Mutation-associated Neoantigens Research Articles

Single-cell landscape reveals the immune heterogeneity of bone marrow involvement in peripheral T-cell lymphoma.

The prognosis of patients with peripheral T-cell lymphoma (PTCL) depends on bone marrow involvement (BMI). The bone marrow (BM) tumor microenvironment in PTCL remains unclear. We performed single-cell RNA sequencing (scRNA-seq) on 11 fresh BM samples from patients with BMI to reveal the associations of immune landscape and genetic variations with the prognosis of PTCL patients. Compared with PTCL not otherwise specified (NOS), angioimmunoblastic T-cell lymphoma (AITL) had a higher number of T cells, lower number of lymphocytes, and greater inflammation. Immune heterogeneity in AITL is associated with prognosis. In particular, specific T-cell receptor (TCR) T cells are enriched in patients with good response to anti-CD30 therapy. We observed RhoA mutation-associated neoantigens. Chidamide-treated patients had a higher number of CD4+ regulatory cells and a better treatment response compared with other patients. In the nonresponder group, T-cell enrichment progressed to secondary B-cell enrichment and subsequently diffuse large B-cell lymphoma. Moreover, AITL patients with lymphoma-associated hemophagocytic syndrome had more T follicular helper (Tfh) cells with copy number variations in CHR5. To our knowledge, this study is the first to reveal the single-cell landscape of BM microenvironment heterogeneity in PTCL patients with BMI. scRNA-seq can be used to investigate the immune heterogeneity and genetic variations in AITL associated with prognosis.

Germline homozygosity and allelic imbalance of HLA-I are common in esophagogastric adenocarcinoma and impair the repertoire of immunogenic peptides

BackgroundThe individual HLA-I genotype is associated with cancer, autoimmune diseases and infections. This study elucidates the role of germline homozygosity or allelic imbalance of HLA-I loci in esophago-gastric adenocarcinoma (EGA)...

Hydrophobic interactions dominate the recognition of a KRAS G12V neoantigen

Specificity remains a major challenge to current therapeutic strategies for cancer. Mutation associated neoantigens (MANAs) are products of genetic alterations, making them highly specific therapeutic targets. MANAs are HLA-presented (pHLA) peptides derived from intracellular mutant proteins that are otherwise inaccessible to antibody-based therapeutics. Here, we describe the cryo-EM structure of an antibody-MANA pHLA complex. Specifically, we determine a TCR mimic (TCRm) antibody bound to its MANA target, the KRASG12V peptide presented by HLA-A*03:01. Hydrophobic residues appear to account for the specificity of the mutant G12V residue. We also determine the structure of the wild-type G12 peptide bound to HLA-A*03:01, using X-ray crystallography. Based on these structures, we perform screens to validate the key residues required for peptide specificity. These experiments led us to a model for discrimination between the mutant and the wild-type peptides presented on HLA-A*03:01 based exclusively on hydrophobic interactions.

Loss of Human Leukocyte Antigen and Immune Escape in Head and Neck Cancer.

Cancer cells evade recognition by the immune system to survive. Head and neck squamous cell carcinoma (HNSCC) is characterized by high levels of immune infiltration and mutation-associated neoantigens; therefore, immune evasion is likely to be an important mechanism in HNSCC tumorigenesis and progression. A commonly employed mechanism of immune evasion is downregulation of human leukocyte antigen (HLA) or loss of heterozygosity (LOH) in tumor cells. The objective of this study was to integrate multi-dimensional genomic and transcriptomic data from HNSCC tumors to better understand the clinical and immunologic implications of HLA LOH. Cross-sectional integrated clinical and genomic analysis. Whole-exome sequencing and RNA-sequencing data from 522 tumors profiled in The Cancer Genome Atlas HNSCC cohort were analyzed and integrated with secondary analyses including immune cell deconvolution data. Associations were analyzed with categorical hypothesis testing and multivariable logistic and Cox regression. HLA LOH was a prevalent event that was identified in 53% of HNSCC tumors; in many cases, more than one class I HLA gene was targeted for LOH. HLA LOH was more common in advanced-stage tumors. Tumors with somatic HLA LOH had tumor microenvironments defined by decreased lymphocyte and T cell infiltration. HLA LOH is one of the most prevalent genetic alterations in HNSCC, and is associated with a cold immune microenvironment, suggesting that HLA LOH is a means of immune evasion. It may have value as a predictive biomarker or potential as a cancer cell-specific therapeutic target. 3 Laryngoscope, 134:160-165, 2024.

