Combination TIGIT/PD-1 blockade enhances the efficacy of neoantigen vaccines in a model of pancreatic cancer.

Background: Cancer neoantigens are important targets of cancer immunotherapy and neoantigen vaccines are currently in development in pancreatic ductal adenocarcinoma (PDAC) and other cancer types. Immune regulatory mechanisms in pancreatic cancer may limit the efficacy of neoantigen vaccines. Targeting immune checkpoint signaling pathways in PDAC may improve the efficacy of neoantigen vaccines. Methods: We used KPC4580P, an established model of PDAC, to test whether neoantigen vaccines can generate therapeutic efficacy against PDAC. We focused on two immunogenic neoantigens associated with genetic alterations in the CAR12 and CDK12 genes. We tested a neoantigen vaccine comprised of two 20-mer synthetic long peptides and poly IC, a Toll-like receptor (TLR) agonist. We investigated the ability of neoantigen vaccine alone, or in combination with PD-1 and TIGIT signaling blockade to impact tumor growth. We also assessed the impact of TIGIT signaling on T cell responses in human PDAC. Results: Neoantigen vaccines induce neoantigen-specific T cell responses in tumor-bearing mice and slow KPC4580P tumor growth. However, KPC4580P tumors express high levels of PD-L1 and the TIGIT ligand, CD155. A subset of neoantigen-specific T cells in KPC4580P tumors are dysfunctional, and express high levels of TIGIT. PD-1 and TIGIT signaling blockade in vivo reverses T cell dysfunction and enhances neoantigen vaccine-induced T cell responses and tumor regression. In human translational studies, TIGIT signaling blockade in vitro enhances neoantigen-specific T cell function following vaccination. Conclusions: Taken together, preclinical and human translational studies support testing neoantigen vaccines in combination with therapies targeting the PD-1 and TIGIT signaling pathways in patients with PDAC.

Optimized polyepitope neoantigen DNA vaccines elicit neoantigen-specific immune responses in preclinical models and in clinical translation.

BACKGROUND: Preclinical studies and early clinical trials have shown that targeting cancer neoantigens is a promising approach towards the development of personalized cancer immunotherapies. DNA vaccines can be rapidly and efficiently manufactured and can integrate multiple neoantigens simultaneously. We therefore sought to optimize the design of polyepitope DNA vaccines and test optimized polyepitope neoantigen DNA vaccines in preclinical models and in clinical translation. METHODS: We developed and optimized a DNA vaccine platform to target multiple neoantigens. The polyepitope DNA vaccine platform was first optimized using model antigens in vitro and in vivo. We then identified neoantigens in preclinical breast cancer models through genome sequencing and in silico neoantigen prediction pipelines. Optimized polyepitope neoantigen DNA vaccines specific for the murine breast tumor E0771 and 4T1 were designed and their immunogenicity was tested in vivo. We also tested an optimized polyepitope neoantigen DNA vaccine in a patient with metastatic pancreatic neuroendocrine tumor. RESULTS: Our data support an optimized polyepitope neoantigen DNA vaccine design encoding long (≥20-mer) epitopes with a mutant form of ubiquitin (Ubmut) fused to the N-terminus for antigen processing and presentation. Optimized polyepitope neoantigen DNA vaccines were immunogenic and generated robust neoantigen-specific immune responses in mice. The magnitude of immune responses generated by optimized polyepitope neoantigen DNA vaccines was similar to that of synthetic long peptide vaccines specific for the same neoantigens. When combined with immune checkpoint blockade therapy, optimized polyepitope neoantigen DNA vaccines were capable of inducing antitumor immunity in preclinical models. Immune monitoring data suggest that optimized polyepitope neoantigen DNA vaccines are capable of inducing neoantigen-specific T cell responses in a patient with metastatic pancreatic neuroendocrine tumor. CONCLUSIONS: We have developed and optimized a novel polyepitope neoantigen DNA vaccine platform that can target multiple neoantigens and induce antitumor immune responses in preclinical models and neoantigen-specific responses in clinical translation.

Breast Cancer Neoantigens Can Induce CD8+ T-Cell Responses and Antitumor Immunity.

