Key words : Antithymocyte globulins, Biopsy-proven acute rejection, Induction immunosuppression, Kidney transplant

Introduction

In the ever-evolving era of transplant immunology, induction of graft tolerance and facilitation of long-term drug-free survival continue to remain the top clinical priorities. Induction immunosuppression is the most modifiable determinant of graft tolerance; its primary aim is to reduce the risk of acute rejection.¹ Induction agents are an inevitable part of renal transplant as endorsed by the Cochrane Collaboration (Kidney Disease: Improving Global Outcomes 2009) guideline.² Conventional armamen-tarium includes lymphocyte-depleting antibodies (eg, rabbit antithymocyte globulins [ATGs], rituximab, bortezomib) and lymphocyte-non-depleting antibodies like interleukin 2 receptor antibodies. However, data on their clinical use remain limited; the drug of choice, their dose, and duration remain controversial, and there is often center-to-center variabilities. It is, thus, imperative to have a tailor-made benefit versus risk assessment done to individualize the regimen, prevent rejections, and still minimize the risk of infections in these patients.

Antithymocyte induction remains a modality of choice in kidney transplant recipients, owing to the lower incidence of acute rejection and successful management of delayed graft function associated with their use.³ Two of the most commonly available rabbit polyclonal antithymocytes are ATG (thymoglobulin), derived from immunization of rabbits with hetero-genic human thymocytes, and anti-T-lymphocyte globulin (ATLG; Grafalon, Neovii Pharmaceuticals AG, which was previously known as ATG-Fresenius). Grafalon is derived from immunization of rabbits with the human lymphoblastoid cell line Jurkat.

However, ATG and ATLG are known to have qualitative differences in the concentrations of antibodies against different target antigens, as well as minor differences among their antigen profile.⁴ Bamoulid and colleagues⁵ reported that ATG contained 522 ?g/mg of antibodies, whereas ATLG contained only 160 ?g/mg of antibodies. This suggests that a 3-fold increase in the dose of ATLG allows an equivalent quantity of antibodies. In addition, because ATLG originates from a T-cell line that is HLA typed, the specificities of anti-HLA antibodies become highly predictable, thus offering a narrower spectrum of activity. Comparative studies have demonstrated efficacy of ATLG similar to that of thymoglobulin in terms of patient and graft outcomes, with lesser long-term T-cell depletion, thus avoiding deleterious consequences like malignancy and posttransplant lymphoproliferative disorder.⁶

In developing economies like India, the cost of immunosuppressive therapy remains a major concern. Hence, identifying both the induction agent and the right dose is imperative to these patients, along with determining anticipated clinical benefits and complications. Antithymocyte globulin has been in use in India since 2005, and the dose and outcomes are also comparable to those shown globally. However, data on ATLG in India are scarce, owing to its limited experience; hence, the dosage and immunosuppression protocol remain inconsistent across various clinical settings. This real-world report aimed to evaluate the optimal dosage of ATLG (Grafalon) as an induction agent for the prevention of rejection and its impact on various posttransplant events in Indian renal transplant recipients.

Materials and Methods

This single-center, retrospective, observational study analyzed patient data available from the medical records of 177 consecutive renal transplant recipients who had received ATLG (Grafalon) as induction agent from September 2016 to March 2018 at Sir Ganga Ram Hospital (New Delhi, India). No control for comparison was applicable, and a formal sample size estimation was not considered owing to the real-world, observational nature of the study. The retrospective study protocol was approved by the Ethics Committee of Sir Ganga Ram Hospital for analysis and publication. Patient consent was considered waived because of the retrospective nature of the study, which was also approved by the Ethics Committee. All the applicable guidelines were followed for the conduct of the study.

As part of the center’s routine practice, all patients had a common posttransplant follow-up schedule (ie, patients were followed weekly until 3 months after discharge, every 2 weeks for the next 2 months, every month thereafter until 1 year, and bimonthly after 1 year posttransplant). For the purpose of this analysis, clinical data, as available from the medical records, from the time of transplant to at least 12 to 18 months posttransplant were retrieved. The cut-off for the last follow-up record was March 2019.

