Heavy Chain Variable Region Research Articles

Clinical and biological features and prognosis of patients with leukemic non-nodal mantle cell lymphoma

Objective: To investigate the clinical, biological and prognostic characteristics of leukemic non-nodal mantle cell lymphoma (nnMCL). Methods: The clinical data of 14 patients with nnMCL and 238 patients with classical mantle cell lymphoma (cMCL) in Blood Diseases Hospital, Chinese Academy of Medical Sciences from November 2000 to October 2020 were retrospectively analyzed. Among the 14 patients with nnMCL, there were 9 males and 5 females, with the age [M (Q1, Q3)] of 57.5 (52.3, 67.0) years. Among the 238 patients with cMCL, there were 187 males and 51 females, with the age of 58.0 (51.0, 65.3) years. The clinical and biological characteristics of the two groups were recorded and compared. Follow-up and efficacy evaluation were conducted by re-examination during hospital stay and telephone follow-up and so on. Results: The proportion of CD200 expression in nnMCL patients was 8/14, which was higher than that in cMCL patients [14.6% (19/130)] (P=0.001). The proportion of CD23 expression in nnMCL patients was 8/14, which was higher than that in cMCL patients [13.5% (23/171)] (P<0.001). The proportion of CD5 expression in nnMCL patients was 10/14, which was lower than that in cMCL patients [97.4% (184/189)] (P=0.001). The proportion of CD38 expression in nnMCL patients was 4/14, which was lower than that in cMCL patients [69.6% (112/161)] (P=0.005). The expression proportion of sex-determining region of Y chromosome-related high-mobility-group box 11 (SOX11) in nnMCL patients was 1/5, which was lower than that in cMCL patients [77.9% (60/77)] (P=0.014). The proportion of immunoglobulin heavy chain variable region (IGHV) mutations in nnMCL patients was 11/11, which was higher than that in cMCL patients [26.0% (13/50)] (P<0.001). As of April 11, 2021, the follow-up time for nnMCL and cMCL patients was 31 (8-89) months and 48 (0-195) months, respectively. Among the 14 nnMCL patients, 6 patients were still under observation, and 8 patients were treated. The overall response rate (ORR) was 8/8, including 4 patients with complete remission and 4 patients with partial response. The median overall survival and median progression-free survival were not reached in nnMCL patients. In the cMCL group, 50.0% (112/224) patients achieved a complete response, 24.6% (55/224) patients achieved a partial response, and ORR was 74.6% (167/224). There was no statistically significant difference in ORR between the two groups (P=0.205). Conclusions: nnMCL patients have an indolent progression, with higher expression rates of CD23 and CD200 and lower expression rates of SOX11, CD5 and CD38. Most patients have IGHV mutations, with a relatively good prognosis, and"watch and wait"approach is an optional treatment.

Contribution of the IGCR1 regulatory element and the 3′Igh CTCF-binding elements to regulation of Igh V(D)J recombination

Immunoglobulin heavy chain variable region exons are assembled in progenitor-B cells, from VH, D, and JH gene segments located in separate clusters across the Igh locus. RAG endonuclease initiates V(D)J recombination from a JH-based recombination center (RC). Cohesin-mediated extrusion of upstream chromatin past RC-bound RAG presents Ds for joining to JHs to form a DJH-RC. Igh has a provocative number and organization of CTCF-binding elements (CBEs) that can impede loop extrusion. Thus, Igh has two divergently oriented CBEs (CBE1 and CBE2) in the IGCR1 element between the VH and D/JH domains, over 100 CBEs across the VH domain convergent to CBE1, and 10 clustered 3'Igh-CBEs convergent to CBE2 and VH CBEs. IGCR1 CBEs segregate D/JH and VH domains by impeding loop extrusion-mediated RAG-scanning. Downregulation of WAPL, a cohesin unloader, in progenitor-B cells neutralizes CBEs, allowing DJH-RC-bound RAG to scan the VH domain and perform VH-to-DJH rearrangements. To elucidate potential roles of IGCR1-based CBEs and 3'Igh-CBEs in regulating RAG-scanning and elucidate the mechanism of the ordered transition from D-to-JH to VH-to-DJH recombination, we tested effects of inverting and/or deleting IGCR1 or 3'Igh-CBEs in mice and/or progenitor-B cell lines. These studies revealed that normal IGCR1 CBE orientation augments RAG-scanning impediment activity and suggest that 3'Igh-CBEs reinforce ability of the RC to function as a dynamic loop extrusion impediment to promote optimal RAG scanning activity. Finally, our findings indicate that ordered V(D)J recombination can be explained by a gradual WAPL downregulation mechanism in progenitor-B cells as opposed to a strict developmental switch.

Characterization of 405B8H3(D-E), a newly engineered high affinity chimeric LAG-3 antibody with potent antitumor activity.

