Antibody-dependent enhancement of toxicity of myotoxin II from Bothrops asper.

Improved therapies are needed against snakebite envenoming, which kills and permanently disables thousands of people each year. Recently developed neutralizing monoclonal antibodies against several snake toxins have shown promise in preclinical rodent models. Here, we use phage display technology to discover a human monoclonal antibody and show that this antibody causes antibody-dependent enhancement of toxicity (ADET) of myotoxin II from the venomous pit viper, Bothrops asper, in a mouse model of envenoming that mimics a snakebite. While clinical ADET related to snake venom has not yet been reported in humans, this report of ADET of a toxin from the animal kingdom highlights the necessity of assessing even well-known antibody formats in representative preclinical models to evaluate their therapeutic utility against toxins or venoms. This is essential to avoid potential deleterious effects as exemplified in the present study.

Re-evaluating the need for chronic toxicity studies with therapeutic monoclonal antibodies, using a weight of evidence approach.

To support registration of monoclonal antibodies (mAbs) for chronic indications, 6-month toxicity studies have historically been conducted. Experience with mAb development has shown a relatively benign and well-understood safety profile for this class, with most toxicity findings anticipated based on pharmacology. We evaluated whether a 6-month toxicity study is necessary to assess the long-term safety of mAbs. Data on First-in-Human (FIH)-enabling and chronic toxicity studies were shared for 142 mAbs submitted by 11 companies. Opportunities to further optimize study designs to reduce animal usage were identified. For 71% of mAbs, no toxicities or no new toxicities were noted in chronic studies compared to FIH-enabling study findings. New toxicities of potential concern for human safety or that changed trial design were identified in 13.5% of cases, with 7% being considered critical and 2% leading to program termination. An iterative, weight-of-evidence model which considers factors that influence the overall risk for a mAb to cause toxicity was developed. This model enables an evidence-based justification, suggesting when 3-month toxicity studies are likely sufficient to support late-stage clinical development and registration for some mAbs.

Regulatory Experience Assessing the Carcinogenic Potential of a Monoclonal Antibody Inhibiting PCSK9, Bococizumab, Including a 2-Year Carcinogenicity Study in Rats.

Bococizumab is an anti-PCSK9 monoclonal antibody that was intended for the treatment of hypercholesterolemia. After reviewing the 6-month rat toxicity study data, in which there was a low spontaneous tumor incidence, unrelated to bococizumab administration, the U.S. FDA granted a carcinogenicity waiver request based on a weight-of-evidence assessment of low carcinogenic risk. Subsequently, after reviewing 6-month rat toxicity study data from another anti-PCSK9 antibody, RN317, with a similar low tumor incidence (unrelated to RN317), the U.S. FDA rescinded the bococizumab carcinogenicity study waiver and requested a full 2-year rat carcinogenicity study be conducted. The resulting 2-year carcinogenicity study demonstrated no bococizumab-related increase in tumors, confirming the weight-of-evidence evaluation and alleviating concerns regarding the carcinogenic potential. Here we report the scientific and regulatory background that led to the request for a rat carcinogenicity study, the feedback on the design of the carcinogenicity study, and the results from this study which affirmed the original weight-of-evidence assessment of low carcinogenic risk.

An Afucosylated Anti-CD32b Monoclonal Antibody Induced Platelet-Mediated Adverse Events in a Human Fcγ Receptor Transgenic Mouse Model and Its Potential Human Translatability.

To assess the safety and tolerability of NVS32b, a monoclonal, afucosylated, anti-CD32b (FCGR2B) antibody, we used a humanized transgenic (Tg) mouse model that expresses all human Fc gamma receptors (FCGRs) while lacking all mouse FCGRs. Prior to its use, we extensively characterized the model. We found expression of all human FCGRs in a pattern similar to humans with some exceptions, such as low CD32 expression on T cells (detected with the pan CD32 antibody but more notably with the CD32b-specific antibody), variation in the transgene copy number, integration of additional human genes, and overall higher expression of all FCGRs on myeloid cells compared to human. Unexpectedly, NVS32b induced severe acute generalized thrombosis in huFCGR mice upon iv dosing. Mechanistic evaluation on huFCGR and human platelets revealed distinct binding, activation, and aggregation driven by NVS32b in both species. In huFCGR mice, the anti-CD32b antibody NVS32b binds platelet CD32a via both Fc and/or complementarity determining region (CDR) causing their activation while in human, NVS32b binding requires platelet preactivation and interaction of platelet CD32a via the Fc portion and an unknown platelet epitope via the CDR portion of NVS32b. We deemed the huFCGR mice to be overpredictive of the NVS32b-associated human thrombotic risk.