Data targeted for retrieval from available patient medical records included the following: pretransplant demographic and clinical profiles (age, sex, history of cardiovascular disease, malignancy, diabetes mellitus), prior renal transplant and panel reactive antibody, relevant donor data (age, serum creatinine, and relationship to recipient), donor and recipient blood type match/mismatch, human leukocyte antigen (HLA) haplotype compatibility, ATLG dosage details, other immunosuppressant treatments and dosage, absolute lymphocyte count (ALC) and/or CD4 counts, details of graft rejection, kidney biopsy results, and posttransplant infection events. Acute rejection was searched for in any case of graft dysfunction, which was defined as at least a 20% increase in creatinine level from baseline. Records with only biopsy-proven acute rejections (BPARs) were considered. All cases of death and its cause were assessed through medical records.

Collected data were analyzed and reported descriptively using Microsoft Excel. Continuous variables were expressed as arithmetic mean ± standard deviation (SD) or as median and range (minimum, maximum). Categorical variables are presented as the number of patients and frequency in percent. Patient and graft outcomes, including incidence of BPARs, treatment-related adverse events, dosage protocol of ATLG, and incidence of infective complications, were also reported.

Results

Pretransplant demographic and clinical charac-teristics
Medical records of 177 consecutive renal transplant recipients, including from both living donors (n = 175) and standard criteria deceased donors (n = 2), seen at our study center over the study period were considered and analyzed. Data were available for a median follow-up of 18 months posttransplant. Among those included, mean age was 41.46 ± 12.67 years (range, 14-68 years), with 85% male patients. Diabetes (33%), hypertension (25%), and chronic glomeru-lonephritis (23%) were the most common underlying indications for renal transplant. The pretransplant demographic and clinical characteristics of the patients are presented in Table 1. The selection and approval of donors were reviewed and confirmed by the Authorization Committee, which was constituted in accordance with the Transplantation of Human Organs Act Amendment 20117 of India. For the 175 living donors, the mean donor age was 50.6 years, with 83% (145/175) being near relatives and 75% (131/175) being females. Donor details are also presented in Table 1.

Retrieved data indicated that all living donor transplant candidates were evaluated with complement-dependent cytotoxicity (CDC) cros-smatch. Screening for HLA antibody was done in all patients by LAB Screen mixed beads kit (Lifecodes Immucor Inc.) for antibodies against class I and class II antigens. Additional crossmatch either by Flow cytometer or Luminex platform was done in case of a sensitized patient or doubtful CDC crossmatch to detect donor-specific antibodies. Single-antigen bead assay was done with class I and II beads to detect and confirm donor-specific antibodies. Typing for HLA (A, B, DRB1 loci) was done by polymerase chain reaction for both patients and donors. At the time of deceased donor renal transplant, CDC crossmatch was done in those patients, and additional solid-phase-based crossmatch (Flow cytometer/Luminex) was done in those who were undergoing a second transplant or for sensitized patients (prior CDC crossmatch positivity and/or having detectable HLA antibody by single-antigen bead assay). ABO incompatibility was observed in 23 of 177 (13%) patients and 77% of the patients had an HLA mismatch of ?3.

All patients (100%) and 95% of the donors tested positive for cytomegalovirus (CMV) immunog-lobulin G and were given standard CMV prophylaxis (100 days); none of the patients had CMV immunoglobulin M positivity. Two patients positive for hepatitis B surface antigen were treated as per the standard protocol and were scheduled for transplant only after the viral load became minimal. Two patients positive for hepatitis C virus received a combination of sofosbuvir (400 mg) plus daclatasvir (60 mg) once daily for up to 4 to 5 days post-transplant once renal function had normalized.

Immunosuppressive treatment protocol
As part of the induction immunosuppression protocol, patients received either tacrolimus 0.1 mg/kg/day (85% patients) or cyclosporine 8 mg/kg/day (15% patients) and mycophenolate mofetil 1.5 to 2 g/day from day -2 (prior to transplant). Patients received methylprednisolone 125 mg/day intravenously on the day of transplant until 3 days posttransplant and oral prednisolone 0.4 mg/kg thereafter. The ABO-incompatible patients had received a single dose of rituximab 500 mg along with tacrolimus 0.5 mg/kg and mycophenolate mofetil 1 g/day starting 14 days before transplant. Therapeutic plasma exchange/immunoadsorption was used for monitoring until ABO antibody titer levels were acceptable (<1:8). Tacrolimus levels and ALC were monitored throughout. Initially, tacrolimus dose adjustments maintained tacrolimus levels at 8 to 9 ng/mL until 1 month posttransplant and then were reduced to 6 to 7 ng/mL thereafter (Figure 1).