Lymphocyte activation gene-3 (LAG-3) is a type I transmembrane protein with structural similarities to CD4. Overexpression of LAG-3 enables cancer cells to escape immune surveillance, while its blockade reinvigorates exhausted T cells and strengthens anti-infection immunity. Blockade of LAG-3 may have antitumor effects. Here, we generated a novel anti-LAG-3 chimeric antibody, 405B8H3(D-E), through hybridoma technology from monoclonal antibodies produced in mice. The heavy-chain variable region of the selected mouse antibody was grafted onto a human IgG4 scaffold, while a modified light-chain variable region was coupled to the human kappa light-chain constant region. 405B8H3(D-E) could effectively bind LAG-3-expressing HEK293 cells. Moreover, it could bind cynomolgus monkey (cyno) LAG-3 expressed on HEK293 cells with a higher affinity than the reference anti-LAG-3 antibody BMS-986016. Furthermore, 405B8H3(D-E) promoted interleukin-2 secretion and was able to block the interactions of LAG-3 with liver sinusoidal endothelial cell lectin and major histocompatibility complex II molecules. Finally, 405B8H3(D-E) combined with anti-mPD-1-antibody showed effective therapeutic potential in the MC38 tumor mouse model. Therefore, 405B8H3(D-E) is likely to be a promising candidate therapeutic antibody for immunotherapy.

Impact of Minimal Residual Disease on Progression-Free Survival Outcomes After Fixed-Duration Ibrutinib-Venetoclax Versus Chlorambucil-Obinutuzumab in the GLOW Study.

In GLOW, fixed-duration ibrutinib + venetoclax showed superior progression-free survival (PFS) versus chlorambucil + obinutuzumab in older/comorbid patients with previously untreated chronic lymphocytic leukemia (CLL). The current analysis describes minimal residual disease (MRD) kinetics and any potential predictive value for PFS, as it has not yet been evaluated for ibrutinib + venetoclax treatment. Undetectable MRD (uMRD) was assessed by next-generation sequencing at <1 CLL cell per 10,000 (<10-4) and <1 CLL cell per 100,000 (<10-5) leukocytes. PFS was analyzed by MRD status at 3 months after treatment (EOT+3). Ibrutinib + venetoclax achieved deeper uMRD (<10-5) rates in bone marrow (BM) and peripheral blood (PB), respectively, in 40.6% and 43.4% of patients at EOT+3 versus 7.6% and 18.1% of patients receiving chlorambucil + obinutuzumab. Of these patients, uMRD (<10-5) in PB was sustained during the first year post-treatment (EOT+12) in 80.4% of patients receiving ibrutinib + venetoclax and 26.3% receiving chlorambucil + obinutuzumab. Patients with detectable MRD (dMRD; ≥10-4) in PB at EOT+3 were more likely to sustain MRD levels through EOT+12 with ibrutinib + venetoclax versus chlorambucil + obinutuzumab. PFS rates at EOT+12 were high among patients treated with ibrutinib + venetoclax regardless of MRD status at EOT+3: 96.3% and 93.3% in patients with uMRD (<10-4) and dMRD (≥10-4) in BM, respectively, versus 83.3% and 58.7% for patients receiving chlorambucil + obinutuzumab. PFS rates at EOT+12 also remained high in patients with unmutated immunoglobulin heavy-chain variable region (IGHV) receiving ibrutinib + venetoclax, independent of MRD status in BM. Molecular and clinical relapses were less frequent during the first year post-treatment with ibrutinib + venetoclax versus chlorambucil + obinutuzumab regardless of MRD status at EOT+3 and IGHV status. Even for patients not achieving uMRD (<10-4), PFS rates remained high with ibrutinib + venetoclax; this is a novel finding and requires additional follow-up to confirm its persistence over time.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

The development of some lymphomas has been associated with infections caused by bacterial species discovered in the 1980s, for example, Helicobacter pylori and Borrelia burgdorferi s.l. The first cases of human infection with a bacterial species that was first described in 2004, the tick-borne bacterium Neoehrlichia mikurensis were published in 2010. Patients with malignant B cell lymphomas emerged as a risk group for severe cases of infection, presenting with fever of unknown cause and venous thromboembolism. The infection is missed by routine microbiologic diagnostic procedures such as blood culture and can only be detected by PCR. Vascular endothelium is a likely target of N. mikurensis infection. Asymptomatic carriage of the infection for months to years appears to occur. We hypothesized that N. mikurensis causes latent infections that can reactivate when B cell defenses are compromised. It was whilst conducting a study of patients with malignant B cell lymphomas scheduled to receive treatment with rituximab that we discovered several patients who had the infection already before the start of treatment. This led us to hypothesize that persistent latent infections with N. mikurensis might contribute to the development of certain cases of malignant B cell lymphoma. Splenic engagement, restricted repertoires of the Immunoglobulin heavy chain variable (IGHV) regions, and mutations of the IGHV regions from the germline configuration featured among these cases of lymphoma, which encompassed chronic lymphocytic leukemia, splenic marginal zone lymphoma, mantle cell lymphoma and non-classified indolent splenic lymphoma. Remarkably, three out of five patients could cease lymphoma therapy after eradication of the infection. Our aim is to confirm and expand these findings of a possible causality between N. mikurensis infection and the development of malignant B cell lymphomas. To this end, we are starting a prospective study of the prevalence of N. mikurensis infection in newly diagnosed patients with malignant B cell lymphomas, and the response of such lymphomas to eradication of the infection. We will also attempt to identify a genetic signature of N. mikurensis infection by studying IGHV gene usage, the mutations induced by somatic hypermutations, as well as the physicochemical properties of the complementarity-determining region 3 (CDR3), the most hypervariable part of the B cell receptor of infected patients. This will help establish if it is mainly post-germinal center B cells that undergo malignant B cell transformation in cases of N. mikurensis-associated lymphoma. The research was funded by: the Swedish Research Council, the Swedish Cancer Society, the Swedish state under the agreement between the Swedish government and the county councils, the ALF-agreement, and the Cancer and Allergy Foundation. Keywords: indolent non-Hodgkin lymphoma, tumor biology and heterogeneity, chronic lymphocytic leukemia (CLL) No conflicts of interests pertinent to the abstract.

