Weak Antigens Research Articles

Genomic characterization of clinically significant blood group variants in Aboriginal Australians.

Hematological disorders are often treated with blood transfusions. Many blood group antigens and variants are population-specific, and for patients with rare blood types, extensive donor screening is required to find suitable matches for transfusion. There is a scarcity of knowledge regarding blood group variants in Aboriginal Australian populations, despite a higher need for transfusion due to the higher prevalence of renal diseases and anemia. In this study, we applied next-generation sequencing and analysis to 245 samples obtained from Aboriginal Australians from South-East Queensland, to predict antigen phenotypes for 36 blood group systems. We report potential weak antigens in blood group systems RH, FY and JR that have potential clinical implications in transfusion and pregnancy settings. These include partial DIII type 4, weak D type 33, and Del RHD (IVS2-2delA). The rare Rh phenotypes D+ C+ E+ c- e+ and D+ C+ E+ c+ e- were also detected. The comprehensive analyses of blood group genetic variant profiles identified in this study will provide insight and an opportunity to improve Aboriginal health by aiding in the identification of appropriate blood products for population-specific transfusion needs.

Molecular testing in a patient with multiple alloantibodies and warm autoimmune hemolytic anemia: A case study

Abstract Introduction/Objective We present a case of RBC alloimmunization and warm autoimmune hemolytic anemia (WAIHA) in a patient with a recent RBC transfusion. Serological testing followed by molecular testing revealed a rare D+ C- E- partial c+ partial e+ VS+ V+ hrS+ hrB+vw/- predicted phenotype. Methods/Case Report A 75-year-old black male with secondary myelofibrosis presented with a sudden onset of chest pain, dyspnea and weakness during an RBC transfusion. The patient was subsequently admitted for further workup. Labs showed low hemoglobin, low haptoglobin, elevated LDH and a positive direct antiglobulin (DAT) test. The patient was started on intravenous immunoglobulin and steroids for suspected WAIHA. On immunohematology serologic testing, blood typed as B-positive and polyspecific DAT positive with anti-IgG positive and anti-C3 negative. Serum plasma studies showed probable warm autoimmune hemolytic anemia and allo anti-S, -E, and -C by previous history. Eluate studies showed panagglutinin and antibody with D-like specificity. Immucor PreciseTypeTM Human Erythrocyte Antigen (HEA) Molecular BeadChip test predicted a partial e antigen and potential hrB-. Genotyping for RHD variants was performed and revealed RHD*01 (homozygous or hemizygous). Targeted genomic testing did not detect variants associated with common partial or weak RhD antigens. Based on this testing, the patient was not predicted to be at risk of allo-anti-D. An RHCE genotype for C, c, E and e variants including hrB and hrS showed RHCE*ce48C,733G/RHCE*ce733G with predicated phenotype of D+ C- E- partial c+ partial e+ VS+ V+ hrS+ hrB+vw/-. Based on the RHCE testing, the patient was predicted to be at risk for allo-anti-c, -e, -f (ce) and possibly anti-hrB. Results (if a Case Study enter NA) NA Conclusion In this case, serologic testing alone did not reveal a precise diagnosis. Molecular testing aided in the diagnosis of WAIHA and will help guide future transfusions. Pathologists must consider molecular testing when faced with a challenging serologic diagnosis.

Conjugation of TLR7 and TLR7/8 agonists onto weak protein antigen via versatile oxime ligation for enhanced vaccine efficacy

Protein-based subunit vaccines are weakly immunogenic, and developing self-adjuvanting vaccines with adjuvant conjugated to antigen is a promising approach for generating optimal immune responses. Here, we report a novel adjuvant-protein conjugate vaccine based on versatile oxime ligation technique. Firstly, the adjuvant properties of a series of TLR7 and TLR7/8 small molecule agonists in self-adjuvanting vaccines were systematically compared by coupling them to proteins in consistent ratio via p-carboxybenzaldehyde (p-CBA) for the first time. All conjugate vaccines induced cytokine secretion in murine and human macrophages in vitro, and promoted specific antibody production in vivo. Notably, a conjugate containing imidazoquinoline TLR7/8 agonist (TLR7/8a1) showed the greatest enhancement in Th1/2 balanced antibody response. To minimize the interference with the protein antigenic integrity, we further developed a systematic glycoconjugation strategy to conjugate this TLR7/8a1 onto the glycan chains of SARS-CoV-2 S1 glycoprotein via oxime ligation, in which S1 containing different numbers of aldehyde groups were obtained by differential periodate oxidation. The resulting TLR7/8a1-S1 conjugate triggered a potent humoral and cellular immunity in vivo. Together these data demonstrate the promise of these TLR7 and TLR7/8 agonists as effective built-in adjuvants, and the versatile oxime ligation strategy might broaden potential applications in designing different conjugate vaccines.