Next-generation sequencing technologies have provided insights into the biology and mutational landscape of cancer. Here, we evaluate the relevance of cancer neoantigens in human breast cancers. Using patient-derived xenografts from three patients with advanced breast cancer (xenografts were designated as WHIM30, WHIM35, and WHIM37), we sequenced exomes of tumor and patient-matched normal cells. We identified 2,091 (WHIM30), 354 (WHIM35), and 235 (WHIM37) nonsynonymous somatic mutations. A computational analysis identified and prioritized HLA class I-restricted candidate neoantigens expressed in the dominant tumor clone. Each candidate neoantigen was evaluated using peptide-binding assays, T-cell cultures that measure the ability of CD8+ T cells to recognize candidate neoantigens, and preclinical models in which we measured antitumor immunity. Our results demonstrate that breast cancer neoantigens can be recognized by the immune system, and that human CD8+ T cells enriched for prioritized breast cancer neoantigens were able to protect mice from tumor challenge with autologous patient-derived xenografts. We conclude that next-generation sequencing and epitope-prediction strategies can identify and prioritize candidate neoantigens for immune targeting in breast cancer. Cancer Immunol Res; 5(7); 516-23. ©2017 AACR.

The Impact of Innovation: How the Changing Nature of Data Will Challenge FDA's Regulatory Framework.

Rapid advances in technology and our understanding of disease will lead to a shift in how the health care system thinks about data, which will in turn challenge current regulatory constructs. In the future, there will be a shift away from milestone-based data to continuous, contextual data; we believe this data shift will impact the current model of medical product regulation, with potential implications across the regulatory landscape, reflecting the convergence of clinical development and clinical practice.

A novel T-cell receptor mimic defines dendritic cells that present an immunodominant West Nile virus epitope in mice.

We used a newly generated T-cell receptor mimic monoclonal antibody (TCRm MAb) that recognizes a known nonself immunodominant peptide epitope from West Nile virus (WNV) NS4B protein to investigate epitope presentation after virus infection in C57BL/6 mice. Previous studies suggested that peptides of different length, either SSVWNATTAI (10-mer) or SSVWNATTA (9-mer) in complex with class I MHC antigen H-2D(b) , were immunodominant after WNV infection. Our data establish that both peptides are presented on the cell surface after WNV infection and that CD8(+) T cells can detect 10- and 9-mer length variants similarly. This result varies from the idea that a given T-cell receptor (TCR) prefers a single peptide length bound to its cognate class I MHC. In separate WNV infection studies with the TCRm MAb, we show that in vivo the 10-mer was presented on the surface of uninfected and infected CD8α(+) CD11c(+) dendritic cells, which suggests the use of direct and cross-presentation pathways. In contrast, CD11b(+) CD11c(-) cells bound the TCRm MAb only when they were infected. Our study demonstrates that TCR recognition of peptides is not limited to certain peptide lengths and that TCRm MAbs can be used to dissect the cell-type specific mechanisms of antigen presentation in vivo.

Decoupling the role of ubiquitination for the dislocation versus degradation of major histocompatibility complex (MHC) class I proteins during endoplasmic reticulum-associated degradation (ERAD).

Aberrantly or excessively expressed proteins in the endoplasmic reticulum are identified by quality control mechanisms and dislocated to the cytosol for proteasome-mediated, ubiquitin-dependent degradation by a process termed endoplasmic reticulum-associated degradation (ERAD). In addition to its role in degradation, ubiquitination has also been implicated in substrate dislocation, although whether direct ubiquitin conjugation of ERAD substrates is required for dislocation has been difficult to ascertain. An obstacle in probing the mechanism of quality control-induced ERAD is the paucity of ERAD substrates being dislocated and detected at any given time. To obviate this problem, we report here the use of a sensitive biotinylation system to probe the dislocation of major histocompatibility complex I (MHCI) heavy chain substrates in the absence of immune evasion proteins. Using this assay system the dislocation of MHCI heavy chains was found not to require potential ubiquitin conjugation sites in the cytoplasmic tail or Lys residues in the ectodomain. By contrast, dislocation of MHCI heavy chains did require deubiquitinating enzyme activity and rapid proteasome-mediated degradation required Lys residues in MHCI heavy chain ectodomain. These combined findings support the model that the endoplasmic reticulum quality control-induced dislocation of MHCI heavy chains may not require direct ubiquitination/deubiquitination as is required for proteasome-mediated degradation post dislocation.

