Immunotherapy for the Treatment of Hodgkin Lymphoma: An Evolving Paradigm

Classical Hodgkin lymphoma (cHL) is one of the most common lymphomas in the Western world. Although most patients are cured with standard first-line therapy, up to 20% of patients will have relapsed or refractory disease. Although the conventional approach to treatment has consisted of chemotherapy, radiation, and for those who relapse, autologous or allogeneic transplantation, newer approaches have become available in recent years, including immunoconjugates and checkpoint inhibitors. These approaches have shown significant efficacy in clinical trials and might be associated with fewer long-term toxicities compared with conventional therapies. In this review we discuss the biology of cHL as it pertains to the immunosuppressive tumor microenvironment and then review the existing clinical trial results of several emerging immunotherapies in this context, including immune checkpoint inhibitors and adoptive cellular therapy. Finally, several clinical practice issues pertaining to the use of immunotherapies are discussed.     

Brentuximab Vedotin and Bendamustine Produce Long-Term Clinical Benefit in Patients With Relapsed or Refractory Classical Hodgkin Lymphoma: A Multicenter Real-Life Experience

BackgroundPatients with relapsed or refractory classical Hodgkin lymphoma (R/R cHL) have limited opportunities for curative therapy. High-dose therapy followed by autologous stem cell transplantation (HDT-ASCT) produces cure rates of 50% to 60%. Patients relapsing after, or ineligible for HDT-ASCT have limited therapeutic options and long-term remission is uncommon. Furthermore, few patients are candidate to allogeneic stem cell transplantation (AlSCT), a potentially curative approach. The combination of brentuximab vedotin and bendamustine (BVB) is a promising treatment for patients with R/R cHL, regardless of SCT eligibility.Patients and methodsWe conducted a real-life study of BVB in 41 patients with R/R cHL after failure of ≥ 1 therapy including ASCT, AlSCT, or BV.ResultsAmong 40 patients evaluable for efficacy, the overall response rate and complete response (CR) rate were 75% and 50%, respectively. No significant differences were observed between patients with primary refractory and relapsed disease, previously treated with ≤ 2 and ≥ 3 lines of therapy, or BV-exposed and BV-naïve. After a median follow-up of 38 months, the median progression free survival (PFS) for the entire population is 26 months; PFS is not reached, 10.5 months, and 4 months for patients achieving CR, partial response and no response, respectively (P < .0001). BVB was well tolerated and no grade 4 toxicity or new safety signals were observed. The most common treatment-emergent adverse events were infections.ConclusionOur experience supports the efficacy and tolerability of the BVB combination in R/R cHL as a bridge to SCT, or as a definitive therapy for SCT-ineligible patients. Larger comparative studies testing BVB against standards of care are warranted in both settings.     

Where Does Brentuximab Vedotin Fit into the Management of Patients with Hodgkin Lymphoma?

Brentuximab vedotin is an antibody-drug conjugate that targets CD30 and links monomethyl auristatin E, a microtubule disrupting agent, to an anti-CD30 monoclonal antibody. A phase II study of brentuximab vedotin in relapsed/refractory classical Hodgkin lymphoma (cHL) showed an impressive overall response rate of 75?% with 34?% complete responses, and median remission duration of 20?months in complete responders. In addition, brentuximab vedotin has very modest toxicity in heavily pretreated patients, with reversible peripheral neuropathy being the most common side effect. Brentuximab vedotin received accelerated FDA approval in August 2011 for use as a salvage therapy in cHL following failure of at least two prior therapies. Brentuximab vedotin is the treatment of choice for patients relapsing after stem cell transplant and for patients refractory to standard salvage regimens pre-transplant. Because of high single-agent activity and limited side effects, brentuximab vedotin has emerged as an ideal drug to test in combination therapy for cHL. Current trials are examining the use of brentuximab vedotin in frontline combination regimens, as salvage therapy prior to stem cell transplant, and as adjuvant treatment post-transplant. Such studies will help clarify the optimal use of brentuximab vedotin in the treatment paradigm for Hodgkin lymphoma.     