ACKR1 gene polymorphisms in Bombay blood group (Oh) individuals of Indian origin

BackgroundACKR1 blood group genes exhibit a high degree of polymorphisms with varying allele distribution seen among different populations and ethnic groups. The study aimed to genotype ACKR1 antigens and to establish FY allele frequency among the individuals with the Bombay (Oh) blood group phenotype. Materials and methodsACKR1 phenotype and genotype frequencies were estimated on 160 individuals typed as Oh and were compared with 100 non-oh blood donors from Mumbai, India by molecularly genotyping via PCR-RFLP. ResultsThe allelic and genotypic frequency of T(−67)C polymorphism showed the dominance of T allele and TT genotype [OR= 3.26 (0.59–17.99)] in both the study groups. The ACKR1 null (Fya-b-) phenotype was not found in the tested group. While the genotypic combination among the Oh group individuals was FYA/FYB (45.3 %), FYA/FYA (42.7 %), and FYB/FYB (12 %), in the non-Oh group donors, it was observed as FYA/FYB (53.3 %), FYA/FYA (39.1 %), and FYB/FYB (7.6 %).The haplotype TGGGC occurred in 38.4 % of the Oh group, but in non-Oh donors, it was found to be 50.9 % [OR = 1.820 (1.196–2.771)], and the difference was statistically significant (p = 0.005). Similarly, the TGGGT haplotype was found at a frequency of 12.7 % in non-Oh donors and 27.1 % in Oh group [OR= 0.411 (0.234–0.722)] (p = 0.001). ConclusionsThis study shows the prevalence of ACKR1 gene polymorphisms, including weak ACKR1 antigens in Oh individuals with a high frequency of haplotype TGGGC. The present study demonstrated for the first time the genotypes FyBweak, FyAweak and Fy Aweak/FyBESon RBC membranes in Indian subjects with Oh phenotype.

Towards optimizing cancer immunotherapeutic index: anti-CD3 Mono-Fab, a novel T cell-based therapeutic approach with less auto-Immunogenic profile.

Abstract Despite advancement of cancer therapeutic approaches with immune checkpoint inhibitors (ICIs) against several tumor types, its anti-tumor benefit is limited by the emergence of immune related adverse events (irAEs). irAEs with an autoimmune phenotype, show a wide spectrum of toxicities that affect almost all organ systems. Several studies suggest a positive correlation between irAEs and anti-tumor response, with higher levels of toxicities seen with combination therapies. Finding strategies that optimize efficacy with less toxicity has been in the frontline of immunotherapy. We have shown a novel therapeutic strategy: anti-CD3e Monovalent Fab fragment (Mono-Fab) that targets the TCR/CD3 complex, strengthening signaling response to moderately weak antigens. Anti-CD3e Mono-Fab as a single therapy and in synergy with combination ICIs is effective against metastatic melanomas. To determine its optimal therapeutic index, we utilized a cell mediated experimental autoimmune encephalomyelitis mouse model as a platform to gauge potential to cause irAEs in the clinic. In this model, anti-CTLA-4 mAb administration to mice increases severity of their EAE symptoms over those observed in mice treated with appropriate IgG control, while anti-CD3e mono-Fab treatment does not. Thus, Anti-CD3e Mono-Fab contrast as an efficient potentially non-toxic cancer immunotherapy when compared with anti-CTLA-4 antibodies that are known to cause irAEs in patients.

Multifunctional Lipidated Protein Carrier with a Built-In Adjuvant as a Universal Vaccine Platform Potently Elevates Immunogenicity of Weak Antigens.

Weak antigens represented by MUC1 are poorly immunogenic, which greatly constrains the development of relevant vaccines. Herein, we developed a multifunctional lipidated protein as a carrier, in which the TLR1/2 agonist Pam3CSK4 was conjugated to the N-terminus of MUC1-loaded carrier protein BSA through pyridoxal 5'-phosphate-mediated transamination reaction. The resulting Pam3CSK4-BSA-MUC1 conjugate was subsequently incorporated into liposomes, which biomimics the membrane structure of tumor cells. The results indicated that this lipidated protein carrier significantly enhanced antigen uptake by APCs and obviously augmented the retention of the vaccine at the injection site. Compared with the BSA-MUC1 and BSA-MUC1 + Pam3CSK4 groups, Pam3CSK4-BSA-MUC1 evoked 22- and 11-fold increases in MUC1-specific IgG titers. Importantly, Pam3CSK4-BSA-MUC1 elicited robust cellular immunity and significantly inhibited tumor growth. This is the first time that lipidated protein was constructed to enhance antigen immunogenicity, and this universal carrier platform exhibits promise for utilization in various vaccines, holding the potential for further clinical application.