Immune Checkpoint Inhibitor-Related Pulmonary Toxicity: A Comprehensive Review, Part II.

The development of immune checkpoint inhibitors (ICIs) has changed the treatment paradigm for cancer. The ICIs nivolumab, pembrolizumab, and cemiplimab target programmed cell death protein 1, and durvalumab, avelumab, and atezolizumab target programmed death ligand 1. Ipilimumab targets cytotoxic T lymphocyte-associated antigen-4. Used as monotherapy or in combination, they have shown remarkable efficacy in melanoma, lung cancer, and many other solid tumors, and indications continue to evolve. These checkpoint inhibitors are typically well tolerated; however, they may cause immune-mediated adverse effects, resulting in inflammation of any organ system. Pulmonary toxicity is vital to recognize, and it can be more challenging to diagnose in patients with lung cancer, given the nature of the disease course and treatment.

Immunogenicity risk assessment for biotherapeutics through in vitro detection of CD134 and CD137 on T helper cells.

Biotherapeutics, which are biologic medications that are natural or bioengineered products of living cells, have revolutionized the treatment of many diseases. However, unwanted immune responses still present a major challenge to their widespread adoption. Many patients treated with biotherapeutics develop antigen-specific anti-drug antibodies (ADAs) that may reduce the efficacy of the therapy or cross-react with the endogenous counterpart of a protein therapeutic, or both. Here, we describe an in vitro method for assessing the immunogenic risk of a biotherapeutic. We found a correlation between clinical immunogenicity and the frequency with which a biotherapeutic stimulated an increase in CD134, CD137, or both cell surface markers on CD4+ T cells. Using high-throughput flow cytometry, we examined the effects of 14 biotherapeutics with diverse rates of clinical immunogenicity on peripheral blood mononuclear cells from 120 donors with diverse human leukocyte antigen class II-encoding alleles. Biotherapeutics with high rates of ADA development in the clinic had higher proportions of CD4+ T cells positive for CD134 or CD137 than biotherapeutics with low clinical immunogenicity. This method provides a rapid and simple preclinical test of the immunogenic potential of a new candidate biotherapeutic or biosimilar. Implementation of this approach during biotherapeutic research and development enables rapid elimination of candidates that are likely to cause ADA-related adverse events and detrimental consequences.

Antibodies to an Epstein Barr Virus protein that cross-react with dsDNA have pathogenic potential.

Pathogens such as the Epstein Barr virus (EBV) have long been implicated in the etiology of systemic lupus erythematosus (SLE). The Epstein Barr virus nuclear antigen I (EBNA-1) has been shown to play a role in the development of anti-nuclear antibodies characteristic of SLE. One mechanism by which EBV may play a role in SLE is molecular mimicry. We previously generated two monoclonal antibodies (mAbs) to EBNA-1 and demonstrated that they cross-react with double-stranded DNA (dsDNA). In the present study, we demonstrate that these mAbs have pathogenic potential. We show that they can bind to isolated rat glomeruli and that binding can be greatly diminished by pretreatment of glomeruli with DNase I, suggesting that these mAbs bind dsDNA in the kidney. We also demonstrate that these antibodies can deposit in the kidney when injected into mice and can induce proteinuria and elicit histopathological alterations consistent with glomerulonephritis. Finally, we show that these antibodies can cross-react with laminin and collagen IV in the extracellular matrix suggesting that direct binding to the glomerular basement membrane or mesangial matrix may also contribute to the antibody deposition in the kidney. In summary, our results indicate that EBNA-1 can elicit antibodies that cross-react with dsDNA, that can deposit in the kidney, and induce kidney damage. These results are significant because they support the role of a viral protein in SLE and lupus nephritis.

A novel target anti-interleukin-13 receptor subunit alpha-2 monoclonal antibody inhibits tumor growth and metastasis in lung cancer.