经典型霍奇金淋巴瘤表观遗传学异常及其对免疫逃逸的影响

经典型霍奇金淋巴瘤（Classic Hodgkin lymphoma,cHL）恶性表型的维持和表观遗传学有一定关系,表观遗传学改变主要包括组蛋白乙酰化异常和DNA甲基化异常,组蛋白去乙酰化酶抑制剂（ His-tone deacetylases inhibitors ,HDACis）的临床研究取得了进展。表观遗传学异常和cHL的免疫逃逸密切相关,甲基转移酶抑制剂、HDACis和免疫检测点阻断剂具有协同作用,采用联合治疗策略对进一步提高cHL的治愈率有重大意义。     

PD-1/PD-L1抑制剂治疗淋巴瘤的研究进展

免疫检查点抑制剂已被批准用于多种难治性实体肿瘤的治疗,如恶性黑色素瘤、肺癌、胰腺癌、肾癌等。其在复发/难治性淋巴瘤的治疗方面也展现出广阔的应用前景。程序性死亡受体-1及其配体(PD-1/PD-L1)通路是免疫检查点抑制治疗(checkpoint blockade therapy,CBT)的关键性通路之一。肿瘤细胞可通过PD-1/PD-L1通路抑制T细胞活性,阻断免疫应答,从而实现免疫逃逸,最终促进肿瘤的发生、发展。新近研究发现PD-1/PD-L1通路在复发/难治性霍奇金淋巴瘤（Hodgkin lymphoma,HL）和部分非霍奇金淋巴瘤（non-Hodgkin lymphoma,NHL）中也发挥着重要作用。本文将简单介绍PD-1/PD-L1通路的生物学活性,总结针对该通路的免疫疗法在恶性淋巴瘤治疗中的研究进展。     

Classical Hodgkin Lymphoma: Clinicopathologic Features,Prognostic Factors,and Outcomes From a 28-Year Single Institutional Experience

IntroductionClassical Hodgkin lymphoma (cHL) is a curable malignancy, with a complete remission rate of approximately 90%. However, relapse remains a significant cause of mortality. Prognostic factors are useful in guiding therapy. This is a large, single-institution study defining the clinicopathologic features, prognostic factors, and treatment outcomes of patients with cHL.Patients and MethodsWe reviewed 727 patients with cHL treated at H. Lee Moffitt Cancer Center and Research Institute from 1990 to 2017. Data on demographics, laboratory studies, and disease statuses were collected from the institutional database and electronic medical records. Statistical analyses, overall survival (OS), progression-free survival (PFS), and multivariate analyses were performed.ResultsThe median age was 35 years. Fifty-four percent of patients were men; 45.6% had advanced stage disease; 82% were treated with ABVD (doxorubicin hydrochloride [adriamycin], bleomycin sulfate, vincristine, and dacarbazine) as frontline therapy; and 70% achieved complete response. The median PFS after first-line treatment was 16.8 years. The median OS of patients with early stage and advanced stage cHL was 19 and 12.9 years, respectively. Poor prognostic factors for OS included older age, advanced stage disease, presence of B symptoms, and a higher International Prognostic Score.ConclusionDespite high cure rates, cHL accounted for the cause of death in 47% of patients who died during follow-up. Prognostic factors, such as age, stage at diagnosis, International Prognostic Score, and B symptoms, are helpful to guide treatment. Outcomes observed in this study are comparable with those reported in previously published studies.     

Trends in Checkpoint Inhibitor Therapy for Advanced Urothelial Cell Carcinoma at the End of Life: Insights from Real-World Practice

Several immune checkpoint inhibitor therapies (CPIs) have been approved to treat metastatic urothelial cell carcinoma (mUC). Because of the favorable toxicity profile of CPI compared with chemotherapy, oncologists may have a low threshold to prescribe CPI to patients near the end of life. We evaluated trends in initiation of end-of-life systemic therapy in 1,637 individuals in the Flatiron Health Database who were diagnosed with mUC between 2015 and 2017 and who died. Rates of systemic therapy initiation in the last 30 and 60 days of life were 17.0% and 29.8%, respectively. The quarterly proportion of patients who initiated CPI within 60 days of death increased from 1.0% to 23% during the study period (p_trend < .001). After CPI approval, end-of-life CPI initiation significantly increased among patients with poor performance status (p_trend = .020) and did not significantly change among individuals with good performance status. The quarterly proportion of patients who initiated any systemic therapy at the end of life doubled (17.4% to 34.8%) during the study period, largely explained by increased CPI use. These findings suggest a dramatic rise in CPI use at the end of life in patients with mUC, a finding that may have important guideline and policy implications.     

