ABSTRACT:The landscape of cancer treatment has been transformed by immune checkpoint inhibitors; however, the failure to benefit a large number of patients with cancer has underlined the need to identify promising targets for more effective interventions. In this study, we leverage 23andMe, Inc.’s large-scale human germline genetic and health database to uncover the previously unknown role of UL16-binding protein 6 (ULBP6), a high-affinity NK group 2D (NKG2D) ligand, in cancer and its promise as an immuno-oncology therapeutic target. We confirm ULBP6 expression in human tumors and demonstrate that soluble ULBP6 shed from tumors circumvents NKG2D activation provided by membrane-anchored NKG2D ligands to inhibit immune cell activation and tumor cell killing. Based on these findings, we developed 23ME-01473, a humanized Fc effector–enhanced antibody that binds to ULBP6 and its closely related family members, ULBP2 and ULBP5. 23ME-01473 effectively blocks soluble ULBP6-mediated immunosuppression to restore the NKG2D axis on NK and T cells to elicit tumor growth control. Moreover, the Fc effector–enhanced design of 23ME-01473 increases its binding affinity to fragment crystallizable gamma receptor IIIa, which, together with 23ME-01473’s binding to membrane-anchored ULBP6/2/5 on cancer cells, allows for augmented antibody-dependent cellular cytotoxicity induction, providing a second activation node for NK cells. Our studies demonstrate the therapeutic potential of an Fc effector–enhanced anti-ULBP6/2/5 antibody to reinvigorate NK cell and T-cell activation and cytotoxicity for the treatment of cancer. SIGNIFICANCE:This study emphasizes the utility of population-based genome-wide assessments for discovering naturally occurring genetic variants associated with lifetime risks for cancer or immune diseases as novel drug targets. We identify ULBP6 as a potential keystone member of the NKG2D pathway, which is important for antitumor immunity. Targeting ULBP6 may hold therapeutic promise for patients with cancer.

Characterization of the binding and blocking activity of 23ME-01473

Abstract Hypertension remains a leading cause of cardiovascular and kidney diseases. Failure to control blood pressure with ≥ 3 medications or control requiring ≥ 4 medications is classified as resistant hypertension (rHTN) and new therapies are needed to reduce the resulting increased risk of morbidity and mortality. Here, we report genetic evidence that relaxin family peptide receptor 2 (RXFP2) is associated with rHTN in men, but not in women. This study shows that adrenal gland gene expression of RXFP2 is increased in men with hypertension and the RXFP2 natural ligand, INSL3, increases adrenal steroidogenesis and corticosteroid secretion in human adrenal cells. To address the hypothesis that RXFP2 activation is an important mechanism in rHTN, we discovered and characterized small molecule and monoclonal antibody (mAb) blockers of RXFP2. The novel chemical entities and mAbs show potent, selective inhibition of RXFP2 and reduce aldosterone and cortisol synthesis and release. The RXFP2 mAbs have suitable rat pharmacokinetic profiles to evaluate the role of RXFP2 in the development and maintenance of rHTN. Overall, we identified RXFP2 activity as a potential new mechanism in rHTN and discovered RXFP2 antagonists for the future interrogation of RXFP2 in cardiovascular and renal diseases.

Bispecific antibodies have gained increasing popularity as therapeutics as they enable novel activities that cannot be achieved with monospecific antibodies. Some of the most popular bispecific formats are molecules in which two Fab arms with different antigen specificities are combined into one IgG-like molecule. One way to produce these bispecific molecules requires the discovery of antibodies against the two antigens of interest that share a common light chain. Here, we present the generation and characterization of a common light chain mouse model, in which the endogenous IGKJ cluster is replaced with a prearranged, modified murine IGKV10-96/IGKJ1 segment. We demonstrate that genetic modification does not impact B-cell development. Upon immunization with ovalbumin, the animals generate an antibody repertoire with VH gene segment usage of a similar diversity to wildtype mice, while the light chain diversity is restricted to antibodies derived from the prearranged IGKV10-96/IGKJ1 germline. We further show that the clonotype diversity of the common light chain immune repertoire matches the diversity of immune repertoire isolated from wildtype mice. Finally, the common light chain anti-ovalbumin antibodies have only slightly lower affinities than antibodies isolated from wildtype mice, demonstrating the suitability of these animals for antibody discovery for bispecific antibody generation.