IL13Rα2 shows high expression in different types of tumors and can be a target for cancer therapy in humans due to its poor prognosis. The aim of our study is to characterize and investigate the effect of interleukin-13 receptor subunit alpha-2monoclonal antibody mAb15D8 on lung cancer cells in vitro and in vivo by blocking its specific epitope in IL13Rα2 antigen. The mAb15D8 blocking epitope was analyzed through the mutagenesis of IL13Rα2 and confirmed with western blot. We found that the IL13Rα2 epitope recognized by mAb15D8 antibody is a new binding site localized in the fibronectin-III domain-1 of IL13Rα2 antigen. Moreover, the mAb15D8 obviously reduced cell proliferation, migration of H460, A549, SKOV3, and B16F10 cells. Treatment with mAb15D8 significantly reduced the H460 xenograft tumor formation and growth in nude mice and inhibited B16F10 tumor metastasis and increased survival in C57BL/6 mice. Pharmacokinetic and toxicological analysis demonstrated the safety of mAb15D8 as a potential therapeutic agent. We developed a novel mouse monoclonal antibody against IL13Rα2 which binds to specific epitope on IL13Rα2 antigen. In vivo treatment with the antibody significantly reduced tumor growth and lung metastasis and prolonged survival. These results suggest mAb15D8 antibody as a potential therapeutic agent for cancer therapy.

Identification of IgG1 isotype phosphorylcholine antibodies for the treatment of inflammatory cardiovascular diseases.

BACKGROUND: Phosphorylcholine (PC) is an important pro-inflammatory damage-associated molecular pattern. Previous data have shown that natural IgM anti-PC protects against cardiovascular disease. We aimed to develop a monoclonal PC IgG antibody with anti-inflammatory and anti-atherosclerotic properties. METHODS: Using various techniques PC antibodies were validated and optimized. In vivo testing was performed in a femoral artery cuff model in ApoE3*Leiden mice. Safety studies are performed in rats and cynomolgus monkeys. RESULTS: A chimeric anti-PC (PC-mAb(T15), consisting of a human IgG1 Fc and a mouse T15/E06 Fab) was produced, and this was shown to bind specifically to epitopes in human atherosclerotic tissues. The cuff model results in rapid induction of inflammatory genes and altered expression of genes associated with ER stress and choline metabolism in the lesions. Treatment with PC-mAb(T15) reduced accelerated atherosclerosis via reduced expression of endoplasmic reticulum stress markers and CCL2 production. Recombinant anti-PC Fab fragments were identified by phage display and cloned into fully human IgG1 backbones creating a human monoclonal IgG1 anti-PC (PC-mAbs) that specifically bind PC, apoptotic cells and oxLDL. Based on preventing macrophage oxLDL uptake and CCL2 production, four monoclonal PC-mAbs were selected, which to various extent reduced vascular inflammation and lesion development. Additional optimization and validation of two PC-mAb antibodies resulted in selection of PC-mAb X19-A05, which inhibited accelerated atherosclerosis. Clinical grade production of this antibody (ATH3G10) significantly attenuated vascular inflammation and accelerated atherosclerosis and was tolerated in safety studies in rats and cynomolgus monkeys. CONCLUSIONS: Chimeric anti-PCs can prevent accelerated atherosclerosis by inhibiting vascular inflammation directly and through reduced macrophage oxLDL uptake resulting in decreased lesions. PC-mAb represents a novel strategy for cardiovascular disease prevention.

Inhibition of IL-34 Unveils Tissue-Selectivity and Is Sufficient to Reduce Microglial Proliferation in a Model of Chronic Neurodegeneration.

The proliferation and activation of microglia, the resident macrophages in the brain, is a hallmark of many neurodegenerative diseases such as Alzheimer's disease (AD) and prion disease. Colony stimulating factor 1 receptor (CSF1R) is critically involved in regulating microglial proliferation, and CSF1R blocking strategies have been recently used to modulate microglia in neurodegenerative diseases. However, CSF1R is broadly expressed by many cell types and the impact of its inhibition on the innate immune system is still unclear. CSF1R can be activated by two independent ligands, CSF-1 and interleukin 34 (IL-34). Recently, it has been reported that microglia development and maintenance depend on IL-34 signaling. In this study, we evaluate the inhibition of IL-34 as a novel strategy to reduce microglial proliferation in the ME7 model of prion disease. Selective inhibition of IL-34 showed no effects on peripheral macrophage populations in healthy mice, avoiding the side effects observed after CSF1R inhibition on the systemic compartment. However, we observed a reduction in microglial proliferation after IL-34 inhibition in prion-diseased mice, indicating that microglia could be more specifically targeted by reducing IL-34. Overall, our results highlight the challenges of targeting the CSF1R/IL34 axis in the systemic and central compartments, important for framing any therapeutic effort to tackle microglia/macrophage numbers during brain disease.

Anti-GD2 induced allodynia in rats can be reduced by pretreatment with DFMO.

