Objective:To evaluate an orthogonal test that uses antibodies in a small blood sample to sensitively detect stage 1 tumors, when standard treatment can save lives. Methods:Sera were studied from 283 dogs with stage 1 hemangiosarcoma (30), lymphoma (34), mast cell tumors (60), osteosarcoma (41), and soft tissue sarcoma (49) and dogs established to be cancer-free (69). Samples were applied to microarrays containing peptides synthesized on silicon wafers using photolithography and tert-butoxycarbonyl chemistry. Serum immunoglobulin G binding was measured. Results:Peptides with antibody-binding activities associated with 1 of the 5 cancers or the cancer-free group were identified. Selections were used to build 2 multiclass models. Test performance was verified by peptide resampling or sample holdouts. A simple model detected the 5 different stage 1 tumors at sensitivities from 68% to 98%; the complex model provided stage 1 sensitivities from 60% to 88%, both at high specificities. Conclusions:Antibody activities to stage 1 tumors can be sensitively detected on peptide microarrays. Two divergent classifier-building approaches yielded similar test results. A field study is needed to validate findings. Clinical Relevance:This blood-based test may address the challenges in detecting stage 1 canine cancers, creating opportunities for improved treatment outcomes.

Preventative anti-cancer vaccination strategies have long been hampered by the challenge of targeting the diverse array of potential tumor antigens, with successes to date limited to cancers with viral etiologies. Identification and vaccination against frameshift neoantigens conserved across multiple species and tumor histologies is a potential cancer preventative strategy currently being investigated. Companion dogs spontaneously develop cancers at a similar incidence to those in people and are a complementary comparative patient population for the development of novel anti-cancer therapeutics. In addition to an intact immune system with tumors that arise in an autochthonous tumor microenvironment, dogs also have a shorter lifespan and temporally compressed tumor natural history as compared to humans, which allows for more rapid evaluation of safety, immunogenicity, and efficacy of cancer vaccination strategies. Here we describe the study protocol for the Vaccination Against Canine Cancer Study (VACCS), the largest interventional cancer clinical trial conducted in companion dogs to date. In addition to safety and immunogenicity, the primary endpoint of VACCS is the cumulative incidence (CI) of dogs developing malignant neoplasia of any type at the end of the study period. Secondary endpoints include changes in incidence of specific tumor types, survival times following neoplasia diagnosis, and all-cause mortality.

Abstract Purpose To evaluate a new class of blood-based biomarkers, anti-frameshift peptide antibodies, for predicting both tumor responses and adverse immune events to immune checkpoint inhibitor (ICI) therapies in advanced lung cancer patients. Experimental design Serum samples were obtained from 74 lung cancer patients prior to palliative PD-(L)1 therapies with subsequently recorded tumor responses and immune adverse events (irAEs). Pretreatment samples were assayed on microarrays of frameshift peptides (FSPs), representing ~ 375,000 variant peptides that tumor cells can be informatically predicted to produce from translated mRNA processing errors. Serum-antibodies specifically recognizing these ligands were measured. Binding activities preferentially associated with best-response and adverse-event outcomes were determined. These antibody bound FSPs were used in iterative resampling analyses to develop predictive models of tumor response and immune toxicity. Results Lung cancer serum samples were classified based on predictive models of ICI treatment outcomes. Disease progression was predicted pretreatment with ~ 98% accuracy in the full cohort of all response categories, though ~ 30% of the samples were indeterminate. This model was built with a heterogeneous sample cohort from patients that (i) would show either clear response or stable outcomes, (ii) would be administered either single or combination therapies and (iii) were diagnosed with different lung cancer subtypes. Removing the stable disease, combination therapy or SCLC groups from model building increased the proportion of samples classified while performance remained high. Informatic analyses showed that several of the FSPs in the all-response model mapped to translations of variant mRNAs from the same genes. In the predictive model for treatment toxicities, binding to irAE-associated FSPs provided 90% accuracy pretreatment, with no indeterminates. Several of the classifying FSPs displayed sequence similarity to self-proteins. Conclusions Anti-FSP antibodies may serve as biomarkers for predicting ICI outcomes when tested against ligands corresponding to mRNA-error derived FSPs. Model performances suggest this approach might provide a single test to predict treatment response to ICI and identify patients at high risk for immunotherapy toxicities.