Abstract Using genome-wide association studies (GWAS) of the 23andMe genetic and healthy survey database across all phenotypes as part of our unique target discovery platform, we identified immuno-oncology (I/O)-related genetic variants as those with opposing directionality of effects on cancer and immunological phenotypes, which comprise the I/O signature. This I/O signature identified ULBP6 as a potential cancer therapeutic target. ULBP6 is a stress-induced cell surface ligand found on cancer cells, that binds to the activating receptor, NKG2D, on NK and T cells to induce tumor cell killing. However, as a mechanism of immune escape, tumors shed ULBP6 to produce a soluble form that attenuates NKG2D activation and is elevated in cancers. Given that the 2 allelic variants of ULBP6 have the highest binding affinity to NKG2D of all human NKG2D ligands, ULBP6 may be the most immunosuppressive soluble NKG2D ligand and a critical regulator of anti-tumor response. Accordingly, soluble ULBP6 exhibited significant suppression of IFNγ release and detectable cell surface NKG2D expression on healthy donor PBMCs using in vitro cell-based assays. Moreover, the overexpression of human ULBP6 in a genetically modified MC38 murine model resulted in decreased infiltration of NK, NKT, and CD8 T cells in vivo. As such, we developed a novel humanized monoclonal antibody, 23ME-01473, that binds with high affinity to ULBP6, and due to the high sequence homology, to ULBP2 and ULBP5, to block their interaction with NKG2D. By preventing the binding of soluble ULBP6 to NKG2D on PBMCs in vitro, 23ME-01473 restored NKG2D activation as measured by increased detectable cell surface NKG2D expression, IFNγ and granzyme B release, and tumor cell killing by PBMCs. To further leverage the binding of 23ME-01473 to ULBP6/2/5, 23ME-01473 was designed to have an afucosylated Fc domain that enhances its binding affinity to the activating Fc receptor, FcγRIIIa, on NK cells to induce ADCC against tumor cells. The combination of NKG2D and FcγRIIIa activation resulted in synergistic PBMC-mediated IFNγ secretion and enhanced tumor cell killing, compared to activation of either NKG2D or FcγRIIIa. Taken together, these results reveal ULBP6 as a novel cancer therapeutic target, and given the dual activation of NKG2D and FcγRIIIa, suggest the potential of 23ME-01473 to activate NK cells and elicit anti-tumor immunity. Citation Format: Joel Benjamin, Abigail Jarret, Shashank Bharill, Shruti Yadav, Dina Ayupova, Clifford Hom, Zahra Bahrami Dizicheh, I-Ling Chen, Anh Diep, Shi Shi, Caroline Bonnans, Danielle Kellar, Germaine Fuh, Maike Schmidt, Kimberline Gerrick, Patrick Koenig, Mauro Poggio. 23ME-01473, a novel anti-ULBP6/2/5 monoclonal antibody, reinvigorates anti-tumor NK cell function through NKG2D and FcγRIIIa activation [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 2375.

VHHs or nanobodies are single antigen binding domains originating from camelid heavy-chain antibodies. They are used as diagnostic and research tools and in a variety of therapeutic molecules. Analyzing variable domain structures from llama and alpaca we found that VHHs can be classified into two large structural clusters based on their CDR-H3 conformation. Extended CDR-H3 loops protrude into the solvent, whereas kinked CDR-H3 loops fold back onto framework regions. Both major families have distinct properties in terms of their CDR-H3 secondary structure, how their CDR-H3 interacts with the framework region and how they bind to antigens. We show that the CDR-H3 conformation of VHHs correlates with the germline from which the antibodies are derived: IGHV3-3 derived antibodies almost exclusively adopt a kinked CDR-H3 conformation while the CDR-H3 adopts an extended structure in most IGHV3S53 derived antibodies. We do not observe any bias stemming from V(D)J recombination in llama immune repertoires, suggesting that the correlation is the result of selection processes during B-cell development. Our findings demonstrate a previously undescribed impact of germline usage on antigen interaction and contribute to a better understanding on how properties of the antibody framework shape the immune repertoire.

