Immunoglobulin Light Chain Research Articles

Evaluation of Sarcopenia in Patients with Monoclonal Gammopathy of Undetermined Significance.

Background: We aimed to determine the prevalence of sarcopenia in patients with monoclonal gammopathy of undetermined significance (MGUS) and to evaluate the links between MGUS and sarcopenia. Methods: Eighty-two patients with a diagnosis of MGUS were enrolled in the study. Muscle strength was measured using the handgrip dynamometer. Physical performance was assessed by assessing gait speed over a 6-minute walking test. Muscle mass was determined using a bioelectrical impedance analyzer. Results: Sarcopenia was confirmed in 34.15% of patients. Male predominance was demonstrated in MGUS subjects with sarcopenia, particularly patients with low hand grip strength, low appendicular skeletal muscle mass (ASMM), or low ASMM index (p < 0.001, 0.013, and 0.001, respectively). Higher age and lower serum free light-chain Lambda levels were shown in MGUS patients with low muscle function scores compared to normal scores (p < 0.001, and 0.014, respectively). In addition, having a low ASMM score was related to low body mass index and high-risk group (p = 0.020, 0.033, respectively). Conclusions: We demonstrated that the frequency of sarcopenia is high in patients with MGUS. Whether sarcopenia has a possible role as a factor contributing to the pathogenesis of MGUS should be supported by further studies containing longitudinal data.

Pulmonary light chain deposition disease from a respiratory physician's perspective

Pulmonary light chain deposition disease (PLCDD) is a rare monoclonal immunoglobulin deposition disease characterized by the deposition of specific immunoglobulin light chains in lung tissue. In its early stages, PLCDD presents with mild clinical symptoms, while pulmonary imaging shows multiple nodules and thin-walled cysts. Pathologically, there is a deposition of eosinophilic amorphous protein-like material in the tissues, which stains negative for Congo red. The exact pathological mechanism of PLCDD remains unclear, and its clinical presentation lacks specificity. Challenges associated with this condition include difficulties in diagnosis, selection and evaluation of treatment options, lack of clear monitoring criteria, and standards for prognosis assessment. Further research is needed to elucidate the pathogenesis of PLCDD, to establish standardized diagnostic and therapeutic protocols, and to evaluate treatment efficacy and prognostic factors.

Kidney Amyloidosis: Updates on Pathogenesis and Therapeutic Frontiers.

Amyloidosis includes a diverse group of rare diseases characterized by the misfolding of native or mutant proteins, leading to extracellular accumulation in various organs. While 42 proteins have been identified to date, their distribution differs between systemic and localized forms. Mass spectrometry analysis of tissue samples in the USA shows immunoglobulin light chain (AL) amyloidosis as the most prevalent systemic type, followed by transthyretin (ATTR). Heart and kidney involvements are common. Although there are 14 recognized types of kidney-related amyloidosis, clinicopathologic studies in the USA have identified 11 types, with AL amyloidosis being the most prevalent cause of kidney involvement. This review focuses on AL, AA, and ATTR amyloidosis due to their common systemic presentations. Recent US-based clinicopathologic studies challenge conventional beliefs that toxicity is primarily driven by amyloid deposition and highlight the role of the complement pathway. Diagnostic methods, particularly mass spectrometry, are crucial for accurate typing. Treatment strategies vary depending on the underlying type, with AL amyloidosis primarily targeting plasma cell clones, AA amyloidosis addressing underlying inflammation with systemic therapies, and ATTR amyloidosis focusing on ATTR stabilization or gene silencing.

Native State Stabilization of Amyloidogenic Proteins by Kinetic Stabilizers: Inhibition of Protein Aggregation and Clinical Relevance.

Proteinopathies or amyloidoses are a group of life-threatening disorders that result from misfolding of proteins and aggregation into toxic insoluble amyloid aggregates. Amyloid aggregates have low clearance from the body due to the insoluble nature, leading to their deposition in various organs and consequent organ dysfunction. While amyloid deposition in the central nervous system leads to neurodegenerative diseases that mostly cause dementia and difficulty in movement, several other organs, including heart, liver and kidney are also affected by systemic amyloidoses. Regardless of the site of amyloid deposition, misfolding and structural alteration of the precursor proteins play the central role in amyloid formation. Kinetic stabilizers are an emerging class of drugs, which act like pharmacological chaperones to stabilize the native state structure of amyloidogenic proteins and to increase the activation energy barrier that is required for adopting a misfolded structure or conformation, ultimately leading to the inhibition of protein aggregation. In this review, we discuss the kinetic stabilizers that stabilize the native quaternary structure of transthyretin, immunoglobulin light chain and superoxide dismutase 1 that cause transthyretin amyloidoses, light chain amyloidosis and familial amyotrophic lateral sclerosis, respectively.