IL-18-secreting multiantigen targeting CAR T cells eliminate antigen-low myeloma in an immunocompetent mouse model

AbstractMultiple myeloma is a plasma cell malignancy that is currently incurable with conventional therapies. Following the success of CD19-targeted chimeric antigen receptor (CAR) T cells in leukemia and lymphoma, CAR T cells targeting B-cell maturation antigen (BCMA) more recently demonstrated impressive activity in relapsed and refractory myeloma patients. However, BCMA-directed therapy can fail due to weak expression of BCMA on myeloma cells, suggesting that novel approaches to better address this antigen-low disease may improve patient outcomes. We hypothesized that engineered secretion of the proinflammatory cytokine interleukin-18 (IL-18) and multiantigen targeting could improve CAR T-cell activity against BCMA-low myeloma. In a syngeneic murine model of myeloma, CAR T cells targeting the myeloma-associated antigens BCMA and B-cell activating factor receptor (BAFF-R) failed to eliminate myeloma when these antigens were weakly expressed, whereas IL-18–secreting CAR T cells targeting these antigens promoted myeloma clearance. IL-18-secreting CAR T cells developed an effector-like T-cell phenotype, promoted interferon-gamma production, reprogrammed the myeloma bone marrow microenvironment through type-I/II interferon signaling, and activated macrophages to mediate antimyeloma activity. Simultaneous targeting of weakly-expressed BCMA and BAFF-R with dual-CAR T cells enhanced T-cell:target-cell avidity, increased overall CAR signal strength, and stimulated antimyeloma activity. Dual-antigen targeting augmented CAR T-cell secretion of engineered IL-18 and facilitated elimination of larger myeloma burdens in vivo. Our results demonstrate that combination of engineered IL-18 secretion and multiantigen targeting can eliminate myeloma with weak antigen expression through distinct mechanisms.

Impact of transcription factors KLF1 and GATA1 on red blood cell antigen expression: a review.

KLF transcription factor 1 (KLF1) and GATA binding protein 1 (GATA1) are transcription factors (TFs) that initiate and regulate transcription of the genes involved in erythropoiesis. These TFs possess DNA-binding domains that recognize specific nucleotide sequences in genes, to which they bind and regulate transcription. Variants in the genes that encode either KLF1 or GATA1 can result in a range of hematologic phenotypes-from benign to severe forms of thrombocytopenia and anemia; they can also weaken the expression of blood group antigens. The Lutheran (LU) blood group system is susceptible to TF gene variations, particularly KLF1 variants. Individuals heterozygous for KLF1 gene variants show reduced Lutheran antigens on red blood cells that are not usually detected by routine hemagglutination methods. This reduced antigen expression is referred to as the In(Lu) phenotype. For accurate blood typing, it is important to distinguish between the In(Lu) phenotype, which has very weak antigen expression, and the true Lunull phenotype, which has no antigen expression. The International Society of Blood Transfusion blood group allele database registers KLF1 and GATA1 variants associated with modified Lutheran expression. Here, we review KLF1 and recent novel gene variants defined through investigating blood group phenotype and genotype discrepancies or, for one report, investigating cases with unexplained chronic anemia. In addition, we include a review of the GATA1 TF, including a case report describing the second GATA1 variant associated with a serologic Lu(a-b-) phenotype. Finally, we review both past and recent reports on variations in the DNA sequence motifs on the blood group genes that disrupt the binding of the GATA1 TF and either remove or reduce erythroid antigen expression. This review highlights the diversity and complexity of the transcription process itself and the need to consider these factors as an added component for accurate blood group phenotyping.

Abstract 6324: Antagonism-enforced braking system to enhance CAR T cell therapeutic specificity

