Multiple Target Antigens Research Articles

Efficacy of MAGE-A4 long peptide as a universal immunoprevention cancer vaccine

BackgroundThe clinical application of peptide vaccines in tumor immunotherapy holds significant promise. Peptide-based tumor vaccines are currently subject to certain limitations in clinical trials, including the challenge of inducing a sustained response from CD4+ T helper cells and cytotoxic T lymphocytes (CTL), as well as human leukocyte antigen (HLA) restrictions.MethodsThrough the utilization of biological information methodology, a screening process was conducted to identify three potential long peptides that are specifically targeted by the MAGE-A4 antigen. The candidate long peptides were subjected to in vitro testing using human peripheral blood lymphocytes as samples to evaluate their immunogenicity and immune function. The antitumor properties and preliminary mechanism of the long peptide vaccine were investigated through the use of a mouse model designed for the prevention of triple negative breast cancer (TNBC).ResultsThree predicted multi-epitope long peptides targeting MAGE-A4 have shown to have a strong immunogenicity, with a total positive rate of 72% across different HLA subtypes in Chinese populations. they can also increase the levels of the costimulatory factor CD137 and tumor necrosis factor-alpha (TNF-α), activate T cells, and boost the cytotoxic activity. Results from an animal study have revealed that the long-peptide vaccine, both on its own and in combination with R848, has displayed impressive anti-tumor and target-specific capabilities. Moreover, it has the ability to increase the expression of effector memory T cells and central memory T cells.ConclusionsThis study was the first to screen three multi-epitope long peptides targeting MAGE-A4 and assess their immunogenicity, immune function, and potential as adjuvant peptides. The results showed that the MAGE-A4 long peptide vaccine can be used as a novel immunoprophylaxis method to prevent TNBC. Moreover, the proposed development model is capable of screening multiple target antigens, which lead to its clinical application.

Abstract 1321: In vivo immune cell programming using mRNA-LNP chimeric antigen receptors

Abstract Introduction: The tumor microenvironment (TME) is a dynamic and multifaceted system that comprises largely myeloid lineage immune cells including monocytes, neutrophils, macrophages, and dendritic cells. Reprogramming the myeloid compartment within the context of cancer therapy presents an attractive strategy to overcome current limitations in solid tumor treatment. We have developed a novel approach that harnesses the natural ability of Myeloid cells to take up mRNA loaded lipid nanoparticles, to reprogram these cells with chimeric antigen receptors (CAR’s) that arm these cells to recognize and kill tumor cells. Methods: Current LNPs technology is sufficient to deliver significant mRNA cargo to numerous cell types, including B cells, T cells and myeloid cells. Leveraging this phenomenon we have developed a new class of chimeric antigen receptors (CARs), using protein biology that imparts targeted immune cell expression and functionality, specifically in Myeloid cells. These CARs harness Immunoreceptor Tyrosine-based Activation Motif (ITAM) signaling adaptors expressed exclusively in immune cells, achieving immune cell-type specific CAR-directed tumor lysis and cytokine responses. With a FcaR I (CD89) based CAR design, we have developed a CAR that is dependent on the common Fc receptor gamma chain, primarily restricted to myeloid cells, for stable expression and function. Results: We have demonstrated specific expression of CD89-based CAR in myeloid cells in mouse and monkey following systemic mRNA/LNP delivery, and in human myeloid cells following in vitro treatment with mRNA/LNPs. Systemic mRNA/LNP delivery of CD89-based CAR has demonstrated robust anti-tumor efficacy against multiple tumor antigens in various xenograft mouse models of human breast cancer, hepatocellular carcinoma, and ovarian cancer. Furthermore, in syngeneic mouse tumor models, robust anti-tumor immune responses, characterized by tumoral infiltration of activated CD8+ T cells, diminished tumor-associated Tregs, systemic enhancement of dendritic cells and anti-tumor IgG production, were observed for treated animals. Conclusions: Taken together, we present CAR designs that achieve expression and function in targeted immune cell populations, capable of eliciting anti-tumor efficacy against multiple target antigens by leveraging innate and adaptive immune responses, and demonstrating a class of novel CAR designs to overcome limitations faced by current in vivo mRNA/LNP delivery strategy. A phase I clinical trial is currently on-going to assess the safety and preliminary efficacy of CD89-based CAR against TROP2+ epithelial malignancies. Citation Format: Shannon Argueta, Franciele K. Melber, Michael Gorgievski, Neha Divangi, Edward Cochran, Thach Chu, Yuxiao Wang, Jian Ding, Daniel Getts. In vivo immune cell programming using mRNA-LNP chimeric antigen receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1321.