The kappa/lambda ratio of surface immunoglobulin light chain as a valuable parameter for MRD assessment in CLL with atypical immunophenotype

In recent years, the significance of detecting minimal/measurable residual disease (MRD) in chronic lymphocytic leukemia (CLL) has increased due to the availability of highly effective therapeutic agents. Flow cytometry provides notable cost-effectiveness and immediacy, with an expected sensitivity level of approximately 10−4. The critical aspect of MRD detection via flow cytometry lies in accurately defining the region containing tumor cells. However, a subset of CLL, known as CLL with atypical immunophenotype, exhibits a distinct cell surface marker expression pattern that can make MRD detection challenging, because these markers often resemble those of normal B cells. To enhance the sensitivity of MRD detection in such atypical cases of CLL, we have capitalized on the observation that cell surface immunoglobulin (sIg) light chains tend to be expressed at a higher level in this subtype. For every four two-dimensional plots of cell surface markers, we used a plot to evaluate the expression of sIg kappa/lambda light chains and identified regions where the kappa/lambda ratio of sIg light chains deviated from a designated threshold within the putative CLL cell region. Using this method, we could detect atypical CLL cells at a level of 10−4. We propose this method as an effective MRD assay.

Resolving haplotype variation and complex genetic architecture in the human immunoglobulin kappa chain locus in individuals of diverse ancestry

Immunoglobulins (IGs), critical components of the human immune system, are composed of heavy and light protein chains encoded at three genomic loci. The IG Kappa (IGK) chain locus consists of two large, inverted segmental duplications. The complexity of the IG loci has hindered use of standard high-throughput methods for characterizing genetic variation within these regions. To overcome these limitations, we use long-read sequencing to create haplotype-resolved IGK assemblies in an ancestrally diverse cohort (n = 36), representing the first comprehensive description of IGK haplotype variation. We identify extensive locus polymorphism, including novel single nucleotide variants (SNVs) and novel structural variants harboring functional IGKV genes. Among 47 functional IGKV genes, we identify 145 alleles, 67 of which were not previously curated. We report inter-population differences in allele frequencies for 10 IGKV genes, including alleles unique to specific populations within this dataset. We identify haplotypes carrying signatures of gene conversion that associate with SNV enrichment in the IGK distal region, and a haplotype with an inversion spanning the proximal and distal regions. These data provide a critical resource of curated genomic reference information from diverse ancestries, laying a foundation for advancing our understanding of population-level genetic variation in the IGK locus.

Use of ultrasensitive RNA in situ hybridization for kappa and lambda light chains assists in the differential diagnosis of B-cell non-Hodgkin and Hodgkin lymphomas in cytopathology practice.

Use of ultrasensitive RNA in situ hybridization for kappa and lambda light chains assists in the differential diagnosis of B-cell non-Hodgkin and Hodgkin lymphomas in cytopathology practice.

Long-term outcomes after cardiac transplantation in AL amyloidosis.