Abstract Development of cancer vaccines is currently focused on using neoantigens arising from mutations in the DNA of tumors. We have proposed, and demonstrated, that errors in RNA processing that create frameshift (FS) neoantigens are also a good source of vaccine components, even in tumors that are DNA mutation poor. Here we directly compare the two vaccine approaches in the mouse ovarian model, ID8. Martin et al. (2016) reported that 7 DNA neoepitopes in the ovarian tumor line (ID8-G7) failed to show any protection when tested individually as peptide vaccines even though the peptides elicited an immune response. We created a pooled peptide vaccine consisting of these 7 neoantigens (NeoAg vax). We compared this vaccine to one composed of 13 RNA-sourced FS neoantigens (FS vax). These FSs had conferred protection when tested in the 4T1 mammary and/or B16 melanoma models. 10 of the FSs arise from mis-transcription thru microsatellites and 3 from mis-splicing of exons. In a prophylactic vaccination protocol, both vaccines induce ~20% extended survival compared to mock controls. The FS vax elicited higher numbers of ELISpots than the ID8 control while the NeoAg vax did not when splenocytes were peptide stimulated. The FS vax and NeoAg vax induced T-cells were only significantly cytotoxic to the ID8 cells if anti-PD-L1/CTLA-4 antibodies were included. In the therapeutic vaccination protocol, both the FS vax and the NeoAg vax conferred extended survival in ~20% of the mice compared to controls. Neither vaccine induced more ELISpots compared to controls using peptides. However, the FS vax did elicit more ELISpots in response to ID8 stimulation and more ID8 cytotoxicity than the NeoAg vax. From this work we first conclude that the DNA-sourced neoantigens that did not protect individually can when pooled. Secondly, and more importantly, the RNA-sourced, FS neoantigens can perform as well as the DNA-sourced neoantigens, at least in this model. This may be important as we have shown that all tumors surveyed to date, in contrast to DNA neoantigens, have abundant RNA-sourced FS neoantigens. Citation Format: Milene Tavares Batista, Sierra Murphy, John Lainson, Lui Shen, Stephen Johnston. A comparison of a DNA-sourced neoantigen vaccine to an RNA-sourced frameshift vaccine in the mouse ovarian cancer model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A28.

Changes in the repertoire of circulating antibodies is a promising biomarker for various diseases. We have developed microarrays containing more than thousands of peptides with random amino acid sequences that can be used to analyze the entire repertoire of circulating antibodies. Using these peptide microarrays, we have shown that in the blood of patients diagnosed with breast cancer (BC) antibodies interacting with certain peptides are found. Moreover, circulating antibodies of patients with different molecular subtypes of BC (luminal A, luminal B and Basal-like) interact with different, but overlapping, panels of peptides. We have selected 634 peptides from 120K peptides presented on microarrays that specifically interact with circulating antibodies of blood plasma from patients with various subtypes of BC. This panel of peptides that specifically interact with the blood plasma immunoglobulins of patients with various molecular BC subtypes can be used for the development of non-invasive diagnostic test systems. Microchips containing thousands of peptides can be used as a tool for molecular screening populations at risk for various diseases. Thus, peptide microarrays can be used to create a new tool for studying the repertoire of circulating antibodies for different diseases evaluation, as well as for studying and evaluating protein-protein interactions.