Abstract 6726: Costimulatory IgM T-cell engagers with enhanced and durable cytotoxicity

Abstract T-cell engagers (TCEs) have established therapeutic effect in treatment of various cancers by harnessing the power of the immune system against tumors in a targeted manner. IgM-based TCEs may offer high avidity, specificity, and safety advantages over other modalities because of their multivalent architecture and unique positioning of the CD3 binding domain on the J-chain. We have built a costimulatory IgM TCE platform that engages both signal 1 and signal 2 on T-cells, with the goal of enabling their optimal activation and survival for more robust and durable cytotoxic activity. Multiple antigen targeting costimulatory IgM TCEs were generated and activity was evaluated both in vitro and in vivo to assess the role of CD28 co-stimulation. Costimulatory IgM TCEs engaged both CD3 and CD28 on the T-cell surface, enhancing in vitro cytotoxicity and T-cell activation. Levels of IL-2 increased significantly with dual signal engagement promoting proliferation and survival of T-cells. In long-term culture assays, costimulatory IgM TCEs promoted better T-cell proliferation and survival compared to IgM TCEs with only a CD3 binder, and was even more pronounced at low E:T ratios. In the absence of CD3 engagement, CD28 binding alone did not induce any T-cell activation, cytotoxicity, or cytokines. Activation of T-cells was also fully dependent on the presence of target cells, underscoring the safety profile of the platform. In Hu-PBMC xenograft tumor models, costimulatory IgM TCEs exhibited stronger in vivo anti-tumor activity compared to TCEs only engaging CD3. Tumor growth inhibition was maintained for longer after dosing was stopped with costimulatory IgM TCEs, indicating the durability of responses. Costimulatory IgM TCEs suppressed tumor growth in xenograft studies using cell lines that expressed both high and low target copy numbers suggestive of their broad utility against multiple tumor types. Enhanced in vivo anti-tumor activity of costimulatory IgM TCEs was associated with significant increases in intra-tumoral CD8/CD3+T-cells.Both peripheral and intra-tumoral T-cells exhibited enhanced anti-apoptotic and cytotoxic phenotypes indicative of the costimulatory activation. IgM-based costimulatory TCEs provide enhanced cytotoxicity through optimal T-cell activation and proliferation/survival. This dual engagement of T-cell activation signals, along with the high avidity target binding offered by IgM platform, could enable the creation of effective therapeutics targeting pathogenic cells in autoimmune diseases and solid tumors that will maintain activity even in conditions with low T-cell counts. Citation Format: Jie Xue, Keyu Li, Poonam Yakkundi, Palak Chadasama, Tina Mao, Zee Malik, Vidhya Rao, Madhura Joglekar, Kristene Mai, Gene Li, Deepal Pandya, Rodnie Rosete, Zhongde Ye, Leyla Tahrani, Jinqiu Wang, Nardeen Hanna, Elizabeth Perez, Yue Wang, Sachi Ahmed, Lusiana Widjaja, Paul Hinton, Krzysztof Bzymek, Bruce Keyt, Miho Oyasu, Liqin Liu, Angus Sinclair, Umesh S. Muchhal. Costimulatory IgM T-cell engagers with enhanced and durable cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6726.

Multiplex Bead Assay for the Serological Surveillance of Measles and Rubella.

There is increasing interest in evaluating antibody responses to multiple antigen targets in a single assay. Immunity to measles and rubella are often evaluated together because immunity is provided through combined vaccines and because routine immunization efforts and surveillance for measles and rubella pathogens are combined in many countries. The multiplex bead assay (MBA) also known as the multiplex immunoassay (MIA) described here combines the measurement of measles- and rubella-specific IgG antibodies in serum quantitatively according to international serum standards and has been successfully utilized in integrated serological surveillance.

Abstract LB_B06: Single cell transcriptomics guided identification of antigen combinations for the design of logic-gated CAR therapies

Abstract Chimeric antigen receptor (CAR) T-cell therapy has demonstrated significant efficacy in treating hematological cancers but faces challenges in treating solid tumors due to antigen heterogeneity and off-tumor, on-target expression in normal tissues. To combat these challenges, we developed a novel genetic algorithm approach to analyze patient tumor single-cell transcriptomics data. This algorithm identifies combinations of cell surface antigens specific to cancer cells by incorporating "AND," "OR," and "NOT" logic. Our method is generalizable to combinations of any size and demonstrates rapid convergence across different parameter choices. When applied to breast cancer datasets, our algorithm identified optimal combinations of triplet antigens that outperformed clinically tested single target antigens in discriminating tumor cells from non-tumor cells across patient samples. In sum, our approach may guide the design of logic-gated CAR therapies aimed at multiple target antigens, thereby increasing the safety and selectivity of these therapies. Citation Format: Sanna Madan, Tiangen Chang, Binbin Wang, Alejandro A. Schäffer, Eytan Ruppin. Single cell transcriptomics guided identification of antigen combinations for the design of logic-gated CAR therapies [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B06.

