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Background: Ending the global COVID-19 pandemic requires efficacious therapies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nevertheless, the emerging Omicron sublineages largely escaped the neutralization of current authorized monoclonal antibody therapies. Here we report a tetravalent bispecific antibody ISH0339, as a potential candidate for long-term and broad protection against COVID-19. Methods: We report here the making of ISH0339, a novel tetravalent bispecific antibody composed of a pair of non-competing neutralizing antibodies that binds specifically to two different neutralizing epitopes of SARS-CoV-2 receptor-binding domain (RBD) and contains an engineered Fc region for prolonged antibody half-life. We describe the preclinical characterization of ISH0339 and discuss its potential as a novel agent for both prophylactic and therapeutic purposes against SARS-CoV-2 infection. Results: ISH0339 bound to SARS-CoV-2 RBD specifically with high affinity and potently blocked the binding of RBD to the host receptor hACE2. ISH0339 demonstrated greater binding, blocking and neutralizing efficiency than its parental monoclonal antibodies, and retained neutralizing ability to all tested SARS-CoV-2 variants of concern. Single dosing of ISH0339 showed potent neutralizing activity for treatment via intravenous injection and for prophylaxis via nasal spray. Preclinical studies following single dosing of ISH0339 showed favorable pharmacokinetics and well-tolerated toxicology profile. Conclusion: ISH0339 has demonstrated a favorable safety profile and potent anti-SARS-CoV-2 activities against all current variants of concern. Furthermore, prophylactic and therapeutic application of ISH0339 significantly reduced the viral titer in lungs. Investigational New Drug studies to evaluate the safety, tolerability and preliminary efficacy of ISH0339 for both prophylactic and therapeutic purposes against SARS-CoV-2 infection have been filed.

Background Currently, cytokine therapy for cancer has demonstrated efficacy in certain diseases but is generally accompanied by severe toxicity. The field of antibody-cytokine fusion proteins (immunocytokines) arose to target these effector molecules to the tumor microenvironment to expand the therapeutic window of cytokine therapy. Therefore, we have developed a novel immunocytokine that binds specifically to programmed death 1 (PD1) and fuses IL15/IL15Rα complex (referred to as IAP0971) for cancer immunotherapy. Methods We report here the making of IAP0971, a novel immunocytokine that binds specifically to PD1 and fuses IL15/IL15Rα complex, and preclinical characterization including pharmacology, pharmacodynamics, pharmacokinetics and toxicology, and discuss its potential as a novel agent for treating patients with advanced malignant tumors. Results IAP0971 bound to human IL2/15Rβ proteins specifically and blocked PD1/PDL1 signaling transduction pathway. IAP0971 promoted the proliferation of CD8 + T cells and natural killer T (NKT) cells, and further activated NK cells to kill tumor cells validated by in vitro assays. In an hPD1 knock-in mouse model, IAP0971 showed potent anti-tumor activity. Preclinical studies in non-human primates following single or repeated dosing of IAP0971 showed favorable pharmacokinetics and well-tolerated toxicology profile. Conclusion IAP0971 has demonstrated a favorable safety profile and potent anti-tumor activities in vivo. A Phase I/IIa clinical trial to evaluate the safety, tolerability and preliminary efficacy of IAP0971 in patients with advanced malignant tumors is on-going (NCT05396391).