A structural and molecular dynamics approach to understanding the peptide-receptive transition state of MHC-I molecules.

The mature conformation of major histocompatibility complex class I (MHC-I) proteins depends on the presence of bound peptides, permitting recognition at the cell surface by CD8(+) T lymphocytes. Newly synthesized MHC-I molecules in the endoplasmic reticulum are maintained in a peptide-receptive (PR) transition state by several chaperones until they are released concomitant with the loading of peptides. By determining the crystallographic structure of a region of an MHC-I molecule that is recognized by a unique monoclonal antibody and comparing this with docking and molecular dynamics simulations with the whole molecule, we demonstrate the movement of a hinged unit supporting the part of the binding groove that interacts with the amino terminal residues of the bound peptide. This unit contains a conserved 310 helix that flips from an exposed "open" position in the PR form to a "closed" position in the peptide-loaded (PL) mature molecule. These analyses indicate how this segment of the MHC-I molecule moves to help establish the A and B pockets critical for tight peptide binding and the stable structure required for antigen presentation and T cell recognition at the cell surface.

The peptide-receptive transition state of MHC class I molecules: insight from structure and molecular dynamics.

MHC class I (MHC-I) proteins of the adaptive immune system require antigenic peptides for maintenance of mature conformation and immune function via specific recognition by MHC-I-restricted CD8(+) T lymphocytes. New MHC-I molecules in the endoplasmic reticulum are held by chaperones in a peptide-receptive (PR) transition state pending release by tightly binding peptides. In this study, we show, by crystallographic, docking, and molecular dynamics methods, dramatic movement of a hinged unit containing a conserved 3(10) helix that flips from an exposed "open" position in the PR transition state to a "closed" position with buried hydrophobic side chains in the peptide-loaded mature molecule. Crystallography of hinged unit residues 46-53 of murine H-2L(d) MHC-I H chain, complexed with mAb 64-3-7, demonstrates solvent exposure of these residues in the PR conformation. Docking and molecular dynamics predict how this segment moves to help form the A and B pockets crucial for the tight peptide binding needed for stability of the mature peptide-loaded conformation, chaperone dissociation, and Ag presentation.

Utilization of a reminder mailing to improve blood glucose log reporting in an outpatient diabetes clinic.

Self-monitored blood glucose (SMBG) offers a strategy used to achieve glycemic control in diabetic patients. However, if SMBG readings are unavailable to clinicians, this strategy will have a limited effect. This study assessed the impact of a reminder mailing on response rates to requests for SMBG logs. Patients were asked to mail completed SMBG logs to the clinic in 2 weeks. For the intervention, a reminder mailing was sent to each patient 1 week before SMBG logs were to be returned. Compliance rates pre and postinterventions were compared. The primary outcome was the percentage of all SMBG logs returned on time. Secondary outcomes included the percentage of SMBG logs returned, percentage fulfilled, percentage of clinic appointments kept, percentage of SMBG logs brought to follow-up appointments, and number of interventions made to antidiabetic therapy. Twenty SMBG requests were made in the preintervention cohort versus 19 in postintervention cohort. A trend toward more on time and fulfilled SMBG requests was observed post vs. preintervention. Overall return rates were similar between groups. A nonsignificant increase in clinic appointments kept and a nonsignificant decrease in interventions made were observed postintervention. Receipt of a reminder mail was not a significant predictor of patients bringing an SMBG log to follow-up appointments. In conclusion, the use of a reminder mail was not associated with an increase in the return rate of SMBG logs, although there were nonsignificant trends toward more on time and fulfilled SMBG logs received during the postintervention period.

Specific mutation of a gammaherpesvirus-expressed antigen in response to CD8 T cell selection in vivo.