Abstract Chimeric Antigen Receptor (CAR) T cell immunotherapy represents a breakthrough in the treatment of hematological malignancies. However, the rarity of cell surface protein targets that are specific to cancerous but not vital healthy tissue has hindered its broad application to solid tumor treatment. While new logic-gated CAR designs have shown reduced toxicity against healthy tissues, the generalizability of such approaches across tumors remains unclear. Here, we harness a universal characteristic of endogenous T cell receptors (TCRs), their ability to discriminate between self and non-self ligands through inhibition of response against self (weak) antigens, to develop a broadly applicable method of enhancing immunotherapeutic precision. We hypothesized that this discriminatory mechanism, known as antagonism, would apply across receptors, allowing for a transfer of specificity from TCRs onto CARs. We therefore systematically mapped out the responses of CAR T cells to joint TCR and CAR stimulations. We first engineered murine T cells with an ovalbumin-specific TCR to express a CAR targeting murine CD19 and discovered that the expression of a strong TCR antigen on CD19+ leukemia enhanced CAR T killing. Importantly though, the presence of a weak TCR antigen antagonized CAR T responses, assessed by in vitro multiplexed dynamic profiling as well as in vivo cytotoxicity. We developed a mathematical model based on cross-receptor inhibitory coupling that accurately predicted the extent of TCR/CAR antagonism across a wide range of immunological settings. This model was validated in a CD19+ B16 mouse melanoma model showing that TCR/CAR antagonism decreased the infiltration of a tumor-reactive T cell cluster, while TCR/CAR agonism enhanced infiltration of this T cell cluster. We then applied our quantitative knowledge of TCR/CAR crosstalk to design an Antagonism-Enforced Braking System (AEBS) for CAR T cell therapy. This was assessed in a model system using a CAR targeting the tyrosine-protein kinase erbB-2 (HER2) together with a hedgehog acyltransferase (HHAT) peptide-specific TCR that binds strongly to mutated tumor neoantigen while retaining weak affinity for the wild-type self-antigen on healthy tissue. We established a humanized in vivo model of CAR T function and found that AEBS CAR T cells maintained high anti-tumor activity against a human lung adenocarcinoma (PC9) but notably, their anti-tissue cytotoxicity against human bronchial epithelial cells (BEAS-2B) was minimized. AEBS CAR T cells therefore sharpen the discriminatory power of synthetic anti-tumor lymphocytes. Our work highlights a novel mechanism by which TCRs can enforce CAR T cell specificity, with practical implications for the rational design of future anti-leukemia immunotherapies. Citation Format: Taisuke Kondo, François X. Bourassa, Sooraj Achar, Justyn DuSold, Pablo Cespedes, Madison Wahlsten, Audun Kvalvaag, Guillaume Gaud, Paul Love, Michael Dustin, Gregoire Altan-Bonnet, Paul François, Naomi Taylor. Antagonism-enforced braking system to enhance CAR T cell therapeutic specificity [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 6324.

Analysis of Blood Group Discrepancy at Dr. Wahidin Sudirohusodo Hospital's Blood Transfusion Unit Makassar

Discrepancy is a lack of compatibility of blood group tests between forward grouping and reverse grouping. Based on the cause, discrepancies are divided into four groups as follows: group I discrepancies, which occur due to weak or missing antibodies, group II discrepancies, which occur due to weak or missing antigens, group III discrepancies, which result in the formation of rouleaux, and group IV discrepancies, which are caused by other problems. A retrospective study was carried out by using ABO blood group data, which were analyzed by the automatic gel test method with the IH-1000 device. Data from January 2019 to December 2021 was collected at the Blood Transfusion Unit, Dr. Wahidin Sudirohusodo Hospital of Makassar, and the data were grouped using SPSS version 22. A total of 21.742 samples were tested. The number of detected ABO blood group discrepancies was 127 (0.58%). There were 68 (51.3%) males and 59 (46.5%) females with an age range divided into toddler (38.6%), child (2.4%), adolescent (13.4%), adult (8.7%), pre-elderly (17.3%), and elderly (6.3 %). Based on the disease, discrepancies were categorized into samples with infectious disease (33.9%), autoimmune disease (3.9%), malignancy (23.6%), chronic disease (11%), and others (27.6%). The discrepancies consisted of group I (70.9%), group II (0%), group III (0.8%), and group IV (28.3%). There was a significant correlation between age and blood group discrepancy with p < 0.001 and moderate correlation strength (0.54). The prevalence of discrepancy in this study was 0.58%. Discrepancies must be resolved before they are reported to minimize transfusion reactions.