7572 Background: Systemic AL amyloidosis can lead to progressive multi-organ dysfunction including advanced heart failure which may require orthotopic heart transplantation (OHT). Hematologic outcomes of AL amyloidosis post-OHT are not well described. Methods: We report outcomes for patients with AL amyloidosis with cardiac involvement post-OHT from Nov 2008 - Jun 2023 at Vanderbilt University Medical Center. Hematologic response was graded per the ISA guidelines. Results: Fourteen pts underwent OHT of whom 13 had lambda light chain disease. Three (21%) pts had isolated AL amyloidosis (AL) without myeloma (MM), while 11 (79%) also had MM: 6 with bone marrow plasma cells (BMPCs) 10-19% (AL-PCMM-10) and 5 with BMPC ≥20% (AL-PCMM-20). One each with AL-PCMM-10 and AL-PCMM-20 had lytic lesions (AL-CRAB). Median age at OHT was 55.5 yrs (range 37-74). Median time from diagnosis to OHT was 18 mo (range 3-62). Five (35%) pts had biopsy-proven extracardiac amyloid involvement. Median difference in serum free light chains at first evaluation was 70 mg/dL (range 9-289). Three pts were Mayo 2012 stage II, 4 stage III, and 7 stage IV. Four pts had t(11;14) and 1 had 1q gain. Ten (71%) pts received triplet therapy (VCd or VRd) and 3 (21%) received quadruplet therapy (Dara-VCd or Isa-VCd) pre-OHT. Six (43%) pts had autologous stem cell transplantation (ASCT); 1 pre-OHT and 5 post-OHT. Of ASCT pts, 1 pt achieved CHR, 1 VGPR, 2 PR, and 1 had PD pre-ASCT. Pre-OHT, 4 pts achieved CHR, 6 VGPR, 3 PR, and 1 had PD. Eleven pts (79%) received maintenance therapy post-OHT (5 proteosome inhibitors, 6 anti-CD38). At last follow-up, 8 pts were in CHR, 3 in VGPR, 1 in PR, and 2 had PD. Four (29%) pts died post-OHT: 2 from AL recurrence (1 each with AL and AL-PCMM-10), 1 from cardiac allograft vasculopathy (AL-CRAB), and 1 from renal failure (AL-CRAB). Three (21%) pts had hematologic relapse 5, 21 and 48 mo post-OHT, and of those, 2 had recurrent cardiac amyloid in graft 37 and 71 mo post-OHT. Of those with hematologic relapse, 2 had AL-PCMM-10 and 1 had AL. One pt had post-transplant lymphoproliferative disorder 126 mo post-OHT, requiring treatment. Median follow-up and survival of the cohort post-OHT was 2.25 yrs (range 0.5-13.8). Conclusions: In this single-center analysis of pts undergoing OHT for cardiac AL amyloidosis, we demonstrate feasibility of excellent long-term outcomes, in predominantly high-risk AL pts where majority harbored high-risk disease with MM overlap, either by AL-CRAB or AL-BMPC. Induction alone led to ≥VGPR response in 10 (71%) pts pre-OHT and further therapy deepened this to 11 (79%) pts. Both AL-CRAB pts died during follow-up, highlighting the overall poor prognosis in this specific subgroup. In our cohort, non-relapse related mortality was seen in only 2 (14%) pts, demonstrating acceptable post-OHT risk and overall ability to achieve far improved outcomes in the high-risk cardiac AL amyloidosis patient population that otherwise suffers from rapid and universal mortality.

Systemic AL kappa chain amyloidosis in a captive Bornean orangutan (Pongo pygmaeus)

Systemic amyloid light-chain (AL) amyloidosis is an infrequent disease in which amyloid fibrils derived from the immunoglobulin light chain are deposited in systemic organs, resulting in functional impairment. This disease has been notably uncommon in animals, and nonhuman primates have not been reported to develop it. In this study, we identified the systemic AL kappa chain amyloidosis in a captive Bornean orangutan (Pongo pygmaeus) and analyzed its pathogenesis. Amyloid deposits were found severely in the submucosa of the large intestine, lung, mandibular lymph nodes, and mediastinal lymph nodes, with milder lesions in the liver and kidney. Mass spectrometry-based proteomic analysis revealed an abundant constant domain of the immunoglobulin kappa chain in the amyloid deposits. Immunohistochemistry further confirmed that the amyloid deposits were positive for immunoglobulin kappa chains. In this animal, AL amyloidosis resulted in severe involvement of the gastrointestinal submucosa and lymph nodes, which is consistent with the characteristics of AL amyloidosis in humans, suggesting that AL amyloid may have a similar deposition mechanism across species. This report enhances the pathological understanding of systemic AL amyloidosis in animals by providing a detailed characterization of this disease based on proteomic analysis.