Adaptor Anti-P329G CAR T Cells for Modular Targeting of AML

Introduction Chimeric antigen receptor (CAR) T cell therapy has achieved promising results for treatment of B cell and plasma cell malignancies, but success has been limited in the treatment of acute myeloid leukemia (AML). Numerous antigens including CD33, CD123 or CSF1R have been proposed for AML therapy. However, clinical success of CAR T cell therapy in AML is impaired by antigen heterogeneity, therapy-associated toxicities, and antigen escape. Adaptor CAR T cells which allow sequential, transient or simultaneous targeting of multiple antigens have the potential to overcome these limitations. These adaptor CAR T cells were combined with tumor antigen-directed CAR-adaptor molecules for indirect tumor binding. We developed a modular P329G-directed CAR targeting the P329G mutation in the Fc part of tumor-targeting human IgG1 antibodies containing P329G L234A/L235A (PGLALA) mutations. These PGLALA mutations render the Fc part inactive for binding to Fc gamma receptors (FcgRs) and to the complement system, and up to now, more than 20 such Fc-silenced therapeutic antibodies including an approved T cell recruiting bispecific antibody have been clinically validated. Compared to other modular CAR T cell platforms this approach does not rely on haptens or artificial tags fused to the targeting antibody and may ease development and testing of the platform. Methods A scFv-based second generation CAR vector system was used in primary human T cells. P329G-targeting CAR T cells were combined with PGLALA-Fc-mutated antibodies targeting CD33, CD123 or CSF1R. Classical, directly binding anti-CD33 and anti-CSF1R CAR T cells have been used as positive controls, while anti-CD19 CAR T cells and untransduced T cells have been used as negative controls. Different AML (MOLM-13, OCI-AML-3, PL-21, Mv4-11, THP-1 and U-937) and an ALL cell line (NALM-6) were used as target cell lines expressing the target antigens in different intensity. Effector function of CAR T cells was also tested against primary human AML blasts derived from patients. Luciferase-based killing assays, ELISA-based and flow-based analysis were used to evaluate CAR effector functions. Results Unlike CD16-CAR T cells, which bind human IgG in a non-selective manner, anti-P329G CAR T cells revealed specific effector functions only when combined with antibodies carrying PGLALA-Fc-mutations. Anti-P329G CAR T cells alone revealed no activation. Anti-P329G CAR T cells could be specifically activated by recombinant protein when combined with antibodies targeting CD33, CD123 and CSF1R leading to upregulation of T cell activation markers. Anti-P329G CAR T cells combined with PGLALA-mutated antibodies targeting CD33, CD123 und CSF1R achieved efficient in vitro cytotoxicity against tested AML cell lines and primary human AML blasts, while antigen-negative ALL cell line NALM-6 was not killed. Remarkably, effector functions of anti-P329G CAR T cells were similar to classical CAR T cells particularly when targeting CD33. This led to specific cytokine production when anti-P329G CAR T cells were co-cultured with respective antibodies and tumor cells. P329G-CAR T cells activated by recombinant CD33, CD123 or CSF1R showed no cytokine production in the presence of an antibody which was not recognizing the respective antigen. However, by switching the antibody after 24 h of stimulation towards the tumor antigen-targeting antibody, CAR T cell activation could be again achieved by the recombinant protein. Depleting the tumor antigen-targeting antibody after 24 h of stimulation decreased the cytokine production after 48 h and 72 h despite ongoing presence of the recombinant protein. This confirms the modularity and reversibility of this adaptor CAR T cell platform. Conclusions Taken together, anti-P329G CAR T cells combined with Fc-silenced tumor antigen-targeting IgG1 antibodies carrying the clinically validated PGLALA-mutations in the Fc part achieved profound effector functions against various human AML cell lines and primary AML blasts. The modular platform has the potential to overcome recent limitations of CAR T cell therapy in AML. These results support the rational for clinical translation of this novel and modular CAR T cell platform.