Blood group antigens visualisation on leukocytes

Background/Aim: The leukocytes have been reported to contain blood group specific antigens, that are clinically relevant, however visualisation of A and B group antigens on leukocytes is a big issue. In cases of ABO discrepancies weak blood group antigens on nuclear cells have been demonstrated by using expensive techniques. Thus, the development of the method of the detection of weak blood group antigens on leukocytes available for any laboratory technician is hardly essential. The study aimed to reveal and analyse A and B blood group specific adsorbing antigens on leukocytes and erythrocytes and to develop a method for visualisation of weak blood group antigens on leukocytes. Methods: Polyclonal and monoclonal anti-A and anti-B antibodies, received from international laboratories according to the program of Workshop IV, held in Paris, 2000, were used for the study. Mixed agglutination reaction was performed as the method for visualisation of weak blood group antigens on leukocytes as nuclear cells. Results: Polyclonal sera from O blood group persons without weak blood group antigens in contrast to monoclonal antibodies demonstrated the ability to reveal weak blood group specific antigens on leukocytes by the method of mixed agglutination reaction. However, the test erythrocytes from the persons with increased levels of platelets and erythrocyte sedimentation rate did not allow to visualise weak antigen expression on the studied leukocytes in contrast to the persons with normal levels of platelets and erythrocyte sedimentation rate, that successfully formed mixed agglutinates with weak blood group antigens on leukocytes in mixed agglutination reaction. The leukocytes suspended in 0.9 % saline (as a diluent) incubated with the mixture of the serum with 0.9 % saline (1:2) led to the formation of specific agglutinates with test erythrocytes. The experiments with different temperature regimes and time of incubation demonstrated the usefulness of the studied method in specific leukocytes antigen visualisation during prolonged incubation at 4 °C. The persons with weak group A and B antigens, revealed on the leukocytes by the studied method, demonstrated decreased level of erythrocytes, platelets, titre of corresponding warm agglutinating antibodies (less than 1:8) and increased erythrocyte sedimentation rate. Conclusion: The mixed agglutination reaction with prolonged incubation at 4 °C and the use of the selected polyclonal sera and test erythrocytes from the donors with normal values of platelets and erythrocyte sedimentation rate may be used for weak blood group antigens detection on leukocytes. The donors of the sera and test erythrocytes used in mixed agglutination reaction should be investigated on common blood analysis, agglutinating titre of corresponding warm group specific antibodies and presence of weak blood group antigens.

A programmable releasing versatile hydrogel platform boosts systemic immune responses via sculpting tumor immunogenicity and reversing tolerogenic dendritic cells

Immunogenicity improvement is a valuable strategy for tumor immunotherapy. However, immunosuppressive factors bestow tolerogenic phenotype on tumor-infiltrating DCs, which exhibit weak antigen presentation and strong anti-inflammatory cytokines secretion abilities, limiting the effectiveness of tumor immunotherapy even if the tumor has adequate immunogenicity. Herein, we designed a programmable releasing versatile hydrogel platform (PIVOT) to sculpt tumor immunogenicity, increase intratumoral DCs and cDC1s abundance, and reverse the tolerogenic phenotype of DCs, thus promoting their maturation for boosting innate and adaptive immune responses. Responsive to tumoral reactive oxygen species (ROS), the hydrogel splits and promotes the activation of DCs and macrophages. Then, oxaliplatin is first released from PIVOT to sculpt tumor immunogenicity by inducing immunogenic cell death (ICD) and causing tumoral DNA fragments exposure simultaneously. Subsequently, the impaired DNA fragments bind to high mobility group protein 1 (HMGB1) forming the DNA-HMGB1 complex. Moreover, exogenous FMS-like tyrosine kinase 3 ligand (Flt-3L) recruits masses of DCs, especially cDC1s, which will endocytose the complex benefiting from TIM-3 blockade (αTIM3) that can reverse tolerogenic DCs. Finally, the endocytosis activates the cGAS-STING pathway of cDC1s, which promotes the secretion of type I IFN that triggers innate immune responses, and CXCL9 which recruits CD8+ effector T cells to initiate the following adaptive immune response against tumor progress. PIVOT achieves nearly 90 % tumor growth inhibition and induces systemic antitumor immune responses. In conclusion, this study focuses on ICD-mediated tumor immunogenicity sculpture and nucleic acid endocytosis-involved tolerogenic DCs reversal, providing a novel paradigm for enhancing DCs-based antitumor immune responses.

Heterologous expression of recombinant proteins and their derivatives used as carriers for conjugate vaccines

The carrier proteins that provide an effective and long-term immune response to weak antigens has become a real breakthrough in disease prevention, making it available to a wider range of patients and making it possible to obtain reliable vaccines against a variety of pathogens. Currently, research is continuing both to identify new peptides, proteins, and their complexes potentially suitable for use as carriers, and to develop new methods for isolation, purification and conjugation of already known and well-proven proteins. The use of recombinant proteins has a number of advantages over isolation from natural sources, such as simpler cultivation of the host organism, the possibility of modifying the genetic construction, the use of numerous promoter variants, signal sequences, and other regulatory elements. This review is devoted to the methods of obtaining both traditional and new recombinant proteins and their derivatives being used or potentially suitable for use as carrier proteins for conjugate vaccines.

