomization, with the primary endpoint occurring at day 90. Participants will be randomized on the same day as enrollment. The investigational agent will be administered on day 1 and day 22. The schedule of events cohort is shown in Appendix 2. During the hospitalization, participants will be assessed on a daily basis. This includes a comprehensive History and Physical Exam and review of daily lab work results to assess for treatment-related AE. On days where testing and investigational agent dosing are both scheduled, testing will be performed first. Accurate measurements of serum IL-6 concentrations by ELISA during treatment with Siltuximab is not feasible. Thus, HS-CRP will be a surrogate measurement of IL-6 activity (76, 77). IL-6 bioavailability will be assessed by reporter gene assay (RGA) which employs an IL-6- responsive reporter cell line (DS-l/SIEIuciferase) that has been shown to have high dynamic range and specificity for IL-6 blockade in the setting of treatment with anti- IL-6 or anti-IL-6R monoclonal antibodies (80). To assess pharmacokinetics, Siltuximab concentrations will be measured using a commercially available ELISA kit specifically designed to measure Siltuximab (Kribiolisa cat # KBI1630) with a minimum detection level of 12 ng/mL and an approximately 100-fold linear detection range. The ELISA kit will also be independently validated with a Siltuximab standard calibration curve generated with healthy human volunteer serum. Timepoints for measuring pharmacokinetics are detailed in the schedule of events above. At every dosing visit, a pre-dose measurement and a post-dose measurement will be made; the post-dose measurement will be made 1 hour after completion of the infusion. To avoid unblinding, pharmacokinetic and HS-CRP measurements will be batched, and the results will only be made available after completion of study follow-up. Additional testing includes baseline serum and exhaled breath condensate (EBC) IL-6 concentrations, and serial measurements of donor-derived cfDNA, DSA, and spirometry as functional biologic assessments of treatment outcome. EBC IL-6 concentrations will be assessed as an exploratory metric at baseline to assess intrapulmonary levels as serum levels may not reflect intrapulmonary production (57, 58, 60). EBC IL-6 concentrations are significantly higher among cigarette smokers and patients with chronic obstructive pulmonary disease (COPD) than healthy controls but have not been measured in LT recipients (81, 82). Fasting lipids and uric acid levels may be affected by IL-6 blockade and will be measured serially. Tests performed for research purposes are HS-CRP, fasting lipids, uric acid, serum and EBC IL-6 concentrations, IL-6 bioavailability, and Siltuximab concentrations. HS-CRP, fasting lipids, and uric acid levels can be measured using the same blood draw as the CMP and do not require additional samples. However, serum IL-6 concentrations, IL-6 bioavailability, and Siltuximab concentrations require separate blood draws; a total of 10 mL of blood will be collected for these research tests at each testing visit. Participants will have 10 mL of blood drawn for research testing at visits 0, 1, 2, 3, 4, 5, 6, 7, and 8 for a total volume of 90 mL during study follow-up. No bronchoscopies or lung biopsies will be performed for study purposes; specifically, the study does not mandate a follow-up biopsy after treatment for several reasons. First, the histologic features of pulmonary AMR are non-specific and improvement in histology has not been defined. Second, patients typically have impaired allograft function after AMR, which increases the risk of serious complications associated with bronchoscopy and biopsy. As a result, a follow-up biopsy after treatment is not done as part of routine clinical care at the sites.

9. Criteria for Participant Completion, Withdrawal, and Replacement

9.1. Participant Completion

Participants have completed the study when they have completed the final visit on day 180.

9.2. Participant withdrawal

Please see Example 2, Section 9.2. In this study participants may be withdrawn from the study if the participant undergoes re-transplantation.

9.3. Participant replacement

After randomization, subjects who withdraw from the study will not be replaced.

10. Safety Monitoring and Reporting

10.1. Safety Definitions

10.1.1. Adverse event, Suspected adverse reaction, Unexpected adverse event and Serious adverse event (SAE)

See Example 2, Section 10.1 and subsections for the definitions of adverse event (AE), suspected adverse reaction (SAR), unexpected adverse event and serious adverse event (SAE).

10.1.2. Serious and unexpected suspected adverse reaction (SUSAR).

A SUSAR is defined as an adverse event that is both serious and unexpected and has a reasonable possibility of a causal relationship with Siltuximab.