Enzymatization of mouse monoclonal antibodies to the corresponding catalytic antibodies

Catalytic antibodies possess a dual function that enables both antigen recognition and degradation. However, their time-consuming preparation is a significant drawback. This study developed a new method for quickly converting mice monoclonal antibodies into catalytic antibodies using site-directed mutagenesis. Three mice type monoclonal antibodies targeting hemagglutinin molecule of influenza A virus could be transformed into the catalytic antibodies by deleting Pro95 in CDR-3 of the light chain. No catalytic activity was observed for monoclonal antibodies and light chains. In contrast, the Pro95-deleted light chains exhibited a catalytic activity to cleave the antigenic peptide including the portion of conserved region of hemagglutinin molecule. The affinity of the Pro95-deleted light chains to the antigen increased approximately 100-fold compared to the wild-type light chains. In the mutants, three residues (Asp1, Ser92, and His93) come closer to the appropriate position to create the catalytic site and contributing to the enhancement of both catalytic function and immunoreactivity. Notably, the Pro95-deleted catalytic light chains could suppress influenza virus infection in vitro assay, whereas the parent antibody and the light chain did not. This strategy offers a rapid and efficient way to create catalytic antibodies from existing antibodies, accelerating the development for various applications in diagnostic and therapeutic applications.

Intraoral hemorrhagic bullae as the first sign of immunoglobulin light chain amyloidosis.

Intraoral hemorrhagic bullae as the first sign of immunoglobulin light chain amyloidosis.

Immunoglobulin repertoire sequencing and de novo sequencing – Powerful tools for identifying free light chains from patients with light chain cast nephropathy

In patients with light chain cast nephropathy (LCCN), abundantly produced monoclonal immunoglobulin free light chains (FLCs) play a vital role in pathogenesis. Determining the precise sequences of patient-derived FLCs is therefore highly desirable. Although immunoglobulin repertoire sequencing (5′ RACE-seq) has been proven to be sensitive enough to provide full-length V(D)J region (variable, diversity and joining genes) of FLCs using bone marrow samples, an invasive and bone marrow independent method is still in demand. Here a de novo sequencing workflow based on the bottom-up proteomics for patient-derived FLCs was established. PEAKS software was used for the de novo sequencing of peptides that were further assembled into full-length FLC sequences. This de novo protein sequencing method can obtain the full-length amino acid sequences of FLCs, and had been shown to be as reliable as 5′ RACE-seq. The two LCCN sequences derived from above the two methods were identical, and they possessed more hydrophobic or nonpolar amino acids compared with the corresponding germline, which may be associated with the pathogenesis.

Cu(II) binding to the λ6aJL2-R24G antibody light chain protein associated with light chain amyloidosis disease: The role of histidines

Light chain amyloidosis is a conformational disease caused by the abnormal proliferation and deposition of antibody light chains as amyloid fibers in organs and tissues. The effect of Cu(II) binding to the model recombinant protein 6aJL2-R24G was previously characterized in our group, and we found an acceleration of the aggregation kinetics of the protein. In this study, in order to confirm the Cu(II) binding sites, histidine variants of 6aJL2-R24G were prepared and the effects of their interaction with Cu(II) were analyzed by circular dichroism, fluorescence spectroscopy, isothermal calorimetry titrations, and molecular dynamics simulations. Confirming our earlier work, we found that His8 and His99 are the highest affinity Cu(II) binding sites, and that Cu(II) binding to both sites is a cooperative event.

AL–AMYLOIDOSIS RELATED ADVANCED HEART FAILURE DIAGNOSED THROUGH F–18–FLUTEMETANOL PET SCAN

Abstract Background Systemic amyloidosis (SA) is a group of rare diseases in which abnormal misfolded proteins, amyloid fibrils, accumulate in tissue. The gold standard for diagnosis is tissue biopsy of the affected organ, but it is invasive and needs specific proteomic or immunohistochemistry analyses to characterize the involved protein. We report a case of a complex diagnosis of SA in an advanced heart failure (HF) patient. Methods and Results A 68–year–old woman with history of recurrent uveitis started to complain dyspnea in 2022, after Covid19. Since her symptoms worsened, in June 2023 she was diagnosed in another center with restrictive cardiomyopathy with preserved left ventricular (LV) ejection fraction. She underwent coronary angiography (unremarkable), cardiac MRI (aspecific fibrosis), and a FDG–PET scan which showed non–homogeneous LV fixation. The patient was treated with steroids for the suspicion of an immunologic–inflammatory disease, without benefit. Fourteen months after the initial presentation, she was admitted to our centre for advanced inotrope–dependent HF. She had mildly increased LV hypertrophy and severe systolic dysfunction with restrictive physiology and, notably, peri–orbital subcutaneous hemorrhages (Figure 1). Hematological screening for amyloidosis showed elevated serum free lambda light chains. Repeated cardiac MRI scan was consistent with infiltrative disease. A total–body PET scan with dual tracer was performed. The control session with F–18–FDG showed mild aspecific cardiac uptake in the basal septum and lateral wall (Figure 2), while off–label use of F–18–Flutemetanol, an Alzheimer‘s disease approved experimental tracer that binds to amyloid fibrils beta sheets with very high sensitivity and specificity, showed intense cardiac, hepatic, pulmonary and muscular uptake, with wash–out–timing diagnostic for AL–SA (Figure 3). Periumbilical fat biopsy confirmed the clinical suspicion of SA. Hematological investigations led to the diagnosis of stage I–A lambda micromolecular multiple myeloma, with systemic amyloidosis and a IIIb cardiac prognostic score. Conclusions Endomyocardial biopsy is an invasive procedure and periumbilical fat biopsy discriminates the amyloid protein type only by proteomic analysis, which is available in very few Italian centres. The off–label use of Alzheimers’ PET tracers could reduce the delay of cardiac AL–SA diagnosis, allowing early disease identification and prompting timely hematological therapy.