The chromatin remodeling protein alpha thalassemia/mental retardation syndrome X-linked (ATRX) is a component of promyelocytic leukemia nuclear bodies (PML-NBs) and thereby mediates intrinsic immunity against several viruses including human cytomegalovirus (HCMV). As a consequence, viruses have evolved different mechanisms to antagonize ATRX, such as displacement from PML-NBs or degradation. Here, we show that depletion of ATRX results in an overall impaired antiviral state by decreasing transcription and subsequent secretion of type I IFNs, which is followed by reduced expression of interferon-stimulated genes (ISGs). ATRX interacts with the transcription factor interferon regulatory factor 3 (IRF3) and associates with the IFN-β promoter to facilitate transcription. Furthermore, whole transcriptome sequencing revealed that ATRX is required for efficient IFN-induced expression of a distinct set of ISGs. Mechanistically, we found that ATRX positively modulates chromatin accessibility specifically upon IFN signaling, thereby affecting promoter regions with recognition motifs for AP-1 family transcription factors. In summary, our study uncovers a novel co-activating function of the chromatin remodeling factor ATRX in innate immunity that regulates chromatin accessibility and subsequent transcription of interferons and ISGs. Consequently, ATRX antagonization by viral proteins and ATRX mutations in tumors represent important strategies to broadly compromise both intrinsic and innate immune responses.

Human fibroblasts represent the most extensively used cell type for the investigation of lytic human cytomegalovirus (HCMV) replication. However, analyzing the function of specific proteins during infection can be challenging since primary cells are difficult to transfect. An alternative approach is the use of lentiviral transduction with vectors for stable or inducible shRNA expression. This approach provides a versatile tool to study the role of host cell factors during HCMV infection. The essential steps to achieve an efficient target protein knockdown are shRNA design, cloning, generation of transgenic lentiviral particles, and, finally, transduction of the cells. However, these steps are highly dependent on the selected vector system. Here we focus on two different vector systems and describe how to successfully generate stable and inducible knockdown fibroblasts. Additionally, we demonstrate different methods to validate the knockdown of the target protein.

Autophagy is a catabolic process contributing to intrinsic cellular defense by degrading viral particles or proteins; however, several viruses hijack this pathway for their own benefit. The role of autophagy during human cytomegalovirus (HCMV) replication has not been definitely clarified yet. Utilizing small interfering RNA (siRNA)-based screening, we observed that depletion of many autophagy-related proteins resulted in reduced virus release, suggesting a requirement of autophagy-related factors for efficient HCMV replication. Additionally, we could show that the autophagy-initiating serine/threonine protein kinase ULK1 as well as other constituents of the ULK1 complex were upregulated at early times of infection and stayed upregulated throughout the replication cycle. We demonstrate that indirect interference with ULK1 through inhibition of the upstream regulator AMP-activated protein kinase (AMPK) impaired virus release. Furthermore, this result was verified by direct abrogation of ULK1 kinase activity utilizing the ULK1-specific kinase inhibitors SBI-0206965 and ULK-101. Analysis of viral protein expression in the presence of ULK-101 revealed a connection between the cellular kinase ULK1 and the viral tegument protein pp28 (pUL99), and we identified pp28 as a novel viral substrate of ULK1 by in vitro kinase assays. In the absence of ULK1 kinase activity, large pp28- and pp65-positive structures could be detected in the cytoplasm at late time points of infection. Transmission electron microscopy demonstrated that these structures represent large perinuclear protein accumulations presumably representing aggresomes. Our results indicate that HCMV manipulates ULK1 and further components of the autophagic machinery to ensure the efficient release of viral particles. IMPORTANCE The catabolic program of autophagy represents a powerful immune defense against viruses that is, however, counteracted by antagonizing viral factors. Understanding the exact interplay between autophagy and HCMV infection is of major importance since autophagy-related proteins emerged as promising targets for pharmacologic intervention. Our study provides evidence for a proviral role of several autophagy-related proteins suggesting that HCMV has developed strategies to usurp components of the autophagic machinery for its own benefit. In particular, we observed strong upregulation of the autophagy-initiating protein kinase ULK1 and further components of the ULK1 complex during HCMV replication. In addition, both siRNA-mediated depletion of ULK1 and interference with ULK1 protein kinase activity by two chemically different inhibitors resulted in impaired viral particle release. Thus, we propose that ULK1 kinase activity is required for efficient HCMV replication and thus represents a promising novel target for future antiviral drug development.