Background: It is widely hoped that personal cancer vaccines will extend the number of patients benefiting from checkpoint and other immunotherapies. However, it is clear creating such vaccines will be challenging. It requires obtaining and sequencing tumor DNA/RNA, predicting potentially immunogenic neoepitopes and manufacturing a one-use vaccine. This process takes time and considerable cost. Importantly, most mutations will not produce an immunogenic peptide and many patient’s tumors do not contain enough DNA mutations to make a vaccine. We have discovered that frameshift peptides (FSP) created from errors in the production of RNA rather than from DNA mutations are potentially a rich source of neoantigens for cancer vaccines. These errors are predictable, enabling the production of a FSP microarray. Previously we found that these microarrays can identify both personal and shared neoantigens. Here, we compared the performance of personal cancer vaccines (PCVs) with that of a shared antigen vaccine, termed Frameshift Antigen Shared Therapeutic (FAST) vaccine , using the 4T1 breast cancer model. Sera from 4T1-tumor bearing mice were assayed on the peptide microarray containing 200 Fs neoantigens, for the PCV, the top 10 candidates were select and personal vaccines constructed and administrated to the respective mice. For the FAST, we selected the top 10 candidates with higher prevalence among all the mice challenged. Seven to 12 days challenged mice were immunized, combined or not with immune checkpoint inhibitor (ICI) (αPD-L1 and αCTLA-4). Primary and secondary tumor clearance and growth were evaluated as well as cellular and humoral immune response against the vaccine targets by IFN-γ ELISPOT and ELISA. Lastly, we analyzed the immune response of the FAST-vaccinated mice by flow cytometry in comparison to the control group. Results: We found that PCVs and FAST vaccines both reduced primary tumor incidence and growth as well as lung metastases when delivered as monotherapies or in combination with ICI. Additionally, the FAST vaccine induces a robust and effective T-cell response. Conclusions: These results suggest that FSPs produced from RNA-based errors are potent neoantigens that could enable production of off-the-shelf shared antigen vaccines for solid tumors with efficacy comparable to that of PCVs.

Therapeutic monoclonal antibodies have the potential to work as biological therapeutics. OKT3, Herceptin, Keytruda and others have positively impacted healthcare. Antibodies evolved naturally to provide high specificity and high affinity once mature. These characteristics can make them useful as therapeutics. However, we may be missing characteristics that are not obvious. We present a means of measuring antibodies in an unbiased manner that may highlight therapeutic activity. We propose using a microarray of random peptides to assess antibody properties. We tested twenty-four different commercial antibodies to gain some perspective about how much information can be derived from binding antibodies to random peptide libraries. Some monoclonals preferred to bind shorter peptides, some longer, some preferred motifs closer to the C-term, some nearer the N-term. We tested some antibodies with clinical activity but whose function was blinded to us at the time. We were provided with twenty-one different monoclonal antibodies, thirteen mouse and eight human IgM. These antibodies produced a variety of binding patterns on the random peptide arrays. When unblinded, the antibodies with polyspecific binding were the ones with the greatest therapeutic activity. The protein target to these therapeutic monoclonals is still unknown but using common sequence motifs from the peptides we predicted several human and mouse proteins. The same five highest proteins appeared in both mouse and human lists.

Gliomas are the most common primary central nervous system tumors occurring in children and adults with neurofibromatosis type 1 (NF1). Over the past decade, discoveries of the molecular basis of low-grade gliomas (LGGs) have led to new approaches for diagnosis and treatments. However, these new understandings have not been fully applied to the management of NF1-associated gliomas. A consensus panel consisting of experts in NF1 and gliomas was convened to review the current molecular knowledge of NF1-associated low-grade “transformed” and high-grade gliomas; insights gained from mouse models of NF1-LGGs; challenges in diagnosing and treating older patients with NF1-associated gliomas; and advances in molecularly targeted treatment and potential immunologic treatment of these tumors. Next steps are recommended to advance the management and outcomes for NF1-associated gliomas.

Parallel measurement of large numbers of antigen-antibody interactions are increasingly enabled by peptide microarray technologies. Our group has developed anin situsynthesized peptide microarray of >400 000 frameshift neoantigens using mask-based photolithographic peptide synthesis, to profile patient specific neoantigen reactive antibodies in a single assay. The system produces 208 replicate mircoarrays per wafer and is capable of producing multiple wafers per synthetic lot to routinely synthesize over 300 million peptides simultaneously. In this report, we demonstrate the feasibility of the system for detecting peripheral-blood antibody binding to frameshift neoantigens across multiple synthetic lots.

