High-Risk HLA-DQ Mismatches Are Associated With Adverse Outcomes After Lung Transplantation.

Human leukocyte antigen (HLA) mismatches (MM) between donor and recipient lead to eplet MM (epMM) in lung transplantation (LTX), which can induce the development of de-novo donor-specific HLA-antibodies (dnDSA), particularly HLA-DQ-dnDSA. Aim of our study was to identify risk factors for HLA-DQ-dnDSA development. We included all patients undergoing LTX between 2012 and 2020. All recipients/donors were typed for HLA 11-loci. Development of dnDSA was monitored 1-year post-LTX. EpMM were calculated using HLAMatchmaker. Differences in proportions and means were compared using Chi2-test and Students' t-test. We used Kaplan-Meier curves with LogRank test and multivariate Cox regression to compare acute cellular rejection (ACR), chronic lung allograft dysfunction (CLAD) and survival. Out of 183 patients, 22.9% patients developed HLA-DQ-dnDSA. HLA-DQ-homozygous patients were more likely to develop HLA-DQ-dnDSA than HLA-DQ-heterozygous patients (p = 0.03). Patients homozygous for HLA-DQ1 appeared to have a higher risk of developing HLA-DQ-dnDSA if they received a donor with HLA-DQB1*03:01. Several DQ-eplets were significantly associated with HLA-DQ-dnDSA development. In the multivariate analysis HLA-DQ-dnDSA was significantly associated with ACR (p = 0.03) and CLAD (p = 0.01). HLA-DQ-homozygosity, several high-risk DQ combinations and high-risk epMM result in a higher risk for HLA-DQ-dnDSA development which negatively impact clinical outcomes. Implementation in clinical practice could improve immunological compatibility and graft outcomes.

Hiding in plain sight: Misinterpretation of immunogenic DPB epitopes within G/P groups. Short running title: The impact of HLA G and P codes on DPB.

The clinical impact of HLA DP antibodies is poorly understood, resulting in variable clinical strategies for transplant candidates and recipients with donor-directed HLA-DP antibodies. Complicating matters further, the DPB naming convention is not based on allelic homology and requires sequence alignments to identify potential immunogenic epitopes. Historically, G and P codes, which consolidated alleles that were identical over Exon 2, were used to simplify the reporting of HLA Class II typing as differences outside of Exon 2 have not been considered immunogenic (i.e., able to induce an antibody response). Herein, we present four cases demonstrating that polymorphisms at codons 96R/K and 170I/T, in Exon 3 of DPB, are targets for alloantibody recognition. These regions "hide in plain sight" due to the current use of G/P code-level typing, potentially leading to incorrect compatibility assessments (i.e., virtual crossmatches) and misinterpreted antibody responses. The unintentional crossing of an HLA-DPB donor-specific antibody (DSA) in a solid organ or hematopoietic stem cell transplant may lead to unforeseen deleterious clinical outcomes. Our data underscore the complexities of DPB histocompatibility assessments and highlight the need for adaptable systems that align with evolving research and clinical outcomes.

Clinical outcomes after a biopsy diagnosis of antibody-mediated rejection in pediatric heart transplant recipients.

BACKGROUND: Extending survival after heart transplant (HT) is of paramount importance for childhood recipients of HT. Acute rejection is a significant event, and biopsy remains the most specific means for distinguishing between cellular (ACR) and antibody-mediated rejection (AMR). METHODS: All children in the Pediatric Heart Transplant Society Registry who underwent HT between January 2015 and June 2022 and had ≥1 rejection episode were included. Survival was compared between AMR and ACR-only. Secondary outcomes of infection, malignancy, and cardiac allograft vasculopathy (CAV) were assessed. Risk factors for graft loss after AMR were identified using Cox proportional hazard modeling. RESULTS: Among 906 children with rejection, 697 (77%) with complete biopsy information were included. AMR was present on biopsy in 261 (37%) patients; ACR-only was present in 436 (63%). Time to rejection was earlier for AMR, median time from HT to rejection 0.11 versus 0.29 years, p = 0.0006. Survival after AMR in the 1st year was lower than survival after ACR-only. Predictors of graft loss after AMR were younger age at HT, congenital heart disease, and rejection with hemodynamic compromise. There was no difference in time to CAV, infection, or malignancy after rejection between groups. CONCLUSIONS: The largest analysis of pediatric HT rejection with biopsy data to identify AMR underscores the continued importance of AMR on survival. AMR is associated with higher graft loss versus ACR when occurring in the first-year post-HT. Predictors of graft loss after AMR identify patients who may benefit from increased surveillance or augmented maintenance immunosuppression.