Immunotherapy has gained great breakthroughs since the 21st century, which however has limitations of relatively low objective response rate, short progression-free survival and drug resistance. The exclusion, desertification and exhaustion of immune cells in the tumor microenvironment (TME) have been found and confirmed in many tumor cases, accounting for the major problems of current PD1/L1-oriented immunotherapy. Cytokines, on the other hand, have been demonstrated efficacious in activating immune cells in TME and achieved great progress in certain diseases, but it is generally accompanied by severe systematic toxicities. The field of antibody-cytokine fusion proteins (immunocytokines) arose to target these effector molecules to the TME to expand the therapeutic window of cytokine therapy. Therefore, we have developed IAP0971 as a novel immunocytokine that binds specifically to PD1 and fuses IL15/IL15Rα complex for cancer immunotherapy. In IAP0971, IL15/IL15Rα complex specifically binds to the IL2/15Rβγ and promotes the proliferation and activation of T cells and NK cells, without activation of Treg cells or inducing apoptosis of activated T cells which are common side effects of IL2-based therapies. Anti-PD1 antibody in IAP0971 can specifically target IL15 into the TME due to the high expression of PD1 in the TME. Both of the components act in the same location and on the same cells at the same time showing great cis-synergy, which will further improve the effectiveness and therapeutic window of IAP0971. We report here the preclinical characterization of IAP0971 including pharmacology, pharmacodynamics, pharmacokinetics and toxicology, and discuss its potential as a novel agent for treating patients with advanced malignant tumors. IAP0971 bound to human IL2/15Rβ specifically and blocked PD1/PDL1 signaling. IAP0971 promoted the proliferation of CD8+T cells and NKT cells, and further activated NK cells to kill tumor cells validated by in vitro assays. In a hPD1 knock-in mouse model, IAP0971 showed potent anti-tumor activity. Preclinical studies in non-human primates following single or repeated dosing of IAP0971 showed favorable pharmacokinetics and well-tolerated toxicology profile. In summary, IAP0971 has demonstrated a favorable safety profile and potent anti-tumor activities in vivo, which has the potential to address both primary non-responsiveness and acquired resistance of current immunotherapy. IAP0971 is the first armed PD1/IL15 immunocytokine with IND approval from both FDA and NMPA. The indications of IAP0971 include lung cancer, cervical cancer, head and neck squamous cell carcinoma, liver cancer, lymphoma, and other malignant tumors. A Phase I/IIa clinical trial to evaluate the safety, tolerability and preliminary efficacy of IAP0971 in patients with locally advanced or metastatic malignant tumors is currently on-going (NCT05396391). Citation Format: Liusong Yin, Xiaoling Jiang, Chongbing Wu, Zi Chen. IAP0971, A novel PD1/IL15 immunocytokine that binds specifically to PD1 and fuses IL15/IL15Rα complex [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 1889.

Exhaustion of immune cells in the tumor microenvironment has been found and confirmed in many tumor cases, accounting for the major problems of current PD1/L1-oriented immunotherapy. There are two types of exhausted T cells present in the tumor microenvironment: progenitor exhausted T cells and terminally exhausted T cells. Data from various mouse syngeneic tumor models suggest that the dominance of progenitor exhausted T cells will be replaced by terminally exhausted T cells as the tumor progresses. The PD1/PDL1 antibodies mainly act on progenitor exhausted T cells and can develop acquired resistance as terminally exhausted T cells become the dominant cells in the tumor microenvironment. Recently, there are studies demonstrating that IL10 can restore the oxidative phosphorylation metabolism of terminally exhausted T cells and restore their potent killing activities of tumor cells. We therefore designed IAE0972, a novel EGFR/IL10 immunocytokine to solve the problems of immune cell exhaustion in current immunotherapy. It is a monovalent anti-EGFR antibody fused with IL10 homodimer. The anti-EGFR antibody can specifically enrich IAE0972 in tumor microenvironment, so that IL10 can reinvigorate antigen specific CD8+ T cells and facilitate its proliferation, while preserving the tumor cell proliferation inhibition activities of EGFR antibody. The design of monovalent form anti-EGFR antibody can also avoid the skin toxicity problem. In this study, we studied the target binding activity, CD8+ T cell activation activity and cancer cell proliferation inhibition activity of IAE0972. We also observed a much higher anti-tumor efficacy of IAE0972 in a MC38 syngeneic tumor model compared with that of Cetuximab. In addition, the molecule has a very good safety profile with MTD over 6 mg/kg and no skin toxicities, which is about 300-fold higher than that of cytokine drugs. In summary, IAE0972 is an innovative EGFR/IL10 immunocytokine with robust preclinical anti-tumor efficacy and favorable safety profile, which has the potential to address acquired resistance of current immunotherapy. IAE0972 is the first EGFR/IL10 immunocytokine with IND approval from both FDA and NMPA. The indications of IAE0972 include colorectal cancer, head and neck squamous cell carcinoma, squamous non-small cell lung cancer, and other EGFR positive tumors. A Phase I/IIa clinical trial to evaluate the safety, tolerability and preliminary efficacy of IAE0972 in patients with locally advanced or metastatic malignant tumors is on-going (NCT05396339). Citation Format: Liusong Yin, Xiaoling Jiang, Chongbing Wu, Zi Chen. IAE0972, A novel EGFR/IL10 immunocytokine of monovalent anti-EGFR antibody fused with IL10 homodimer [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 1888.