Pipeline to identify neoantigens exposed by radiation.

Mutation-associated neoantigens are key targets of tumor-specific T cells and thus play a major role in driving responses to immune checkpoint blockade (ICB) therapy in tumors with high mutational burden. However, only a small number of mutated peptides are actually presented by MHC molecules and only a minority can induce T cell responses. In addition, the recognition of these neoantigens by T cells is limited by the level of expression of the mutated gene product in the tumor cells. Preclinical studies have shown that radiation can convert the irradiated tumor into an in situ vaccine, leading to the priming of tumor-specific T cells and to the rejection of otherwise ICB-resistant tumors. There is now preclinical and clinical evidence that radiation can upregulate the expression of genes containing immunogenic mutations and expose them to the immune system. Therefore, the identification of neoantigens upregulated by radiation could help to predict which patients might benefit from treatment with combinations of radiotherapy and ICB and could also be incorporated into personalized neoantigen vaccination strategies. In this chapter, we present the pipeline that we used to identify relevant radiation-upregulated neoantigens in a poorly immunogenic mouse model of metastatic breast cancer.

Phase 1 studies of personalized neoantigen vaccine TG4050 in ovarian carcinoma (OC) and head and neck squamous cell carcinoma (HNSCC).

2637 Background: Mutation-associated neoantigens (MANAs) constitute attractive antigens for the design of cancer vaccines. However, clinical implementation remains challenging because of the patient specific nature of MANAs, hereby requiring that a bespoke vaccine is designed for each subject. We developed a pipeline for the genomic characterization and the design of tailored vaccine using a modified vaccinia Ankara viral vector. Methods: Immunogenicity, safety and early clinical activity of personalized cancer vaccines are being assessed in two phase I trials, respectively in high grade serous OC and HPV-negative HNSCC. Patients (Pts) at high risk of relapse are enrolled in the study being in remission following surgical primary treatment. High risk is defined as stage IIIC or IVA for OC and stage III or IVA for HNSCC. A vaccine is manufactured for each patient. OC pts are treated at asymptomatic relapse based on radiological finding or elevation of CA-125 with the vaccine alone; HNSCC pts in complete remission after surgery and adjuvant therapy are randomized to receive the vaccine either at the end of locoregional treatment or in combination with standard of care at relapse. The vaccine was administered weekly for 6 weeks and a booster dose every three weeks over a year or until progression, whichever occurred first. Results: At the time of the data cut-off, a total of 8 pts were treated: 4 relapsing OC pts, 3 HNSCC pts in complete remission and 1 relapsing HNSCC pt. AE attributable to the vaccine were mainly grade 1 injection site reactions and fatigue. One OC pt displayed an objective response 6 weeks after initiation of vaccine and for 9 months with a normalization of CA-125 until death from an unrelated cause; 1 other OC pt remains on treatment with a stable radiological disease for 11 months. The 2 other OC progressed at the first evaluation on day 43. HNSCC pts treated in complete remission received respectively 20, 15 and 4 doses, and remain on treatment and disease free. One HNSCC patient received 9 doses of vaccine after relapse in conjunction with chemotherapy and anti-PD-1 therapy and remains on treatment with stable disease after 5 months. Immune monitoring demonstrates priming of a polyepitopic T cell response against class I and II antigens. Responses were observed regardless of HLA and without cross-reactivity to the germline protein. Adaptive response was associated with a shift of CD4 and CD8 T cells toward an effector phenotype and innate cellular immunity was activated with a strong maturation and activation of NK cells. Immune changes were stronger in pts with controlled disease versus progressors. Conclusions: Data demonstrate that TG4050 is safe, well tolerated and able to induce T cell responses whatever the HLA haplotype. Early signs of clinical activity were observed in OC pt. These data pave the way for further development and synergistic immunotherapeutic combinations. Clinical trial information: NCT04183166.

