KRAS G12V neoantigen specific T cell receptor for adoptive T cell therapy against tumors.

KRAS mutations are broadly recognized as promising targets for tumor therapy. T cell receptors (TCRs) can specifically recognize KRAS mutant neoantigens presented by human lymphocyte antigen (HLA) and mediate T cell responses to eliminate tumor cells. In the present study, we identify two TCRs specific for the 9-mer KRAS-G12V mutant neoantigen in the context of HLA-A*11:01. The TCR-T cells are constructed and display cytokine secretion and cytotoxicity upon co-culturing with varied tumor cells expressing the KRAS-G12V mutation. Moreover, 1-2C TCR-T cells show anti-tumor activity in preclinical models in female mice. The 9-mer KRAS-G12V mutant peptide exhibits a distinct conformation from the 9-mer wildtype peptide and its 10-mer counterparts. Specific recognition of the G12V mutant by TCR depends both on distinct conformation from wildtype peptide and on direct interaction with residues from TCRs. Our study reveals the mechanisms of presentation and TCR recognition of KRAS-G12V mutant peptide and describes TCRs with therapeutic potency for tumor immunotherapy.

Limited Cross-Linking of 4-1BB by 4-1BB Ligand and the Agonist Monoclonal Antibody Utomilumab.

Monoclonal antibodies (mAbs) targeting the co-stimulatory molecule 4-1BB are of interest for tumor immunotherapy. We determined the complex structures of human 4-1BB with 4-1BB ligand (4-1BBL) or utomilumab to elucidate the structural basis of 4-1BB activation. The 4-1BB/4-1BBL complex displays a typical TNF/TNFR family binding mode. The structure of utomilumab/4-1BB complex shows that utomilumab binds to dimeric 4-1BB with a distinct but partially overlapping binding area with 4-1BBL. Competitive binding analysis demonstrates that utomilumab blocks the 4-1BB/4-1BBL interaction, indicating the interruption of ligand-mediated signaling. The binding profiles of 4-1BBL and utomilumab to monomeric or dimeric 4-1BB indicate limited cross-linking of 4-1BB molecules. These findings provide mechanistic insight into the binding of 4-1BB with its ligand and its agonist mAb, which may facilitate the future development of anti-4-1BB biologics for tumor immunotherapy.

Remarkably similar CTLA-4 binding properties of therapeutic ipilimumab and tremelimumab antibodies.

Monoclonal antibody based immune checkpoint blockade therapies have achieved clinical successes in management of malignant tumors. As the first monoclonal antibody targeting immune checkpoint molecules entered into clinics, the molecular basis of ipilimumab-based anti-CTLA-4 blockade has not yet been fully understood. In the present study, we report the complex structure of ipilimumab and CTLA-4. The complex structure showed similar contributions from VH and VL of ipilimumab in binding to CTLA-4 front ß-sheet strands. The blockade mechanism of ipilimumab is that the strands of CTLA-4 contributing to the binding to B7-1 or B7-2 were occupied by ipilimumab and thereafter prevents the binding of B7-1 or B7-2 to CTLA-4. Though ipilimumab binds to the same epitope with tremelimumab on CTLA-4 with similar binding affinity, the higher dissociation rate of ipilimumab may indicate the dynamic binding to CTLA-4, which may affect its pharmacokinetics. The molecular basis of ipilimumab-based anti-CTLA-4 blockade and comparative study of the binding characteristics of ipilimumab and tremelimumab would shed light for the discovery of small molecular inhibitors and structure-based monoclonal antibody optimization or new biologics.

CTL immunogenicity of Rv3615c antigen and diagnostic performances of an ESAT-6/CFP-10/Rv3615c antigen cocktail for Mycobacterium tuberculosis infection.