Anti-T-lymphocyte globulin (Grafalon) dosage
Anti-T-lymphocyte globulin was administered at 4 to 6 mg/kg in either 2 or 3 equally divided doses starting from day 0, day 1, and day 2 and titrated to target an ALC of <200/?L. Details of the actual ATLG doses used in this group are provided in Table 2. The average dose of ATLG used in the study group was 5.81 ± 1.95 mg/kg. The minimum dose was 2.41 mg/kg, and the maximum dose was 10.07 mg/kg. Overall, 4 patients (2%) received a single dose of ATLG, 92 (52%) received 2 doses, 76 (43%) received 3 doses, 3 (1.7%) received 4 doses, and 2 (1%) received 5 doses of ATLG.

Graft dysfunction, rejection, and survival
The mean serum creatinine values before and after transplant at various time points until 12 months are presented in Table 3. The mean creatinine values continued to remain low after discharge (from 1.24 to 1.36 mg/dL) during follow-up visits throughout 12 months posttransplant. Graft dysfunction, defined as at least a 20% increase in creatinine levels, was observed in 26 patients (14%), with two-thirds of these occurring within 3 months posttransplant. Of this, acute tubular necrosis was observed in 11 patients (6.2%), BPARs in 11 patients (6.2%), and calcineurin inhibitor toxicity in 4 patients (2.2%).

Of the 11 incidences of BPAR, 7 (4%) were antibody-mediated rejections (AMRs) and 4 (2.2%) were acute cellular rejections (2 rejections had Banff criteria IA, and 2 rejections had Banff criteria IIB); all of these rejections occurred within month 1 posttransplant and were treated with intravenous methylprednisolone 500 mg consecutively for 3 days. In cases of AMRs, plasma exchanges with intravenous immunoglobulins were also sup-plemented as part of antirejection therapy. All of the acute cellular rejections and 3 cases of AMR responded well to the treatment with complete and good recovery of renal function. The remaining 4 patients with BPAR continued to progress to end-stage renal disease and returned to maintenance hemodialysis.

In the study group, there were 7 deaths. Reasons for death included fungal pneumonia, bacterial pneumonia, and acute coronary syndrome in 2 patients each and urinary tract infection (UTI) with septicemia in 1 patient. Death-censored graft survival was 100% until month 12 and 98% until month 18 of follow-up. Overall patient survival was 96% until the end of the study analysis.

Infection episodes and other side effects
No incidence of malignancy was reported over our follow-up period. There were 58 infection episodes in 40 patients (22.5%), with the most common being UTI in 32 patients (18%) and Escherichia coli being the most common organism. Other common infections included lower respiratory tract infection in 3 patients, CMV and BK virus reactivation in 1 patient each, and other infections in 3 patients. There were no major adverse events related to ATLG except for fever and chills observed in 4 patients (2%) and postural hypotension in 1 patient (only during the infusion period); these did not lead to treatment discontinuation in any of the patients.

Discussion

Despite multiple reports demonstrating the benefits of induction therapy with ATGs and ATLGs in recipients with high immunologic risk globally,^8-12 clinical experiences, optimal dosage, and safety and effectiveness of these agents in Indian transplant recipients remain particularly limited. This real-world experience attempted to bridge this gap in knowledge and to provide details of ATLG dosage and clinical outcomes in Indian renal transplant recipients with moderate immunologic risk. In our analysis, a mean dose of 5.81 ± 1.95 mg/kg ATLG in combination with a tacrolimus-based steroid-containing triple maintenance immunosuppression was effective in protecting the transplant recipients from acute rejection, with rate of BPARs of 6.2%. Overall patient survival and death-censored graft survival at a median follow-up of 18 months were 96% and 98%, respectively.

In an earlier single-center isolated study13 that evaluated ATLG in renal transplant induction in India, the sample size was extremely limited and may have compromised interpretation of findings (only 11 recipients were studied). Patients in the study used ATLG at a dose of 4 mg/kg as induction, and all patients had also received steroid pulse therapy. The study reported a high graft rejection rate of 36.3%, which could have been primarily affected from the slightly lower and fixed ATLG dose that was used. In contrast to this report, rate of BPARs was only 6.2% in our patient group. The mean serum creatinine levels were also generally lower for the first month posttransplant and continued to remain so until the end of our study period. For the initial 4 to 5 days, tacrolimus levels were high and the dose was titrated. However, levels were reduced 7 to 10 days later along with ATLG dose completion (which is also seen with ATG and interleukin 2 receptor antagonists, although reasons remain unknown).