PD-1/PD-L1 expression in pancreatic cancer and its implication in novel therapies.

Pancreatic cancer is the seventh leading cause of death in developed countries and it still has a poor prognosis despite intense research in the last 20 years. Immunotherapy is a relatively new strategy in cancer treatment. The aim of immunotherapy is to block the immunosuppressive effect of tumoral cells. The PD1/PD-L1 axis has an important role in the inhibition of effector T cells and the development of regulatory T cells (Tregs). Blocking these checkpoints, and also inhibitory signals, leads to apoptosis of Tregs and increased immune response of effector T cells against tumoral antigens. Unfortunately, pancreatic cancer is generally considered to be a non-immunogenic tumor. Thus PD-1/PD-L1 inhibitors demonstrated poor results in pancreatic cancer, excepting some patients with MSI/dMMR (microsatellite instability/deficient mismatch repair). Furthermore, pancreatic cancer has a particular microenvironment with a strong desmoplastic reaction, increased interstitial fluid pressure, hypoxic conditions, and acidic extracellular pH, which promote tumorigenesis and progression of the tumor. Mismatch repair deficiency (dMMR) is correlated with a high level of mutation-associated neoantigens, most recognized by immune cells which could predict a favorable response to anti-PD-1/PD-L1 therapy. PD-1/PD-L1 molecules could be also found as soluble forms (sPD-1, sPD-L1). These molecules have a potential role in the prognosis and treatment of pancreatic cancer.

A Sampling of Highlights from the Literature: Article Recommendations from Our Deputy and Senior Editors.

