Long-lasting and highly effective malaria vaccines or monoclonal antibodies (mAbs) remain urgently needed, as current interventions show age-dependent benefits rather than broad protection across all ages. Recently, two highly protective human mAbs 224 (IGHV3-49/IGLV1-40) and 7088 (IGHV3-33/IGKV1-5), encoded by distinct germline genes, were re-engineered and renamed MAM01 and MS-1805, respectively, to extend serum half-life and enable low-cost, large-scale manufacturing in alignment with WHO guidelines. MAM01 completed Phase I and IIb clinical trials (safety/PK/challenge) in US healthy naive adults and is now in Phase I trials (age-de-escalation studies) in Uganda. Here, we determined crystal structures of the antigen-binding fragments (Fabs) of engineered MAM01, MS-1805, and 7088 in complex with different regions of the Plasmodium falciparum circumsporozoite protein (PfCSP), including junctional, minor, and major repeat regions. Notably, Fab 7088 features an extended CDRL3 comprising 10 amino acids (CDRL3:10) instead of the canonical 8 amino acid CDRL3 (CDRL3:8) typically observed in VH3-33/VK1-5-encoded mAbs, indicating unique folding within its germline context. In addition, cryo-EM structures of Fabs 7088 and MS-1805, in complex with recombinant shortened CSP (rsCSP), revealed a regular spiral configuration stabilized by homotypic Fab-Fab interactions, whereas 224 and MAM01 did not form such assemblies. Structural comparisons demonstrated that engineered antibodies retained key molecular and hydrogen-bond interactions without significant conformational changes, thereby maintaining antigen recognition and preserving binding affinity. These findings demonstrate that targeted framework engineering can enhance therapeutic potential while preserving structural integrity and function, providing insights for next-generation antibody-based interventions against malaria.

The Plasmodium falciparum circumsporozoite protein ( Pf CSP) is the major surface antigen on Pf sporozoites. WHO-recommended vaccines RTS,S/AS01 E and R21/Matrix-M target the Pf CSP major repeat region and C-terminal domain (ctCSP). Although multiple studies associated protection with antibody responses to ctCSP, only a few ctCSP-specific monoclonal antibodies (mAbs) have been characterized. Here, crystal structures of 11 Fab-ctCSP complexes reveal how mAbs against the conserved β-epitope region achieve diverse modes of strain-transcending recognition, in contrast to mAbs to the hypervariable ⍺-epitope. Consistent with previous studies, ctCSP on sporozoites could be unmasked by mAbs that bind CSP repeats, with unmasking dependent on the mAb fine-specificity and binding mode. In vitro, ctCSP mAbs promoted stronger Fc-receptor signaling, cellular cytotoxicity, and phagocytosis than repeat region mAbs, while mAb combinations targeting distinct Pf CSP epitopes modulated Fc-signaling and cellular cytotoxicity. This study provides a rationale for optimization of Pf CSP-based immunogens to enhance Fc-mediated contributions to malaria vaccine efficacy.

Long-lasting and effective vaccines or monoclonal antibodies (mAbs) for malaria prevention are highly beneficial for people of all ages living in malaria-endemic regions. Recently, two highly protective human mAbs AB000224 (IGHV3-49/IGLV1-40) and AB007088 (IGHV3-33/IGKV1-5), which are encoded by different germline genes, were re-engineered and renamed as MAM01 and MS-1805, respectively, to improve their half-life, developability (including manufacturing), and cost-effectiveness according to WHO guidelines. MAM01 has completed phase 1 and 1b clinical trials (safety / PK / challenge) in US healthy naive adults and is in phase 1 trials (age-de-escalation studies) in Uganda. Here, we determined crystal structures of the antigen-binding fragments (Fabs) of engineered MAM01, MS-1805, and 7088 in complex with different regions of the Plasmodium falciparum (Pf) circumsporozoite protein (CSP), including junctional, minor, and major repeat regions. Notably, Fab 7088 features an extended CDRL3 comprising 10 amino acids (CDRL3:10) instead of the typical 8 amino acid CDRL3 (CDRL3:8) for VH3-33/VK1-5-encoded mAbs, revealing unique folding within its germline context. Structural comparisons showed that engineered antibodies retain the key molecular and hydrogen bond interactions with no significant conformational changes or loss in binding affinity. These findings demonstrate that the efficacy and affordability of human mAbs can be enhanced by selectively mutating residues in antibody framework regions without compromising binding affinity or epitope interaction. ### Competing Interest Statement M.J., S.A., G.G.-P, R.M. and I.A.W. declare that they have no competing interests. K.L.W. and R.R.K. have worked for Atreca, Inc. and Just-Evotec Biologics, respectively. D.E.E. is a consultant for Atreca, Inc. Gates Foundation, https://ror.org/0456r8d26, INV-004923, INV-056202