COL6A6 Peptide Vaccine Alleviates Atherosclerosis through Inducing Immune Response and Regulating Lipid Metabolism in Apoe-/- Mice.

Atherosclerosis is an autoimmune disease characterized by lipid imbalances and chronic inflammation within blood vessels, with limited preventive and treatment options currently available. In this study, a vaccine prepared with COL6A6 peptide (named the Pep_A6 vaccine) was administered to immunize Apoe-/- mice, and the immune mechanism of the Pep_A6 vaccine against atherosclerosis was first investigated. The results of arterial oil red O staining demonstrated that the Pep_A6 vaccine significantly reduced the atherosclerotic plaque area in Apoe-/- mice fed with a high-fat diet for 20 weeks. A flow cytometry analysis revealed that the Pep_A6 vaccine inhibited Th1 cell differentiation and increased the proportion of Treg cells. Furthermore, there was a significant increase in Ly6Clow monocytes observed in the vaccinated group. The ELISA results showed that the Pep_A6 vaccine induced a significant expression of Pep_A6-specific antibody IgG and IgG1 in mouse serum. Additionally, we found that the Pep_A6 vaccine significantly decreased serum LDL-C content and regulated the expression of genes related to liver lipid metabolism. Together, our findings suggest that the Pep_A6 vaccine alleviates atherosclerosis by inducing a positive immune response and regulating lipid metabolism, providing new insights into potential prevention strategies for atherosclerosis as an innovative vaccine.

The role of C4d and donor specific antibodies in face and hand transplantation-a systematic review.

To date, little is known about the mechanisms of rejection in vascularized composite allotransplantation, particularly for antibody mediated rejection. Additionally, no clear guidelines exist for the diagnosis and management of antibody-mediated rejection in vascularized composite allotransplantation. A systematic review of electronic databases (Embase and PubMed) was conducted to evaluate the relationship of donor specific antibodies and C4d deposition in correlation with cellular rejection following hand and face transplantation reported by centers between 1998 and July 2023. We extracted data on serum donor specific antibodies at the time of biopsy proven rejection according to Banff classification and C4d staining of target tissues. Mann-Whitney U tests were performed to compare rejection grade between groups divided by status of C4d deposition and serum donor specific antibodies, and Fisher's Exact test was used to assess association between the two markers. This review adhered to PRISMA guidelines. A total of 26 patients (5 face, 21 hand) were identified and data on 90 acute rejection episodes with information on Banff grade, donor specific antibody status, and C4d deposition were available. Donor specific antibodies were found to be associated with higher rejection grade (p = 0.005). C4d was not found to be associated with higher rejection grade (p = 0.33). Finally, no significant association was found between concurrent status of the two markers (p = 0.23). These findings suggest that the presence of donor specifc antibodies may be associated with higher grades of acute cellular rejection following hand and face transplantation. More consistent reporting on rejection episodes is needed in order to better understand antibody-mediated rejection in vascularized composite allotransplantation.

Activation and Regulation of Indirect Alloresponses in Transplanted Patients With Donor Specific Antibodies and Chronic Rejection.

Following transplantation, human CD4+T cells can respond to alloantigen using three distinct pathways. Direct and semi-direct responses are considered potent, but brief, so contribute mostly to acute rejection. Indirect responses are persistent and prolonged, involve B cells as critical antigen presenting cells, and are an absolute requirement for development of donor specific antibody, so more often mediate chronic rejection. Novel in vitro techniques have furthered our understanding by mimicking in vivo germinal centre processes, including B cell antigen presentation to CD4+ T cells and effector cytokine responses following challenge with donor specific peptides. In this review we outline recent data detailing the contribution of CD4+ T follicular helper cells and antigen presenting B cells to donor specific antibody formation and antibody mediated rejection. Furthermore, multi-parametric flow cytometry analyses have revealed specific endogenous regulatory T and B subsets each capable of suppressing distinct aspects of the indirect response, including CD4+ T cell cytokine production, B cell maturation into plasmablasts and antibody production, and germinal centre maturation. These data underpin novel opportunities to control these aberrant processes either by targeting molecules critical to indirect alloresponses or potentiating suppression via exogenous regulatory cell therapy.