Elimination of Medium/High Molecular Weight Solutes. Comparison of High Flow Hemodialysis with Extended Hemodialysis

Introduction: Post-dilution online hemodiafiltration is the most efficient extracorporeal depurative treatment of CKD. Recently a new type of membrane has been developed, with a higher cut-off point also called a medium cut-off point, which has the capacity to eliminate higher molecular weight molecules in hemodialysis. The hemodialysis performed with these membranes has been called “Expanded Hemodialysis”. Objective: Compare the purifying efficacy of medium and high molecular weight molecules in patients dialyzed with high-flux hemodialysis, VitaPES 210HF, and with patients treated with expanded hemodialysis with the medium cut-off dialyzer, Elisio-HX. We also assessed the effect that the increased removal of inflammatory mediators by MCO hemodialysis had on fecal Calprotectin levels. Patients and methods: This is a prospective observational cross-over study in which 8 prevalent hemodialysis patients were followed for two months. Blood levels of IL-6, C-reactive protein (CRP), β2-microglobulin, Kappa and Lambda immunoglobulin light chains, and serum albumin were determined before and after each hemodialysis session. Results: The percentage of reduction of medium and higher molecular weight molecules: β2microglobulin, IL-6, Kappa and Lambda chains and CRP were higher with the Elisio-21HX dialyzer compared to the VitaPES 210HF dialyzer. There was no difference in albumin clearance between the two dialyzers. Fecal calprotectin levels were lower in patients dialyzed with Elisio-21HX. Conclusion: The medium cutoff dialyzer, Elisio-HX, is more efficient in the elimination of medium/high molecular weight molecules than the VitaPES HF high-flux dialyzer, with the same albumin elimination. Improving inflammation at the local intestinal level with lower levels of fecal Calprotectin.

Serum monoclonal immunoglobulin light-chain detection differs between immunofixation electrophoresis methods in patients with AL amyloidosis.

Serum immunofixation electrophoresis (IFE) is often performed for screening monoclonal proteins (M proteins) in immunoglobulin light-chain amyloidosis (AL amyloidosis). However, the performance of serum IFE for detecting M protein in AL amyloidosis patients is often insufficient. In this study, we examined the detection rate of serum M protein in newly diagnosed AL amyloidosis patients and analyzed differences in M protein detection between IFE methods. Among 60 patients newly diagnosed with AL amyloidosis, 22 had undetectable serum M protein by IFE with the Epalyzer2 system. Samples with undetectable M protein had significantly lower involved serum-free light-chain (iFLC) and a smaller difference between involved and uninvolved serum-free light-chain (dFLC) values than samples with IFE-detectable monoclonal light chains. When samples that tested negative for M protein by the Epalyzer2 system were retested by IFE with the HYDRASYS 2 system, 50% had IFE-detectable monoclonal light chains. The IFE system and reagents used may affect serum monoclonal immunoglobulin light-chain detection in AL amyloidosis patients, especially those with low iFLC or low dFLC samples. More attention should be paid to the performance of IFE systems, since it may affect the diagnostic and therapeutic evaluation of AL amyloidosis patients.

Localized Urinary Bladder Amyloidosis as Urothelial Cancer Mimicker.