Over 75% of malaria-attributable deaths occur in children under the age of 5 years. However, the first malaria vaccine recommended by the World Health Organization (WHO) for pediatric use, RTS,S/AS01 (Mosquirix), has modest efficacy. Complementary strategies, including monoclonal antibodies, will be important in efforts to eradicate malaria. Here we characterize the circulating B cell repertoires of 45 RTS,S/AS01 vaccinees and discover monoclonal antibodies for development as potential therapeutics. We generated >28,000 antibody sequences and tested 481 antibodies for binding activity and 125 antibodies for antimalaria activity in vivo. Through these analyses we identified correlations suggesting that sequences in Plasmodium falciparum circumsporozoite protein, the target antigen in RTS,S/AS01, may induce immunodominant antibody responses that limit more protective, but subdominant, responses. Using binding studies, mouse malaria models, biomanufacturing assessments and protein stability assays, we selected AB-000224 and AB-007088 for advancement as a clinical lead and backup. We engineered the variable domains (Fv) of both antibodies to enable low-cost manufacturing at scale for distribution to pediatric populations, in alignment with WHO’s preferred product guidelines. The engineered clone with the optimal manufacturing and drug property profile, MAM01, was advanced into clinical development.

Significance A target-agnostic approach that harnesses the human antitumor immune response to find potential anticancer lead antibodies and their targets was used to generate ATRC-101, an engineered version of a tumor-targeting antibody identified from a patient with non-small cell lung cancer experiencing an ongoing antitumor immune response. ATRC-101 is an antibody that targets an extracellular, tumor-specific ribonucleoprotein complex. Here, we describe the extracellular binding of this complex and antitumor activity of ATRC-101 in murine models. Preclinical data suggest a mechanism of action in which ATRC-101 activates myeloid cells of the innate immune system, leading to an adaptive immune response that yields its antitumor activity. These data have led to an ongoing phase 1 trial in patients with advanced solid tumors.

Recent advances in high-throughput single cell sequencing have opened up new avenues into the investigation of B cell receptor (BCR) repertoires. In this study, PBMCs were collected from 17 human participants vaccinated with the split-inactivated influenza virus vaccine during the 2016–2017 influenza season. A combination of Immune Repertoire Capture (IRC TM ) technology and IgG sequencing was performed on ~7,800 plasmablast (PB) cells and preferential IgG heavy-light chain pairings were investigated. In some participants, a single expanded clonotype accounted for ~22% of their PB BCR repertoire. Approximately 60% (10/17) of participants experienced convergent evolution, possessing public PBs that were elicited independently in multiple participants. Binding profiles of one private and three public PBs confirmed they were all subtype-specific, cross-reactive hemagglutinin (HA) head-directed antibodies. Collectively, this high-resolution antibody repertoire analysis demonstrated the impact evolution can have on BCRs in response to influenza virus vaccination, which can guide future universal influenza prophylactic approaches.