Modulating RNA splicing can augment antitumor T-cell responses (by Srosenbe via Wikimedia Commons)Altered RNA splicing in cancer cells can give rise to neoantigens, but it is unclear whether this is clinically relevant and therapeutically targetable. Using B16-F10 and MC38 tumor models, Lu et al. find several compounds that modulate RNA splicing can inhibit tumor growth and improve responses to anti-PD1 checkpoint blockade. These effects are dependent on T cells and MHC class I peptide presentation. Neoepitopes induced by pharmacologic modulation of RNA splicing in B16-F10 cells can trigger antitumor T-cell responses in mice. The data suggest potential new approaches to enhancing cancer immunotherapy.Lu SX, …, Bradley RK. Cell 2021 Jul 22;184:4032–47.e31.Arming CAR T cells with BATF enhances efficacy (from Spezadams via Wikimedia Commons)The transcriptional networks underlying T-cell exhaustion, including exhaustion in CAR T cells, are not fully known. Seo et al. show that basic leucine zipper ATF-like transcription factor (BATF) and interferon regulatory factor 4 (IRF4) act in a cooperative manner to offset T-cell exhaustion. Overexpression of BATF in CAR T cells increases cell persistence and cytokine production and decreases expression of inhibitory receptors and exhaustion-related markers. These effects are dependent on interactions between BATF and IRF4. The data highlight potential therapeutic targets to improve CAR T-cell therapy.Seo H, …, Hogan PG. Nat Immunol 2021 Aug 1;22:983–95.TILs with differing specificities have different functional states (from Brandon Dilbeck via Wikimedia Commons)Understanding of the relationship between the functional state of tumor-infiltrating lymphocytes (TIL) and the specificity of their TCRs is limited, but advances in single-cell technology are changing this. Using such technologies, Caushi et al. show that non–small cell lung cancer TILs with TCRs specific for mutation-associated neoantigens (MANA) have a unique transcriptional profile that is largely characteristic of tissue-resident memory cells and that MANA-specific T cells from tumors nonresponsive to anti-PD1 express significantly reduced levels of genes associated with T-cell dysfunction. Oliveira et al. show that melanoma-reactive TILs are predominantly in an exhausted state and only rarely acquire memory properties, and that disease persistence correlates with persistence of exhausted melanoma-reactive cells in peripheral blood. These data will inform further development of cancer immunotherapy.Caushi JX, …, Smith KN. Nature 2021 Aug 5;596:126–32.Oliveira G, …, Wu CJ. Nature 2021 Aug 5;596:119–25.A targeted radionuclide and checkpoint inhibition combination enhances antitumor immunity (by MichaelFrey via Wikimedia Commons)External beam radiotherapy enhances responses to immune checkpoint blockade (ICB) in preclinical models. Patel et al. show that a targeted radionuclide therapy comprising the alkylphosphocholine analogue NM600 chelated to yittrium-90 (90Y) enhances responses to ICB in mouse models of melanoma, neuroblastoma, and breast cancer without causing toxicity to normal tissues. The 90Y-NM600 plus ICB combination increases immune-cell infiltration into the tumor, and production of proinflammatory cytokines and clonal expansion of CD8+ T cells in the tumor, highlighting the immunologic mechanisms behind this potential new therapeutic combination.Patel RB, …, Morris ZS. Sci Transl Med 2021 Jul 14;13:eabb3631.TFR cells can suppress antitumor responses induced by ICB (from Fig. 1C of Rodriguez and Engelhard, Cancer Immunol Res 2020)The mechanisms behind the effects of immune checkpoint blockade (ICB) are not completely understood. Eschweiler et al. show that follicular regulatory T (TFR) cells are present in tumor-associated tertiary lymphoid structures (TLS) and that they are more suppressive than regulatory T cells. Anti-PD1 can increase the number of TFR cells in tumors, and administration of anti-CTLA4 to deplete regulatory cells prior to anti-PD1 improves treatment efficacy in mice and humans.Eschweiler S, …, Vijayanand P. Nat Immunol 2021 Aug 1;22:1052–63.β2M can accumulate in the tumor microenvironment (β2M staining, from Fig. 1A of Roemer et al., Cancer Immunol Res 2016)β2-microglobulin (β2M) concentrations increase during progression of multiple myeloma (MM), but β2M's role in disease is not well-known. Hofbauer et al. find that MM-associated macrophages can phagocytose β2M, which then leads to NLRP3-mediated inflammasome activation. The resulting cytokine secretion and inflammation contribute to MM severity. Inhibition of the inflammasome or related cytokines ameliorates disease in a mouse model of MM, thus highlighting potential therapeutic targets for the treatment of MM.Hofbauer D, …, Bruns H. Immunity 2021 Jul 20. DOI: 10.1016/j.immuni.2021.07.002.

Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers

PD-1 blockade unleashes CD8 T cells1, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell transcriptomics have revealed global T cell dysfunction programs in tumour-infiltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens2, and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay3 in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a ‘barcode’ to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We find both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein–Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TIL express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL-7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signalling, are largely confined to HOBIThigh TRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade.

Abstract 1571: Neoantigen vaccine plus CD38 blockade suppress the proliferation of murine cold lung cancer LLC1