Myalgia and Arthralgia Immune-related Adverse Events (irAEs) in Patients With Genitourinary Malignancies Treated With Immune Checkpoint Inhibitors

BackgroundMyalgia and arthralgia immune-related adverse events (irAEs) in patients treated with checkpoint inhibitors (CPIs) present a clinical challenge. We describe the clinical characteristics and treatment of myalgia and arthralgia irAEs in CPI-treated patients with genitourinary (GU) malignancies.Patients and MethodsPatients with GU malignancies who were treated with CPIs and developed myalgia and arthralgia irAEs that resulted in interruption or discontinuation of CPI therapy were reviewed. Patient-, disease-, and irAE-related data were collected and analyzed.ResultsTwenty-one patients were identified. Eighteen (86%) had renal cell carcinoma; 3 (14%) had urothelial carcinoma. The majority (71%) were male; the median age at diagnosis was 56 years (range, 36-78 years). CPI therapy included anti-programmed death-ligand 1 (29%), anti-programmed cell death protein 1 (48%), and combined programmed cell death protein 1/cytotoxic T-lymphocyte-associated protein 4 antibodies (24%). The median time from CPI initiation to myalgia and arthralgia irAE was 5.1 months (range, 0.23-50.5 months). All patients were treated with prednisone with a median initial dose of 40 mg/d (range, 10-90 mg/d) for a median duration of 64 weeks (range, 3-242 weeks). Treatment with methotrexate (14%), infliximab (14%), tocilizumab (10%), gabapentin (10%), and etanercept (5%) was also required in some patients. Six (29%) patients restarted CPI therapy following symptom improvement, 3 (15%) switched to a subsequent therapy, and 12 (55%) patients had an ongoing sustained response to therapy (median, 14.5 months; range, 3-55 months) despite no subsequent anti-cancer therapy.ConclusionMyalgia and arthralgia irAEs in CPI-treated patients with GU malignancies vary in timing of presentation, severity, and treatment. Multidisciplinary teams that include a rheumatologist are critical for optimal management. Durable response to CPIs can be maintained even after therapy discontinuation.     

How to Approach a Hodgkin Lymphoma Patient With Relapse After Autologous SCT: Allogeneic SCT

Hodgkin lymphoma (HL) is a highly curable B-cell lymphoma, and ～90% of patients who present with early-stage (stage I-II) disease and 70% of patients who present with late-stage disease will be cured with standard frontline treatment. For patients with relapsed or refractory (r/r) disease after initial therapy, the standard of care is salvage chemotherapy, followed by autologous transplantation (autoSCT). Although this approach will cure a significant proportion of patients, upto 50% of patients will experience disease progression after autoSCT, and this population has historically had a very poor prognosis. In the past, further salvage chemotherapy, followed by allogeneic transplantation (alloSCT), has been the only option associated with a significant probability of long-term survival, owing to a graft-versus-lymphoma effect. However, this approach has been complicated by high rates of treatment-related morbidity and mortality and a high risk of disease relapse. Furthermore, many patients have been unable to proceed to alloSCT because of disease refractoriness, poor performance status, or the lack of a donor. However, significant therapeutic advances in recent years have greatly expanded the options for patients with post-autoSCT r/r HL. These include the anti-CD30 antibody–drug conjugate brentuximab vedotin and the checkpoint inhibitors nivolumab and pembrolizumab, as well as increasing experience with alternative donor alloSCT, especially from haploidentical donors. In the present review, we discuss the current role of alloSCT in the treatment of HL after autoSCT relapse.     

Real-World Patient Characteristics,Treatment Patterns,and Outcomes for Patients With Stage III or IV Classic Hodgkin Lymphoma Treated With Frontline ABVD: A Retrospective Database Review in the United States