Our study included a larger group of 177 recipients from our high-volume center who have used ATLG since its introduction in India in 2016. The dose of ATLG was individualized based on benefit-risk profile and titrated with ALC count monitoring (if ALC was <200/?L, a third dose was not given); these factors could have resulted in good clinical outcomes. Our BPAR rate of 6.2% in this real-world experience in India was also in agreement with a recently published cohort study in which acute rejections rates were 4.7% and 7.5% in dose cohorts of ?7 mg/kg and <7 mg/kg ATLG, respectively.¹² Our patient survival rates (96%) and death-censored graft survival rates (98%) at 18 months were similar to those reported by Yilmaz and colleagues,¹⁰ who reported rates of 93% and 94.8%, respectively, at 1 year in deceased-donor renal transplant recipients.

Although the ATG dose and its optimization to alleviate acute rejection, facilitate recovery of the immune system, and improve outcomes for recipients have always been emphasized, the impact of different ATG doses has shown inconsistency.^14-16 Moreover, recommended doses for ATLG are higher compared with thymoglobulin but still determined only empirically.5 In previous studies,^10,17,18 different dosing regimens of ATLG have been used, varying from 3 to 21 mg/kg. Multiple reports have shown the efficacy of ATLG at a single high dose of 9 mg/kg for the prevention of acute rejection responses and good survival rates in renal transplant recipients^19-23; however, the corresponding increased risk of serious adverse effects, including high mortality linked to cardiovascular or infectious episodes or advanced malignancies, cannot be overlooked.²³ On the other hand, Chen and colleagues¹¹ have reported a cumulative dose of 6 mg/kg as effective induction therapy and further indicated that a lower dose might be suitable for Chinese patients in their study, which suggests that ethnic, genetic, and hereditary factors may play roles in the efficacy of these therapies and the need for locally researched regimens. A recent retrospective Indian study reported the use of 6 mg/kg Grafalon as an induction agent in renal transplantation. The patient survival and death-censored graft survival rates (99%) with Grafalon were comparable to thymoglobulin (3 mg/kg) at 22 months follow-up but the rate of BPARs was significantly higher in Grafalon group (12.8% vs. 5.1%) in this study.²⁴ The survival rates in our study are comparable to these findings, with a lower BPAR of 6.2% in our patient group. In our study, a dose of 4 to 6 mg/kg titrated with ALC monitoring and given in divided doses was associated with good clinical outcomes in Indian patients. This is suggestive of a tailor-made approach to patient dose requirements, as some may need a higher or a lower dose, a scenario that we consider to be applicable in an Indian setting. Our findings from this real-world experience for India were also in agreement with a recently published cohort study, wherein a <7 mg/kg dose of ATLG reduced the risk of infections without an effect on the induction efficacy or increased risk of acute rejections.¹²

The most common concern with ATG and ATLG use is overimmunosuppression and severe infections. Gaber and colleagues²⁵ reported a CMV infection rate of 4.2% with thymoglobulin induction in living donor transplant recipients in the TAILOR study where 1 patient had invasive CMV infection. Bamoulid and colleagues,⁵ however, reported a CMV infection rate of 9% in patients who received ATLG. In our study, the dose of 4 to 6 mg/kg was associated with a marked reduction in incidence of infections, with the most common infection being UTIs (in 18% of patients), which is expected with ATLG use. Only 1 case each of CMV and BK virus reactivation was reported.

This was a single-arm, single-center retrospective study, thus purely descriptive in nature with a small cohort size and no comparator arm. The missing or incomplete data in the records and short-term follow up are limitations. Nonetheless, the report is reflective of a real-world scenario, showing a tailor-made approach to induction protocols and outcomes in Indian renal transplant recipients, data for which are particularly lacking. Future prospective comparisons in controlled settings with long-term follow-up and varied subsets of patients, including elderly patients, can help investigate the benefits of these induction protocols in a larger population across centers.

Conclusions

Many transplant centers are hesitant to use potent induction therapies like ATLG because of the risks of infection or malignancy, and there is a lack of local clinical evidence supporting the role of ATLG in long-term graft survival. Reports such as ours can provide evidence from real-world practice on the effectiveness and safety of ATLG as an induction therapy, where an individualized dosing approach was shown to lead to good clinical outcomes in living-donor transplant patients with moderate immunological risk.

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