MHC class I (MHC I)–restricted virus-specific CTLs are implicated as critical components in the control of this naturally occurring lentivirus and in the protective immune response to the successfully applied attenuated equine infectious anemia virus vaccine in the horse. Nevertheless, the structural basis for how the equine MHC I presents epitope peptides remains unknown. In this study, we investigated the binding of several equine infectious anemia virus–derived epitope peptides by the ability to refold recombinant molecules and by thermal stability, and then by determining the x-ray structure of five peptide–MHC I complexes: equine MHC class I allele (Eqca)-N*00602/Env-RW12, Eqca-N*00602/Gag-GW12, Eqca-N*00602/Rev-QW11, Eqca-N*00602/Gag-CF9, and Eqca-N*00601/Gag-GW12. Although Eqca-N*00601 and Eqca-N*00602 differ by a single amino acid, Eqca-N*00601 exhibited a drastically different peptide presentation when binding a similar CTL epitope, Gag-GW12; the result makes the previously reported function clear to be non–cross-recognition between these two alleles. The structures plus Eqca-N*00602 complexed with a 9-mer peptide are particularly noteworthy in that we illuminated differences in apparent flexibility in the center of the epitope peptides for the complexes with Gag-GW12 as compared with Env-RW12, and a strict selection of epitope peptides with normal length. The featured preferences and unconventional presentations of long peptides by equine MHC I molecules provide structural bases to explain the exceptional anti-lentivirus immunity in the horse. We think that the beneficial reference points could serve as an initial platform for other human or animal lentiviruses.
β(2)-Microglobulin (β(2)m) is an essential subunit of the major histocompatibility complex (MHC) class I molecule that helps to stabilize the structure of peptide-MHC I (pMHC I). It is also one of the typical immunoglobulin superfamily (IgSF) molecules in the adaptive immune system (AIS). Sharks belong to the cartilaginous fish, which are the oldest jawed vertebrate ancestors with an AIS to exist in the world. Thus, the study of cartilaginous fish β(2)m would help in understanding the evolution of IgSF molecules. In order to demonstrate this, β(2)m from a cartilaginous fish, nurse shark (Ginglymostoma cirratum), was expressed, refolded, purified and crystallized. Diffraction data were collected to a resolution of 2.3 Å. The crystal belonged to space group P3(2)21, with unit-cell parameters a = b = 88.230, c = 67.146 Å. The crystal structure contained two molecules in the asymmetric unit. The results will provide structural information for study of the evolution of β(2)m and IgSF in the AIS.
The emergence of adaptive immunity in jawed vertebrates depended on the appearance of variable immune receptors, BCRs and TCRs, which exhibit variable-J-constant (V-C)-type Ig superfamily folds. Hitherto, however, the structures of IgV-J-IgC-type molecules had never been characterized in invertebrates, leaving the origin of BCR/TCR-type molecules unknown. Using x-ray crystallography, the structure of a V-C2 molecule, named AmpIgV-C2, was determined in amphioxus (). The first domain shows typical V folding, including the hydrophobic core, CDR analogs, and eight conserved residues. The second domain is a C2-type Ig superfamily domain, as defined by its short length and the absence of β-strand D- and C1-typical motifs. AmpIgV-C2 molecules form homodimers, using "three-layer packing dimerization," as described for TCRs and BCRs. The AmpIgV-C2 V domain harbors a diglycine motif in β-strand G and forms a β-bulge structure participating in V-V intermolecular interaction. By immunohistochemistry, AmpIgV-C2 molecules were primarily found in mucosal tissues, whereas PCR and sequence analysis indicated considerable genetic variation at the single-gene level; these findings would be consistent with an immune function and a basic ability to adapt to binding different immune targets. Our results show a BCR/TCR-ancestral like molecule in amphioxus and help us to understand the evolution of the adaptive immune system.