Herpesviruses are thought to be highly genetically stable, and their use as vaccine vectors has been proposed. However, studies of the human gammaherpesvirus, Epstein-Barr virus, have found viral isolates containing mutations in HLA class I-restricted epitopes. Using murine gammaherpesvirus 68 expressing ovalbumin (OVA), we examined the stability of a gammaherpesvirus antigenic locus under strong CD8 T cell selection in vivo. OVA-specific CD8 T cells selected viral isolates containing mutations in the OVA locus but minimal alterations in other genomic regions. Thus, a CD8 T cell response to a gammaherpesvirus-expressed antigen that is not essential for replication or pathogenesis can result in selective mutation of that antigen in vivo. This finding may have relevance for the use of herpesvirus vectors for chronic antigen expression in vivo.

Patient preferences in choosing a primary care physician.

BACKGROUND AND AIMS: Studies have identified factors important to patients in consideration of a primary care physician (PCP). Few have explored relevant differences in choosing between family medicine (FM) and internal medicine (IM) physicians. The objective of this study was to identify differences in rating of factors perceived to be important to racially diverse FM and IM patients in the selection of a PCP, and to determine patient knowledge of PCP training. SETTINGS, DESIGN, METHODS, AND MATERIAL: This observational study used self-administered questionnaires to obtain information from adult participants at 2 continuity clinics, FM and IM. Participants rated 16 factors on their importance in selecting a PCP. Demographics and information regarding participants' knowledge of PCP training were collected. STATISTICAL ANALYSES USED AND RESULTS: 857 surveys were completed. Data were analyzed using descriptive statistics, Student t test, χ(2), and multivariate logistic regression. Sixty-five percent and 32% of participants were Caucasian or African American, respectively. Combined responses from both clinics revealed good patient care as the factor ranked highest in importance for selecting a PCP, followed by good communication skills. Forty-eight percent and 35% of FM and IM participants, respectively, did not know whether their PCP was trained in IM or FM. More than 50% of participants were not familiar with the scope of their physicians' practice. CONCLUSIONS: Our results suggest that good patient care and communication are similarly important to all patients, regardless of race. Practices should maintain focus on these qualities, as well as on patient education regarding the relevant differences between FM and IM physicians. Results from this study are consistent with prior research on these issues in more racially homogenous populations.

Endogenous MHC-related protein 1 is transiently expressed on the plasma membrane in a conformation that activates mucosal-associated invariant T cells.

The development of mucosal-associated invariant T (MAIT) cells is dependent upon the class Ib molecule MHC-related protein 1 (MR1), commensal bacteria, and a thymus. Furthermore, recent studies have implicated MR1 presentation to MAIT cells in bacteria recognition, although the mechanism remains undefined. Surprisingly, however, surface expression of MR1 has been difficult to detect serologically, despite ubiquitous detection of MR1 transcripts and intracellular protein. In this article, we define a unique mAb capable of stabilizing endogenous mouse MR1 at the cell surface, resulting in enhanced mouse MAIT cell activation. Our results demonstrated that under basal conditions, endogenous MR1 transiently visits the cell surface, thus reconciling the aforementioned serologic and functional studies. Furthermore, using this approach, double-positive thymocytes, macrophages, and dendritic cells were identified as potential APCs for MAIT cell development and activation. Based on this pattern of MR1 expression, it is intriguing to speculate that constitutive expression of MR1 may be detrimental for maintenance of immune homeostasis in the gut and/or detection of pathogenic bacteria in mucosal tissues.

Comparison of short-acting intramuscular antipsychotic medication: impact on length of stay and cost.

A retrospective cohort study was conducted to determine if there is an association between short-acting intramuscular (SAIM) antipsychotics used for acute agitation and length of stay (LOS). Patients with a diagnosis of schizophrenia or schizoaffective disorder who were dispensed at least one dose of a SAIM antipsychotic were divided into groups based on the initial SAIM antipsychotic received once admitted to a psychiatric unit. Electronic records were used to gather demographic information, LOS, and number of injections received during an admission. Cost was calculated from the number of injections received. One-hundred and thirty-six patients were enrolled. When comparing the haloperidol group to the second generation antipsychotic group, there was no statistically significant difference, in LOS 16.98 ± 9.56 days versus 17.59 ± 11.52 days (P = 0.75), respectively. There was a statistically significant difference in both cost and number of injections between groups, favoring the haloperidol group. Ziprasidone was associated with a shorter LOS compared with olanzapine, 13.57 and 19.10 days, respectively (P = 0.026). Patient characteristics should be evaluated when determining an agent for acute agitation. However, because literature indicates second generation SAIM antipsychotics are only noninferior to haloperidol; other factors should also be evaluated; including impact on LOS and impact on hospital resources. This study indicates use of a second generation SAIM antipsychotic for acute agitation is more costly, requires more injections, and was not associated with a shorter length of stay when compared with SAIM haloperidol.