Localized amyloidosis of the bladder is rare and often mimics bladder malignancy. It is typically associated with the extracellular deposition of monoclonal light chains, either κ or λ. The cause is unknown, but it is thought to be due to chronic inflammation/cystitis. To highlight the importance of localized urinary bladder amyloidosis as a rare mimicker of urothelial malignancy and elucidate its clinical, histopathologic, and cytopathologic manifestations. Cases of urinary bladder amyloidosis diagnosed during 2000-2023 were retrieved retrospectively from pathology archives. Electronic medical records, including cystoscopy findings and pathology slides including Congo red stain, were reviewed. Here we present 6 patients with localized urinary bladder amyloidosis. Four of the 6 patients were women, with ages ranging from 46 to 69 years, and a mean age of 58 years. Five of 6 patients presented with hematuria, while in 1 patient, bladder amyloidosis was discovered incidentally. Cystoscopy findings invariably were concerning for malignancy, with raised erythema in 5 patients and fungating mass protruding into the bladder lumen in 1 patient. Bladder biopsies and urine cytology were negative for malignancy in all cases. Congo red-positive amyloid deposits involved lamina propria with sparing of the detrusor muscle. In 5 cases, the deposits were typed as derived from the λ light chain, whereas no information was available for 1 patient. Subsequent clinical workup ruled out systemic amyloidosis. These cases of urinary bladder amyloidosis highlight the importance of considering rare amyloidosis in the differential diagnosis of hematuria and cystoscopy with a lesion mimicking malignancy.

The Real-World Data on Patients With Cardiac Stage IIIb AL Amyloidosis

BackgroundMorbidity and mortality of patients with immunoglobulin light chain (AL) amyloidosis are strongly associated with the severity of cardiac involvement, especial in patients with cardiac stage IIIb, but the real-world data on these patients is still limited. Patients and MethodsA retrospective analysis was conducted on 77 patients diagnosed with cardiac stage IIIb AL amyloidosis at our center. We analyzed the clinical characteristics, treatment and outcome of the patients. ResultsThe median age of patients was 57 years and 49.4% were male. Median serum N-terminal pro-brain natriuretic peptide (NT-proBNP) and cardiac troponin T (cTnT) were 13,384 ng/L and 0.166 ug/L, and 42 (54.5%) patients had heart failure at diagnosis. Fifty-seven (74.0%) patients received antiplasma cell treatment, and the main treatment options include bortezomib or thalidomide combined with dexamethasone. The hematologic overall response rate was 70% (28/40), and at 6-month landmark analysis, patients with hematologic responses had a higher survival rate. Cardiac and renal responses were achieved in 14 (37.8%) and 13 (32.5%) patients, respectively. After a median follow-up of 10 months (range 1-115 months), median overall survival (OS) was 18 months, and the estimated survival rates at 3, 6, and 12 months were 79.9%, 75.6%, and 54.5%, respectively. In Cox regression models, age, hypotension and cTnT were independently predictive of mortality after adjusting for heart failure. ConclusionThe hematologic, cardiac and renal responses were relative lower in patients with cardiac stage IIIb AL amyloidosis. The overall prognosis of patients was poor, and age, hypotension, and cTnT can be used to predict mortality.