Good response to cadonilimab as first-line treatment in superaged patient with advanced gastric cancer: a case report.

Gastric cancer remains a considerable global health burden, with limited treatment options available for advanced cases, especially for superaged patients. Cadonilimab, a first-in-class bi-specific antibody (BsAb), offer a promising immunotherapy approach by targeting PD-1/CTLA-4 simultaneously. Herein, we present a case report of an 85-year-old patient with HER2-negative advanced gastric cancer who received first-line treatment with cadonilimab combined with chemotherapy, but cadonilimab was discontinued due to the observation of immune-related pneumonitis during treatment. Despite these changes, the patient still exhibited a stable disease condition for a year until now. This case report highlights the potential of cadonilimab in the treatment of superaged patients with advanced gastric cancer, while the efficacy and safety of it need to be further evaluated.

At present, immunotherapy plus chemotherapy is the standard first-line treatment for patients with advanced gastric cancer. However, immunotherapy is generally not used in elderly patients over 75 years of age due to safety concerns and the treatment options for these patients are very limited. In this paper, we report a case of successful treatment of cadonilimab (novel immunotherapy drug) plus chemotherapy in an 85-year-old male patient with advanced gastric cancer. Until now, the survival time of this patient is over 12 months, and the quality of life is satisfactory.

High resolution HLA genotyping with third generation sequencing technology-A multicentre study.

Molecular HLA typing techniques are currently undergoing a rapid evolution. While real-time PCR is established as the standard method in tissue typing laboratories regarding allocation of solid organs, next generation sequencing (NGS) for high-resolution HLA typing is becoming indispensable but is not yet suitable for deceased donors. By contrast, high-resolution typing is essential for stem cell transplantation and is increasingly required for questions relating to various disease associations. In this multicentre clinical study, the TGS technique using nanopore sequencing is investigated applying NanoTYPE™ kit and NanoTYPER™ software (Omixon Biocomputing Ltd., Budapest, Hungary) regarding the concordance of the results with NGS and its practicability in diagnostic laboratories. The results of 381 samples show a concordance of 99.58% for 11 HLA loci, HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, -DPA1 and -DPB1. The quality control (QC) data shows a very high quality of the sequencing performed in each laboratory, 34,926 (97.15%) QC values were returned as 'passed', 862 (2.4%) as 'inspect' and 162 (0.45%) as 'failed'. We show that an 'inspect' or 'failed' QC warning does not automatically lead to incorrect HLA typing. The advantages of nanopore sequencing are speed, flexibility, reusability of the flow cells and easy implementation in the laboratory. There are challenges, such as exon coverage and the handling of large amounts of data. Finally, nanopore sequencing presents potential for applications in basic research within the field of epigenetics and genomics and holds significance for clinical concerns.

Tripokin: A multi-specific immunocytokine for cancer immunotherapy.

Antibodies that target the tumor microenvironment can be used to deliver pro-inflammatory payloads, such as cytokines. Cytokines are small proteins able to modulate the activity of the immune system, and antibody-cytokine fusion proteins have been tested in preclinical and clinical settings. In this study, we describe Tripokin, a novel multi-specific fusion protein that combines interleukin-2 and a single amino acid mutant of tumor necrosis factor. The two pro-inflammatory payloads were fused to the L19 antibody, a clinical-grade antibody against the extradomain B of fibronectin. The human payloads were used for clinical applications, while the corresponding murine cytokines were used for preclinical studies. The resulting fusion proteins were produced in mammalian cells and purified to homogeneity. The murine Tripokin product was well tolerated in tumor-bearing mice at three doses of 30 µg in a 2-day interval and promoted rapid tumor eradication in murine models, more efficiently than single-agent immunocytokines. Tripokin induced rapid tumor necrosis and stimulated a robust immune response, impacting innate and adaptive immune pathways. In addition, the combination with immune checkpoint inhibitors further boosted the therapeutic efficacy of our molecule. Tripokin represents a promising clinical candidate for the simultaneous delivery of interleukin-2 and tumor necrosis factor to neoplastic sites.