T cell immune responses have played pivotal roles in host immune protection against Mycobacterium tuberculosis (MTB) infection. MTB specific antigen, Rv3615c (EspC), was identified to be as immunodominant as the well-known ESAT-6 and CFP-10, and has brought promising expectations to more sensitive T-cell based diagnosis and vaccine development. However, limited knowledge about the immunogenicity and diagnostic values of this antigen has restricted its application in clinical practice. Herein, the Rv3615c antigen was identified as a robust CTL immunoantigen with broadly cross-human leucocyte antigen (HLA) allele recognized peptides which may contribute to the broad recognition of Rv3615c antigen among the population. A three-antigen-cocktail (3-Ag-cocktail) comprising of ESAT-6, CFP-10 and Rv3615c was investigated in a multicenter, randomized and double-blinded study to evaluate its clinical diagnostic performances. A significantly improved sensitivity was demonstrated against the 3-Ag-cocktail compared with that against ESAT-6 and CFP-10. Both responsive magnitude and sensitivity were significantly lower in patients concurrently suffering from cancer, indicating its restriction in diagnosis of immunocomprised patients. In conclusion, inclusion of the Rv3615c antigen with multiple HLA restricted CTL epitopes would benefit the T-cell based diagnosis of MTB infection.

Crystal clear: visualizing the intervention mechanism of the PD-1/PD-L1 interaction by two cancer therapeutic monoclonal antibodies.

Antibody-based PD-1/PD-L1 blockade therapies have taken center stage in immunotherapies for cancer, with multiple clinical successes. PD-1 signaling plays pivotal roles in tumor-driven T-cell dysfunction. In contrast to prior approaches to generate or boost tumor-specific T-cell responses, antibody-based PD-1/PD-L1 blockade targets tumor-induced T-cell defects and restores pre-existing T-cell function to modulate antitumor immunity. In this review, the fundamental knowledge on the expression regulations and inhibitory functions of PD-1 and the present understanding of antibody-based PD-1/PD-L1 blockade therapies are briefly summarized. We then focus on the recent breakthrough work concerning the structural basis of the PD-1/PD-Ls interaction and how therapeutic antibodies, pembrolizumab targeting PD-1 and avelumab targeting PD-L1, compete with the binding of PD-1/PD-L1 to interrupt the PD-1/PD-L1 interaction. We believe that this structural information will benefit the design and improvement of therapeutic antibodies targeting PD-1 signaling.

Seeing is believing: anti-PD-1/PD-L1 monoclonal antibodies in action for checkpoint blockade tumor immunotherapy.

Structural immunology, focusing on structures of host immune related molecules, enables the immunologists to see what the molecules look like, and more importantly, how they work together. Antibody-based PD-1/PD-L1 blockade therapy has achieved brilliant successes in clinical applications. The recent breakthrough of the complex structures of checkpoint blockade antibodies with their counterparts, pembrolizumab with PD-1 and avelumab with PD-L1, have made it clear how these monoclonal antibodies compete the binding of PD-1/PD-L1 and function to blockade the receptor-ligand interaction. Herein, we summarize the structural findings of these two reports and look into the future for how this information would facilitate the development of more efficient PD-1/PD-L1 targeting antibodies, small molecule drugs, and other protein or non-protein inhibitors.

Structure of measles virus hemagglutinin bound to its epithelial receptor nectin-4.

Measles virus is a major public health concern worldwide. Three measles virus cell receptors have been identified so far, and the structures of the first two in complex with measles virus hemagglutinin (MV-H) have been reported. Nectin-4 is the most recently identified receptor in epithelial cells, and its binding mode to MV-H remains elusive. In this study, we solved the structure of the membrane-distal domain of human nectin-4 in complex with MV-H. The structure shows that nectin-4 binds the MV-H ß4-ß5 groove exclusively via its N-terminal IgV domain; the contact interface is dominated by hydrophobic interactions. The binding site in MV-H for nectin-4 also overlaps extensively with those of the other two receptors. Finally, a hydrophobic pocket centered in the ß4-ß5 groove is involved in binding to all three identified measles virus receptors, representing a potential target for antiviral drugs.

Design and analysis of post-fusion 6-helix bundle of heptad repeat regions from Newcastle disease virus F protein.