10.1.3. Unanticipated problems (UP)

UP are defined as any event that meets all of the following criteria:

1. Unexpected in terms of nature, severity, or frequency given the research procedures and subject population, and

2. Related or probably related to participation in the study or if the event probably or definitely affects the safety, rights and welfare of current participants, and

3. Suggests that the research places subjects at greater risk of harm than was previously recognized. 10.2. Grading of adverse events

See Example 2, Section 10.2. In this study, grading will be performed by the principal investigators (Pls).

ABBREVIATIONS

ACR Acute Cellular Rejection

ADL Activities of Daily Living

AE Adverse Event

ALT Alanine Transaminase

AMR Antibody-Mediated Rejection

ANC Absolute Neutrophil Count

AST Aspartate Aminotransferase

ATG Anti-Thymocyte Globulin

BAL Bronchoalveolar Lavage

BOS Bronchiolitis Obliterans Syndrome

BSA Body Surface Area

BSF-2 B-cell Stimulating Factor 2

C4d Complement Component 4d

CARV Community-Acquired Respiratory Virus

CBC Complete Blood Count

CEAC Clinical Events Adjudication Committee cfDNA Donor-Derived Cell Free-DNA

CLAD Chronic Lung Allograft Dysfunction

CMP Comprehensive Metabolic Profile

CMV Cytomegalovirus

CNI Calcineurin Inhibitor

COPD Chronic Obstructive Pulmonary Disease

CRF Case Report Form

CRP C-Reactive Protein

CRS Cytokine Release Syndrome

CTCAE Common Terminology Criteria for Adverse Events

DCC Data Coordinating Center

DLT Dose Limiting Toxicity

DSA Donor-Specific Antibodies

DSMB Data Safety Monitoring Board

EBC Exhaled Breath Condensate FDA Food and Drug Administration

FEVi Forced Expiratory Volume in One Second

FVC Forced Vital Capacity

GD Gestation Days

GERD Gastro-Esophageal Reflux Disease

GFR Glomerular Filtration Rate

GI Gastrointestinal

GRAfT Genomic Research Alliance for Transplantation gpl30 Glycoprotein 130

HHV-8 Human Herpesvirus-8

HIV Human Immunodeficiency Virus

HLA Human Leukocyte Antigens

HS-CRP High-sensitivity C-reactive protein

IB Investigator Brochure

ICANS Immune Effector Cell Associated Neurotoxicity Syndrome

IDE Investigational Device Exemption

IgGlK Immunoglobulin G1K

IL-6 Interleukin-6

IL-6R Interleukin-6 Receptor

IND Investigational New Drug

IP Investigational Product

IRB Institutional Review Board

ISHLT International Society for Heart and Lung Transplantation

IVIG Intravenous Immune Globulin

JAK Janus Kinase

LT Lung Transplantation

MCD Multicentric Castleman's Disease

MFI Mean Fluorescence Intensity

MM Multiple Myeloma

MMF Mycophenolate Mofetil

MTD Maximum Tolerated Dose

NCI National Cancer Institute

NHP Non-Human Primate

PCR Polymerase Chain Reaction

PGD Primary Graft Dysfunction

PHI Protected Health Information

PI Principal Investigator

PLEX Plasma Exchange PE Polyethylene

PES Polyethersulfone

PO Polyolefin

PP Polypropylene PU Polyurethane

PVC Polyvinyl Chloride

RAS Restrictive Allograft Syndrome

RCT Randomized Controlled Trial

RGA Reporter Gene Assay SAE Serious Adverse Event

SAR Suspected Adverse Reaction

SIL-6R Soluble Interleukin-6 Receptor

SOC Standard of Care

STAT Signal Transducer and Activator of Transcription SUSAR Serious and Unexpected Suspected Adverse Reaction

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100. Levilimab, Wikipedia entry, "https://en.wikipedia.org/wiki/Levilimab" accessed 19 April 2024 APPENDIX 1

Table 5 - Cohort A Table 6 - Cohort A2

Table 7 - Cohort B

Table 8 - Cohort C

Table 9 - Cohort D APPENDIX 2

X* indicates 2 measurements of pharmacokinetics on days that an investigational agent dose is given: a pre-dose measurement and a postdose measurement 1 hour after completing the infusion.