Antibody signatures in hospitalized hand, foot and mouth disease patients with acute enterovirus A71 infection.

Enterovirus A71 (EV-A71) infection is a major cause of severe hand, foot and mouth disease (HFMD) in young children. The characteristics of EV-A71 neutralizing antibodies in HFMD patients are not well understood. In this study, we identified and cloned EV-A71-neutralizing antibodies by single cell RNA and B cell receptor sequencing of peripheral blood mononuclear cells. From 145 plasmablasts, we identified two IgG1 monoclonal antibodies (mAbs) and six IgM mAbs that neutralized EV-A71. Four of the IgM mAbs harbor germline variable sequences and neutralize EV-A71 potently. Two genetically similar IgM antibodies from two patients have recurrent heavy chain variable domain gene usage and similar complementarity-determining region 3 sequences. We mapped the residues of EV-A71 critical for neutralization through selection of virus variants resistant to antibody neutralization in the presence of neutralizing mAbs. The residues critical for neutralization are conserved among EV-A71 genotypes. Epitopes for the two genetically similar antibodies overlap with the SCARB2 binding site of EV-A71. We used escape variants to measure the epitope-specific antibody response in acute phase serum samples from EV-A71 infected HFMD patients. We found that these epitopes are immunogenic and contributed to the neutralizing antibody response against the virus. Our findings advance understanding of antibody response to EV-A71 infection in young children and have translational potential: the IgM mAbs could potentially be used for prevention or treatment of EV-A71 infections.

CLINICAL AND BIOLOGICAL FEATURES AND OUTCOMES OF PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA: A REAL‐WORLD STUDY OF 2005 PATIENTS IN CHINA

Introduction: Chronic lymphocytic leukemia (CLL) is less common in Han Chinese compared with persons of Europeans. The data about the clinical and biological features and outcomes of Chinese with CLL are limited, thus promoting us to perform this analysis on a large cohort of 2005 patients with CLL in China. Methods: Clinical data of 2005 patients with CLL were analyzed in Blood Diseases Hospital, Chinese Academy of Medical Sciences from February 2000 to December 2020. Results: In this cohort, the median age of patients at diagnosis was 61(20-92) years, which was younger than that for Western CLL patients (65-70 years). Patients showed similar male predominance as Europeans with CLL with an M:F ratio of about 1.8. 69.3% of patients had Binet stage B or C at diagnosis, higher than those of predominately European descent (20%-40%). A higher proportion of mutated immunoglobulin heavy chain variable region (IGHV) (64.0%) was shown in patients with CLL in this cohort than Europeans. IGHV4-34 rather than IGHV1-69 was the most commonly used fragment. Compared with Europeans, patients in this cohort had a higher proportion of subset eight stereotype and a lower proportion of subset two. The most frequent abnormality detected by FISH was RB1 deletion (21.1%), followed by trisomy 12 (20.4%), IGH translocation (16.8%), del(17p) (11.4%) and del(11q) (10.9%), respectively. It was shown that the frequencies of the chromosomal abnormalities in this study population were similar to the frequencies in Western countries. Of the 1476 patients for whose treatment data were available, 1191 (80.7%) received treatment. Median time to first treatment (TTFT) for CLL patients was 7 months. Among patients treated, chemotherapy, immunochemotherapy and targeted therapy accounted for 69.6%, 14.1% and 16.3% of first line, yielding 68.4%, 90.0% and 94.2% of overall response rate (ORR), respectively. After median 75-month follow up, median progression free survival (PFS) was 42 months, 5-year and 10-year PFS rate was 35.5% and 12.5%, respectively. Median overall survival (OS) was 130 months, 5-year and 10-year OS rate was 74.9% and 52.3%, respectively. The outcomes were similar to those reported in previous Chinese and Western studies. As expected, PFS and OS were better in CLL patients receiving immunochemotherapy and targeted therapy than those receiving chemotherapy. In addition, patients who achieved partial remission (PR) or complete remission (CR) after first-line treatment had a longer PFS and OS. Interestingly, females with CLL had a longer OS than males. Conclusions: This is the largest real-world study involving Chinese patients with CLL so far, which might lay a foundation for clinical investigation of Chinese CLL in future. Although the patients were younger, had a later stage at diagnosis and had different genetic backgrounds compared with Europeans, the outcomes were not different from those reported in Western studies. Keyword: chronic lymphocytic leukemia (CLL) No conflicts of interests pertinent to the abstract.

The Longitudinal Analysis of Convergent Antibody VDJ Regions in SARS-CoV-2-Positive Patients Using RNA-Seq.