C-reactive protein (CRP) is an acute phase protein that is found in blood, the concentration of which in plasma rises rapidly in response to inflammation. It functions as a pattern-recognition molecule, recognizing dead cells and various pathogenic agents and eliminating them by utilizing the classical complement pathway and activating macrophages. CRP is phylogenetically highly conserved in invertebrates and mammals. To date, information on the CRP gene has been reported from numerous species of animals, but little is known about the structure of CRP from species other than humans. In order to solve the structure of CRP from bony fish, the CRP gene from zebrafiah (Danio rerio) was cloned and expressed in Escherichia coli. The zebrafish CRP (Dare-CRP) was then purified and crystallized. The crystal diffracted to 2.3 Å resolution and belonged to space group R3, with unit-cell parameters a = b = 114.7, c = 61.0 Å. The Matthews coefficient and solvent content were calculated to be 3.28 Å(3) Da(-1) and 62.55%, respectively. Determination of the zebrafish CRP structure should be helpful in investigating the evolution of CRPs in the innate immune system.
The generation of a potent humoral immune response by B cells relies on the integration of signals induced by the B cell receptor, toll-like receptors and both negative and positive co-receptors. Several reports also suggest that integrin signaling plays an important role in this process. How integrin signaling is regulated in B cells is however still partially understood. Integrin activity and function are controlled by several mechanisms including regulation by molecular adaptors of the paxillin family. In B cells, Leupaxin (Lpxn) is the most expressed member of the family and in vitro studies suggest that it could dampen BCR signaling. Here, we report that Lpxn expression is increased in germinal center B cells compared to naïve B cells. Moreover, Lpxn deficiency leads to decreased B cell differentiation into plasma cells in vitro. However, Lpxn seems dispensable for the generation of a potent B cell immune response in vivo. Altogether our results suggest that Lpxn is dispensable for T-dependent and T-independent B cell immune responses.
In order to clarify the structure and the peptide-presentation characteristics of the equine major histocompatibility complex (MHC) class I molecule, a complex of equine MHC class I molecule (ELA-A1 haplotype, 7-6 allele) with mouse 2 -microglobulin and the cytotoxic T lymphocyte (CTL) epitope Env-RW12 (RVEDVTNTAEYW) derived from equine infectious anaemia virus (EIAV) envelope protein (residues 195-206) was refolded and crystallized. The crystal, which belonged to space group P2 1 , diffracted to 2.3 Å resolution and had unitcell parameters a = 82.5, b = 71.4, c = 99.8 Å , = 102.9 . The crystal structure contained two molecules in the asymmetric unit. These results should help to determine the first equine MHC class I molecule structure presenting an EIAV CTL epitope.
We developed a single domain VHH multi-specific antibody format. Multispecific antibodies have multiple mechanisms of action which may work independently or together, to achieve better clinical outcomes in cancers with high unmet medical need such as SCLC. Here we describe the preclinical development of a trispecific antibody (KB-436) that targets Dopamine Receptor 2 (DRD2), PD-1 and CD47. DRD2 is a G protein-coupled receptor upregulated in many cancer types where it correlates with decreased patient survival. In pre-clinical studies DRD2 is associated with cancer cell stemness and tumor growth. Clinical responses were achieved with small molecules targeting DRD2 and dopaminergic drugs. In SCLC, representing 15% of lung cancers, 60-70% of patients showed high expression of DRD2. Checkpoint inhibition has shown some efficacy in lung cancer where PD-L1 inhibitors were approved as first line therapy in SCLC . SCLC patients rapidly fail chemotherapy, develop resistance to treatment including to immunotherapy associated with lack of tumor infiltrating immune cells, appearance of metastases and large numbers of circulating tumor cells. These observations suggest a link between DRD2 expression and resistance to treatment, making this receptor an attractive target for a multispecific therapy. CD47 is overexpressed by many cancers and is also expressed on lymphocytes. Here, targeting CD47 is integral to T-cell engagement and redirection, in a mechanism distinct from CD3 mediated T-cell engagement. The VHH modules of KB-436 (anti-DRD2, anti-PD-1 and anti-CD47) mediate multiple mechanisms of action to achieve anti-tumor effect. The anti-DRD2 VHH induces intracellular signaling, the anti-PD-1 VHH restores T cell function, and