The authors wish to add the following acknowledgment to their Research paper: "The automated DNA sequencing for antibody generation was performed in Vermont Integrative Genomics Resource DNA Facility and was supported by University of Vermont Cancer Center, Lake Champlain Cancer Research Organization, and the UVM Larner College of Medicine." Longitudinal analysis of acute and convalescent B cell responses in a human primary dengue serotype 2 infection modelOur data suggest overall functional alignment of DENV2-specific responses from the plasmablast, through the MBC and LLPC compartments following primary DENV2 inflection. These results provide enhanced resolution of the temporal and specificity of the B cell compartment in viral infection and serve as framework for evaluation of B cell responses in challenge models. Full-Text PDF Open Access

BackgroundAcute viral infections induce a rapid and transient increase in antibody-secreting plasmablasts. At convalescence, memory B cells (MBC) and long-lived plasma cells (LLPC) are responsible for long-term humoral immunity. Following an acute viral infection, the specific properties and relationships between antibodies produced by these B cell compartments are poorly understood.MethodsWe utilized a controlled human challenge model of primary dengue virus serotype 2 (DENV2) infection to study acute and convalescent B-cell responses.FindingsThe level of DENV2 replication was correlated with the magnitude of the plasmablast response. Functional analysis of plasmablast-derived monoclonal antibodies showed that the DENV2-specific response was dominated by cells producing DENV2 serotype-specific antibodies. DENV2-neutralizing antibodies targeted quaternary structure epitopes centered on domain III of the viral envelope protein (EDIII). Functional analysis of MBC and serum antibodies from the same subjects six months post-challenge revealed maintenance of the serotype-specific response in both compartments. The serum response mainly targeted DENV2 serotype-specific epitopes on EDIII.InterpretationOur data suggest overall functional alignment of DENV2-specific responses from the plasmablast, through the MBC and LLPC compartments following primary DENV2 inflection. These results provide enhanced resolution of the temporal and specificity of the B cell compartment in viral infection and serve as framework for evaluation of B cell responses in challenge models.FundingThis study was supported by the Bill and Melinda Gates Foundation and the National Institutes of Health.

Abstract Background: The role of B cells and antibodies in anticancer immune responses may correlate with improved prognosis in several types of cancer. Indeed, tumor-reactive antibodies are detected in the blood of cancer patients, tumor-infiltrating B cells have been shown to produce tumor-reactive antibodies, and tumor-reactive antibodies can cause tumor regression in several mouse models. Taken together, these observations support further identification, isolation and characterization of antitumor antibodies from patients demonstrating effective anticancer responses and defining the cognate targets and mechanisms whereby they contribute to tumor control. Methods: We identified cohorts of patients with nonprogressing metastatic cancer who had received checkpoint immunotherapy and isolated their circulating plasmablasts. Antibody heavy and light chain paired sequences were obtained from individual cells using Atreca's Immune Repertoire Capture (IRCTM) technology. The expressed antibodies were then analyzed for their ability to bind to tumor cells as well as tumor tissue and their ability to mediate antitumor activity was explored in syngeneic mouse tumor models. Results: Elevated plasmablast levels were observed in individuals with nonprogressing metastatic cancer, and analysis of plasmablast antibody sequences revealed clonal families of B cells that persisted over time with hallmarks of affinity maturation and class switching. We also identified antibody sequences with features common to more than one patient, consistent with convergent antibody selection. In particular, one antibody (AB-213) isolated from a NSCLC patient was found to demonstrate binding to unrelated human tumors as well as the mouse EMT6 tumor. When AB-213 was expressed with a mouse IgG2a constant region the chimeric antibody showed efficacy in vivo by reducing tumor volume and increasing survival in Balb/c mice harboring the syngeneic EMT6 model. Antitumor activity of the chimeric antibody was observed to be dose-dependent when administered as monotherapy or in combination with checkpoint inhibitors. We feel, based on these data, AB-213 could become a very important clinical therapeutic. Citation Format: Gilson Baia, Amy Manning-Bog, Alexander Scholz, Jeff DeFalco, Michael Harbell, Danhui Zhang, Felix Chu, Beatriz Millare, May Sumi, Patricia Zuno, Judevin Lugar Sapugay, Dongkyoon Kim, Yvonne Leung, Shuwei Jiang, Xiaobin Tang, Kevin Williamson, Xiaomu Chen, Sean Carroll, Christine Dowd, Ish Dhawan, Jonathan Benjamin, Gregg Espiritu Santo, Nicole Haaser, Ngan Nguyen, Eldar Giladi, David Minor, Yann Chong Tan, Jeremy B. Sokolove, Lawrence Steinman, Tito Serafini, Guy Cavet, Norman M. Greenberg, Jacob Glanville, Wayne Volkmuth, Daniel E. Emerling, William H. Robinson. Mining the cancer immuno-responsome: The identification of functional antitumor antibodies from patients receiving checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3966.