Multispecific Hybrid T Cell Receptors for Sensitive Targeting of Cancer

CAR T cells represent a promising therapy for a growing list of tumors. Despite high initial response rates, 50-60% of patients relapse within 1 year after CAR T cell infusion. Heterogeneity of target antigen expression can lead to outgrowth of tumor cells with low or negative antigen levels. Conventional CARs require high antigen levels for efficient T cell activation and tumor elimination. By contrast, T cell receptors (TCRs) are 10-100 times more sensitive than CARs. To achieve greater antigen sensitivity, we and others designed synthetic hybrid T cell receptors by fusing antibody-based recognition domains to TCRa and TCRb constant chains. Here we extend the initial monospecific design to incorporate two antigen recognition domains. We evaluated 2 monospecific hybrid receptors, one with VH and VL domains fused to TCRa and TCRb constant chains respectively (Split TCR/CAR) and a second where the entire scFv (VL-linker-VH) is fused only to the TCRa chain (Full TCR/CAR) leaving TCRb available for a second scFv. We used based editing to knock out endogenous TCRa and b chains to avoid mispairing. Using CD19 as a model target we found that both TCR/CAR designs lacked tonic signaling, induced TCR-like changes in Ca+ flux, more efficiently phosphorylated LAT, and exhibited superior recognition of Nalm-6 clones expressing a range of CD19 levels compared to conventional CD19 28z or 4-1BBz CARs. Interestingly, the Full TCR/CAR outperformed the Split format in these assays. Using soluble bilayer and TIRF microscopy, we demonstrated that unlike CARs, TCR/CAR T cells recapitulate a classical TCR-like immunological synapse. I n vivo studies using NSG mice engrafted with CD19+ Raji cells demonstrated potent anti-tumor efficacy, and the Full format outperformed the Split TCR/CAR. Capitalizing on the superiority of the Full TCR/CAR format, we next designed bispecific hybrid receptors composed of two scFvs of different specificities fused to the TCRa and TCRb chains. Multiple Myeloma is particularly suitable for bispecific targeting as relapse following CAR T cell therapy is common and multiple target antigens have been described. We designed bispecific Full TCR/CARs specific for pairs of myeloma antigens (BCMA/SLAMF7 and BCMA/GPRC5D) and expressed them in primary T cells. We used base editing to knock out SLAMF-7 which can be expressed in T cells and could cause fratricide. Bispecific TCR/CAR receptors conferred sensitive T cell recognition of target cells expressing BCMA and SLAMF-7 as measured by cytokine production, proliferation, and cytotoxicity. T cells equipped with these novel bispecific receptors maintained function when exposed to L363 cells BCMA ko or SLAMF-7 ko demonstrating that both antigen binding domains can trigger T cell functions against target cells. In vivo studies using NSG mice implanted with a mixture composed of 33% of Nalm-6 BCMA+, Nalm-6 SLAMF-7+ and Nalm-6 BCMA+SLAMF-7+ cells to mimic target antigen expression heterogeneity and antigen loss showed superior anti-tumor efficacy for bispecific TCR/CAR cells compared to conventional BCMA and SLAMF-7 BBz CARs. Our hybrid bispecific TCR/CAR also demonstrated superior anti-tumor efficacy compared to a previously described, and systematically optimized bispecific CAR format (Zah et al., 2020). Bispecific hybrid T cell receptors embedded with natural TCR signaling machinery represent a promising therapeutic option to address antigen downmodulation/loss and antigen heterogeneity for treatment of cancer.

Conditional Control of Universal CAR T Cells by Cleavable OFF-Switch Adaptors.

As living drugs, engineered T cell therapies are revolutionizing disease treatment with their unique functional capabilities. However, they suffer from limitations of potentially unpredictable behavior, toxicities, and nontraditional pharmacokinetics. Engineering conditional control mechanisms responsive to tractable stimuli such as small molecules or light is thus highly desirable. We and others previously developed "universal" chimeric antigen receptors (CARs) that interact with coadministered antibody adaptors to direct target cell killing and T cell activation. Universal CARs are of high therapeutic interest due to their ability to simultaneously target multiple antigens on the same disease or different diseases by combining with adaptors to different antigens. Here, we further enhance the programmability and potential safety of universal CAR T cells by engineering OFF-switch adaptors that can conditionally control CAR activity, including T cell activation, target cell lysis, and transgene expression, in response to a small molecule or light stimulus. Moreover, in adaptor combination assays, OFF-switch adaptors were capable of orthogonal conditional targeting of multiple antigens simultaneously, following Boolean logic. OFF-switch adaptors represent a robust new approach for the precision targeting of universal CAR T cells with potential for enhanced safety.

Targeting of multiple tumor-associated antigens by individual T cell receptors during successful cancer immunotherapy

The Tcells of the immune system can target tumors and clear solid cancers following tumor-infiltrating lymphocyte (TIL) therapy. We used combinatorial peptide libraries and a proteomic database to reveal the antigen specificities of persistent cancer-specific Tcell receptors (TCRs) following successful TIL therapy for stage IV malignant melanoma. Remarkably, individual TCRs could target multiple different tumor types via the HLA A∗02:01-restricted epitopes EAAGIGILTV, LLLGIGILVL, and NLSALGIFST from Melan A, BST2, and IMP2, respectively. Atomic structures of a TCR bound to all three antigens revealed the importance of the shared x-x-x-A/G-I/L-G-I-x-x-x recognition motif. Multi-epitope targeting allows individual Tcells to attack cancer in several ways simultaneously. Such "multipronged" Tcells exhibited superior recognition of cancer cells compared with conventional Tcell recognition of individual epitopes, making them attractive candidates for the development of future immunotherapies.

One antigen closer to understanding membranous nephropathy

Membranous nephropathy is an immune-mediated kidney disease characterized by subepithelial immune deposits. Through the utilization of advanced proteomic techniques, multiple target antigens have been identified, including those associated with primary and secondary etiologies. Nonsteroidal anti-inflammatory drugs represent an important secondary cause of membranous nephropathy. In this study by Sethi etal., advanced proteomic techniques identify proprotein convertase subtilisin/kexin type 6 as a target antigen in nonsteroidal anti-inflammatory drug-associated membranous nephropathy.