Abstract Immune checkpoint inhibitors are now the mainstay for the treatment of lung cancer. However, so-called cold tumors are known to be resistant to checkpoint inhibitors. Neoantigens associated with genetic mutations of the tumor have the potential to stimulate a potent and highly specific immune response. To develop effective immunotherapy for cold lung cancer, we examined whether neoantigen-based immunotherapy can develop immune responses and display anti-tumor effects on the cold lung cancer model, Lewis lung carcinoma cells (LLC1) resistant to anti-CTLA-4 or anti-PD-1 therapy. LLC1 cells were subjected to whole-exome and RNA sequencing. Mutation-associated neoantigens were predicted and prioritized by their affinity to MHC class I molecules and their expression. Using netMHCpan, sixty mutated peptides with IC50 <200 nM and 68 mutated peptides with IC50>200 nM, but the wild to corresponding mutated peptide ratio of binding affinity>10 were selected as candidate neoantigens. C57BL/6 mice were immunized with dendritic cells pulsed with these neoantigen short peptides (8-10mer). Twenty-five out of 128 short mutated peptides induced peptide-specific CD8+ T cell response to some extent. Next, 25 long peptides (21mer), which incorporated corresponding immunogenic short peptide sequences, were synthesized and used to immunize mice. DC vaccines pulsed with neoantigen in long peptide format (LP) induced both CD4+ and CD8+ T cell response ex vivo. Of them, DC pulsed with LP82 partly delayed the LLC1 growth in vivo. By RNA-Seq, CD38 was highly expressed in LLC1. Thereby, an anti-CD38 antibody was administered in LLC1-bearing mice immunized with DC pulsed with LP82. The tumor growth was suppressed in the combination treatment. Although CD38 plays an important role in converting nicotinamide adenine dinucleotide (NAD+) to adenosine, adenosine receptor antagonist could not inhibit the tumor growth in mice immunized with LP82. Alternatively, NAD+ accumulation due to CD38 blockade decreased regulatory T cells in the tumor immune microenvironment, consistent with the decreased Foxp3+ transcription and increased IFNg and IL2 production of CD4+ T cells sorted from tumor immune microenvironment. Our study suggested that the prioritization of neoantigens that can induce anti-tumor response is critical for developing neoantigen-targeting immunotherapy. Combining an effective neoantigen vaccine and precise immune modulator could enhance anti-tumor activities even in cold lung tumors. Citation Format: Changbo Sun, Koji Nagaoka, Akihiro Hosoi, Yukari Kobayashi, Jun Nakajima, Kazuhiro Kakimi. Neoantigen vaccine plus CD38 blockade suppress the proliferation of murine cold lung cancer LLC1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1571.

Abstract 1617: Sex-specific genomic determinants of response to immunotherapy

Abstract Introduction: The magnitude of benefit from immune checkpoint inhibitors (ICI) may be sex-dependent, however the mechanisms underlying this sex dimorphism in ICI response are unknown. Methods: To explore sex-specific genomic features linked with ICI response, we analyzed whole-exome sequence data of 89 patients with non-small cell lung cancer (NSCLC) treated with ICI. Sequence alteration, mutation signatures and clonality analyses were combined with HLA class I and II genotyping, and computationally derived mutation-associated neoantigens were evaluated based on the peptide-HLA binding affinity (<500nM) and the relative binding affinity between mutated and wild-type peptides [quotient differential aggretopic index (qDAI) >2]. Missense sequence alterations were characterized as putatively immunogenic mutations (IMM), and HLA class I and II matched IMM loads were calculated. Durable clinical benefit ≥6 months was used to define response. Our findings were validated in an independent cohort of ICI treated NSCLC (n=34). Results: There were no differences in the genomic features assessed or in clinical outcomes between male (n=46) and female (n=43) patients. Female responders had higher tumor mutational burden (TMB; p=0.004), in contrast to male patients (p=0.15). Similarly, a mutation spectra-derived smoking signature was associated with response in females (p=0.001) but not in males (p=0.2). Responding female patients had a more clonal TMB compared to non-responders (p=0.008). Importantly, female responders harbored a significantly higher class I and II IMM load (p=0.008 and p=0.004 respectively), which was not appreciated in males. These findings were supported by analyses of a second independent cohort, where HLA class I and II IMM load differentiated responders from non-responders only in the female patient group (p=0.008 and p=0.004 for class I and II IMM, respectively). In the primary cohort, germline HLA class I zygosity was not associated with outcome for either sex, however a trend towards HLA DQ-B1 homozygosity and worse outcome was evident only in females. Importantly, when HLA class II diversity was combined with class II IMM load, a survival benefit was noted for tumors harboring a high IMM load and maximal HLA II germline diversity in males (log rank p=0.015) but not in females. Similarly, HLA class I diversity was important in males when combined with class I IMM load, such that low IMM load combined with reduced antigen presentation capacity was predictive of non-durable clinical benefit (p=0.003 vs p=0.68 for males and females respectively) and was linked with shorter overall survival (log rank p= 0.062 vs p=0.58 for males and females respectively). Conclusions: Our findings support a sex dimorphism in the genomic determinants of response to immune checkpoint blockade, which may point to differences in immune surveillance between males and females in the context of therapy and may be used to tailor therapeutic delivery. Citation Format: Susan C. Scott, Xiaoshan M. Shao, Noushin Niknafs, Archana Balan, Mara Lanis, James White, Patrick Forde, Kristen Marrone, Vincent Lam, Josephine Feliciano, Benjamin Levy, Julie Brahmer, David Ettinger, Victor Velculescu, Rachel Karchin, Christine Hann, Jarushka Naidoo, Valsalmo Anagnostou. Sex-specific genomic determinants of response to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1617.