Harnessing TCR/CAR Antagonism to Enhance Immunotherapeutic Precision

Chimeric Antigen Receptor (CAR) T cell immunotherapy represents a conceptual breakthrough in the treatment of hematological malignancies. However, the rarity of cell surface protein targets specific to cancerous but not vital tissue has hindered its broad application to solid tumor treatment. While new logic-gated CAR designs have shown reduced toxicity against healthy tissues, the generalizability of such approaches across tumors remains unclear. Here, we harness a universal characteristic of endogenous T cell receptors (TCRs), their ability to discriminate between self and non-self ligands through inhibition of response against self (weak) antigens, to develop a broadly applicable method of enhancing immunotherapeutic precision. We hypothesized that this discriminatory mechanism, known as antagonism, would apply across receptors, allowing for a transfer of specificityfrom TCRs onto CARs. We therefore systematically mapped out the responses of CAR T cells to joint TCR and CAR stimulations. We transduced ovalbumin-specific TCR T cells with mouse CD19 CAR to produce T cells expressing both TCR and CAR and evaluated the response of TCR/CAR T cells using in vivo and in vitro leukemia models ( Figure 1A). We discovered that strong TCR antigen enhanced CAR T killing of CD19 + leukemia, while weak TCR antigen antagonized CAR T responses as assessed in vivo cytotoxicity and in vitro multiplexed dynamic profiling ( Figure 1B). We developed a mathematical model based on cross-receptor inhibitory coupling that accurately predicted the extent of TCR/CAR antagonism across a wide range of immunological settings. This model was validated in a CD19 + B16 mouse melanoma model showing that TCR/CAR antagonism decreased the infiltration of a tumor-reactive T cell cluster (cluster 1), while TCR/CAR agonism enhanced infiltration of this cluster1 ( Figure 1C). We then applied our quantitative knowledge of TCR/CAR crosstalk to design an Antagonism-Enforced Braking System (AEBS) for CAR T cell therapy. This was assessed in a model system using a CAR targeting the tyrosine-protein kinase erbB-2 (HER2), expressed on a subset of patients with both B-ALL and AML together with a hedgehog acyltransferase (HHAT) specific TCR, which responds strongly to mutated peptides presented on tumor cells and weakly to wild-type peptides presented on healthy tissue. Consistent with our discovery of the TCR/CAR antagonism, TCR signals against healthy cells expressing wild-type HHAT peptide antigen antagonized HER2 CAR T cell responses, minimizing on-target/off-tumor cytotoxicity against healthy cells. Notably though, AEBS-CAR T cells exhibited high anti-tumor cytotoxicity against tumor cells expressing HER2 and mutated HHAT peptides ( Figure 1D). AEBS CAR T cells sharpen the discriminatory power of synthetic anti-tumor lymphocytes, laying the groundwork for future studies to engineer complex logic into cells with minimal numbers of receptors ( Figure 1E). Our work highlights a novel mechanism by which TCRs can enforce CAR T cell specificity, with practical implications for the rational design of future anti-leukemia immunotherapies.

In Vitro Efficacy of Anti-HER2 CAR T-Cells in HER2 + BCP-ALL

Introduction: Around 30% of adults and 15% of children with B cell precursor acute lymphoblastic leukemia (BCP-ALL) relapse after a first line of treatment. The prognosis of these patients remains poor. Recent advances have involved targeted therapies with monoclonal antibodies and chimeric antigen receptor (CAR) T-lymphocytes (anti-CD19 CAR T-cells), which have produced very encouraging results in the high risk BCP-ALL setting(Maude et al. 2018). Nevertheless, half of these patients relapse after such therapies. In a third of cases, the disease no longer expresses the antigen previously targeted. New targets are therefore needed. HER2 is expressed in above one third of adult BCP-ALL (Chevallier et al. 2004) and 10% of childhood BCP-ALL (Camuset et al. 2021). HER2 expression is associated with chemoresistance and poor prognosis (Chevallier et al. 2004) (Camuset et al. 2021). The anti-HER2 antibody trastuzumab was prospectively tested in adult patients with HER2+ BCP-ALL, but yielded disappointing results (Chevallier et al. 2012). Anti-HER2 CAR T-cells could be an interesting alternative. Indeed, they have shown encouraging preclinical efficacy in solid tumors expressing HER2/neu (Budi et al. 2022). The present work was designed to evaluate, in vitro, the sensitivity of HER2+ BCP-ALL to anti-HER2 CAR engineered Epstein Barr virus (EBV) specific T-cells. Methods: DMSO-frozen cell-samples from 6 HER2+ BCP-ALL patients were available for this study. Patient characteristics are shown in Table 1. Blast cells from patient samples were preferred to cell lines in order to be more representative of BCP-ALL biology. level of HER2 expression on BCP-ALL samples was determined by flow cytometry and compared to the HER2 expressing breast-cancer cell line BT474. Anti-EBV cytotoxic T lymphocytes (CTLs), armed with a trastuzumab-based scFv-CH2-CH3-IgG2A-FcεRIγ chimeric receptor as previously described (Clémenceau et al. 2015) were used for long-term cytoxicity assays. For 4 samples, non-transduced anti-EBV CTLs were used as controls. Cytotoxic activity was assessed at a 3-to-1 effector-to-target ratio, in a flow cytometry assay. Cells were harvested at H0, H4 and H24 and stained with a viability stain (VS780). Further staining with anti-CD22 or anti-CD24 (depending on the BCP-ALL immunophenotype) and anti-CD3 antibodies allowed to characterize blast cells and CAR T-cells respectively. The study was approved by the local review board and two alive patients provided informed consent. Results: The level of HER2 expression by the 6 HER2+ BCP-ALL samples was variable with a median relative fluorescence intensity ratio (RFI) of 9 (range 1.8 to 16). These levels of expression were lower than those of the BT474 cell line (RFI= 79). No BCP-ALL lysis was observed when cells were incubated with trastuzumab alone (not shown). When co-cultured with non-transduced anti-EBV CTLs (CAR negative) (n=4), a decrease of residual viable BCP-ALL cells was observed at variable levels (Figure 1A). The lysis observed at 24h was possibly due to allogeneic recognition since anti-EBV CTLs were not autologous to the leukemias tested. Conclusion: This study first reveals that, although CTLs with TCRs restricted to EBV antigens and therefore intrinsically low allo-specificity were used, CAR-independent lysis was observed in 24 hours co-culture with BCP-ALL cells. These results underline the need for cautious interpretation of cytotoxicity test results in an allogeneic setting. Secondly, these preliminary data suggest that an anti-HER2 CAR T-cell approach could be promising for BCP-ALL even with weak antigen expression. Anti-EBV CTLs have already been used as a platform for CAR-engineered T-cells (Rossig et al. 2017). Should the cytotoxic activity of allogeneic EBV-CTL be proven to be specifically directed against BCP-ALL, the synergy between TCR and anti-HER2-CAR might be beneficial, and adoptive transfer of anti-HER2 CAR EBV CTLs could be a promising therapeutic option for patients with HER2+ BCP-ALL. * This research is funded by the non-profit organization Etoile de Martin/SFCE.