Tumor necrosis factor receptor 2 (TNFR2) is expressed on highly suppressive tumor-infiltrating T regulatory cells (Treg), activated T effector cells (Teff), as well as many tumor cells in the tumor microenvironment, which makes TNFR2 a novel and very promising target for cancer immunotherapy. We have identified and characterized a wide panel of first-in-class highly selective TNFR2 agonist and antagonist antibodies, from murine hybridoma and camelid phage display campaigns. Our Treg antagonist lead candidate IAT0981-1A8 is a humanized single domain antibody (VHH), which showed tight TNFR2 binding with low-digit nanomolar affinity. IAT0981-1A8 potently blocked ligand TNFa binding to TNFR2, and elicited strong Treg proliferation inhibition activities. Our Teff agonist lead candidate IAT0981-231 is a humanized monoclonal antibody with sub-nanomolar affinity to TNFR2, which potently stimulated CD8+ T cell activation, proliferation and cytokine secretion, without blocking endogenous TNFa activity. Both IAT0981-1A8 and IAT0981-231 did not cross-react with TNFR1, and exhibited extremely potent tumor growth inhibition activities as single agent in multiple tumor models in TNFR2 transgenic animals, better than tested benchmarks. Our epitope binning studies demonstrated that IAT0981-1A8 and IAT0981-231 bound to totally different epitopes on TNFR2. We also evaluated several bispecific antibodies composing of tumor associated antigens or other immune cell modulators in tumor microenvironment, with either IAT0981-1A8 or IAT0981-231. We have generated high quality commercial stable cell line for the Teff agonist lead candidate IAT0981-231, and completed several batches of 200L non-GMP and GMP production. Our preclinical cynomolgus monkey toxicity studies revealed extremely good safety profile for IAT0981-231, with no significant toxicity observed even with high 200mg/kg repeated dose. We are currently performing IND-enabling studies for the agonist lead candidate IAT0981-231, and aim to enter clinical studies in the first half of 2022, with first-in-class potential as monotherapy and I/O combination therapy for advanced cancers. Citation Format: Xiaoling Jiang, Chonging Wu, Liusong Yin. First-in-class TNFR2 agonist & antagonist antibodies as Treg-Teff modulator for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5549.

Ag presentation by MHC class II (MHC II) molecules to CD4+ T cells plays a key role in the regulation of the adaptive immune response. Loading of antigenic peptides onto MHC II is catalyzed by HLA-DM (DM), a nonclassical MHC II molecule. The mechanism of DM-facilitated peptide loading is an outstanding problem in the field of Ag presentation. In this study, we systemically explored possible kinetic mechanisms for DM-catalyzed peptide association by measuring real-time peptide association kinetics using fluorescence polarization assays and comparing the experimental data with numerically modeled peptide association reactions. We found that DM does not facilitate peptide association by stabilizing peptide-free MHC II against aggregation. Moreover, DM does not promote transition of an inactive peptide–averse conformation of MHC II to an active peptide–receptive conformation. Instead, DM forms an intermediate with MHC II that binds peptide with faster kinetics than MHC II in the absence of DM. In the absence of peptides, interaction of MHC II with DM leads to inactivation and formation of a peptide-averse form. This study provides novel insights into how DM efficiently catalyzes peptide loading during Ag presentation.