FLT3-directed UniCAR T-cell therapy of acute myeloid leukaemia.

Adaptor chimeric antigen receptor (CAR) T-cell therapy offers solutions for improved safety and antigen escape, which represent main obstacles for the clinical translation of CAR T-cell therapy in myeloid malignancies. The adaptor CAR T-cell platform 'UniCAR' is currently under early clinical investigation. Recently, the first proof of concept of a well-tolerated, rapidly switchable, CD123-directed UniCAR T-cell product treating patients with acute myeloid leukaemia (AML) was reported. Relapsed and refractory AML is prone to high plasticity under therapy pressure targeting one single tumour antigen. Thus, targeting of multiple tumour antigens seems to be required to achieve durable anti-tumour responses, underlining the need to further design alternative AML-specific target modules (TM) for the UniCAR platform. We here present the preclinical development of a novel FMS-like tyrosine kinase 3 (FLT3)-directed UniCAR T-cell therapy, which is highly effective for in vitro killing of both AML cell lines and primary AML samples. Furthermore, we show in vivo functionality in a murine xenograft model. PET analyses further demonstrate a short serum half-life of FLT3 TMs, which will enable a rapid on/off switch of UniCAR T cells. Overall, the presented preclinical data encourage the further development and clinical translation of FLT3-specific UniCAR T cells for the therapy of AML.

Bispecific Antibodies in Multiple Myeloma: Opportunities to Enhance Efficacy and Improve Safety.

Multiple myeloma (MM) is the second most common haematological neoplasm of adults in the Western world. Overall survival has doubled since the advent of proteosome inhibitors (PIs), immunomodulatory agents (IMiDs), and monoclonal antibodies. However, patients with adverse cytogenetics or high-risk disease as determined by the Revised International Staging System (R-ISS) continue to have poorer outcomes, and triple-refractory patients have a median survival of less than 1 year. Bispecific antibodies (BsAbs) commonly bind to a tumour epitope along with CD3 on T-cells, leading to T-cell activation and tumour cell killing. These treatments show great promise in MM patients, with the first agent, teclistamab, receiving regulatory approval in 2022. Their potential utility is hampered by the immunosuppressive tumour microenvironment (TME), a hallmark of MM, which may limit efficacy, and by undesirable adverse events, including cytokine release syndrome (CRS) and infections, some of which may be fatal. In this review, we first consider the means of enhancing the efficacy of BsAbs in MM. These include combining BsAbs with other drugs that ameliorate the effect of the immunosuppressive TME, improving target availability, the use of BsAbs directed against multiple target antigens, and the optimal time in the treatment pathway to employ BsAbs. We then discuss methods to improve safety, focusing on reducing infection rates associated with treatment-induced hypogammaglobulinaemia, and decreasing the frequency and severity of CRS. BsAbs offer a highly-active therapeutic option in MM. Improving the efficacy and safety profiles of these agents may enable more patients to benefit from these novel therapies and improve outcomes for patients with high-risk disease.

Magnetic Nanoparticle-Based Semi-automated Panning for High-Throughput Antibody Selection.

Bio-panning is a common process involved in recombinant antibody selection against defined targets. The biopanning process aims to isolate specific antibodies against an antigen via affinity selection from a phage display library. In general, antigens are immobilized on solid surfaces such as polystyrene plastic, magnetic beads, and nitrocellulose. For high-throughput selection, semi-automated panning selection allows simultaneous panning against multiple target antigens adapting automated particle processing systems such as the KingFisher Flex. The system setup allows for minimal human intervention for pre- and post-panning steps such as antigen immobilization, phage rescue, and amplification. In addition, the platform is also adaptable to perform polyclonal and monoclonal ELISA for the evaluation process. This chapter will detail the protocols involved from the selection stage until the monoclonal ELISA evaluation with important notes attached at the end of this chapter for optimization and troubleshooting purposes.

Utility of the pattern-based approach using BIOCHIP-indirect immunofluorescence in the evaluation of subepidermal bullous disorders.

Subepidermal bullous disorders (SEBD) are a heterogeneous group of vesiculobullous diseases because of antibody-mediated destruction of proteins of the dermo-epidermal junction. Direct immunofluorescence (DIF) is the gold standard for diagnosis. BIOCHIP-indirect immunofluorescence (IIF) is a novel serological test that combines multiple target antigens in a single field. The present study aimed to evaluate the utility of the pattern-based approach in BIOCHIP-IIF for the diagnosis of SEBD. Seventy cases of BIOCHIP-IIF that showed clinical, histopathological, and/or DIF features favoring SEBD were included in the study. The interpretation in the BIOCHIP was categorized into one of the following patterns. Pattern I: basement membrane zone (BMZ) staining in monkey esophagus (ME), primate salt-split skin (SSS)-roof staining, BP180+ and/or BP230+; Pattern II: roof staining in SSS, BP180- and BP230- with or without BMZ staining in ME; Pattern III: floor staining in SSS, BP180- and BP230-; and pattern IV: negative in SSS and other substrates. The findings were correlated with histopathology and/or DIF. Fifty (71.5%) cases showed pattern I or the typical bullous pemphigoid (BP) pattern. Eight (11.4%) cases showed pattern II. Patterns III and IV were observed in seven (10%) and five (7.1%) cases, respectively. BP was the most common diagnosis in patterns I and II, while anti-p200 pemphigoid was most common in pattern III, as confirmed by immunoblotting. The sensitivity of pattern I in the diagnosis of BP was 96%. BIOCHIP-IIF showed a good correlation with DIF and histopathology in the diagnosis of SEBD. This can be used as a first-line investigation in case of bullous disorders.