A single peptide-MHC complex positively selects a diverse and specific CD8 T cell repertoire.

Pathogen recognition by T cells is dependent on their exquisite specificity for self-major histocompatibility complex (MHC) molecules presenting a bound peptide. Although this specificity results from positive and negative selection of developing T cells in the thymus, the relative contribution of these two processes remains controversial. To address the relation between the selecting peptide-MHC complex and the specificity of mature T cells, we generated transgenic mice that express a single peptide-MHC class I complex. We demonstrate that positive selection of CD8 T cells in these mice results in an MHC-specific repertoire. Although selection on a single complex is peptide promiscuous, mature T cells are highly peptide specific. Thus, positive selection imparts MHC and peptide specificity on the peripheral CD8 T cell repertoire.

Act on early warnings.

Two conditions establish the threshold for protective action in the presence of scientific uncertainty; 1. Credible evidence that a synthetic chemical can cause biological changes that are known to result in unintended harmful outcomes to human health or the environment in some cases; 2. The presence of such a chemical where it does not belong and where it can cause damage to biological systems (such as human bodies). Acting with foresight takes many forms. We must create and strengthen human health and wildlife monitoring programs to detect and predict harm; take steps to prevent, eliminate, and mitigate exposure when credible evidence of harm is found; monitor novel technologies; consider clusters of problems to be early warnings of harm; and open toxic tort records. All action taken must be based on precautionary definitions of "harm" and "credible evidence" and must include public participation. Significant precautionary actions may be taken on the state and local level in advance of a precautionary national chemicals policy.

Antigen-specific cytotoxic T lymphocytes protect against lethal West Nile virus encephalitis.

Infection with West Nile virus (WNV) causes fatal encephalitis in immunocompromised animals. Previous studies in mice have established that T cell protection is required for clearance of WNV infection from tissues and preventing viral persistence. The current study assessed whether specific WNV peptide epitopes could elicit a cytotoxic T lymphocyte (CTL) response capable of protecting against virus infection. Hidden Markov model analysis was used to identify WNV-encoded peptides that bound the MHC class I proteins K(b) or D(b). Of the 35 peptides predicted to bind MHC class I molecules, one immunodominant CTL recognition peptide was identified in each of the envelope and non-structural protein 4B genes. Addition of these but not control peptides to CD8(+) T cells from WNV-infected mice induced IFN-gamma production. CTL clones that were generated ex vivo lysed peptide-pulsed or WNV-infected target cells in an antigen-specific manner. Finally, adoptive transfer of a mixture of envelope- and non-structural protein 4B-specific CTL to recipient mice protected against lethal WNV challenge. Based on this, we conclude that CTL responses against immundominant WNV epitopes confer protective immunity and thus should be targets for inclusion in new vaccines.

Applications of major histocompatibility complex class I molecules expressed as single chains.

Generation of CD8 T-cell responses to pathogens and tumors requires optimal expression of class I major histocompatibility complex/peptide complexes, which, in turn, is dependent on host cellular processing events and subject to interference by pathogens. To create a stable structure that is more immunogenic and resistant to immune evasion pathways, we have engineered class I molecules as single-chain trimers (SCTs), with flexible linkers connecting peptide, beta2m, and heavy chain. Herein we extend our earlier studies with SCTs to the K(b) ligand derived from vesicular stomatitis virus (VSV) to characterize further SCTs as probes of immune function as well as their potential in immunotherapy. The VSVp-beta2m-K(b) SCTs were remarkably stable at the cell surface, and immunization with DNA encoding SCTs elicited complex-specific antibody. In addition, SCTs were detected by cytotoxic T-lymphocytes specific for the native molecule, and the covalently bound peptide was highly resistant to displacement by exogenous peptide. SCTs can also prime CD8 T-cells in vivo that recognize the native molecule. Furthermore, SCTs were resistant to downregulation by the immune evasion protein mK3 of gamma herpesvirus 68. Moreover, owing to their preassembled nature, SCTs should be resistant to other immune evasion proteins that restrict peptide supply. Thus, SCTs possess therapeutic potential both for prophylactic treatment and for the treatment of ongoing infection.