HLA-DM mediates the exchange of peptides loaded onto MHCII molecules during antigen presentation by a mechanism that remains unclear and controversial. Here, we investigated the sequence and structural determinants of HLA-DM interaction. Peptides interacting nonoptimally in the P1 pocket exhibited low MHCII binding affinity and kinetic instability and were highly susceptible to HLA-DM-mediated peptide exchange. These changes were accompanied by conformational alterations detected by surface plasmon resonance, SDS resistance assay, antibody binding assay, gel filtration, dynamic light scattering, small angle x-ray scattering, and NMR spectroscopy. Surprisingly, all of those changes could be reversed by substitution of the P9 pocket anchor residue. Moreover, MHCII mutations outside the P1 pocket and the HLA-DM interaction site increased HLA-DM susceptibility. These results indicate that a dynamic MHCII conformational determinant rather than P1 pocket occupancy is the key factor determining susceptibility to HLA-DM-mediated peptide exchange and provide a molecular mechanism for HLA-DM to efficiently target unstable MHCII-peptide complexes for editing and exchange those for more stable ones.

HLA-DM (DM) functions as a peptide editor that mediates the exchange of peptides loaded onto MHCII molecules by accelerating peptide dissociation and association kinetics. The relative DM-susceptibility of peptides bound to MHCII molecules correlates with antigen presentation and immunodominance hierarchy, and measurement of DM-susceptibility has been a key effort in this field. Current assays of DM-susceptibility, based on differential peptide dissociation rates measured for individually labeled peptides over a long time base, are difficult and cumbersome. Here, we present a novel method to measure DM-susceptibility based on peptide binding competition assays performed in the presence and absence of DM, reported as a delta-IC(50) (change in 50% inhibition concentration) value. We simulated binding competition reactions of peptides with various intrinsic and DM-catalyzed kinetic parameters and found that under a wide range of conditions the delta-IC(50) value is highly correlated with DM-susceptibility as measured in off-rate assay. We confirmed experimentally that DM-susceptibility measured by delta-IC(50) is comparable to that measured by traditional off-rate assay for peptides with known DM-susceptibility hierarchy. The major advantage of this method is that it allows simple, fast and high throughput measurement of DM-susceptibility for a large set of unlabeled peptides in studies of the mechanism of DM action and for identification of CD4+ T cell epitopes.

Immunization with vaccinia virus elicits a protective Ab response that is almost completely CD4+ T cell dependent. A recent study in a rodent model observed a deterministic linkage between Ab and CD4+ T cell responses to particular vaccinia virus proteins suggesting that CD4+ T cell help is preferentially provided to B cells with the same protein specificity (Sette et al. 2008. Immunity 28: 847–858). However, a causal linkage between Ab and CD4+ T cell responses to vaccinia or any other large pathogen in humans has yet to be done. In this study, we measured the Ab and CD4+ T cell responses against four vaccinia viral proteins (A27L, A33R, B5R, and L1R) known to be strongly targeted by humoral and cellular responses induced by vaccinia virus vaccination in 90 recently vaccinated and 7 long-term vaccinia-immunized human donors. Our data indicate that there is no direct linkage between Ab and CD4+ T cell responses against each individual protein in both short-term and long-term immunized donors. Together with the observation that the presence of immune responses to these four proteins is linked together within donors, our data suggest that in vaccinia-immunized humans, individual viral proteins are not the primary recognition unit of CD4+ T cell help for B cells. Therefore, we have for the first time, to our knowledge, shown evidence that CD4+ T cells provide intermolecular (also known as noncognate or heterotypic) help to generate robust Ab responses against four vaccinia viral proteins in humans.