Safety and Efficacy of Humanized Versus Murinized CD19 and CD22 CAR T-Cell Cocktail Therapy for Refractory/Relapsed B-Cell Lymphoma.

CD19 chimeric antigen receptor T-cell (CAR-T) therapy is efficacious for refractory/relapsed (R/R) B-cell hematological malignancies, yet relapse due to CD19 antigen escape remains a challenge. Our trial explored simultaneous targeting of multiple B-cell antigens as a therapeutic approach that may reduce the risk of relapse. We tested the safety and efficacy of CAR19/22 T-cell cocktail therapy including murinized and humanized products among patients with R/R aggressive B-cell lymphoma. In the group that received the humanized product, 11/12 (91.7%) patients achieved an objective response, including 9/12 (75%) complete responses (CRs) by day 28. The overall response rate and CR rate in the murinized group was 92.9% (13/14) and 42.9% (6/14), respectively. Nine of 12 (75%) patients in the humanized group maintained CR at month 3 following infusion, compared to 5/14 patients (35.7%) in the murinized group. Progression-free survival (PFS) was more favorable in the humanized compared to the murinized group. Most patients had mild cytokine release syndrome (CRS) (grade 1-2) in both groups. This study demonstrates that CAR19/22 T-cell cocktail therapy is safe and effective for R/R B-cell lymphoma and that patients treated with a humanized CAR-T exhibited better efficacy compared to patients treated with a murinized CAR-T therapy.

Development of a 23-color flow cytometry panel for the characterization of myelomonocytic differentiation with applications for MRD testing.

Abstract It is well agreed that measurable residual disease (MRD) testing in acute leukemia patients following induction chemotherapy has profound prognostic implications. Flow cytometry is well suited to be the mainstay for MRD testing due to the heterogenous immunophenotypic and molecular natures of leukemia. Current flow cytometry MRD testing involves examination of more than 15 antigens, requiring multiple tubes to be run for each patient as current clinical flow cytometers are limited to examining 12 parameters per tube. Because multiple tubes are required, hematopathologists must infer antigen expression between tubes, which can be difficult. As MRD testing has such prognostic implications and defines treatment stratifications and outcomes, MRD testing requires very high sensitivity. While several groups have improved sensitivity by increasing the number of cells analyzed, we propose that increasing the antigenic targets in a single immunophenotyping assay will improve sensitivity by enhancing our ability to differentiate normal from abnormal hematopoietic cells. To test this, we utilized spectral flow cytometry, which allows for targeting more parameters with the same number of lasers for a single instrument. We developed a 23-color flow cytometry panel which was designed to thoroughly examine myelomonocytic differentiation and included multiple other antigenic targets which are known to be aberrantly expressed on leukemia cells. The panel also included a live-dead stain to exclude dead cells from analysis. All data was collected with a 3-laser Cytek Northern Lights spectral flow cytometer, which can analyze 10 million events in ~10 minutes. We tested this panel on more than 50 bone marrow aspirate samples, including normal samples, multiple myeloid leukemias, myelodysplastic samples, as well as more than 20 aspirate samples which also had MRD testing completed at a reference laboratory. Our results for MRD testing had excellent concordance with results from the gold-standard reference laboratory method. As our method only required one tube to be run per sample, we were routinely able to analyze at least 10 million cells per sample. Using in silico modelling from our data, we determined the sensitivity of our assay to be an average of 0.005% (range 0.002 – 0.015%) depending on the specific immunophenotype, when analyzing 10 million cells. As we examined all marker expression in a single tube, we were able to model sensitivity as if only 10-parameters were collected per tube and inference was required between tubes for analysis as is the current standard practice. We found that our 23-color assay was significantly more sensitive (p =0.02) compared to running multiple 10-color tubes and inferring between samples. Overall, higher parameter flow cytometric assays (>20 markers) allow for a more sensitive and robust MRD analysis than current gold-standard methods and should be explored for utilization in the clinical setting.