Analysis of B-cell receptor repertoire to evaluate immunogenicity of monovalent Omicron XBB.1.5 mRNA vaccines.

Monovalent Omicron XBB.1.5 mRNA vaccines were newly developed and approved by the FDA in Autumn 2023 for preventing COVID-19. However, clinical efficacy for these vaccines is currently lacking. We previously established the quantification of antigen-specific antibody sequence (QASAS) method to assess the response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination at the mRNA level using B-cell receptor (BCR) repertoire assay and the coronavirus antibody database (CoV-AbDab). Here, we used this method to evaluate the immunogenicity of monovalent XBB.1.5 vaccines. We analyzed repeated blood samples of healthy volunteers before and after monovalent XBB.1.5 vaccination (BNT162b2 XBB.1.5 or mRNA-1273.815) for the BCR repertoire to assess BCR/antibody sequences that matched SARS-CoV-2-specific sequences in the database. The number of matched unique sequences and their total reads quickly increased 1 week after vaccination. Matched sequences included those bound to the Omicron strain and Omicron XBB sublineage. The antibody sequences that can bind to the Omicron strain and XBB sublineage revealed that the monovalent XBB.1.5 vaccines showed a stronger response than previous vaccines or SARS-CoV-2 infection before the emergence of XBB sublineage. The QASAS method was able to demonstrate the immunogenic effect of monovalent XBB.1.5 vaccines for the 2023-2024 COVID-19 vaccination campaign.

Heart Transplant Rejection: From the Endomyocardial Biopsy to Gene Expression Profiling.

Heart transplant prolongs life for patients with end-stage heart failure but rejection remains a complication that reduces long-term survival. The aim is to provide a comprehensive overview of the current status in HT rejection. EMB is an invasive diagnostic tool, consisting in the sampling of a fragment of myocardial tissue from the right ventricular septum using fluoroscopic guidance. This tissue can later be subjected to histopathological, immunohistochemical or molecular analysis, providing valuable information for cardiac allograft rejection, but this procedure is not without complications. To increase the accuracy of the rejection diagnosis, EMB requires a systematic evaluation of endocardium, myocardium, interstitium and intramural vessels. There are three types of rejection: hyperacute, acute or chronic, diagnosed by the histopathological evaluation of EMB as well as by new diagnostic methods such as DSA, ddcfDNA and gene expression profiling, the last having a high negative predictive value. More than 50 years after the introduction of EMB in medical practice, it still remains the "gold standard" in monitoring rejection in HT recipients but other new, less invasive diagnostic methods reduce the number of EMBs required.

Assessing N-terminal acetylation status of cellular proteins via an antibody specific for acetylated methionine.

N-terminal acetylation is being recognized as a factor affecting protein lifetime and proteostasis. It is a modification where an acetyl group is added to the N-terminus of proteins, and this occurs in 80 % of the human proteome. N-terminal acetylation is catalyzed by enzymes called N-terminal acetyltransferases (NATs). The various NATs acetylate different N-terminal amino acids, and methionine is a known target for some of the NATs. Currently, the acetylation status of most proteins can only be assessed with a limited number of methods, including mass spectrometry, which although powerful and robust, remains laborious and can only survey a fraction of the proteome. We here present testing of an antibody that was developed to specifically recognize Nt-acetylated methionine-starting proteins. We have used dot blots with synthetic acetylated and non-acetylated peptides in addition to protein analysis of lysates from NAT knockout cell lines to assess the specificity and application of this anti-Nt-acetylated methionine antibody (anti-NtAc-Met). Our results demonstrate that this antibody is indeed NtAc-specific and further show that it has selectivity for some subtypes of methionine-starting N-termini, specifically potential substrates of the NatC, NatE and NatF enzymes. We propose that this antibody may be a powerful tool to identify NAT substrates or to analyse changes in N-terminal acetylation for specific cellular proteins of interest.

Challenges and opportunities for designing clinical trials for antibody mediated rejection.