HLA-DM (DM) is a nonclassical MHC class II (MHC II) protein that acts as a peptide editor to mediate the exchange of peptides loaded onto MHC II during Ag presentation. Although the ability of DM to promote peptide exchange in vitro and in vivo is well established, the role of DM in epitope selection is still unclear, especially in human response to infectious disease. In this study, we addressed this question in the context of the human CD4 T cell response to vaccinia virus. We measured the IC50, intrinsic dissociation t1/2, and DM-mediated dissociation t1/2 for a large set of peptides derived from the major core protein A10L and other known vaccinia epitopes bound to HLA-DR1 and compared these properties to the presence and magnitude of peptide-specific CD4+ T cell responses. We found that MHC II–peptide complex kinetic stability in the presence of DM distinguishes T cell epitopes from nonrecognized peptides in A10L peptides and also in a set of predicted tight binders from the entire vaccinia genome. Taken together, these analyses demonstrate that DM-mediated dissociation t1/2 is a strong and independent factor governing peptide immunogenicity by favoring the presentation of peptides with greater kinetic stability in the presence of DM.

The hemagglutinin protein (HA) of the influenza virus family is a major antigen for protective immunity. Thus, it is a relevant target for developing vaccines. Here, we describe a human CD4 + T cell epitope in the influenza virus HA that lies in the fusion peptide of the HA. This epitope is well conserved in all 16 subtypes of the HA protein of influenza A virus and the HA protein of influenza B virus. By stimulating peripheral blood mononuclear cells (PBMCs) from a healthy adult donor with peptides covering the entire HA protein based on the sequence of A/Japan/305/1957 (H2N2), we generated a T cell line specific to this epitope. This CD4 + T cell line recognizes target cells infected with influenza A virus seasonal H1N1 and H3N2 strains, a reassortant H2N1 strain, the 2009 pandemic H1N1 strain, and influenza B virus in cytotoxicity assays and intracellular-cytokine-staining assays. It also lysed target cells infected with avian H5N1 virus. We screened healthy adult PBMCs for T cell responses specific to this epitope and found individuals who had ex vivo gamma interferon (IFN-γ) responses to the peptide epitope in enzyme-linked immunospot (ELISPOT) assays. Almost all donors who responded to the epitope had the HLA-DRB1*09 allele, a relatively common HLA allele. Although natural infection or standard vaccination may not induce strong T and B cell responses to this highly conserved epitope in the fusion peptide, it may be possible to develop a vaccination strategy to induce these CD4 + T cells, which are cross-reactive to both influenza A and B viruses.

Following primary infection, human herpesvirus 6 (HHV-6) establishes a persistent infection for life. HHV-6 reactivation has been associated with transplant rejection, delayed engraftment, encephalitis, muscular dystrophy, and drug-induced hypersensitivity syndrome. The poor understanding of the targets and outcome of the cellular immune response to HHV-6 makes it difficult to outline the role of HHV-6 in human disease. To fill in this gap, we characterized CD4 T cell responses to HHV-6 using peripheral blood mononuclear cell (PBMC) and T cell lines generated from healthy donors. CD4 + T cells responding to HHV-6 in peripheral blood were observed at frequencies below 0.1% of total T cells but could be expanded easily in vitro . Analysis of cytokines in supernatants of PBMC and T cell cultures challenged with HHV-6 preparations indicated that gamma interferon (IFN-γ) and interleukin-10 (IL-10) were appropriate markers of the HHV-6 cellular response. Eleven CD4 + T cell epitopes, all but one derived from abundant virion components, were identified. The response was highly cross-reactive between HHV-6A and HHV-6B variants. Seven of the CD4 + T cell epitopes do not share significant homologies with other known human pathogens, including the closely related human viruses human herpesvirus 7 (HHV-7) and human cytomegalovirus (HCMV). Major histocompatibility complex (MHC) tetramers generated with these epitopes were able to detect HHV-6-specific T cell populations. These findings provide a window into the immune response to HHV-6 and provide a basis for tracking HHV-6 cellular immune responses.