1415P Phase II trial of a DNA vaccine with nivolumab in patients with PSA-recurrent, castration-sensitive prostate cancer (nmCSPC)

We previously reported a trial of a DNA vaccine encoding prostatic acid phosphatase (PAP, pTVG-HP) with pembrolizumab in patients with metastatic, castration-resistant prostate cancer. The current trial evaluated a similar approach in patients with earlier stage prostate cancer, without evidence of metastases (nmCSPC). Patients with nmCSPC were treated with pTVG-HP and nivolumab every 2 weeks for 12 weeks, and then every 4 weeks, for 1 year total. Patients were then followed an additional year off treatment. The trial was designed as a single-arm 2-stage phase 2 trial with the primary endpoints being safety and complete PSA response rate. Secondary objectives included immune evaluations, 2-year MFS, median rPFS, and changes in PSA. No patients achieved a complete PSA response, and the trial was stopped after 19 patients were accrued to the first phase. Treatment was without unexpected toxicity. Median PSA doubling time (DT) pre-treatment was 5.9 months, and significantly increased (25.6 months, p=0.001) during the 12-month treatment period. PSA DT decreased in the 1-year post-treatment period (14.2 months). 3 patients (16%) had PSA declines from baseline > 50%, and 5 (26%) had PSA declines > 25%. 8/19 (42%) patients developed PAP-specific IFNγ- and/or granzyme B-secreting T cells, which tended to occur in patients with favorable change in PSA DT. While long-term follow up continues for several patients, with a median follow-up time of 13.8 months, 2 patients (11%) have had radiographic progression within 2 years. PD-1 pathway inhibitors alone have demonstrated little clinical activity for prostate cancer. Our findings demonstrate that combining PD-1 blockade with the T-cell activating agent pTVG-HP is safe, and can augment tumor-specific T cells that can result in prolonged stable disease. The absence of complete PSA responses, and the finding that PSA rise recurred after treatment, suggest that either longer therapy, or additional treatments, are required to optimize response. Ongoing and planned trials are assessing vaccines encoding multiple target antigens and/or using combined T-cell checkpoint blockade.

Abstract 4190: Combining dual CAR iPSC-derived immune cells with antibody for multi-antigen targeting to overcome clonal resistance in multiple myeloma

Abstract Resistance to CAR-T cell therapy through antigen loss and clonal heterogeneity is a major limiting factor in achieving durable responses, even in highly persistent cell therapies. Simultaneous targeting of multiple tumor antigens with a single therapeutic modality offers the potential to treat heterogeneous tumor populations, prevent antigen escape, and induce durable clinical remission. Here, we demonstrate the application of a unique dual-CAR approach simultaneously targeting two tumor associated antigens (TAA) for the treatment of multiple myeloma (MM) using an off-the-shelf induced pluripotent stem cell (iPSC) derived NK cell platform. The iPSC background has been functionally enhanced and can be combined with therapeutic antibodies. A CAR targeting B cell maturation antigen (BCMA), a well-defined TAA in MM, was designed utilizing a previously published high-affinity binding sequence shown to exhibit high selectivity to BCMA with enhanced recognition of low-BCMA expressing myeloma cells (Bluhm et al., Molec Ther 2018). This CAR was combined with a companion CAR targeting the pan-TAAs, MICA and MICB. The CAR binding sequence targets the conserved α3 domain of MICA/MICB, which we have previously shown to inhibit MICA/B shedding by blocking the putative cleavage site (Andrade et al., Science 2018). The designed anti-MICA/B-α3 CAR exhibits selective targeting potential against a broad range of tumor types. To determine the suitability of co-targeting BCMA and MICA/B in MM, we surveyed surface expression of BCMA and MICA/B on a variety of MM cancer cell lines and observed a complimentary pattern of co-expression compatible with a dual-CAR to broaden the targeting approach of malignant plasma cells. In a reductionist approach, dual CAR-iNK cells exhibited antigen-specific activation, degranulation and cytotoxicity against a Nalm6 target line constitutively expressing the BCMA and MICA/B. Similar trends were observed in a series of long-term cytotoxicity assays against several MM lines, consistent with antibody staining on target cells, illustrating that co-targeting of MICA/B and BCMA expands the breadth of coverage against MM. Known modulators of antigen expression were tested for their ability to provide further depth of response, and therapeutic antibodies such as anti-CD38 were tested in combination to exploit the non-cleavable CD16 and CD38KO edits in the iPSC backbone. These combinations were further tested in vivo against a disseminated model of multiple myeloma where BCMA/MICA dual CAR-iNK cells demonstrated superior tumor control relative to single-CAR controls, and TGI was augmented with the addition of Daratumamab. These data highlight the applicability of a multi-targeted approach in MM patients, whereby MM dual-CAR NK and/or T cells maintain responsiveness to malignant cells that shed or downregulate tumor antigens to evade treatment. Citation Format: Ketan Mathavan, John Reiser, Sajid Mahmood, Yijia Pan, Bryan Hancock, Robert Blum, Wen-I Yeh, Andrew Houk, Chia-Wei Chang, Tom Lee, Bobby Goulding, Jode Goodridge, Ryan Bjordahl, Bahram Valamehr, Uta Hoepken, Armin Rehm, Kai Wucherpfennig. Combining dual CAR iPSC-derived immune cells with antibody for multi-antigen targeting to overcome clonal resistance in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4190.