Claims

1. A method of treatment of antibody mediated rejection (AMR) of a lung transplant in a subject in need thereof, the method comprising a combination therapy of:

2. The method according to Claim 1, wherein the subject is a human.

3. The method according to Claim 1 or 2, wherein the method is initiated after the transplant of a lung from a donor.

4. The method according to any one of Claims 1-3, wherein the lung transplant is a single or bilateral lung transplant.

5. The method according to any one of Claims 1-4, wherein the antibody or fragment which is capable of inhibiting human IL-6 is a chimeric, humanized or CDR grafted antibody or fragment thereof.

6. The method according to any one of Claims 1-5, wherein the antibody or fragment which is capable of inhibiting human IL-6 is a chimeric, humanized or CDR grafted antibody or fragment thereof comprising a heavy chain variable region in which CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and a light chain variable region in which CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; and a constant region derived from a human IgG antibody.

7. The method according to any one of Claims 1-6, wherein the antibody or fragment which is capable of inhibiting human IL-6 is selected from siltuximab, olokizumab, elsilimomab, mAb 1339, clazakizumab, sirukumab, levilimab, SK2, MH166, and ARGX-109.

8. The method according to any one of Claims 1-7, wherein the antibody or fragment which is capable of inhibiting human IL-6 is siltuximab.

9. The method according to any one of Claims 1-8, wherein the administration to the subject of the antibody or fragment which is capable of inhibiting human IL-6 is in a dose of 1 mg/kg to 15 mg/kg, optionally wherein the dose is 5.5 mg/kg or 11 mg/kg.

10. The method according to Claim 9, wherein the antibody or fragment which is capable of inhibiting human IL-6 is siltuximab, and is administered in a dose of 5.5 mg/kg or 11 mg/kg.

11. The method according to any one of Claims 8-10, wherein the siltuximab is administered as 2 or more doses, optionally as 3 doses.

12. The method according to Claim 11, wherein a first dose of siltuximab is administered at 11 mg/kg on day 1 of treatment, and a second dose of siltuximab is administered at 11 mg/kg on day 8 or day 22 of treatment.

13. The method according to Claim 11, wherein a first dose of siltuximab is administered at 11 mg/kg on day 1 of treatment, a second dose of siltuximab is administered at 11 mg/kg on day 8 of treatment, and a third dose of siltuximab is administered at 11 mg/kg on day 22 of treatment.

14. The method according to Claim 11, wherein a first dose of siltuximab is administered at 5.5 mg/kg on day 1 of treatment, and a second dose of siltuximab is administered at 5.5 mg/kg on day 8 or day 22 of treatment, optionally wherein the second dose is on day 8 of treatment and a third dose of siltuximab is administered at 5.5 mg/kg on day 22 of treatment.

15. The method according to any one of Claims 1-14, wherein the administration to the subject of the antibody or fragment which is capable of inhibiting human IL-6 is by intravenous administration, optionally by infusion, optionally wherein the infusion is over the course of one hour.

16. The method according to any one of Claims 1-15, wherein the AMR comprises the presence of donor-specific antibodies to human leukocyte antigens in the subject and/or wherein the AMR comprises allograft dysfunction.

17. The method according to any one of Claims 1-16, wherein the AMR is probable AMR or definite AMR.

18. The method according to any one of Claims 1-17, wherein the administration to the subject of the antibody or fragment which is capable of inhibiting human IL-6 is given in a first treatment dose initiated within the period 24 hours before or after initiation of the immunosuppressive therapy, optionally wherein administration of the first treatment dose is initiated within the period 2 hours before or after initiation of the immunosuppressive therapy, optionally wherein administration of the first treatment dose is initiated within the period one hour before or after initiation of the immunosuppressive therapy, optionally wherein administration of the first treatment dose is initiated substantially at the same time as initiation of the immunosuppressive therapy.

19. The method according to any one of Claims 1-18, wherein the immunosuppressive therapy is administered 30 to 60 minutes prior to an or each administration to the subject of the antibody or fragment which is capable of inhibiting human IL-6; optionally wherein the antibody or fragment which is capable of inhibiting human IL-6 is siltuximab.