The complexity of the serological response to the four serotypes of the dengue virus (DENV1-4) is a key factor confounding rational vaccine development for dengue. To refine our understanding of the basis of the humoral response to DENV, we used high-coverage approaches to unravel the early and late B cell response at the clonal level to a single controlled infection with DENV2 in humans. In peripheral blood specimens we identified the peak DENV2-induced early plasmablast response. By analyzing paired immunoglobulin heavy (IGH) and light (IGL) chains derived from peak plasmablasts we found broad evidence of lineage expansion, somatic hypermutation, and variable gene usage. We selected 92 IGH/IGL pairs from expanded lineages and found that 54 (59%) of these monoclonal antibodies (mAb) bound DENV2, five of which also neutralized virus. In these same subjects we assessed the memory B cell (MBC) repertoire at six months post infection by screening IgG from MBCs immortalized by genetic reprogramming to a germinal center-like state. Compared to the early plasmablast response, we found a drastic reduction in the DENV-specific population in the memory phase of the B cell response. There was a unexpectedly high degree of DENV2 type-specificity in the convalescent MBC pool, though heterotypic clones were also observed. MBC-derived mAbs mainly reacted to epitopes present on whole virions. Our results suggest that controlled DENV2 infection elicits a broad and complex early B cell response to DENV that contracts to yield a DENV-specific MBC compartment. These results provide insights into the basis for long-term serotype-specific humoral immunity and provide additional metrics by which to assess DENV vaccines.

There is significant debate regarding whether B cells and their antibodies contribute to effective anti-cancer immune responses. Here we show that patients with metastatic but non-progressing melanoma, lung adenocarcinoma, or renal cell carcinoma exhibited increased levels of blood plasmablasts. We used a cell-barcoding technology to sequence their plasmablast antibody repertoires, revealing clonal families of affinity matured B cells that exhibit progressive class switching and persistence over time. Anti-CTLA4 and other treatments were associated with further increases in somatic hypermutation and clonal family size. Recombinant antibodies from clonal families bound non-autologous tumor tissue and cell lines, and families possessing immunoglobulin paratope sequence motifs shared across patients exhibited increased rates of binding. We identified antibodies that caused regression of, and durable immunity toward, heterologous syngeneic tumors in mice. Our findings demonstrate convergent functional anti-tumor antibody responses targeting public tumor antigens, and provide an approach to identify antibodies with diagnostic or therapeutic utility.

Antagonists of the TRPV4 receptor were identified using a focused screen, followed by a limited optimization program. The leading compounds obtained from this exercise, RN-1665 23 and RN-9893 26, showed moderate oral bioavailability when dosed to rats. The lead molecule, RN-9893 26, inhibited human, rat and murine variants of TRPV4, and showed excellent selectivity over related TRP receptors, such as TRPV1, TRPV3 and TRPM8. The overall profile for RN-9893 may permit its use as a proof-of-concept probe for in vivo applications.

Fatty acid amide hydrolase (FAAH) inhibition is a promising target for the treatment of pain, anxiety and depression. The vast majority of FAAH inhibitors contain an electrophilic moiety and are known to react covalently with the enzyme. Herein we present the discovery of potent inhibitors, such as RN-450 29, which are based upon a novel tetrahydropyridopyridine scaffold lacking an obvious electrophilic site, and which appear to inhibit FAAH in a reversible and non-covalent manner.