Dipeptidase 3 (DPEP3) is one of three glycosylphosphatidylinositol-anchored metallopeptidases potentially involved in the hydrolytic metabolism of dipeptides. While its exact biological function is not clear, DPEP3 expression is normally limited to testis, but can be elevated in ovarian cancer. Antibody drug conjugates targeting DPEP3 have shown efficacy in preclinical models with a pyrrolobenzodiazepine conjugate, SC-003, dosed in a phase I clinical trial (NCT02539719). Here we reveal the novel atomic structure of DPEP3 alone and in complex with the SC-003 Fab fragment at 1.8 and 2.8 angstrom, respectively. The structure of DPEP3/SC-003 Fab complex reveals an eighteen-residue epitope across the DPEP3 dimerization interface distinct from the enzymatic active site. DPEP1 and DPEP3 extracellular domains share a conserved, dimeric TIM (beta/alpha)8-barrel fold, consistent with 49% sequence identity. However, DPEP3 diverges from DPEP1 and DPEP2 in key positions of its active site: a histidine to tyrosine variation at position 269 reduces affinity for the beta zinc and may cause substrate steric hindrance, whereas an aspartate to asparagine change at position 359 abolishes activation of the nucleophilic water/hydroxide, resulting in no in vitro activity against a variety of dipeptides and biological substrates (imipenem, leukotriene D4 and cystinyl-bis-glycine). Hence DPEP3, unlike DPEP1 and DPEP2, may require an activating co-factor in vivo or may remain an inactive, degenerate enzyme. This report sheds light on the structural discriminants between active and inactive membrane dipeptidases and provides a benchmark to characterize current and future DPEP3-targeted therapeutic approaches.

Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo.

Monoclonal antibodies developed for therapeutic or diagnostic purposes need to demonstrate highly defined binding specificity profiles. Engineering of an antibody to enhance or reduce binding to related antigens is often needed to achieve the desired biologic activity without safety concern. Here, we describe a deep sequencing-aided engineering strategy to fine-tune the specificity of an angiopoietin-2 (Ang2)/vascular endothelial growth factor (VEGF) dual action Fab, 5A12.1 for the treatment of age-related macular degeneration. This antibody utilizes overlapping complementarity-determining region (CDR) sites for dual Ang2/VEGF interaction with K-D in the sub-nanomolar range. However, it also exhibits significant (KD of 4 nM) binding to angiopoietin-1, which has high sequence identity with Ang2. We generated a large phage-displayed library of 5A12.1 Fab variants with all possible single mutations in the 6 CDRs. By tracking the change of prevalence of each mutation during various selection conditions, we identified 35 mutations predicted to decrease the affinity for Ang1 while maintaining the affinity for Ang2 and VEGF. We confirmed the specificity profiles for 25 of these single mutations as Fab protein. Structural analysis showed that some of the Fab mutations cluster near a potential Ang1/2 epitope residue that differs in the 2 proteins, while others are up to 15 angstrom away from the antigen-binding site and likely influence the binding interaction remotely. The approach presented here provides a robust and efficient method for specificity engineering that does not require prior knowledge of the antigen antibody interaction and can be broadly applied to antibody specificity engineering projects.

The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with subnanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies.

A mono-specific antibody may recruit a second antigen binding specificity, thus converting to a dual-specific Two-in-One antibody through mutation at the light chain complementarity-determining regions (CDRs). It is, however, unknown whether mutation at the heavy chain CDRs may evolve such dual specificity. Herein, we examined the CDRs of a humanized interleukin 4 (IL4) antibody using alanine scanning and structural modeling, designed libraries of mutants in regions that tolerate mutation, and isolated dual specific antibodies harboring mutation at the heavy chain CDRs only. We then affinity improved an IL4/IL5 dual specific antibody to variants with dissociation constants in the low nanomolar range for both antigens. The results demonstrate the full capacity of antibodies to evolve dual binding specificity.

Phage display is a powerful tool to isolate specific binders from a large and diverse combinatorial library. Here we provide a step-by-step protocol in how to set up a successful phage panning experiment in order to isolate useful antibodies. The protocol includes testing antigens for their suitability in the phage panning procedure and optimizing the panning conditions and alternative screening methods to minimize nonspecific binding. We describe example phage panning experiments starting from the library transformation to the phage clone screening.