In newly diagnosed stage III/IV classic Hodgkin lymphoma (cHL), A+AVD (brentuximab vedotin, doxorubicin, vinblastine, dacarbazine) improved overall survival (OS) versus ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine). As clinical trial and real-world populations may differ, real-world treatment characteristics and OS (rwOS) were assessed for patients with stage III/IV cHL treated with frontline ABVD. This retrospective, observational analysis of deidentified electronic health record data (1/1/2011-8/31/2020) evaluated baseline disease and clinical characteristics, treatment patterns, and rwOS in patients with stage III/IV cHL treated with frontline ABVD. Data for 167 patients were analyzed. A median of 6 ABVD cycles were received. Baseline/interim positron emission tomography (PET) scans were obtained for 60.5%/89.8% of patients. Of patients diagnosed in 2016 or later (n = 73), 89% received an interim PET scan; 15/46 patients with no documented Deauville score, 6/15 with a score of 1 to 3, and 3/4 with a score of 4 to 5 de-escalated to AVD. Following frontline ABVD, 55.1% of patients received subsequent systemic therapy and 31.7% stem cell transplantation (SCT). At a median follow-up of 31.8 months, 82.0% of patients were alive (median rwOS, 101.2 months). Patients with stage III/IV cHL treated with frontline ABVD in the real world versus in clinical trials receive more subsequent therapy, including SCTs. Interim PET scans and Deauville scores were not universally obtained after treatment cycle 2, yet treatment de-escalation was observed. Patients with stage III/IV cHL may benefit from frontline A+AVD versus ABVD, as it improves OS and reduces the burden of subsequent therapy, including SCTs.     

Pembrolizumab for the Treatment of Relapsed and Refractory Classical Hodgkin Lymphoma After Autologous Transplant and in Transplant-Naïve Patients

IntroductionCheckpoint inhibitors demonstrated significant efficacy in relapsed/refractory Hodgkin's Lymphoma (R/R cHL) resulting in high responses and prolonged progression free survival in patients, who relapse after or are ineligible for autologous stem cell transplantation (auto-SCT). We aimed to assess the efficacy and toxicity of Pembrolizumab before auto-SCT and in transplant naïve patients and calculate survival outcomes.Patients and MethodsFifty-five patients with R/R cHL were included. Patients demographics, including age, sex, risk stratification, therapy received and details pertaining transplantation, were collected.ResultsMedian age was 28 years (range, 16-62 years). The median follow-up was 15.3 months and the median number of previous treatments was 3 (1-10). The best objective response was 74.5% (CR 32.7%, SD 5.5%) with reasonable safety profile. Twenty-nine of the responding patients received subsequent auto-SCT and 9 allogeneic stem cell transplantation (allo-SCT), 6 are currently alive with ongoing response. At the time of analysis, 6 patients remained on Pembrolizumab and the rest discontinued. The main reason for discontinuation was disease progression (n-49). Twelve-months overall survival and progression free survival (PFS) was 92% (95% CI: 76%-95%) and 51% (95% CI, 39%-67%) respectively. Twelve-month PFS for patients, who achieved CR or PR or PD was 88% (95% CI: 07%-75%); PR 60% (95% CI: 21%-29%) and 5% (95% CI: 5%-0%). Though the number of patients who received auto-SCT after Pembrolizumab was small (n-15), 12 months overall survival and PFS 100% and PFS 92%. 11 patients (20%) deceased during the follow-up and none was regarded to be treatment-related.ConclusionCheckpoint inhibitors are effective in heavily pretreated cHL patients with reasonable survival outcomes. The results supporting the concept of auto and/or allo-SCT after checkpoint inhibitors use.     

中国霍奇金淋巴瘤的诊断与治疗指南(2022年版)

  目的  探讨新药时代自体造血干细胞移植（autologous hematopoietic stem cell transplantation,ASCT）治疗复发或难治性（relapse or refractory,R/R）经典型霍奇金淋巴瘤（classical Hodgkin's lymphoma,cHL）的疗效。  方法  回顾性分析2010年1月至2022年12月在中国医学科学院血液病医院诊治的56例挽救治疗敏感、序贯ASCT治疗的R/R cHL患者,根据挽救治疗是否包含维布妥昔单抗（brentuximab vedotin,BV）或程序性死亡受体1（programmed death-1,PD-1）抑制剂分为新药组32例和非新药组24例,分析两组患者的临床特征及疗效,使用Kaplan-Meier法进行生存分析。  结果  56例患者中男性35例,女性21例,中位移植年龄29（11～55）岁。中位随访时间56（ 2～137）个月,移植后预期5年总生存（overall survival,OS）率和无进展生存（progression- free survival,PFS）率分别为94.3%和75.8%。新药组患者的5年PFS更优（90.1% vs. 59.1%,HR=0.23,95%CI：0.07～0.71,P=0.011）,但OS差异无统计学意义（93.5% vs. 95.5%,HR=1.2,95%CI：0.14～10.34,P=0.873）。  结论  对于挽救治疗敏感的R/R cHL患者,ASCT仍然是标准的巩固治疗策略。新药在移植前、后的应用可进一步提高ASCT治疗R/R cHL患者的生存。     