Enhanced immune presentation of a single-chain major histocompatibility complex class I molecule engineered to optimize linkage of a C-terminally extended peptide.

Major histocompatibility complex class I molecules can be expressed as single polypeptides wherein the antigenic peptide, beta2-microglobulin, and heavy chain are attached by flexible linkers. These molecules, single-chain trimers (SCTs), are remarkably stable at the cell surface compared with native (noncovalently attached) class I molecules. In this study, we used a structure-based approach to engineer an F pocket variant SCT of the murine class I molecule Kb that presents the SIINFEKL epitope of ovalbumin. Mutation of heavy chain residue Tyr84 (Y84A) in the SCT resulted in enhanced serological and cytolytic CD8 T cell recognition of the covalently linked peptide due to better accommodation of the linker extending from the C terminus of the peptide. These SCTs exhibit significant cell-surface stability, which we hypothesize is rendered by their ability to continuously and efficiently rebind the covalently attached peptide. In addition, we demonstrate that SCT technology can be applied to tetramer construction using recombinant SCTs expressed in Escherichia coli. SCT-based tetramers could have applications for the enumeration of T and natural killer cells that recognize peptide.class I complexes prone to dissociation.

Homology between an alloantigen and a self MHC allele calibrates the avidity of the alloreactive T cell repertoire independent of TCR affinity.

The self-restricted T cell repertoire exhibits a high frequency of alloreactivity. Because these alloreactive T cells are derived from the pool of cells selected on several different self MHC alleles, it is unknown how development of the alloantigenic repertoire is influenced by homology between a self MHC allele and an alloantigen. To address this, we used the 2C transgenic TCR that is selected by K(b), is alloreactive for L(d), and cross-reacts with L(q). L(q) is highly homologous to L(d) and binds several of the same peptide ligands, including p2Ca, the peptide recognized by 2C. We find that L(d)/p2Ca is a high avidity agonist ligand, whereas L(q)/p2Ca is a low avidity agonist ligand for 2C T cells. When mice transgenic for the 2C TCR are bred to L(q)-expressing mice, 2C(+) T cells develop; however, they express lower levels of either the 2C TCR or CD8 and require a higher L(d)/p2Ca ligand density to be activated than 2C(+) T cells selected by K(b). Furthermore, the 2C T cells selected in the presence of L(q) fail to detect L(q)/p2Ca complexes even at high ligand density. Thus, despite possessing the identical TCR, there is a functional avidity difference between 2C(+) T cells selected in the presence of L(q) vs K(b). These data provide evidence that homology between the selecting ligand and an alloantigen can influence the avidity of the T cell repertoire for the alloantigen, and suggest that thymic selection can fine tune T cell avidity independent of intrinsic TCR affinity.

Peptide induction of surface expression of class I MHC.

This unit describes a method for comparing the relative binding of different peptides to the same MHC class I (MHC-I) molecule using live cells. Live cells expressing suboptimally loaded MHC-I proteins are incubated with medium containing diluted amounts of synthetic peptides to be tested for binding to class I. After overnight incubation with peptide, surface class I expression is monitored by flow cytometry using an allele-specific MAb. Relative binding affinity of peptide reliably correlates with the amount of surface induction of the class I molecule to which it specifically binds. The mechanistic basis of this assay is that surface MHC-I molecules become conformationally unstable shortly after peptide dissociation. However, the binding of an exogenous peptide can stabilize the surface class I molecule, prevent conformational instability, and thus increase class I surface expression in an allele-specific manner.