Personal Neoantigens From Patients With NSCLC Induce Efficient Antitumor Responses.

ObjectiveTo develop a neoantigen-targeted personalized cancer treatment for non-small cell lung cancer (NSCLC), neoantigens were obtained from collected human lung cancer samples, and the utility of neoantigen and neoantigen-reactive T cells (NRTs) was assessed.MethodsTumor specimens from three patients with NSCLC were obtained and analyzed by whole-exome sequencing, and neoantigens were predicted accordingly. Dendritic cells and T lymphocytes were isolated, NRTs were elicited and IFN-γ ELISPOT tests were conducted. HLA-A2.1/Kb transgenic mice were immunized with peptides from HLA-A*02:01+patient with high immunogenicity, and NRTs were subjected to IFN-γ, IL-2 and TNF-α ELISPOT as well as time-resolved fluorescence assay for cytotoxicity assays to verify the immunogenicity in vitro. The HLA-A*02:01+lung cancer cell line was transfected with minigene and inoculated into the flanks of C57BL/6nu/nu mice and the NRTs induced by the immunogenic polypeptides from autologous HLA-A2.1/Kb transgenic mice were adoptively transfused to verify their immunogenicity in vivo.ResultsMultiple putative mutation-associated neoantigens with strong affinity for HLA were selected from each patient. Immunogenic neoantigen were identified in all three NSCLC patients, the potency of ACAD8-T105I, BCAR1-G23V and PLCG1-M425L as effective neoantigen to active T cells in suppressing tumor growth was further proven both in vitro and in vivo using HLA-A2.1/Kb transgenic mice and tumor-bearing mouse models.ConclusionNeoantigens with strong immunogenicity can be screened from NSCLC patients through the whole-exome sequencing of patient specimens and machine-learning-based neoantigen predictions. NRTs shown efficient antitumor responses in transgenic mice and tumor-bearing mouse models. Our results indicate that the development of neoantigen-based personalized immunotherapies in NSCLC is possible.PrecisNeoantigens with strong immunogenicity were screened from NSCLC patients. This research provides evidence suggesting that neoantigen-based therapy might serve as feasible treatment for NSCLC.

Abstract 6541: Accounting for immune escape mechanisms in personalized and shared neoantigen cancer vaccine design