Detection and phenotype analysis of a novel Ael blood group allele.

The presence of blood subtypes may lead to difficulties in blood group identification; however, third-generation sequencing (TGS) can help in accurately identifying difficult blood groups, and study the serological characteristics and molecular mechanism of Ael subtypes. ABO blood group was identified by the standard serological technique, weak blood group antigen was identified by adsorption-elution experiments, ABH substance in the saliva was determined and glycosyltransferase activity of A and B was detected. The ABO gene full-length sequence and promoter region were amplified by specific primers using single-molecule real-time sequencing, with the amplified products being sequenced directly and analysed in real time. The patient was serologically identified as Ael subtype, and TGS analysis revealed new intron mutations in Ael patients (c.467C>T; c.29-10T>A). The discovery of the new allele and the identification of ABO subtypes can be combined with serological characterization and molecular biological methods.

Identification of HLA Class I Alleles in Patients with Paroxysmal Nocturnal Hemoglobinuria with Different Clone Sizes

Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is a hematological disease characterized by complement-mediated hemolysis, thrombosis, and bone marrow failure. Human leukocyte antigen (HLA) evolutionary divergence and mutations were widely analyzed in aplastic anemia (AA). However, there were few studies in PNH thus far. Methods Patients with newly diagnosed PNH at Peking Union Medical College Hospital (PUMCH) were recruited from April 2019 to April 2021 according to International PNH Interest Group standards. They were analyzed the genotypes of classical HLA class I loci (A, B, C) using whole-exome sequencing (WES). Patients were further stratified into four groups according to the clone size (Group A: <10%; Group B: 10% ≤ to ≤50%; Group C: 50% < to ≤80%; Group D: >80%) to identify the association between HLA class I alleles and PNH clone size. Result Samples from 40 patients were enrolled, including 20 classic PNH and 20 bone marrow failure (BMF)/PNH. There were 29 males and 11 females. The median age was 32 years (range, 16-70). The median PNH clone size measured by flaer-granulocyte ratio was 72% (range 1-99%). Among the 59 identified alleles in PNH, HLA-B genotypes, which were reported to be associated with severe AA, accounted for the highest percentage (46%). A*02:01, the most common one and found in all the four groups, was reported as one of the risk alleles overrepresents in AA. The alleles found in group A were significantly different from those in group B, C, and D ( P = 0.004, 0.006, and < 0.001, respectively). But there was no significant difference between B, C, and D groups ( P > 0.05). 47% of the alleles identified in group A were reported in AA, whereas only 26%, 23%, and 26% of the detected alleles in Group B, C, and D were reported in AA, respectively. The other alleles in B, C, D groups were mainly for the susceptibility of autoimmune diseases or drug-related adverse reactions. Among them, HLA-B*46:01, the most common and found in B, C and D groups, may indicate weak antigen presentation and immune ability. B*44, B*57:01, C*07:01 were unique in Group B, B*38:02 were identified in Group C and HLA-B*27 expressed most in Group D, which were mainly on cell survival, drug hypersensitivity, and complement activation. Conclusion Significantly different alleles were observed between PNH with smaller clone sizes (< 10%) and PNH with larger clone sizes (≥10%). Patients with smaller clone sizes had similar HLA class Ⅰ alleles to AA; whereas those with larger clone sizes had numerous unique alleles which may contribute to the pathogenesis of PNH. Keywords: paroxysmal nocturnal hemoglobinuria, human leukocyte antigens, clone size, class I allele, pathogenesis