Significant progress has been made in kidney transplantation, with 1-year graft survival nearing 95%. However, long-term allograft survival remains suboptimal, with a 10-year overall graft survival rate of only 53.6% for deceased donor transplant recipients. Chronic active antibody-mediated rejection (ABMR) is a leading cause of death-censored graft loss, yet no therapy has demonstrated efficacy in large, randomized trials, despite substantial investment from pharmaceutical companies. Several clinical trials aimed to treat chronic ABMR in the past decade have yielded disappointing results or were prematurely terminated, attributed to factors including incomplete understanding of disease mechanisms, heterogeneous patient populations with comorbidities, slow disease progression, and limited patient numbers. This review aims to discuss opportunities for improving retrospective and prospective studies of ABMR, focusing on addressing heterogeneity, outcome measurement, and strategies to enhance patient enrollment to inform study design, data collection, and reporting.

Is Component-Specific Antibody Testing Sufficient to Replace the Oral Food Challenge in the Diagnostics of Peanut-Sensitized Children? A Proof-of-Concept Study.

(1) Peanut allergy is associated with high risk of anaphylaxis which could be prevented by oral immunotherapy. Patients eligible for immunotherapy are selected on the basis of a food challenge, although currently the assessment of antibodies against main peanut molecules (Ara h 1, 2, 3 and 6) is thought to be another option. (2) The current study assessed the relationship between the mentioned antibodies, challenge outcomes, skin tests and some other parameters in peanut-sensitized children. It involved 74 children, divided into two groups, based on their response to a food challenge. (3) Both groups differed in results of skin tests, levels of component-specific antibodies and peanut exposure history. The antibody levels were then used to calculate thresholds for prediction of challenge results or symptom severity. While the antibody-based challenge prediction revealed statistical significance, it failed in cases of severe symptoms. Furthermore, no significant correlation was observed between antibody levels, symptom-eliciting doses and the risk of severe anaphylaxis. Although in some patients it could result from interference with IgG4, the latter would not be a universal explanation of this phenomenon. (4) Despite some limitations, antibody-based screening may be an alternative to the food challenge, although its clinical relevance still requires further studies.

B-Cell Induction Therapies in Intestinal Transplantation.

Despite advancements in short-term outcomes since the inception of intestinal transplant, significant long-term graft failure persists. Early successes are attributed to the utilization of tacrolimus for maintenance therapy, coupled with T-cell modulating induction regimens, which effectively reduce the incidence of acute cellular rejection. However, the challenge of chronic allograft injury remains unresolved. There is increasing evidence indicating a correlation between donor-specific antibodies and the survival of visceral allografts. Strategies aimed at reducing the presence or load of these antibodies may potentially enhance long-term outcomes. Consequently, our focus is now turning toward B-cell induction therapies as a possible solution.

Glycolysis Changes in Alloreactive Memory B Cells in Highly Sensitized Kidney Transplant Recipients Undergonig Desensitization Therapy.

Despite the growing use of desensitization strategies, hyperimmune patients remain at high risk of antibody-mediated rejection suggesting that, even when donor-specific antibodies (DSA) are effectively depleted, anti-donor specific B cells persist. We included 10 highly sensitized recipients that underwent desensitization with plasmapheresis and B cell depletion prior to kidney transplantation. We quantified changes in DSA (luminex), total B-cell subsets (flow cytometry), anti-donor HLA B cells (fluorospot), and single-cell metabolism in serially collected samples before desensitization, at the time of transplant, and at 6 and 12 months thereafter. Desensitization was associated with a decrease in DSA and total memory B cell and naive B cell percentage, while plasma cells and memory anti-donor HLA circulating B cells persisted up to 12 months after transplant. At 12-month post-transplantation, memory B cells increased their glycolytic capacity, while proliferative KI67+ plasma cells modified their metabolism by increasing fatty acid and amino acid oxidation capacity and decreasing their glucose dependence. Despite effective DSA depletion, anti-donor B cells persist in kidney transplant recipients. Due to the reliance of these cells on glycolysis, glycolysis-targeting therapies might represent a valuable treatment strategy.

Differential decline of SARS-CoV-2-specific antibody levels, innate and adaptive immune cells, and shift of Th1/inflammatory to Th2 serum cytokine levels long after first COVID-19.