Measurements of thermal stability by circular dichroism (CD) spectroscopy have been widely used to assess the binding of peptides to MHC proteins, particularly within the structural immunology community. Although thermal stability assays offer advantages over other approaches such as IC50 measurements, CD-based stability measurements are hindered by large sample requirements and low throughput. Here we demonstrate that an alternative approach based on differential scanning fluorimetry (DSF) yields results comparable to those based on CD for both class I and class II complexes. As they require much less sample, DSF-based measurements reduce demands on protein production strategies and are amenable for high throughput studies. DSF can thus not only replace CD as a means to assess peptide/MHC thermal stability, but can complement other peptide-MHC binding assays used in screening, epitope discovery, and vaccine design. Due to the physical process probed, DSF can also uncover complexities not observed with other techniques. Lastly, we show that DSF can also be used to assess peptide/MHC kinetic stability, allowing for a single experimental setup to probe both binding equilibria and kinetics.

The repertoire of peptides displayed in vivo by MHC II molecules derives from a wide spectrum of proteins produced by different cell types. Although intracellular endosomal processing in dendritic cells and B cells has been characterized for a few antigens, the overall range of processing pathways responsible for generating the MHC II peptidome are currently unclear. To determine the contribution of non-endosomal processing pathways, we eluted and sequenced over 3000 HLA-DR1-bound peptides presented in vivo by dendritic cells. The processing enzymes were identified by reference to a database of experimentally determined cleavage sites and experimentally validated for four epitopes derived from complement 3, collagen II, thymosin β4, and gelsolin. We determined that self-antigens processed by tissue-specific proteases, including complement, matrix metalloproteases, caspases, and granzymes, and carried by lymph, contribute significantly to the MHC II self-peptidome presented by conventional dendritic cells in vivo. Additionally, the presented peptides exhibited a wide spectrum of binding affinity and HLA-DM susceptibility. The results indicate that the HLA-DR1-restricted self-peptidome presented under physiological conditions derives from a variety of processing pathways. Non-endosomal processing enzymes add to the number of epitopes cleaved by cathepsins, altogether generating a wider peptide repertoire. Taken together with HLA-DM-dependent and-independent loading pathways, this ensures that a broad self-peptidome is presented by dendritic cells. This work brings attention to the role of "self-recognition" as a dynamic interaction between dendritic cells and the metabolic/catabolic activities ongoing in every parenchymal organ as part of tissue growth, remodeling, and physiological apoptosis.

Therapeutic protein products (TPPs) are of considerable value in the treatment of a variety of diseases, including cancer, hemophilia, and autoimmune diseases. The success of TPP mainly results from prolonged half-life, increased target specificity and decreased intrinsic toxicity compared with small molecule drugs. However, unwanted immune responses against TPP, such as generation of anti-drug antibody, can impact both drug efficacy and patient safety, which has led to requirements for increased monitoring in regulatory studies and clinical practice, termination of drug development, or even withdrawal of marketed products. We present an overview of current knowledge on immunogenicity of TPP and its impact on efficacy and safety. We also discuss methods for measurement and prediction of immunogenicity and review both product-related and patient-related risk factors that affect its development, and efforts that may be taken to mitigate it. Lastly, we discuss gaps in knowledge and technology and what is needed to fill these.

In this paper, we give an overview for the shared task at the 4th CCF Conference on Natural Language Processing & Chinese Computing NLPCC 2015: Chinese word segmentation and part-of-speech POS tagging for micro-blog texts. Different with the popular used newswire datasets, the dataset of this shared task consists of the relatively informal micro-texts. The shared task has two sub-tasks: 1 individual Chinese word segmentation and 2 joint Chinese word segmentation and POS Tagging. Each subtask has three tracks to distinguish the systems with different resources. We first introduce the dataset and task, then we characterize the different approaches of the participating systems, report the test results, and provide a overview analysis of these results. An online system is available for open registration and evaluation at http://nlp.fudan.edu.cn/nlpcc2015.