Preliminary assessment of adjuvant activities of Glycine Max (L.) Merr saponin extract in BALB/c mice immunized with hepatitis B virus vaccine

Background: Vaccine adjuvants are used to increase the immunogenicity of weak antigens. Some saponins have adjuvant effects that are exerted via their immune-stimulatory effects and production of cytokines. Soybean (Glycine max (L.) Merr.) contains saponins that could provide affordable vaccine adjuvants. This study aimed to assess the effect of the saponin extracts of soybean on the immune system in BALB/c mice immunized with the hepatitis B virus (HBV) vaccine and hepatitis B surface antigen (HBsAg). Methods: Saponins were extracted from soybean meal and their presence confirmed by foam generation and Fourier-transform infrared methods. A total of 51 mice were immunized in triplicate with 50 µL of various regimens of concentrations of the extracts and either HBV vaccine or HBsAg. The plasma anti-HBsAg antibody titre was determined using an enzyme immunosorbent assay (ELISA) 14 days post-immunization. Gene expression levels of interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-α), and haematological profile of the blood samples were determined. Results: When the two immunogens were co-administered with the soybean extract, immune response was slightly higher (0.799 + 0.013 for HBsAg and 0.758 + 0.012 for vaccine) than with the Revac B vaccine used alone. However, this difference was not statistically significant (p-value > 0.467, and 0.416 respectively). Unexpectedly, mice immunized with the vaccine expressed less IL-6 levels than the untreated mice (0.603- and 1.469-fold change in transcription (FCT), respectively). Mice treated with the vaccine expressed higher TNF-α levels than the untreated group (28.84 –and 1.00 FCT respectively) while adding the extract significantly reduced the expression of TNF-α (p < 0.063). Administration of immunogens and extract reduced neutrophil (P-value = 0.027) and platelet (p-value > 0.592) counts. Conclusions: The study demonstrated that soybean extract lacked marked adjuvant activity for HBV vaccine, that HBV vaccine suppressed expression of IL-6 and promoted expression of TNF-α.

Heterologous Expression of Recombinant Proteins and Their Derivatives Used as Carriers for Conjugate Vaccines.

Carrier proteins that provide an effective and long-term immune response to weak antigens has become a real breakthrough in the disease prevention, making it available to a wider range of patients and making it possible to obtain reliable vaccines against a variety of pathogens. Currently, research is continuing both to identify new peptides, proteins, and their complexes potentially suitable for use as carriers, and to develop new methods for isolation, purification, and conjugation of already known and well-established proteins. The use of recombinant proteins has a number of advantages over isolation from natural sources, such as simpler cultivation of the host organism, the possibility of modifying genetic constructs, use of numerous promoter variants, signal sequences, and other regulatory elements. This review is devoted to the methods of obtaining both traditional and new recombinant proteins and their derivatives already being used or potentially suitable for use as carrier proteins in conjugate vaccines.

Polymer Chemistry Defines Adjuvant Properties and Determines the Immune Response against the Antigen or Vaccine.

Activation of the immune system is a needed for designing new antigen/drug delivery systems to develop new therapeutics and for developing animal disease models to study the disease pathogenesis. A weak antigen alone is insufficient to activate the immune system. Sometimes, assistance in the form of polymers is needed to control the release of antigens under in vivo conditions or in the form of an adjuvant to activate the immune system efficiently. Many kinds of polymers from different functional groups are suitable as microbial antigens for inducing therapeutic immune responses against infectious diseases at the preclinical level. The choice of the functionality of polymer varies as per the application type. Polymers from the acid and ester groups are the most common types investigated for protein-based antigens. However, electrostatic interaction-displaying polymers like cationic polymers are the most common type for nucleic acid-based antigens. Metal coordination chemistry is commonly used in polymers designed for cancer immunotherapeutic applications to suppress inflammation and induce a protective immune response. Amide chemistry is widely deployed in polymers used to develop antigen-specific disease models like the experimental autoimmune arthritis murine model.