20. The method according to any one of Claims 1-19, wherein the immunosuppressive therapy comprises a multi-drug regimen.

21. The method according to any one of Claims 1-20, wherein the immunosuppressive therapy comprises administering one or more of a baseline immunosuppression maintenance therapy, and/or a standard of care treatment for AMR.

22. The method according to Claim 21, wherein the immunosuppressive therapy comprises a standard of care treatment for AMR comprising one or more of a corticosteroid, intravenous immune globulin (IVIG), Rituximab, Bortezomib, Carfilzomib, anti-thymocyte globulin (ATG), plasma exchange (PLEX), tacrolimus, cyclosporine A, Mycophenolate Mofetil (MMF), azathioprine, prednisone, and methylprednisolone, and optionally acetaminophen (paracetamol) and/or diphenhydramine.

23. The method according to Claim 22, wherein the corticosteroid is selected from one or more of prednisolone, prednisone, methylprednisolone, cortisone, dexamethasone, betamethasone and hydrocortisone.

24. The method according to any one of Claims 20-23, wherein the immunosuppressive therapy comprises acetaminophen, diphenhydramine, and methylprednisolone; optionally wherein the acetaminophen, diphenhydramine, and methylprednisolone are administered 30-60 minutes prior to an or each administration to the subject of the antibody or fragment which is capable of inhibiting human IL-6; optionally wherein the antibody or fragment which is capable of inhibiting human IL-6 is siltuximab.

25. The method according to Claim 24, wherein the immunosuppressive therapy comprises administering acetaminophen 650 or 1000 mg by mouth, diphenhydramine 25 or 50 mg by mouth, and methylprednisolone 60 mg intravenously, and wherein the administration of the antibody or fragment which is capable of inhibiting human IL-6 is administration of siltuximab; optionally wherein the acetaminophen, diphenhydramine, and methylprednisolone are administered 30-60 minutes prior to an or each administration of the siltuximab.

26. The method according to any one of Claims 1-25, wherein the method further comprises administration to the subject of a prophylactic medication, wherein the prophylactic medication is an antiviral and/or antimicrobial medication, optionally wherein the prophylactic medication comprises one or more of Valganciclovir, Trimethoprim-Sulfamethoxazole, Voriconazole, Posaconazole, and Isavuconazonium.

27. The method according to any one of Claims 21-26, wherein the baseline immunosuppression maintenance therapy comprises one or more of tacrolimus, cyclosporine A, Mycophenolate Mofetil (MMF), azathioprine, and prednisone.

28. The method according to Claim 27, wherein the baseline immunosuppression maintenance therapy comprises:

(a) tacrolimus, or cyclosporine A;

(b) Mycophenolate Mofetil (MMF), or azathioprine; and

(c) optionally prednisone.

29. The method according to any one of Claims 1-28, wherein the administration of the combination therapy reduces one or more of the severity of allograft dysfunction, the severity of abnormal lung histology, and the amount of donor-specific antibodies in the subject.

30. An anti-IL-6 antibody or fragment for use in treatment of an antibody mediated rejection (AMR) of a lung transplant, wherein the treatment comprises a combination therapy of administration of the antibody or fragment and administration of an immunosuppressive therapy, wherein the immunosuppressive therapy does not comprise any antibody or fragment thereof which is capable of inhibiting human IL-6.

31. Use of an anti-IL-6 antibody or fragment in treatment of antibody mediated rejection (AMR) of a lung transplant, wherein the treatment comprises a combination therapy of administration of the antibody or fragment and administration of an immunosuppressive therapy, wherein the immunosuppressive therapy does not comprise any antibody or fragment thereof which is capable of inhibiting human IL-6.

32. Use of an anti-IL-6 antibody or fragment for the manufacture of a medicament for treatment of antibody mediated rejection (AMR) of a lung transplant, wherein the medicament is to be administered in a combination therapy with administration of an immunosuppressive therapy, wherein the immunosuppressive therapy does not comprise any antibody or fragment thereof which is capable of inhibiting human IL- 6.

33. An anti-IL-6 antibody or fragment for use according to Claim 30, or use of an anti- IL-6 antibody or fragment according to Claim 31 or 32, having any of the additional features of any of Claims 1 to 29.