Abstract PO4-06-08: ZP4 AS A NOVEL TARGET FOR CAR T CELL THERAPY IN TRIPLE NEGATIVE BREAST CANCER

Abstract Triple negative breast cancer (TNBC) has the worst prognosis of BC subtypes, largely due to lack of approved targeted therapies. Chimeric antigen receptor (CAR) T cells have demonstrated great success in certain hematological malignancies; however, in the treatment of solid cancers, CAR T cells are faced with a variety of obstacles that limit their efficacy, including on-target/off-tumor toxicity. To minimize these toxicities, the ideal CAR T cell target must be overexpressed by the tumor and absent from vital organs. Cancer/germline (CG) antigens satisfy this need, as their expression is restricted to male or female germ cells. Using a bioinformatic pipeline to identify novel CG antigens expressed in TNBC and other solid cancers, we have identified zona pellucida glycoprotein 4 (ZP4) as a candidate CG antigen target for CAR T cell therapy. This protein is a component of the human zona pellucida matrix, an extracellular structure that surrounds the oocyte and functions in folliculogenesis, species-specific fertilization, and early development. We have identified ZP4 expression in approximately 10% of TNBC cases from available RNAseq and proteomic databases and have confirmed ZP4 expression in several TNBC PDX models. The overall purpose of this study is to redirect T cells against ZP4 by engineering and validating a second generation ZP4-specific CAR. We have generated a library of monoclonal antibody (mAb) candidates against the extracellular portion of human ZP4. Antibody clones were screened, and their specificity validated via ELISA and flow cytometry, and the three strongest binders (z108, z128, and z164) were selected for further investigation. By IHC, we confirmed that the ZP4 mAb clones stained the area surrounding normal oocytes and did not show binding to any of the 23 normal tissues screened. The variable regions of the antibody heavy and light chains were sequenced using 5’RACE and used to assemble second generation CAR constructs composed of the variable heavy and light chain fragments separated by a linker, a CH2/CH3 hinge domain, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and the CD3z signaling domain. Activated T cells transduced with retroviral supernatant express the ZP4 CAR constructs at high levels. While all ZP4 CAR T cells demonstrate antigen-specific cytotoxic killing of ZP4+ target tumor cells in short-term in vitro assays, z128 ZP4 CAR T cells demonstrate superior persistence and efficacy in long-term repeat tumor challenge in vitro assays and orthotopic in vivo mouse models. In summary, ZP4 is a promising target for TNBC and we have successfully engineered ZP4 specific CAR T cells. ZP4 CAR T cells effectively target and kill ZP4+ cells in in vitro cytotoxicity assays but perform differently in repeat tumor challenge assays and in vivo. Future studies will elucidate the precise differences between our three candidate CAR constructs and further test the effector function, trafficking, proliferation, and persistence of z128 ZP4 CAR T cells in vivo using ZP4+ PDX models. This work has exciting potential to bring a novel CAR T cell therapy to patients with TNBC, without concerns for on-target off-tumor toxicity. Citation Format: Lauren Somes, Jonathan Lei, Paul Shafer, Xinpei Yi, Diego Chamorro, Emily Madaras, Michael Lewis, Bing Zhang, Valentina Hoyos. ZP4 AS A NOVEL TARGET FOR CAR T CELL THERAPY IN TRIPLE NEGATIVE BREAST CANCER [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-06-08.

Expert Center for cardiac amyloidosis: reality and perspectives

Aim. To evaluate the features of diagnosis of amyloid cardiomyopathy (ACMP), differential diagnosis of different types of amyloidosis and its clinical manifestations. Materials and methods. Were analyzed 150 cases of patients who consulted at the Expert Center for Amyloidosis with suspicion of the presence of ACMP. 63 patients were diagnosed with ACMP: 25 (39.7%) – women, 38 (60.3%) – men, with an average age of 64.1±1.5. 36 (57.1%) patients had AL-amyloidosis (immunoglobulin amyloid light-chain amyloidosis), 25 (39.7%) – ATTR-amyloidosis (transthyretin amyloidosis), 2 (3.2%) – AA-amyloidosis with heart failure (reactive systemic amyloidosis caused by hypersecretion of á-globulin). The analysis of clinical manifestations depending on the type of amyloidosis, data of laboratory and instrumental methods of diagnosis is carried out. Results. In most cases, 53 (84.1%) patients, amyloidosis manifested as signs of heart failure. Among cardiac manifestations, shortness of breath (95.2%), general weakness (93.7%), lower limb edema (76.2%) were the most common. To confirm the diagnosis, despite the high accuracy of the speckle-tracking echocardiography and magnetic resonance imaging of the heart with gadolinium, in rare cases a biopsy is required (e.g. there is a combination of clinical signs of several types of amyloidosis). Biopsy of the affected organ was performed in 31 (49.2%) patients. The strategy for further pathogenetic treatment depends on the determination of the type of amyloidosis. Free light chains of immunoglobulins were detected in 57.1% of cases, which allowed diagnosis of AL-amyloidosis. In 17 (38.6%) patients myocardial scintigraphy with 99mTc-pyrophosphate showed signs of ATTR-amyloidosis, which with a negative result of immunochemical studies allows non-invasive diagnosis of it. Conclusion. ACMP is a disease with an extremely adverse prognosis. Raising the awareness of specialists about ACMP is an important goal. With timely diagnosis, pathogenetic therapy can be started early, which will improve the quality of life and prognosis of patients with ACMP.