BACKGROUND: Anti-GD2 therapy with dinutuximab is effective in improving the survival of high-risk neuroblastoma patients in remission and after relapse. However, allodynia is the major dose-limiting side effect, hindering its use for neuroblastoma patients at higher doses and for other GD2-expressing malignancies. As polyamines can enhance neuronal sensitization, including development of allodynia and other forms of pathological pain, we hypothesized that polyamine depletion might prove an effective strategy for relief of anti-GD2 induced allodynia. METHOD: Sprague-Dawley rats were allowed to drink water containing various concentrations of difluoromethylornithine (DFMO) for several days prior to behavioral testing. Anti-GD2 (14G2a) was injected into the tail vein of lightly sedated animals and basal mechanical hindpaw withdrawal threshold assessed by von Frey filaments. Endpoint serum DFMO and polyamines, assessed 24h after 14G2a injection, were measured by HPLC and mass spectrometry. RESULTS: An i.v. injection of 14G2a causes increased paw sensitivity to light touch in this model, a response that closely mimics patient allodynia. Animals allowed to drink water containing 1% DFMO exhibited a significant reduction of 14G2a-induced pain sensitivity (allodynia). Increasing the dosage of the immunotherapeutic increased the magnitude (intensity and duration) of the pain behavior. Administration of DFMO attenuated the enhanced sensitivity. Consistent with the known actions of DFMO on ornithine decarboxylase (ODC), serum putrescene and spermidine levels were significantly reduced by DFMO, though the decrease in endpoint polyamine levels did not directly correlate with the behavioral changes. CONCLUSIONS: Our results demonstrate that DFMO is an effective agent for reducing anti-GD2 -induced allodynia. Using DFMO in conjunction with dinutuximab may allow for dose escalation in neuroblastoma patients. The reduction in pain may be sufficient to allow new patient populations to utilize this therapy given the more acceptable side effect profile. Thus, DFMO may be an important adjunct to anti-GD2 immunotherapy in addition to a role as a potential anti-cancer therapeutic.

Enabling speed to clinic for monoclonal antibody programs using a pool of clones for IND-enabling toxicity studies.

The importance of speed to clinic for medicines that may address unmet medical needs puts pressure on product development timelines. Historically, both toxicology and first-in-human clinical materials are generated using the same clonal-derived cells to ensure safety and minimize any development risks. However, cell line development with single cell cloning is time consuming, and aggravated by the time needed to screen for a lead clone based on cell line stability and manufacturability. In order to achieve faster timelines, we have used pools of up to six clones for earlier production of drug substance for regulatory filing-enabling toxicology studies, and then the final single clone was selected for production of clinical materials. This approach was enabled by using platform processes across all stages of early development, including expression vectors, host cell lines, media, and production processes. Through comprehensive cell culture and product quality analysis, we demonstrated that the toxicology material was representative of the clinical material for all six monoclonal antibody programs evaluated. Our extensive development experience further confirmed that using a pool of clones for toxicology material generation is a reliable approach to shorten the early development timeline.

Immune complex disease in a chronic monkey study with a humanised, therapeutic antibody against CCL20 is associated with complement-containing drug aggregates.

Despite the potential for the chemokine class as therapeutic targets in immune mediated disease, success has been limited. Many chemokines can bind to multiple receptors and many receptors have multiple ligands, with few exceptions. One of those exceptions is CCL20, which exclusively pairs to CCR6 and is associated with several immunologic conditions, thus providing a promising therapeutic target. Following successful evaluation in a single dose, first time in human clinical study, GSK3050002-a humanized IgG1 monoclonal antibody against human CCL20-was evaluated in a 26-week cynomolgus monkey toxicology study. A high incidence of unexpected vascular and organ inflammation was observed microscopically, leading to the decision to halt clinical development. Here we report a dose-responsive increase in the incidence and severity of inflammation in multiple organs from monkeys receiving 30 and 300 mg/kg/week by either subcutaneous or intravenous injection. Histomorphological changes resembled an immune complex-mediated pathology, which is often due to formation of anti-drug antibodies in monkeys receiving a human protein therapeutic and thus not predictive of clinical outcome. However, the presentation was atypical in that there was a clear dose response with a very high incidence of inflammation with a low incidence of ADA that did not correlate well individually. Additionally, the immunohistologic presentation was atypical in that the severity and distribution of tissue inflammation was greater than the numbers of associated immune complexes (i.e., granular deposits). An extensive ex vivo analysis of large molecular weight protein complexes in monkey serum from this study and in human serum samples demonstrated a time-dependent aggregation of GSK3050002, that was not predicted by in vitro assays. The aggregates also contained complement components. These findings support the hypothesis that immune complexes of drug aggregates, not necessarily including anti-drug antibodies, can fix complement, accumulate over time, and trigger immune complex disease. A situation which may have increased clinical relevance than typical anti-drug antibody-associated immune complex disease in monkeys administered human antibody proteins.