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) is an ongoing pandemic that continues to evolve and reinfect individuals. To understand the convergent antibody responses that evolved over the course of the pandemic, we evaluated the immunoglobulin repertoire of individuals infected by different SARS-CoV-2 variants for similarity between patients. We utilized four public RNA-seq data sets collected between March 2020 and March 2022 from the Gene Expression Omnibus (GEO) in our longitudinal analysis. This covered individuals infected with Alpha and Omicron variants. In total, from 269 SARS-CoV-2-positive patients and 26 negative patients, 629,133 immunoglobulin heavy-chain variable region V(D)J sequences were reconstructed from sequencing data. We grouped samples based on the SARS-CoV-2 variant type and/or the time they were collected from patients. Our comparison of patients within each SARS-CoV-2-positive group found 1011 common V(D)Js (same V gene, J gene and CDR3 amino acid sequence) shared by more than one patient and no common V(D)Js in the noninfected group. Taking convergence into account, we clustered based on similar CDR3 sequence and identified 129 convergent clusters from the SARS-CoV-2-positive groups. Within the top 15 clusters, 4 contain known anti-SARS-CoV-2 immunoglobulin sequences with 1 cluster confirmed to cross-neutralize variants from Alpha to Omicron. In our analysis of longitudinal groups that include Alpha and Omicron variants, we find that 2.7% of the common CDR3s found within groups were also present in more than one group. Our analysis reveals common and convergent antibodies, which include anti-SARS-CoV-2 antibodies, in patient groups over various stages of the pandemic.

Empirical Study on Exploring the Role of CD180 and MD-1 Prognostic Indicators for the Chronic Lymphocytic Leukaemia (CLL) Disease.

&amp;lt;b&amp;gt;Background and Objective:&amp;lt;/b&amp;gt; Chronic Lymphocytic Leukaemia (CLL) is a frequent type of leukaemia disease. This study was focused on investigating the role of prognostic indicators, such as CD180 and MD-1 for Chronic Lymphocytic Leukaemia (CLL) pathogenesis because they involve cell signalling and proliferation. &amp;lt;b&amp;gt;Materials and Methods:&amp;lt;/b&amp;gt; A total of 12 normal controls and 52 patients were taken to determine the expressions of CD180 and MD-1 with different variations in comparison with the IgVH (Immunoglobulin Heavy Chain variable region gene) mutational status, FISH (fluorescence &amp;lt;i&amp;gt;in situ&amp;lt;/i&amp;gt; hybridization) and Rai staging. &amp;lt;b&amp;gt;Results:&amp;lt;/b&amp;gt; The quantitative data findings were evident that CD180 and MD-1 expressions showed insignificant differences among CLL patients at the protein level based on SPSS results. On the contrary, they resulted in significant differences for subgroups of established biomarkers like Rai staging (stages 0, I, II and III), FISH (13q and non-13q deletions) and IgVH (mutated and unmutated). &amp;lt;b&amp;gt;Conclusion:&amp;lt;/b&amp;gt; The CD180 and MD-1 have been used as prognostic indicators to evaluate the outcomes relevant to the cell cycle and survival rate of CLL cells.

Immunoglobulin genes and stereotyped antigenic receptors in chronic lymphocytic leukemia and other lymphoproliferative diseases

Introduction. The mutational status of immunoglobulin heavy chain variable region genes (IGHV) is the most important prognostic factor in chronic lymphocytic leukemia (CLL). Furthermore, a significant narrowing of the IGHV gene repertoire is found in CLL and other lymphoproliferative diseases. Aim — to review the publication data on the IGHV genes repertoire and mutational status in CLL and other lymphoproliferative diseases regarding their clinical significance. General information. Nucleotide sequence of rearranged IGHV genes is a unique marker of a tumor clone. CLL patients with unmutated IGHV genes have an extremely unfavorable disease outcome in contrast to the patients with mutated IGHV genes. Patients with mutated IGHV genes benefit from conventional immunochemotherapy, while non-mutated IGHV patients require therapy escalation with new targeted drugs. The study of IGHV genes and stereotyped antigen receptors repertoire makes possible to identify additional groups of CLL patients with specific genetic and clinical features. Stereotype receptors are also detected in other lymphoproliferative diseases, but their clinical significance has not yet been defined. However, stereotyped receptors are found to be disease-specific.

Abstract B039: BIO-PROTAC: Application of cell penetrating scFv to treat undruggable KRAS mutant cancer