the anti-CD47 VHH recruits T cells without their generalized activation and blocks interaction of CD47 with SIRPa. Treatment with anti-DRD2 antibody significantly suppressed tumor growth in the DRD2-positive NCI-H510A SCLC model in SCID mice. KB-436 anti-tumor efficacy was tested in several in vivo immuno-oncology xenograft models of human SCLC and other solid tumors , reconstituted with human PBMC or with CD34+ hemopoietic stem cells. Treatment suppressed tumor growth, enhanced the in vivo effect of cisplatin-treatment in a less chemosensitive NCI-H69 variant, blocked metastases formation in CD34+ humanized NCG mice bearing established NCI-H69 tumors, and blocked metastases formation and increased survival in tail vein metastatic models. Trispecific KB-436 has a half-life in mice of around 5 days similar to that of other antibodies. It is produced at high yield (6 g/L) in a manufacturing cell line, conventional purification yields 99% purity and notably displays high stability under accelerated stability testing. In conclusion, the trispecific KB-436 antibody, has strong in vivo anti-tumor activity mediated via multiple mechanisms of action, is easily expressed and purified and is very stable. Together, this data supports the clinical development of KB-436 in advanced metastatic solid cancer indications, including SCLC. Citation Format: Shugang Yao, Yun Cui, Anna Kazanats, Liying Gong, Claire Bonfils, Dominic Hou, Emily Chen, Elijus Undzys, Jacynthe Toulouse, Milica Krstic, Hiba Zahreddine, Israel Matos, Alex Zhou, Aniel Moya-Torres, Richard Wargachuk, Carl Gay, Lauren Byers, Gordon Ngan, Luis da Cruz, David Young. Pre-clinical development of a dopamine receptor 2, PD-1 and CD47 trispecific antibody for treatment of small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P200.
Trophoblast cell surface antigen-2 (TROP2) is a membrane-bound protein expressed during development and at lower levels in normal adult tissues. In many solid tumors TROP2 upregulation is associated with increased tumor aggressiveness, metastasis, and decreased survival in difficult-to-treat cancers, making it an attractive target for cancer therapy. TROP2 ADCs have been developed and one has been approved in breast cancer and advanced bladder cancer. The toxicity associated with the toxin payload and the development of resistance to the payload limits their use. We previously described an anti-TROP2 heavy chain-only antibody (HCAb), KJ-103. KJ-103 has demonstrated high potency in in vivo preclinical human models, including breast cancer, colon cancer, esophageal carcinoma and pancreatic cancer. In vivo efficacy of KJ-103 was strongly diminished by use of a IgG4-FALA variant which has negligible interaction with Fc gamma receptors indicating that effector function is the primary mechanism of action. Activity in mouse strains with deficiency in different immune cell types showed that macrophages were the predominant effector cell responsible for in vivo activity. Analysis of the xenograft microenvironment from different cancer models established a clear correlation between the in vivo efficacy and the degree of macrophage infiltration that was independent of the CD47 IHC score. In vitro, KJ-103 induces the phagocytosis by human macrophages of M0, M1 and M2 subtypes. KJ-103 also induced phagocytosis by mouse macrophages, that reflected human CD47 (huCD47)/mouse SIRPα (mSIRPα) affinity. Importantly, in vivo tumor regression was observed in NCG mouse models where there is nM huCD47/mSIRPα alpha affinity. In vivo dose response in SCID and NCG mice which have different huCD47/mSIRPα affinity showed tumor regression at doses levels of 2-10mg/kg in both strains. The huCD47/mSIRPα affinity effect was only obvious at low dose levels (0.5mg/kg). In a dose range finding study in cynomolgus monkeys KJ-103 was very well tolerated with no abnormal observations in clinical parameters and blood clinical chemistry and hematology. KJ-103 has been humanized and has the same degree of activity as the parental antibody. Immunohistochemistry showed that the KJ-103 epitope was expressed across many tumor types, including breast, colon, ovarian, and head and neck among others. KJ-103 is a HCAB that has demonstrated high levels of preclinical potency and safety making it suitable for clinical development for TROP2 positive tumors. Citation Format: Shugang Yao, Hiba Zahreddine, Amit Subedi, Liying Gong, Amogh Nair, Alex Zhou, Tiffany Cheng, Elijus Undzys, Dominic Hou, Lucy Lai, Luis A. Da Cruz, David Young. Functional characterization of KJ-103, a novel therapeutic anti-TROP2 heavy chain-only antibody [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 2931.
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