The insertion of proteins into and the transport across outer membranes in cyanobacteria has a strong semblance to protein import into eukaryotic organelles, with a swap in directionality [1]. Specific transporters mediate the transfer across and the insertion into bio-membranes. We focus on the study of beta-barrel proteins that reside in the outer membranes and periplasmatic factors that mediate protein recognition and transport. The beta barrel proteins have a varying number of socalled POTRA domains (POlypeptideTRansport-Associated), with inherent flexibility between individual POTRA domains [2]. These recognise target proteins and facilitate protein transport through the beta barrel [2], [3]. Chaperones similar to protobacterial SurA and Skp/DegP assist cyanobacterial protein transport through the periplasm. The 3D structure of the cyanobacterial chaperone Tic22 has a “butterfly” shape revealing a repeat likely caused by gene duplication [4], [5]. Four helices point orthogonal at each other, adding up their dipole moments in a central cavity. The surface of the structure is dotted with hydrophobic pockets in which we identified bound solvent molecules. These likely represent binding sites for protein substrates. We demonstrate that Tic22 is present in the cyanobaterial periplasm as well as in thylakoids, and it can be functionally replaced by knock-in of a plant orthologue [4]. In the apicoplast organelle of unicellular parasites such as Plasmodium and Toxoplasma, Tic22 is essential for parasite survival and protein import into the apicoplast stroma [5]. The structural clues together with the functional data suggest that Tic22 can have a function in both, protein import or protein insertion, depending on the organism where it is found. The protein is conserved in bacteria, plants, and unicellular organisms and links these protein transporters to a common ancestry.

In Streptomyces griseus, AdpA is the central transcriptional factor in the A-factor regulatory cascade and activates approximate thousands of genes required for both secondary metabolism and morphological differentiation, leading to onset of streptomycin biosynthesis as well as aerial mycelium formation and sporulation [1].AdpA consists of two domains-the dimerization domain at its N-terminal portion and the DNA-binding domain at its C-terminal portion-and belongs to the AraC/XylS family of transcriptional regulators.The DNA-binding domain of AdpA is characterized by two helix-turn-helix DNA-binding motifs [2] and shows a low nucleotide sequence specificity [3].To reveal the molecular basis of the low nucleotide sequence specificity, we have determined the crystal structure of the complex of DNA-binding domain of AdpA (AdpA-DBD) and a 14-mer duplex DNA with two-nucleotide overhangs at 5'-ends at 2.9-Å resolution.The crsytal belonged to the space group C222 1 , with unit cell parameters a = 77.0Å, b = 101.3Å, and c = 101.5 Å, and contained one complex ofAdpA-DBD and dsDNA in an asymmetric unit.The crystal structure was solved by molecular replacement.The model was refined to R factor /R free values of 19.7/25.2%.AdpA-DBD comprises two helix-turn-helix (HTH) motifs linked by a long a-helix.The N-terminal HTH motif engages the major groove of the binding site, whereas the C-terminal HTH motif only binds to phosphate groups and half-inserts into adjacent major groove of the dsDNA.The linker helix is also involved in interactions with the DNA and imposes the orientation and distance restraints on the two HTH motifs for proper binding.These interactions stabilize the complex of AdpA-DBD and dsDNA and reflect the sequence specificity of AdpA-DBD.By comparing the sequence specificity and the crystal structure of AdpA-DBD with those ofother AraC/XylS family members, MarA and Rob.we discovered that AdpA-DBD possesses the lowest DNA-binding specificity, which results in AdpA directly controls many more genes than these global transcriptional regulators for bacterial differentiation.To the best of our knowledge, the AdpAregulon seems to be the largest one in bacteria.

Mitochondria and chloroplasts are of endosymbiotic origin. Their integration into cells entailed the development of protein translocons, partially by recycling bacterial proteins. We demonstrate the evolutionary conservation of the translocon component Tic22 between cyanobacteria and chloroplasts. Tic22 in Anabaena sp. PCC 7120 is essential. The protein is localized in the thylakoids and in the periplasm and can be functionally replaced by a plant orthologue. Tic22 physically interacts with the outer envelope biogenesis factor Omp85 in vitro and in vivo, the latter exemplified by immunoprecipitation after chemical cross-linking. The physical interaction together with the phenotype of a tic22 mutant comparable with the one of the omp85 mutant indicates a concerted function of both proteins. The three-dimensional structure allows the definition of conserved hydrophobic pockets comparable with those of ClpS or BamB. The results presented suggest a function of Tic22 in outer membrane biogenesis.