Abstract The paucity of mutations in pediatric and adult brain tumors has limited the ability to design therapeutic personal cancer vaccines. However, we have discovered that, in contrast to mutations in DNA, RNA is a rich source of frameshift neoantigens produced by mis-splicing of exons or indels from transcription through micro satellites. We have found that in GBM there are an average of 4000 such FS neoantigens per patient and in DIPG an average of at least 500 FS neoantigens/patient. Approximately 20% of these neoantigens are frequently recurrent across different patients. This has allowed us to design pre-synthesized vaccines for each tumor type. The vaccines consist of 20 -30 FS epitopes that are predicted to be present in at least 20% of all patient’s tumor. Each patient would have a 90% chance of having at least 50% of the peptides in their tumor. This type of “FAST” vaccine is much less expensive than a personal vaccine, any patient could be treated and there would be no delay in producing the vaccine. We have directly compared the efficacy of a FAST and personal vaccines in a mouse model of breast cancer. We find that both approaches have comparable protection though there are some interesting differences. We suggest that FAST vaccines may be a useful approach to developing therapeutic vaccines for brain cancers. We also demonstrate that an array of the 400K possible FS neoantigen peptides can be used to assay which neoantigens the patient has developed antibodies to. This array was useful in discovering the FAST vaccine components and could also be used to determine the pre-existing overlap of a FAST vaccine with the patient’s immune response.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Immunotherapy with immune check point inhibitors (CPI) has dramatically transformed cancer therapy. However, only ~25% cancer patients have a positive clinical response to CPI. Moreover, the cost of the treatment and the risk of severe side effects makes it necessary to develop biomarkers to predict the benefit of the treatment. It has been shown that the tumor neoantigen load correlates with a positive response to treatment. This indicates that pre-existing anti-tumor immune responses to neoantigens can be used for the CPI response prediction. We have discovered a new source of frameshift (FS) neoantigens created by errors in RNA production in tumor cells, including the insertion and deletion (INDEL) of microsatellite regions during the RNA transcription and the mis-splicing of exons. These errors can generate FS neoantigens, which are highly immunogenic and can elicit both T cell and B cell immune response in cancer patients. We have shown that, although most antibody reactivity to FS peptides (FSPs) are personal, there are common antibodies reactive in different cancer patients, even across different cancer types. The FSPs with positive reactive antibodies can offer protection in mouse tumor models as vaccines. We thus hypothesize that antibodies reactive to FSPs in cancer patients can be used for predicting the clinical benefit of cancer immunotherapy. There is a total of~ 220,000 potential FS neoantigens that can be generated by INDELs of transcription and mis-splicing of genes. These neoantigens can be represented by ~400,000 FSPs, 15-amino acids peptides. We have created arrays of by in-situ synthesis of these FSPs. We used these array to test our hypothesis with pre-treatment serum of 40 cancer patients, from 26 different cancer types in clinical trials with CPI treatments. A total of 13 patients had a clinical response to CPI treatment. Similar to ELISA, diluted serum were applied to the FSP array, and total IgG were detected by fluorescent labeled antibody. IgG reactive to each FSP was measured by the fluorescent intensity and then median normalized within each array for the analysis. As predicted, there are common IgG antibodies reactive to FSPs in the response patients. By selecting 100 to 500 most significantly different reactive FSPs between two group patients, and trained with prediction models, such as SVM, our FSP array can reach up to 96% accuracy in the prediction of clinical response with leave-one-out validation. We hypothesize that the FSPs with positive IgG reactive in response patients may be related to anti-tumor immune response, which is need to be further investigated. We also showed that the FSP array can potentially predict the patients who may have high grade immune related adverse events with the CPI treatment. Our preliminary data indicates that the FSP array is a promising technology for predicting the clinical benefit of immunotherapy. We will expand our sample size to further evaluate this technology. Citation Format: Luhui Shen, Jianfen Chen, Stephen Albert Johnston, David Hong, Jianjun Gao, Aung Naing. A simple blood base test for predicting clinical benefit of cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2231.