Ocular toxicity of intravitreal golimumab in a rabbit model

Background/aim: To investigate the effect of intravitreal golimumab on rabbit retina histopathology. Materials and methods: Sixteen albino New Zealand rabbits were divided into three groups. The right eye of each rabbit in groups I, II, and III received a single intravitreal injection of 5 mg/0.05 mL (6 eyes), 10 mg/0.1 mL (6 eyes), or 20 mg/0.2 mL (4 eyes) golimumab, while left eyes served as controls with the same volume of a balanced salt solution injection. All animals were examined using slit-lamp biomicroscopy and indirect ophthalmoscopy before and after intravitreal injection and at days 1 and 7. Animals were euthanized on day 7 and the eyes were enucleated for immunohistochemistry evaluation and electron microscopic examination of the retinas. Results: For groups I, II, and III, the number of cells in the outer nuclear layer and the inner nuclear layer was decreased compared to those in the control groups. In group I, the percentage of caspase-3 staining of the outer nuclear layer was significantly higher than that in the control. For groups II and III, TUNEL and caspase-3 staining percentages in the outer and inner nuclear layers were found to be significantly higher than those for the control groups. In the ganglion cell layer, for groups I, II, and III, neither TUNEL nor caspase-3 staining percentages showed any significant difference between two groups. No significant dose-dependent relationship was found for increasing doses of golimumab in all layers. Myelin figures and karyorrhexis in the photoreceptor cells were prominent in electron microscopy of the golimumab-injected eyes. Conclusion: Golimumab caused apoptosis in both photoreceptors and bipolar cells of the rabbit retina. Potential retinal toxicity of intravitreal golimumab should be considered if an intravitreal administration is planned.

Immunotoxin SS1P is rapidly removed by proximal tubule cells of kidney, whose damage contributes to albumin loss in urine.

Recombinant immunotoxins (RITs) are chimeric proteins composed of an Fv and a protein toxin being developed for cancer treatment. The Fv brings the toxin to the cancer cell, but most of the RITs do not reach the tumor and are removed by other organs. To identify cells responsible for RIT removal, and the pathway by which RITs reach these cells, we studied SS1P, a 63-kDa RIT that targets mesothelin-expressing tumors and has a short serum half-life. The major organs that remove RIT were identified by live mouse imaging of RIT labeled with FNIR-Z-759. Cells responsible for SS1P removal were identified by immunohistochemistry and intravital two-photon microscopy of kidneys of rats. The primary organ of SS1P removal is kidney followed by liver. In the kidney, SS1P passes through the glomerulus, is taken up by proximal tubular cells, and transferred to lysosomes. In the liver, macrophages are involved in removal. The short half-life of SS1P is due to its very rapid filtration by the kidney followed by degradation in proximal tubular cells of the kidney. In mice treated with SS1P, proximal tubular cells are damaged and albumin in the urine is increased. SS1P uptake by kidney is reduced by coadministration of l-lysine. Our data suggests that l-lysine administration to humans might prevent SS1P-mediated kidney damage, reduce albumin loss in urine, and alleviate capillary leak syndrome.

Mechanisms of anti-GPIbα antibody-induced thrombocytopenia in mice.

Immune thrombocytopenia (ITP) is an acquired bleeding disorder characterized by antibody-mediated platelet destruction. Different mechanisms have been suggested to explain accelerated platelet clearance and impaired thrombopoiesis, but the pathophysiology of ITP has yet to be fully delineated. In this study, we tested 2 mouse models of immune-mediated thrombocytopenia using the rat anti-mouse GPIbα monoclonal antibody 5A7, generated in our laboratory. After a single IV administration of high-dose (2 mg/kg) 5A7, opsonized platelets were rapidly cleared from the circulation into the spleen and liver; this was associated with rapid upregulation of thrombopoietin (TPO) messenger RNA. In contrast, subcutaneous administration of low-dose 5A7 (0.08-0.16 mg/kg) every 3 days gradually lowered the platelet count; in this case, opsonized platelets were observed only in the spleen, and TPO levels remained unaltered. Interestingly, in both models, the 5A7 antibody was found on the surface of, as well as internalized to, bone marrow megakaryocytes. Consequently, platelets generated in the chronic phase of repeated subcutaneous 5A7 administration model showed reduced GPIbα membrane expression on their surface. Our findings indicate that evaluation of platelet surface GPIbα relative to platelet size may be a useful marker to support the diagnosis of anti-GPIbα antibody-induced ITP.