2,5-Disubstituted benzoxazole derivatives were evaluated for their ability to inhibit hFAAH. Structure–activity studies indicated that an isoindoline group at the 2-position of the benzoxazole ring gave rise to particularly potent inhibitors. Further refinement resulted in compounds, such as 50, with low nanomolar potencies against hFAAH. Preliminary biochemical experiments revealed that model benzoxazole FAAH inhibitors inhibited the enzyme in a manner consistent with a reversible mechanism. Additionally, the species dependency of FAAH inhibition was measured. Of the species tested, inhibition of rabbit FAAH, but not rat and guinea pig FAAH, appeared to show close alignment to human FAAH. These results may suggest similarities between the active sites of the FAAH enzyme for these two species, and may also suggest that rabbit could be a viable species with which to conduct preclinical testing with benzoxazole FAAH inhibitors.

The functional activity of a number of ion channels is highly sensitive to large changes in temperature. Foremost among these are the thermosensing TRP channels which include cold- (TRPM8, TRPA1), warmth- (TRPV3, TRPV4), and heat-sensing (TRPV1, TRPV2) members. TRPV1, also known as the vanilloid receptor (VR1), is activated by ligands such as capsaicin, acidic pH, and heat (an increase in temperature to approximately 42 degrees C will lead to channel opening). Screening against the thermal gating of TRPV1 is generally performed using perfusion systems or water baths for temperature control, in conjunction with electrophysiology or Ca2 + influx readouts for direct functional assessment. These approaches are very useful, but have limited throughput or minimal thermo-temporal control. A standard real-time PCR machine with standard microplates allowed us to combine fluorescent Ca2 + detection with precise temperature manipulation to develop a homogeneous (Z' = 0.53), cell-based assay that uses temperature as the agonist. A temperature response curve of TRPV1 was obtained, which provided a T50 of 46.1 degrees C, and IC50 values against heat agonism were determined for known TRPV1 antagonists. Furthermore, we expanded this approach to a cold-activated ion channel, TRPM8. We developed and validated an analytical technique with broad applications for the study and screening of temperature-gated ion channels.

TRPV4, a close relative of the vanilloid receptor TRPV1, is activated by diverse modalities such as endogenous lipid ligands, hypotonicity, protein kinases and, possibly, mechanical inputs. While its multiple roles in vivo are being explored with KO mice and selective agonists, there is a dearth of selective antagonists available to examine TRPV4 function. Herein we detail the use of a focused library of commercial compounds in order to identify RN-1747 and RN-1734, a pair of structurally related small molecules endowed with TRPV4 agonist and antagonist properties, respectively. Their activities against human, rat and mouse TRPV4 were characterized using electrophysiology and intracellular calcium influx. Significantly, antagonist RN-1734 was observed to completely inhibit both ligand- and hypotonicity-activated TRPV4. In addition, RN-1734 was found to be selective for TRPV4 in a TRP selectivity panel including TRPV1, TRPV3 and TRPM8, and could thus be a valuable pharmacological probe for TRPV4 studies.

AP18 1 was recently disclosed as an antagonist of the TRPA1 receptor by the research group of Patapoutian. However, no detailed structure–activity relationships around 1 have been disclosed. Thus, a small number of oximes related to AP18 were examined in order to characterize the determinants of TRPA1 activity. Congeners of AP18 were found to possess both agonist and antagonist activity, suggesting that AP18 may behave as a covalent antagonist of the TRPA1 ion-channel.

The screening of known medicinal agents against new biological targets has been shown to be a valuable approach for revealing new pharmacology of marketed compounds. Recently, carbamate, urea and ketone inhibitors of fatty acid amide hydrolase (FAAH) have been described as promising treatments for pain, anxiety, depression and other CNS-related conditions. In order to find novel FAAH inhibitors, a focused screen of molecules containing potentially reactive moieties or having in vivo effects that are possibly relevant to the biology of FAAH was conducted. These studies revealed phenmedipham 13 and amperozide 14 to be inhibitors of human FAAH, with an IC(50) of 377 nM and 1.34 microM, respectively.