Targeting CD30 in Anaplastic Large Cell Lymphoma

Anaplastic large cell lymphoma (ALCL) is a lymphoid neoplasm characterized by strong and uniform expression of the CD30 antigen on the cell surface. Current standard frontline therapy of ALCL is anthracycline-based combination chemotherapy, usually CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) or CHOP-like regimens. Despite aggressive chemotherapy a significant number of patients relapse. Newer agents and strategies are needed in the management of this challenging disease especially in ALK-negative and high-risk ALK-positive patients who tend to have a poor prognosis. In this review we discuss the different approaches to targeting CD30 including naked antibodies, ??enhanced antibodies??, antibody drug?Ctoxin conjugates, radioimmunoconjugates, CD30-ligand?Ctoxin conjugates, bispecific antibodies and T cell-based immune therapies.     

Matrix metalloproteinase-9 is consistently expressed in Hodgkin/Reed-Sternberg cells and has no impact on survival in patients with Epstein–Barr virus (EBV)-related and non-related Hodgkin lymphoma in Brazil

Clinical and histological features of classical Hodgkin lymphoma (cHL) are primarily due to the effects of cytokines and enzymes produced by Hodgkin/Reed-Sternberg (HRS) cells and their surrounding inflammatory cells. In EBV-related cancers, the expression of viral latent membrane protein 1 correlates with an increased MMP9 expression. In this study, we evaluated the prognostic relevance of MMP9 expression and EBV status in HRS cells in patients with cHL in Brazil. We selected 97 patients with cHL for EBV and MMP9 detection. EBV was detected in 52.5%, and MMP9 expression positivity was found in 87.6%. Of all cases, there was no correlation between MMP9 expression and EBV status. Response to treatment and relapse rate was independent of MMP9 expression and EBV status. MMP9 positivity did not influence overall survival and event-free survival. The consistent and increased intensity of MMP9 expression in HRS cells make this enzyme a potential target for therapy.     

Where Do the New Drugs Fit in for Relapsed/Refractory Hodgkin Lymphoma?

The standard approach for relapsed or refractory (rel/ref) Hodgkin lymphoma (HL) following frontline treatment failure is salvage therapy followed by consolidation with high-dose therapy and autologous stem cell transplant (HDT/ASCT). While this overall treatment paradigm has been in place for several decades, recent studies have aimed to improve the efficacy and tolerability of salvage therapies by incorporating newer drugs, such as brentuximab vedotin (BV) and checkpoint inhibitors. Following HDT/ASCT, survival is improved due to the availability of BV and the checkpoint inhibitors, nivolumab and pembrolizumab; however, for patients responding to checkpoint inhibition, the appropriate length of treatment and the role of allogeneic stem cell transplant are unclear. In this review, we discuss our management of rel/ref HL, with particular focus on how BV, nivolumab, and pembrolizumab are currently incorporated into the treatment paradigms for rel/ref HL.     

Mechanistic overview of immune checkpoints to support the rational design of their combinations in cancer immunotherapy

Checkpoint receptor blockers, known to act by blocking the pathways that inhibit immune cell activation and stimulate immune responses against tumor cells, have been immensely successful in the treatment of cancer. Among several checkpoint receptors of immune cells, cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), programmed cell death protein-1 (PD-1), T-cell immunoglobulin and ITIM domain (TIGIT), T-cell immunoglobulin-3 (TIM-3) and lymphocyte activation gene 3 (LAG-3) are the most commonly targeted checkpoints for cancer immunotherapy. Six drugs including one CTLA-4 blocker (ipilimumab), two PD-1 blockers (nivolumab and pembrolizumab) and three PD-L1 blockers (atezolizumab, avelumab and durvalumab) are approved for the treatment of different types of cancers including both solid tumors such as melanoma, lung cancer, head and neck cancer, bladder cancer and Merkel cell cancer as well as hematological tumors such as classic Hodgkin's lymphoma. The main problem with checkpoint blockers is that only a fraction of patients respond to the therapy. Insufficient immune activation is considered as one of the main reason for low response rates and combination of checkpoint blockers has been proposed to increase the response rates. The combination of checkpoint blockers was successful in melanoma but had significant adverse events. A combination that is selected based on the mechanistic differences between checkpoints and the differences in expression of checkpoints and their ligands in the tumor microenvironment could have a synergistic effect in a given cancer subtype and also have a manageable safety profile. This review aims to help in design of optimal checkpoint blocker combinations by discussing the mechanistic details and outlining the subtle differences between major checkpoints targeted for cancer immunotherapy.     