Case Report: An EGFR-Targeted 4-1BB-agonistic Trimerbody Does Not Induce Hepatotoxicity in Transgenic Mice With Liver Expression of Human EGFR.

Agonistic monoclonal antibodies (mAbs) targeting the co-stimulatory receptor 4-1BB are among the most effective immunotherapeutic agents across pre-clinical cancer models. However, clinical development of full-length 4-1BB agonistic mAbs, has been hampered by dose-limiting liver toxicity. We have previously developed an EGFR-targeted 4-1BB-agonistic trimerbody (1D8N/CEGa1) that induces potent anti-tumor immunity without systemic toxicity, in immunocompetent mice bearing murine colorectal carcinoma cells expressing human EGFR. Here, we study the impact of human EGFR expression on mouse liver in the toxicity profile of 1D8N/CEGa1. Systemic administration of IgG-based anti-4-1BB agonist resulted in nonspecific immune stimulation and hepatotoxicity in a liver-specific human EGFR-transgenic immunocompetent mouse, whereas in 1D8N/CEGa1-treated mice no such immune-related adverse effects were observed. Collectively, these data support the role of FcγR interactions in the major off-tumor toxicities associated with IgG-based 4-1BB agonists and further validate the safety profile of EGFR-targeted Fc-less 4-1BB-agonistic trimerbodies in systemic cancer immunotherapy protocols.

Brij-58, a potential injectable protein-stabilizer used in therapeutic protein formulation.

Polysorbates (PSs) are common protein stabilizers used in biotherapeutic formulations. However, PSs are heterogeneous and unstable in liquid protein formulations [1,2]. The purpose of this work is to explore possible alternatives for polysorbate replacements that demonstrate superior protein protection, superior self-stability, low toxicity, and wide applicability. For this purpose, 8 non-ionic surfactants that have not yet been used as excipients in marketed biotherapeutic products were investigated with PS20/80 as the benchmark. Compared with PS20/80, Brij-58 showed better protein protection ability in the mAb1 formulation under forced degradation conditions when examined by visual inspection, SEC, and dynamic lighting scanning. Additionally, Brij-58 has a better inherent stability than PS20/80 in the protein formulation when detected by UPLC-CAD. Moreover, Brij-58 is an inert excipient that does not affect protein bioactivity and conformation. In addition, the LD50 and hemolysis concentration of Brij-58 were determined, which is relatively safe when used as a parenteral injection. Furthermore, Brij-58 was also an effective protein stabilizer for the other two antibody products (IgG4 subtype and bispecific antibody) in the shaking study. In summary, Brij-58 stands out as a promising PS replacement in biotherapeutic formulations with a safe, stable and effective protein-protection profile among candidate surfactants.

Toxicity associated with PD-1 blockade after allogeneic haematopoietic cell transplantation.

Cancer immunotherapy with immune checkpoint inhibitors (ICIs) such as programmed death ligand-1 (PD-L1) blockers offers pronounced clinical benefit with durable responses and a manageable safety profile. Patients with a high risk of immune-related adverse events are generally excluded from clinical trials testing ICI therapy. Thus, only a little information on the safety and clinical outcome of patients treated with an ICI after allogeneic haematopoietic cell transplantation (HCT) is currently available. Here, we report the characteristics and outcomes of six patients with, respectively, clear cell renal carcinoma, diffuse large cell B-cell lymphoma, Hodgkin lymphoma, a microsatellite instable colorectal cancer and melanoma who were treated with PD-1 blocking antibodies. All patients had previously undergone allogeneic HCT. Severe grade 3–5 immune-related adverse events were observed in three of five patients who received full-dose ICI therapy. One patient received a lower dose of PD-1 blocking antibody. Only one patient had an objective response, whereas all the other patients had progressive disease. The high toxicity of a full- dose anti-PD-1 treatment regimen suggests that other treatment approaches for patients after allogeneic HCT are needed outside of the context of relapsed Hodgkin disease. In cases where ICI therapy is the only treatment option, reduced dosing should be explored.