Peptide binding to major histocompatibility complex class II (MHCII) molecules is a key process in antigen presentation and CD4+ T cell epitope selection. This unit describes a fairly simple but powerful fluorescence polarization-based binding competition assay to measure peptide binding to soluble recombinant MHCII molecules. The binding of a peptide of interest to MHCII molecules is assessed based on its ability to inhibit the binding of a fluorescence-labeled probe peptide, with the strength of binding characterized as IC50 (concentration required for 50% inhibition of probe peptide binding). Data analysis related to this method is discussed. In addition, this unit includes a support protocol for fluorescence labeling peptide using an amine-reactive probe. The advantage of this protocol is that it allows simple, fast, and high-throughput measurements of binding for a large set of peptides to MHCII molecules.

Peptides presented by major histocompatibility complex class II (MHCII) molecules to CD4+ T cells play a central role in the initiation of adaptive immunity. This antigen presentation process is characterized by the proteolytic cleavage of foreign and self proteins, and loading of the resultant peptides onto MHCII molecules. Loading and exchange of antigenic peptides is catalyzed by a non-classical MHCII molecule, HLA-DM. The impact of HLA-DM on epitope selection has been appreciated for a long time. However, the molecular mechanism by which HLA-DM mediates peptide exchange remains elusive. Here, we review recent efforts in elucidating how HLA-DM works, highlighted by two recently solved co-structures of HLA-DM bound to HLA-DO (a natural inhibitor of HLA-DM), or to HLA-DR1 (a common MHCII). In light of these efforts, a model for HLA-DM action in which HLA-DM utilizes conformational flexibility around the P1 pocket of the MHCII-peptide complex to catalyze peptide exchange is proposed.

HLA-DM (DM) mediates the exchange of peptides loaded onto MHC II. However, the determinants of DM-mediated peptide release remain unclear and controversial. In this study, we synthesized a series of peptides derived from HLA-A2104-117 and measured their kinetic stabilities when bound to HLA-DR1 (DR1) in the absence or presence of DM. As expected from previous work, we found that peptides with non-optimal pocket 1 residues were highly DM susceptible. Surprisingly we found that substitution of the pocket 9 residue can counteract the low binding affinity, low kinetic stability and high DM-susceptibility of peptides containing non-optimal pocket 1 residues. Surface Plasmon Resonance demonstrated that DM bound well to DR1 loaded with a peptide variant with pocket 1 alanine (A1), while no binding was observed for a rescue variant with leucine at pocket 9 (A1L9) or for the wild-type peptide (W1Q9). We determined the crystal structure of DR1-A1L9 to 2.3Å resolution, confirming the expected binding frame with alanine at pocket 1. DR1-A1 appeared to adopt a different conformation which can be better detected and edited by DM, as evidenced by the sodium dodecyl sulfate-resistance and recognition by a conformation-specific monoclonal antibody UL-5A1. Together with the results that multiple substitutions influence DM-susceptibility, our data suggest that conformation of MHC II-peptide complex constrained by interactions throughout the peptide binding site determines DM-susceptibility.

HLA-DM (DM) is a non-classic major histocompatibility complex II (MHC II), which mediates the exchange of peptides loading to MHC II during antigen presentation. However, the role of DM-mediated peptide exchange in epitope selection is still unclear. In this study, we addressed this question systematically using overlapping peptides from vaccinia virus protein A10L. We measured the binding affinity, intrinsic half life, DM-mediated half life and immunogenecity of 126 A10L peptides bound to HLA-DR1. Correlation coefficient analysis shows that immunogenecity of peptides is very weakly correlated with either binding affinity or intrinsic half life, while strongly correlating with half life in the presence of DM. Moreover, receiver operating characteristics analysis (ROC) indicates that DM-mediated half life is a very strong predictor for epitopes, while binding affinity and intrinsic half life can only mildly predicts epitopes. Taken together, our present quantitative and qualitive analysises demonstrate that DM-susceptibility is a strong and independent factor governing peptide’s immunogenicity.