BACKGROUND: SARS-CoV-2 has triggered a pandemic and contributes to long-lasting morbidity. Several studies have investigated immediate cellular and humoral immune responses during acute infection. However, little is known about long-term effects of COVID-19 on the immune system. METHODS: We performed a longitudinal investigation of cellular and humoral immune parameters in 106 non-vaccinated subjects ten weeks (10 w) and ten months (10 m) after their first SARS-CoV-2 infection. Peripheral blood immune cells were analyzed by multiparametric flow cytometry, serum cytokines were examined by multiplex technology. Antibodies specific for the Spike protein (S), the receptor-binding domain (RBD) and the nucleocapsid protein (NC) were determined. All parameters measured 10 w and 10 m after infection were compared with those of a matched, noninfected control group (n = 98). RESULTS: Whole blood flow cytometric analyses revealed that 10 m after COVID-19, convalescent patients compared to controls had reduced absolute granulocyte, monocyte, and lymphocyte counts, involving T, B, and NK cells, in particular CD3+CD45RA+CD62L+CD31+ recent thymic emigrant T cells and non-class-switched CD19+IgD+CD27+ memory B cells. Cellular changes were associated with a reversal from Th1- to Th2-dominated serum cytokine patterns. Strong declines of NC- and S-specific antibody levels were associated with younger age (by 10.3 years, p < .01) and fewer CD3-CD56+ NK and CD19+CD27+ B memory cells. Changes of T-cell subsets at 10 m such as normalization of effector and Treg numbers, decline of RTE, and increase of central memory T cell numbers were independent of antibody decline pattern. CONCLUSIONS: COVID-19 causes long-term reduction of innate and adaptive immune cells which is associated with a Th2 serum cytokine profile. This may provide an immunological mechanism for long-term sequelae after COVID-19.

Positive Long-Term Outcome of Kidney Allocation via Acceptable Mismatch Program in Highly Sensitized Patients.

Introduction: Eurotransplant established the acceptable mismatch (AM) program to facilitate timely kidney transplantations of highly sensitized patients, but long-term granular clinical and immunological outcomes regarding overall graft survival and de novo DSA (dnDSA) formation are still intensively researched. The right choice of induction therapy in patients with differing immunological risk is not conclusively determined, as well as the impact of human leukocyte antigen (HLA) epitope matching on dnDSA formation. Methods: This monocentric, retrospective study analyzed 94 patients transplanted within the AM program between 2000 and 2019 compared to case-control matched cohorts of non- (PRA 0-5%; PRA-0) and intermediately sensitized (PRA 6-84%; PRA-6/84) patients transplanted through Eurotransplant Kidney Allocation System. Results: Estimated 10-year overall graft survival between the PRA-0 and AM cohorts was similar, whereas PRA-6/84 was significantly disadvantageous compared to PRA-0. Estimated 10-year incidence of antibody-mediated rejection rates was significantly lower in the PRA-0 group compared to AM and PRA-6/84 groups. Compared to the AM group, estimated incidence of de novo donor-specific antibody (dnDSA) was significantly lower in PRA-0 patients, with no differences between the AM and PRA-6/84 cohorts. The PRA-6/84 cohort was the only subgroup in which interleukin-2 receptor antagonist (IL2RA) induction was associated with longer overall graft survival, patient survival, and graft survival compared to depleting induction (ATG or OKT3). Broad HLA-A, -B, -DR mismatches (mmABDR) and HLA epitope mismatches determined by Eplets and PIRCHE-II were predictive for dnDSA formation in the total cohort, and the AM subgroup. Discussion: The high efforts expended on AM patients are justified to allow timely organ transplantation with acceptable risk profile and non-inferior outcomes. IL2RA induction in intermediately sensitized patients is associated with superior overall graft survival, patient survival, and graft survival compared to ATG/OKT3 induction, without negative effects on rejection episodes or dnDSA formation. In silico epitope matching might further help reduce dnDSA formation, particularly in high-risk AM patients.

Bi-specific T-cell engagers (BiTEs) in prostate cancer and strategies to enhance development: hope for a BiTE-r future.

Metastatic castrate resistant prostate cancer (mCRPC) continues to have poor survival rates due to limited treatment options. Bi-specific T cell engagers (BiTEs) are a promising class of novel immunotherapies with demonstrated success in haematological malignancies and melanoma. BiTEs developed for tumour associated antigens in prostate cancer have entered clinical testing. These trials have been hampered by high rates of treatment related adverse events, minimal or transient anti-tumour efficacy and generation of high titres of anti-drug antibodies. This paper aims to analyse the challenges faced by the different BiTE therapy constructs and the mCRPC tumour microenvironment that result in therapeutic resistance and identify possible strategies to overcome these issues.