It is widely hoped that personal cancer vaccines will extend the number of patients benefiting from checkpoint and other immunotherapeutics. However, it is clear creating such vaccines will be challenging. It requires obtaining and sequencing tumor DNA/RNA, predicting potentially immunogenic neoepitopes and manufacturing a one-use vaccine. This process takes time and considerable cost. Importantly, most mutations will not produce an immunogenic peptide and many patient’s tumors do not contain enough DNA mutations to make a vaccine. We have discovered that frameshift peptides (FSP) produced in tumors though errors in RNA production are a rich source of neoantigens. There are ~220K bioinformatically predictable possible FSP allowing us to make arrays representing them as 15aa peptides. These arrays can then be used to screen cancer patient blood antibodies for reactivity to the arrays. In screening many cancer patients blood on these array, we found both personal and cancer-type specific peptides. This suggests a new type of vaccine consisting of pre-made FSP components for a specific type of cancer. We term these FAST vaccines. Here we use the mouse 4T1 breast cancer model to test the relative effectiveness of a FAST and a PERSONAL vaccine. To create the vaccines, we initially challenged mice subcutaneously with 4T1 tumor cells and, seven days later, sera were collected. Pre-challenge and 7-days sera were assayed on peptide microarrays containing 200 FS neoantigens. For the PERSONAL vax, the top 10 candidates (higher median intensity fluorescence) were select and personal vaccines constructed and administrated to respective mice (n=10). For the FAST vax, we selected the top 10 candidates with higher prevalence among all the mice challenged (n=24), a common Breast cancer FAST vax was constructed (mBC FAST-vax). Mice were challenged with 4T1 cells subcutaneously. Vaccines were then, administrated twice with one-week interval, combined or not with checkpoint inhibitor (CPI) (anti- PD-L1/ CTLA-4). Our results demonstrated that both vaccine approaches, FAST and PERSONAL vax, alone reduced tumor growth as well as increased animal survival. Nonetheless, the FAST vax protected 70 % of mice (7/10 - tumor free) even after re-challenge, 29 days after vaccine regimen. For the Personal vax group, co-administration with CPI resulted in enhancement of tumor control with 57 % of the mice strongly controlling the tumor. The FAST vax performance was not improved by CPI. Both vaccine approaches elicited a robust and homogenous B- and T- cell immune response against both vaccine peptides and tumor cells. Additionally, use of Non-reactive FSPs and a Non-Breast cancer FAST vax were not able to control tumor development. We conclude that the FAST technology may open new opportunities to develop a low cost, feasible and efficacious vaccines against cancer. Citation Format: Milene Tavares Batista, Sierra Nicole Murphy, Ji'an Zhang, Luhui Shen, Phillip Stafford, Stephen A. Johnston. FAST vaccines based on frameshift neoantigens may have advantages over personal vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1463.

A random-sequence peptide microarray can interrogate serum antibodies in a broad, unbiased fashion to generate disease-specific immunosignatures. This approach has been applied to cancer detection, diagnosis of infections, and interrogation of vaccine response. We hypothesized that there is an immunosignature specific to ME/CFS and that this could aid in the diagnosis. We studied two subject groups meeting the Canadian Consensus Definition of ME/CFS. ME/CFS (n=25) and matched control (n=25) sera were obtained from a Canadian study. ME/CFS (n=25) sera were obtained from phase 1/2 Norwegian trials (NCT01156909). Sera from six healthy controls from the USA were included in the analysis. Canadian cases and controls were tested for a disease immunosignature. By combining results from unsupervised and supervised analyses, a candidate immunosignature with 654 peptides was able to differentiate ME/CFS from controls. The immunosignature was tested and further refined using the Norwegian and USA samples. This resulted in a 256-peptide immunosignature with the ability to separate ME/CFS cases from controls in the international data sets. We were able to identify a 256-peptide signature that separates ME/CFS samples from healthy controls, suggesting that the hit-and-run hypothesis of immune dysfunction merits further investigation. By extending testing of both our signature and one previously reported in the literature to larger cohorts, and further interrogating the specific peptides we and others have identified, we may deepen our understanding of the origins of ME/CFS and work towards a clinically meaningful diagnostic biomarker.