Abstract Activation of mutations in RAS has been found in high ratio in human cancers, inducing downstream critical effectors corresponding tumorigenesis. In recent years, RAS-RAF-ERK1/2 pathway is known as a crucial target for the anti-cancer drug development because of high prevalence of ERK activation in human cancers. The attempts in direct targeting to activated RAS, which is GTP bound, has been somewhat successful in the treatment of certain type of cancer, however, still beset by the resistance. Our aim is to develop inhibitor that cell-penetrating scFv (single-chain variable fragment) which targets mutated KRAS (mKBscFv; mutated KRAS binding scFv). The Fv fragment is the smallest unit composed of the variable region of heavy chain and light chain in an immunoglobulin molecule, that has a function of antigen-binding activity. The scFv can specifically bind to an antigen, thereby reducing side effects of non-specific binding seen in chemical drugs. However, due to the lack of cell penetrating ability of scFv, a delivery carrier is often required. In this study, the mKBscFv was measured binding affinity toward wild type KRAS and mutated KRAS using surface plasmon resonance (SPR). The scFv has higher affinity to mutated KRAS than wild type KRAS. A cell-penetrating peptide (CPP) was developed for drug delivery and was expressed at the C-terminus of mKBscFv for transferring cells. The mKBscFv-CPP was tagged with His tag to express in CHO cells, which was purified by FPLC. The purity of mKBscFv-CPP was confirmed by SDS PAGE and immunoblotting. The mKBscFv-CPP treated KRAS mutated cancer cells decreased cell viability in a concentration-dependent manner, and decreased expression of mutated GTP-bound KRAS. In addition to the bioactivity of the mKBscFv, it has further advantage as a proteolysis-targeting chimera (PROTAC). The plasmid DNA was prepared by adding a VHL-expressing sequence to the C-terminus of mKBscFv (mKBscFv-Vh). This gene was transfected into KRAS mutated cancer cells to express the protein, and as a result mutated GTP-bound KRAS was reduced. A CPP peptide domain was added to the C-terminus of this gene to express it as a protein in CHO cells. The mutated GTP-bound KRAS was reduced by mKBscFv-Vh-CPP in KRAS mutated cacner cells. Taken together, mKBscFv that binds to mutated KRAS was developed, and it can be expected to become a new protein anticancer drug effective against mutated KRAS-mediated cancer cells by fusing cell penetrating peptide and PROTAC technology. Citation Format: Gookjin Yoon, Beom Soo Jo, Dong Woo Lee, Jinwook Yang, Min-Ho Park, Sanghui Seok, Jue Yeon Lee, Chong Pyoung Chung, Yoon Shin Park, Yoon Jeong Park. BIO-PROTAC: Application of cell penetrating scFv to treat undruggable KRAS mutant cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B039.

Selective targeting of pathogenic auto-reactive VH4–34 B cells with a rationally developed anti-VH4–34 antibody offers a new therapeutic approach for autoimmune disorders

Abstract B cells expressing B cell receptors (BCRs) specific for self-antigens are normally eliminated, or made anergic, through negative selection during B cell development. VH4–34 is an intrinsically autoreactive antibody (and BCR) heavy chain variable region that includes a germline encoded hydrophobic patch in framework region 1 (FR1). Autoantigens recognized by VH4–34 antibodies include N-acetyl lactosamine chains displayed on RBCs. In healthy individuals, &amp;lt;1% of B cells express VH4–34 encoded BCRs but during the loss of self-tolerance, characteristic of pathogenic disorders, the hydrophobic region of VH4–34 reacts to self-antigen, erroneously driving BCR activation and expansion. Expansion of VH4–34+ B cells and increased serum concentrations of VH4–34 antibodies has been implicated in systemic lupus erythematosus (50%) and virtually all cases of cold agglutinin disease. To date there have been no therapies against VH4–34 due to challenges of specifically targeting VH4–34 while avoiding other VH regions. We applied Hummingbird Bioscience’s Rational Antibody Discovery approach to develop anti-VH4–34 antibodies that bind to a computationally predicted and lineage conserved epitope in the hydrophobic FR1 region. The lead antibody, HMBD-011, demonstrates selective and high affinity binding to all tested VH4–34 antibodies, with no off-target binding. The binding epitope of HMBD-011 was confirmed by mutagenesis to be within the FR1 region of VH4–34. Further, HMBD-011 shows selective binding to cells that express VH4–34 BCRs. In vivo, HMBD-011 treatment resulted in rapid elimination of VH4–34+ antibodies and cells. In conclusion, HMBD-011 represents a promising precision therapy for VH4–34 driven autoimmune disorders.

A new class of monoclonal Aβ antibodies selectively targets and triggers deposition of Aβ protofibrils.

Aggregation and accumulation of amyloid-β peptide (Aβ) are a critical trigger for the onset of Alzheimer's disease (AD). While the plaques are the most outstanding Aβ pathological feature, much of the recent research emphasis has been on soluble Aβ species because of their diffusible, proinflammatory, and toxic properties. The focus on soluble aggregated Aβ species has also increased the interest in antibodies that are selective for different Aβ conformations. In the current study, we developed and characterized a new class of monoclonal antibodies (referred to as mAbSL) that are selective for Aβ protofibrils. Cloning and sequencing of the heavy and light chain variable regions for multiple antibodies identified sequence characteristics that may impart the conformational selectivity by the antibodies. Transfection of FreeStyle 293F cells with the plasmids permitted in-house expression and purification of mAbSL antibodies along with non-conformation-selective Aβ monoclonal antibodies (Aβ mAbs). Several of the purified mAbSL antibodies demonstrated significant affinity and selectivity for Aβ42 protofibrils compared with Aβ42 monomers and Aβ42 fibrils. Competition ELISA assays assessing the best overall antibody, mAbSL 113, yielded affinity constants of 7 nM for the antibody-Aβ42 protofibril interaction, while the affinity for either Aβ42 monomers or Aβ42 fibrils was roughly 80 times higher. mAbSL 113 significantly inhibited Aβ42 monomer aggregation by a unique mechanism compared with the inhibition displayed by Aβ mAb 513. Aβ42 protofibril dynamics were also markedly altered in the presence of mAbSL 113, whereby insoluble complex formation and protofibril deposition were stimulated by the antibody at low substoichiometric molar ratios. As the field contemplates the therapeutic effectiveness of Aβ conformation-selective antibodies, the findings presented here demonstrate new information on a monoclonal antibody that selectively targets Aβ protofibrils and impacts Aβ dynamics.