Fusion of paramyxovirus to the cell involves receptor binding of the HN glycoprotein and a number of conformational changes of F glycoprotein. The F protein is expressed as a homotrimer on the virus surface. In the present model, there are at least three conformations of F protein, i.e. native form, pre-hairpin intermediate and the post-fusion state. In the post-fusion state, the two highly conserved heptad repeat (HR) regions of F protein form a stable 6-helix coiled-coil bundle. However, no crystal structure is known for this state for the Newcastle disease virus, although the crystal structure of the F protein native form has been solved recently. Here we deployed an Escherichia coli in vitro expression system to engineer this 6-helix bundle by fusion of either the two HR regions (HR1, linker and HR2) or linking the 6-helix [3 x (HR1, linker and HR2)] together as a single chain. Subsequently, both of them form a stable 6-helix bundle in vitro judging by gel filtration and chemical cross-linking and the proteins show salient features of an alpha-helix structure. Crystals diffracting X-rays have been obtained from both protein preparations and the structure determination is under way. This method could be used for crystallization of the post-fusion state HR structures of other viruses.

Crystallization and preliminary X-ray crystallographic analysis of the trimer core from measles virus fusion protein.

Two heptad-repeat regions (HR1 and HR2) are highly conserved in paramyxovirus fusion proteins and form a stable helical trimer of heterodimers [(HR1-HR2)(3)] after the fusion between viral and cellular membranes. In this study, two HR regions of the fusion protein of measles virus, a member of the paramyxoviruses, were selected and overexpressed as a single chain (named 2-Helix) connected by an amino-acid linker using a GST-fusion expression system in Escherichia coli. Crystals of 2-Helix protein (GST removed) could be obtained from many conditions using the sitting- or hanging-drop vapour-diffusion method. A complete data set was collected in-house to 1.9 A resolution from a single crystal. The crystal belongs to space group P6, with unit-cell parameters a = b = 51.637, c = 67.058 A. To facilitate the crystal structure solution, SeMet-substituted 2-Helix crystals, grown under similar conditions to the native, were also obtained and diffracted X-rays to 1.8 A using synchrotron radiation.

The fusion protein core of measles virus forms stable coiled-coil trimer.

Recent studies have shown that paramyxovirus might adopt a similar molecular mechanism of virus entry and fusion in which the attachment glycoprotein binds receptor/s and triggers the conformational changes of the fusion protein. There are two conserved regions of heptad repeat (HR1 and HR2) in the fusion protein and they were shown with fusion-inhibition effects in many paramyxoviruses, including measles virus. They also appear to show characteristic structure in the fusion core: the HR1/HR2 forms stable six-helix coiled-coil centered by HR1 and is surrounded by HR2 (trimer of HR1/HR2), which represents the post-fusion conformational structure. In this study, we expressed the HR1 and HR2 of measles virus fusion protein as a single chain (named 2-Helix) and subsequently tested its formation of trimer. Indeed, the results do show that the HR1 and HR2 interact with each other and form stable six-helix coiled-coil bundle. This is the first member in genus Morbillivirus of family Paramyxoviridae to be confirmed with this characteristic structure and provides the basis for the HR2-inhibition effects on virus fusion/entry for measles virus.

Six-helix bundle assembly and characterization of heptad repeat regions from the F protein of Newcastle disease virus.

Paramyxoviruses may adopt a similar fusion mechanism to other enveloped viruses, in which an anti-parallel six-helix bundle structure is formed post-fusion in the heptad repeat (HR) regions of the envelope fusion protein. In order to understand the fusion mechanism and identify fusion inhibitors of Newcastle disease virus (NDV), a member of the Paramyxoviridae family, we have developed an E. coli system that separately expresses the F protein HR1 and HR2 regions as GST fusion proteins. The purified cleaved HR1 and HR2 have subsequently been assembled into a stable six-helix bundle heterotrimer complex. Furthermore, both the GST fusion protein and the cleaved HR2 show virus-cell fusion inhibition activity (IC(50) of 1.07-2.93 microM). The solubility of the GST-HR2 fusion protein is much higher than that of the corresponding peptide. Hence this provides a plausible method for large-scale production of HR peptides as virus fusion inhibitors.