We demonstrate a platform to screen a virus pseudotyped with Ebola virus glycoprotein (GP) against a library of peptides that contain non-natural amino acids to develop GP affinity ligands. This system could be used for rapid development of peptide-based antivirals for other emerging or neglected tropical infectious diseases.

It has been demonstrated that DNA mutations generating neo-antigens are important for an effective immune response to tumors as evident from recent clinical studies of immune checkpoint inhibitors (ICIs). Further, it was shown that frameshift peptides (FSP) generated in tumors from insertions and deletions (INDELs) of microsatellites (MS) in coding region are a very good correlate of positive response to PD1 treatment. However, these types of DNA-sourced FSPs are infrequent in cancer. We hypothesize that tumors may also generate FSPs in transcription errors through INDELs in MS or by exon mis-splicing. Since there are a finite number of predictable sequences of such possible FSPs in the genome, we propose that peptide arrays with all possible FSPs could be used to analyze antibody reactivity to FSPs in patient sera as a FS neo-antigen screen. If this were the case it would facilitate finding common tumor neoantigens for cancer vaccines. Here we test this proposal using an array of 377 predicted FS antigens. The results of screening 9 types of dog cancer sera indicate that cancer samples had significantly higher antibody responses against FSPs than non-cancer samples. Both common reactive FSPs and cancer-type specific immune responses were detected. In addition, the protection of a common reactive FSP was tested in mouse tumor models, comparing to the non-reactive FSPs. The mouse homologs non-reactive FSPs did not offer protection in either the mouse melanoma or breast cancer models while the reactive FSP did in both models. The tumor protection was positively correlated to antibody response to the FSP. These data suggest that FSP arrays could be used for cancer neo-antigen screening.

Membrane proteins are the molecular interface of the cell and its environs; however, studies of membrane proteins are highly technically challenging, mainly due to instability of the isolated protein. Towards the production of antibodies that recognize properly folded and stabilized forms of membrane protein antigen, we describe a DNA-based immunization method for mice that expresses the antigen in the membranes of dendritic cells, thus allowing direct presentation to the immune system. This genetic immunization approach employs a highly efficient method of biolistic delivery based on DNA-gold micronanoplexes, which are complexes of micron-sized gold particles that allow dermal penetration and nanometer-sized gold particles that provide a higher surface area for DNA binding than micron gold alone. In contrast to antibodies derived from immunizations with detergent-solubilized protein or with protein fragments, antibodies from genetic immunization are expected to have a high capacity for binding conformational epitopes and for modulating membrane protein activity. © 2018 by John Wiley & Sons, Inc.

There are an increasing variety of applications in which peptides are both synthesized and used attached to solid surfaces. This has created a need for high throughput sequence analysis directly on surfaces. However, common sequencing approaches that can be adapted to surface bound peptides lack the throughput often needed in library-based applications. Here we describe a simple approach for sequence analysis directly on solid surfaces that is both high speed and high throughput, utilizing equipment available in most protein analysis facilities. In this approach, surface bound peptides, selectively labeled at their N-termini with a positive charge-bearing group, are subjected to controlled degradation in ammonia gas, resulting in a set of fragments differing by a single amino acid that remain spatially confined on the surface they were bound to. These fragments can then be analyzed by MALDI mass spectrometry, and the peptide sequences read directly from the resulting spectra.

We have previously shown that the diversity of antibodies in an individual can be displayed on chips on which 130,000 peptides chosen from random sequence space have been synthesized. This immunosignature technology is unbiased in displaying antibody diversity relative to natural sequence space, and has been shown to have diagnostic and prognostic potential for a wide variety of diseases and vaccines. Here we show that a global measure such as Shannon’s entropy can be calculated for each immunosignature. The immune entropy was measured across a diverse set of 800 people and in 5 individuals over 3 months. The immune entropy is affected by some population characteristics and varies widely across individuals. We find that people with infections or breast cancer, generally have higher entropy values than non-diseased individuals. We propose that the immune entropy as measured from immunosignatures may be a simple method to monitor health in individuals and populations.