Immunosensor for Rapid and Sensitive Detection of Digoxin.

Digoxin is a cardiac glycosylated steroid-like drug with a positive inotropic effect and has been widely used in treating congestive heart failure, atrial fibrillation, atrial flutter, and other heart diseases. Digoxin is also a dangerous drug, which can cause drug poisoning at a low blood drug concentration (2.73-3.9 nmol/L, i.e., 2.14-3.05 ng/mL). Therefore, the timely detection of a patient's blood drug concentration plays a significant role in controlling blood drug concentration, reducing the occurrence of drug poisoning events, and maximizing the role of drug therapy. In this study, a DNA vector for the expression of the antidigoxin antibody Fab fragment was constructed. With the vector, Fab was expressed in E. coli and purified, and 1.2 mg of antibodies was obtained from 100 mL of culture. An immunofluorescent sensor based on the mechanism of photoinduced electron transfer was constructed by labeling additional cysteines in the heavy chain variable region and light chain variable region of the antibody Fab fragment with fluorescent dyes. The assay for digoxin with the immunosensor could be finished within 5 min with a limit of detection of 0.023 ng/mL, a detectable range of 0.023 ng/mL to 100 μg/mL, and an EC50 of 0.256 ng/mL. A new approach for the rapid detection of digoxin was developed and will contribulte to therapeutic drug monitoring.

Obinutuzumab plus bendamustine for relapsed/refractory chronic lymphocytic leukemia and predictive and prognostic impact of genetic alterations: the phase II GABRIELL study

GABRIELL was a phase II single-arm study to evaluate the efficacy and safety of obinutuzumab plus bendamustine for relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL). Seventy-two patients with active disease received treatment for up to six 28-day cycles. Overall response rate was 78.6% with a median progression-free survival (PFS) of 26 months, and overall survival (OS) not reached at the end of follow-up (36 months). Undetectable measurable residual disease (≤0.01%; 36.4% in bone marrow and 53.4% in peripheral blood) correlated with a significantly longer PFS and OS (vs. >0.01). Common grade ≥3 adverse events (76.4%) were neutropenia (58.3%), thrombocytopenia (26.4%) and febrile neutropenia (11.1%). TP53 disruption was the only independent predictive factor for response (Hazard ratio; HR: 0.228). Unmutated immunoglobulin heavy chain variable region (HR: 16.061) was a negative prognostic factor for PFS. In conclusion, the combination of obinutuzumab plus bendamustine is an active and generally adequately-tolerated treatment for R/R CLL.

Abstract CT021: Assessing clinical and pharmacodynamic (PD) profiles of patients (pts) with chronic lymphocytic leukemia (CLL) on ianalumab (VAY736) + ibrutinib