Treating Hodgkin lymphoma in the new millennium: Relapsed and refractory disease

Although the majority of patients with Hodgkin lymphoma are cured with initial therapy, 10% to 15% of patients with early stage disease and 15% to 30% of patients with advanced disease have primary refractory or relapsed lymphoma. For younger patients whose disease is sensitive to second‐line therapy, more than half of patients will experience long‐term disease control with high‐dose chemotherapy/autologous stem cell rescue (ASCT). For those patients who are chemotherapy refractory, relapse after, or are ineligible for ASCT, brentuximab vedotin, and checkpoint, inhibitors are highly active, although the majority of patients will ultimately experience recurrent lymphoma. Allogeneic transplant is curative in a subset of patients but may be associated with significant toxicity. Novel targeted and immunotherapy approaches, including chimeric antigen receptor T‐cell therapy, are currently being studied in clinical trials with promising early results.     

Evaluation of the antibody response to the EBV proteome in EBV-associated classical Hodgkin lymphoma

The humoral immune response to Epstein–Barr virus (EBV) in classical Hodgkin lymphoma (cHL) stratified by EBV tumor status is unclear. We examined IgG and IgA antibody responses against 202 protein sequences representing 86 EBV proteins using a microarray and sera from 139 EBV-positive cHL cases, 70 EBV-negative cHL cases and 141 population-based controls frequency matched to EBV-positive cHL cases on sex and age by area (UK, Denmark and Sweden). We leveraged existing data on the proportion of circulating B-cells infected by EBV and levels of serum CCL17, a chemokine secreted by cHL tumor cells, from a subset of the cHL cases in the UK. Total IgG but not IgA response level was significantly different between EBV-positive cHL cases and controls. The distinct serological response included significant elevations in 16 IgG antibodies and 2 IgA antibodies, with odds ratios_{highest vs. lowest tertile > 3} observed for the following EBV proteins: LMP1 (oncogene), BcLF1 (VCAp160, two variants) and BBLF1 (two variants). Our cHL IgG signature correlated with the proportion of circulating EBV-infected B-cells, but not serum CCL17 levels. We observed no differences in the anti-EBV antibody profile between EBV-negative cHL cases and controls. BdRF1(VCAp40)-IgG and BZLF1(Zta)-IgG were identified as the serological markers best able to distinguish EBV-positive from EBV-negative cHL tumors. Our results support the hypothesis that differences in the EBV antibody profile are specific to patients with EBV-positive cHL and are not universally observed as part of a systematically dysregulated immune response present in all cHL cases.     

Efficacy of an adapted granzyme B-based anti-CD30 cytolytic fusion protein against PI-9-positive classical Hodgkin lymphoma cells in a murine model

Tumors develop when infiltrating immune cells contribute growth stimuli, and cancer cells are selected to survive within such a cytotoxic microenvironment. One possible immune-escape mechanism is the upregulation of PI-9 (Serpin B9) within cancer cells. This serine proteinase inhibitor selectively inactivates apoptosis-inducing granzyme B (GrB) from cytotoxic granules of innate immune cells. We demonstrate that most classical Hodgkin lymphoma (cHL)-derived cell lines express PI-9, which protects them against the GrB attack and thereby renders them resistant against GrB-based immunotherapeutics. To circumvent this disadvantage, we developed PI-9-insensitive human GrB mutants as fusion proteins to target the Hodgkin-selective receptor CD30. In contrast to the wild-type GrB, a R201K point-mutated GrB construct most efficiently killed PI-9-positive and -negative cHL cells. This was tested in vitro and also in vivo whereby a novel optical imaging-based tumor model with HL cell line L428 was applied. Therefore, this variant, as part of the next generation immunotherapeutics, also named cytolytic fusion proteins showing reduced immunogenicity, is a promising molecule for (targeted) therapy of patients with relapsing malignancies, such as cHL, and possibly other PI-9-positive malignancies, such as breast or lung carcinoma.