Multiplex Serology for Measurement of IgG Antibodies Against Eleven Infectious Diseases in a National Serosurvey: Haiti 2014-2015.

BackgroundIntegrated surveillance for multiple diseases can be an efficient use of resources and advantageous for national public health programs. Detection of IgG antibodies typically indicates previous exposure to a pathogen but can potentially also serve to assess active infection status. Serological multiplex bead assays have recently been developed to simultaneously evaluate exposure to multiple antigenic targets. Haiti is an island nation in the Caribbean region with multiple endemic infectious diseases, many of which have a paucity of data for population-level prevalence or exposure.MethodsA nationwide serosurvey occurred in Haiti from December 2014 to February 2015. Filter paper blood samples (n = 4,438) were collected from participants in 117 locations and assayed for IgG antibodies on a multiplex bead assay containing 15 different antigens from 11 pathogens: Plasmodium falciparum, Toxoplasma gondii, lymphatic filariasis roundworms, Strongyloides stercoralis, chikungunya virus, dengue virus, Chlamydia trachomatis, Treponema pallidum, enterotoxigenic Escherichia coli, Entamoeba histolytica, and Cryptosporidium parvum.ResultsDifferent proportions of the Haiti study population were IgG seropositive to the different targets, with antigens from T. gondii, C. parvum, dengue virus, chikungunya virus, and C. trachomatis showing the highest rates of seroprevalence. Antibody responses to T. pallidum and lymphatic filariasis were the lowest, with <5% of all samples IgG seropositive to antigens from these pathogens. Clear trends of increasing seropositivity and IgG levels with age were seen for all antigens except those from chikungunya virus and E. histolytica. Parametric models were able to estimate the rate of seroconversion and IgG acquisition per year for residents of Haiti.ConclusionsMultiplex serological assays can provide a wealth of information about population exposure to different infectious diseases. This current Haitian study included IgG targets for arboviral, parasitic, and bacterial infectious diseases representing multiple different modes of host transmission. Some of these infectious diseases had a paucity or complete absence of published serological studies in Haiti. Clear trends of disease burden with respect to age and location in Haiti can be used by national programs and partners for follow-up studies, resource allocation, and intervention planning.

Evaluation of a Multiplex Bead Assay against Single-Target Assays for Detection of IgG Antibodies to SARS-CoV-2.

ABSTRACTSerological assays for SARS-CoV-2 antibodies must be validated for performance with a large panel of clinical specimens. Most existing assays utilize a single antigen target and may be subject to reduced diagnostic specificity. This study evaluated a multiplex assay that detects antibodies to three SARS-CoV-2 targets. Human serum specimens (n = 323) with known previous SARS-CoV-2 exposure status were tested on a commercially available multiplex bead assay (MBA) measuring IgG to SARS-CoV-2 spike protein receptor-binding domain (RBD), nucleocapsid protein (NP), and RBD/NP fusion antigens. Assay performance was evaluated against reverse transcriptase PCR (RT-PCR) results and also compared with test results for two single-target commercial assays. The MBA had a diagnostic sensitivity of 89.8% and a specificity of 100%, with serum collection at >28 days following COVID-19 symptom onset showing the highest seropositivity rates (sensitivity: 94.7%). The MBA performed comparably to single-target assays with the ability to detect IgG against specific antigen targets, with 19 (5.9%) discrepant specimens compared to the NP IgG assay and 12 (3.7%) compared to the S1 RBD IgG assay (kappa coefficients 0.92 and 0.88 compared to NP IgG and S1 RBD IgG assays, respectively. These findings highlight inherent advantages of using a SARS-CoV-2 serological test with multiple antigen targets; specifically, the ability to detect IgG against RBD and NP antigens simultaneously. In particular, the 100.0% diagnostic specificity exhibited by the MBA in this study is important for its implementation in populations with low SARS-CoV-2 seroprevalence or where background antibody reactivity to SARS-CoV-2 antigens has been detected.IMPORTANCE Reporting of SARS-CoV-2 infections through nucleic acid or antigen based diagnostic tests severely underestimates the true burden of exposure in a population. Serological data assaying for antibodies against SARS-CoV-2 antigens offers an alternative source of data to estimate population exposure, but most current immunoassays only include a single target for antibody detection. This report outlines a direct comparison of a multiplex bead assay to two other commercial single-target assays in their ability to detect IgG against SARS-CoV-2 antigens. Against a well-defined panel of 323 serum specimens, diagnostic sensitivity and specificity were very high for the multiplex assay, with strong agreement in IgG detection for single targets compared to the single-target assays. Collection of more data for individual- and population-level seroprofiles allows further investigation into more accurate exposure estimates and research into the determinants of infection and convalescent responses.