Abstract Introduction VAY736 is an afucosylated, human monoclonal antibody engineered to enhance antibody-dependent cellular cytotoxicity that targets BAFF-R+ B cells for elimination. In preclinical CLL models, VAY736 showed antileukemic activity and, when combined with ibrutinib, significantly reduced disease burden, which may allow some pts to discontinue ibrutinib. Methods This Phase Ib dose-escalation (ESC)/-expansion (EXP) trial (NCT03400176) enrolled pts with CLL who did not achieve a complete response (CR) after &amp;gt;1 year of ibrutinib or had developed a resistance mutation to ibrutinib. Pts received IV VAY736 (ESC: 0.3-9 mg/kg; EXP: 3 mg/kg) once every 2 weeks and oral ibrutinib (420 mg) once daily for up to 8 28-day cycles. Pts achieving undetectable MRD (uMRD) at C9D1 could discontinue ibrutinib at investigator discretion. The study aimed to characterize the safety and tolerability of VAY736 + ibrutinib, assess antitumor activity, PK, and characterize PD profiles. Results By Jul 29, 2022, 39 pts were enrolled (ESC: n=15; EXP: n=24). Table 1 shows pt characteristics, safety, and efficacy data. The overall response at C9D1 for 37 evaluable pts was 40.5% CR + CRi and 16.2% PR (1L: 63.6% CR + CRi and 18.2% PR). At C9D1, 17 pts (45.9%) had uMRD in blood or bone marrow (BM). In the 2-year follow-up period, 16 pts discontinued ibrutinib and were off therapy for 4.9-19.8 months. Frequency of peripheral NKp46+ NK cells increased at least 50% after VAY736 in over 50% of pts. Preliminary coverage-based limiting-cell experiment analysis of RNAseq (CLEAR) data from 10 pts supports peripheral NK cell activation with VAY736. Conclusions VAY736 + ibrutinib appears highly active and has an acceptable safety profile. Multiple pts attained uMRD in blood or BM. Biomarker data suggest NK cell activation with VAY736. More pts will be included in the RNAseq analysis at presentation. Future development of VAY736 for CLL is strongly indicated based on these promising data. Table 1. Patient characteristics, safety, and efficacy results. All patients (N=39) Patient demographics and prior treatment Median age, years (range) 65.0 (39-82) ECOG performance status, n (%) 0 36 (92.3) 1 3 (7.7) No prior regimens excluding ibrutinib, n (%) 12 (30.8) Median number of prior regimens, n (range) 1.0 (0.0-14.0) Median duration of ibrutinib, years (range) 2.95 (0.2-8.3) Patient baseline characteristics Dohner risk by FISH,a n (%) 17p deletion 6 (15.4) 11q deletion 9 (23.1) Trisomy 12 3 (7.7) 13q deletion 10 (25.6) IGHV mutant status, n (%) Non-mutant 32 (82.1) Complex karyotype, n (%) Yes 20 (51.3) Safety Dose-limiting toxicities, n (%) 0 Patients with at least one AE, any grade, n (%) 38 (97.4) Patients with at least one Grade ≥3 AE, n (%) 13 (33.3) Most common (occurring in ≥2 patients) Grade ≥3 AEs, n (%) Neutrophil count decreased 5 (12.8) Lymphocyte count decreased 2 (5.1) Hypophosphatemia 2 (5.1) Lipase increased 2 (5.1) Efficacy 1Lb n=11 R/R n=26 Evaluable patients N=37 Overall response at C9D1 or before discontinuation,c n (%) Complete response 6 (54.5) 8 (30.8) 14 (37.8) Complete response with incomplete marrow recovery 1 (9.1) 0 1 (2.7) Partial response 2 (18.2) 4 (15.4) 6 (16.2) Stable disease 2 (18.2) 8 (30.8) 10 (27.0) Progressive disease 0 5 (19.2) 5 (13.5) uMRD response at C9D1 or before discontinuation,c n (%) Bone marrow uMRD 6 (54.5) 6 (23.1) 12 (32.4) Blood uMRD 7 (63.6) 10 (38.5) 17 (45.9) Blood or bone marrow uMRD 7 (63.6) 10 (38.5) 17 (45.9) Patients elected to discontinue ibrutinib after achieving CR or uMRD, n (%) 16 (43.2) aThe categories were: patients with a 17p deletion; patients with an 11q deletion without a 17p deletion; patients with trisomy 12 without a 17p deletion or an 11q deletion; and patients with a 13q deletion without a 17p deletion, trisomy 12, or an 11q deletion; bPatients with no prior therapies excluding ibrutinib; cFor evaluable patients (N=37). 1L, first line; AE, adverse event; CR, complete response; C, cycle; D, day; ECOG, Eastern Cooperative Oncology Group; FISH, fluorescence in situ hybridization; IGHV, immunoglobulin heavy chain variable region; R/R, relapsed/refractory; uMRD, undetectable minimal residual disease. Citation Format: Kerry Anne Rogers, Pearlly Yan, Ian W. Flinn, Deborah M. Stephens, Thomas J. Kipps, Sarah M. Larson, Laura Martz, Xi Chen, Huabao Wang, Ethan Hopping, Ralf Bundschuh, Alexandra Acosta, Daniela Baldoni, Anwesha Chaudhury, Jeanne Whalen, Nadia B. Hassounah, Nina Orwitz, Janghee Woo, John C. Byrd. Assessing clinical and pharmacodynamic (PD) profiles of patients (pts) with chronic lymphocytic leukemia (CLL) on ianalumab (VAY736) + ibrutinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT021.

Abstract 1325: Novel recombinant rabbit monoclonal antibodies for cancer biology research

Abstract Recombinant monoclonal antibodies (rAbs) are superior tools for cancer biology research as they offer distinct advantages over standard polyclonal and hybridoma-generated monoclonal antibody reagents. Recombinant technology produces antibodies that can be defined at the primary sequence level and are characterized by consistent performance and supply. These rAb features are in stark contrast to traditional polyclonal and hybridoma-generated monoclonal antibodies, whose lack of dependability has been widely documented as a primary reason for data irreproducibility plaguing biomedical research. In an effort to respond to this challenge, GeneTex has established a recombinant antibody production platform combined with more rigorous validation protocols. The goal is to develop reliable recombinant rabbit monoclonal antibodies for reproducible performance in various experimental applications utilized routinely by cancer biologists. GeneTex’s rAb production protocol is based on the approach described by Starkie et al. (2016). This involves a multi-parameter fluorescence-activated single cell sorting (FACS)-based methodology to identify and isolate antigen-specific IgG+ memory B cells obtained from an immunized rabbit. The heavy and light chain variable region genes from selected cells are PCR-amplified and cloned into plasmids to produce full-length heavy and light chains for a single IgG. These constructs are subsequently introduced together into mammalian cells for expression, meaning that natural pairing of the chains is maintained. This manufacturing strategy allows application-specific testing (e.g., for western blot (WB), immunohistochemistry (IHC), and immunocytochemistry (ICC/IF), etc.) of clones during the antibody production process. Validation of antibodies is performed inhouse and, when possible, in relationships with academic or industry/pharma institutions where ideal samples and reagents are frequently more available. This recombinant monoclonal antibody production platform has created a series of well-validated antibodies against many important targets for cancer biology research. This includes reagents to study tumor immunobiology (e.g., PD-L1), hormone receptors (e.g., ER alpha and androgen receptor variants like ARV7), oncogenic RAS protein mutants (e.g., RAS G12D), transcription factors (e.g., NRF2), and epithelial-mesenchymal transition (EMT) (e.g., E-cadherin), among many others. Extensive validation for multiple applications was performed, with knockdown or CRISPR-based knockout cell lysates employed when possible to establish specificity. In summary, GeneTex is utilizing a FACS-based recombinant rabbit monoclonal antibody production approach to generate and thoroughly validate antibody reagents that will hopefully facilitate reproducible cancer biology research. Citation Format: Alexander Ball, Yung Lin Hsieh, Chun Kai Huang, Sega Huang, Ping Kuan Hsieh, Shang Ru Wu, Yue Yun Chang, Jiming Liu, Po Shin Peng, Chia Yi Lin. Novel recombinant rabbit monoclonal antibodies for cancer biology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1325.

B cell M-CLL clones retain selection against replacement mutations in their immunoglobulin gene framework regions.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia, accounting for 30-40% of all adult leukemias. The dynamics of B-lymphocyte CLL clones with mutated immunoglobulin heavy chain variable region (IgHV) genes in their tumor (M-CLL) can be studied using mutational lineage trees. Here, we used lineage tree-based analyses of somatic hypermutation (SHM) and selection in M-CLL clones, comparing the dominant (presumably malignant) clones of 15 CLL patients to their non-dominant (presumably normal) B cell clones, and to those of healthy control repertoires. This type of analysis, which was never previously published in CLL, yielded the following novel insights. CLL dominant clones undergo - or retain - more replacement mutations that alter amino acid properties such as charge or hydropathy. Although, as expected, CLL dominant clones undergo weaker selection for replacement mutations in the complementarity determining regions (CDRs) and against replacement mutations in the framework regions (FWRs) than non-dominant clones in the same patients or normal B cell clones in healthy controls, they surprisingly retain some of the latter selection in their FWRs. Finally, using machine learning, we show that even the non-dominant clones in CLL patients differ from healthy control clones in various features, most notably their expression of higher fractions of transition mutations. Overall, CLL seems to be characterized by significant loosening - but not a complete loss - of the selection forces operating on B cell clones, and possibly also by changes in SHM mechanisms.

A novel mAb broadly neutralizes SARS-CoV-2 VOCs in vitro and in vivo, including the Omicron variants.

Novel immune escape variants have emerged as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread worldwide. Many of the variants cause breakthrough infections in vaccinated populations, posing great challenges to current antiviral strategies targeting the immunodominance of the receptor-binding domain within the spike protein. Here, we found that a novel broadly neutralizing monoclonal antibody (mAb), G5, provided efficient protection against SARS-CoV-2 variants of concern (VOCs) in vitro and in vivo. A single dose of mAb G5 could significantly inhibit the viral burden in mice challenged with the mouse-adapted SARS-CoV-2 or SARS-CoV-2 Omicron BA.1 variant, as well as the body weight loss and cytokine release induced by mouse-adapted SARS-CoV-2. The refined epitope recognized by mAb G5 was identified as 1148 FKEELDKYF1156 in the stem helix of subunit S2. In addition, a human-mouse chimeric mAb was generated based on the variable region of heavy chain and VL genes of mAb G5. Our study provides a broad antibody drug candidate against SARS-CoV-2 VOCs and reveals a novel target for developing pan-SARS-CoV-2 vaccines.

Orelabrutinib in relapsed or refractory chronic lymphocytic leukemia/small lymphocytic lymphoma patients: Multi-center, single-arm, open-label, phase 2 study.

Orelabrutinib is a novel, small molecule, selective irreversible Bruton's tyrosine kinase inhibitor. The aim of this study was to evaluate the efficacy and safety in patients with refractory or relapsed chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). This is single-arm, multi-center, open-label, phase 2 study in 80 eligible Chinese patients, who were treated with monotherapy of orelabrutinib at 150 mg once daily. Overall response rate evaluated by an independent review committee was the primary endpoint, and secondary endpoints include progression-free survival, overall survival, and safety. Independent review committee assessed overall response rate was 92.5% (74/80); complete response 21.3% (17/80), partial response 60.0% (48/80), partial response with lymphocytosis 11.3% (9/80). At a 32.3-month median follow-up, the median progression-free survival had not been achieved, while the 30-month progression-free survival rate and overall survival rates were 70.9% (95% confidence interval [CI], 59.5-79.6) and 81.3% (95% CI, 70.8-88.2), respectively. Orelabrutinib also revealed substantial response in patients with high prognostic risks: overall response rates of patients carrying positive TP53 mutational status or del(17p), del(11q), as well as unmutated immunoglobulin heavy-chain variable region gene were 100%, 94.7%, and 93.9%, respectively. Most adverse events were in low grade, with 86.8% of AEs being Grade 1 or 2. Nearly 67% of patients were still receiving orelabrutinib after almost a 3-year follow-up. In conclusion, Orelabrutinib demonstrated compelling efficacy as well as safety profiles, with a noteworthy number of patients obtaining complete response in refractory or relapsed CLL/SLL.