Abstract The purpose of this study is to evaluate the importance of immune escape mechanisms (IEMs) when designing cancer immunotherapeutic strategies. Cancer IEMs induced by negative selective pressure on tumor cells can have dramatic effects on immunotherapy treatment outcome. IEMs occurring during cancer evolution have been shown to facilitate loss of mutation-associated neoantigens (Anagnostou et al. Cancer Discov 2017). These mechanisms include: Loss of Heterozygosity (LOH), underexpression of HLA genes and mutations in the neoantigen presentation machinery. During cancer evolution, the initially arising clonal neoantigens may be shared across many patients, followed by predominantly subclonal private neoantigens. The propensity of shared neoantigens to be clonal makes them promising candidates for the development of off-the-shelf cancer vaccines. In order to optimize patient treatment strategies and qualify patients for either shared or private neoantigen-directed therapy, it is crucial to correctly assess the indication-specific IEMs and neoantigen landscape in both hot and cold tumors. We carry out an extensive analysis of tumor neoantigen landscape from different indications based on TCGA, on Parkhurst et al. Cancer Discov. 2019 and other available data for selected indications (including MEL, NSCLC, CRC, and GBM). Using bioinformatics methods we investigate IEMs and their potential effects on the plasticity of neoantigen repertoires including enrichment of immunogenic neoantigens within the predicted vaccine composition, computed per patient. Immunogenicity is assessed using advanced deep learning models for neoantigen presentation and functional T-cell response prediction (Mazzocco et al., poster presented at SITC 2019). We present the results of the comparison between different cancer indications including CRC, MEL, NSCLC and GBM using TCGA patient data and GI-tract cancer patients with validated T-cell responses. We present the frequency of particular immune evasion events, pointing out the differences in the number and quality of immunogenic neoepitopes (both shared and private) under those conditions and provide summary statistics. As a result, we draw conclusions for vaccine design strategies, which are informed by the investigation of immune evasion events and are tested on selected datasets including experimentally-validated neoantigen responses. Citation Format: Alexander Myronov, Iga Niemiec, Katarzyna Gruba, Giovanni Mazzocco, Anna Sanecka-Duin, Piotr Skoczylas, Michał Drwal, Jan Kaczmarczyk, Piotr Stepniak. Accounting for immune escape mechanisms in personalized and shared neoantigen cancer vaccine design [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6541.

A method of screening highly common neoantigens with immunogenicity in colorectal cancer based on public somatic mutation library

Colorectal cancer (CRC) is a malignant cancer with high incidence and mortality in the world. Immunotherapy targeting neoantigens can induce durable tumor regression in cancer patients, but is almost limited to personalized precision therapy, due to the individual differences of unique neoantigens. With the discovery of many common oncogenic mutations, and such mutation-associated neoantigens could cover more patients, and hence are valuable in clinical field. However, whether the common neoantigens can be identified in CRC is unknown. Combining the somatic mutations data from 321 CRC patients with a filter standard and 7 predicted algorithms, we screened and obtained 25 HLA-A*1101-restricted common neoantigens with a high binding affinity (IC50<50 nmol/L) and presentation score (>0.90). Besides the positive epitope KRAS_G12V8-16, 11 out of 25 common neoantigens specifically induced in vitro pre- stimulated cytotoxic lymphocyte (CTL) to secrete interferon gamma (IFN-γ). Moreover, combining cell-sorting technology and single-cell RNA sequencing, the immune repertoire profiles of C1orf170_S418G413-421 and KRAS_G12V8-16-specific CTL were analyzed and validated. Their related T-cell receptor engineered T cell (TCR-T) cells could also recognize the neoantigens and secrete IFN-γ. Hence, we have established a method to screen for common neoantigens with immunogenicity in CRC based on the public somatic mutation library. It can provide essential peptide and TCR information for immunotherapies, such as peptides, dendritic cells (DC) vaccines, TCR-like antibodies, TCR-T, etc., for the CRC and other cancers, which has practical application value in the clinics.

Discovery of a novel shared tumor antigen in human lung cancer.

3087 Background: While there has been much attention on mutation-associated neoantigens in tumors, there is less known about non-mutated tumor antigens that are shared across individuals. Understanding tumor-infiltrating T cell recognition of shared tumor antigens is important for understanding cancer immune recognition and escape, and may reveal novel targets for therapy. Methods: We have established a novel approach for discovering shared tumor antigens in human lung cancer. This approach involves identifying candidate T cell receptor (TCR) alpha/beta pairs that are predicted to exhibit specificity for shared tumor antigens in the context of a given human leukocyte antigen (HLA). We then screen the T cell receptor for binding to yeast display libraries of peptide-HLA. The Mark Davis lab at Stanford has previously developed an algorithm that groups T cell receptors into antigen specificity groups based on shared motifs within the TCR complementarity-determining region 3 (CDR3) sequences. Leveraging a dataset of over 700K CDR3 sequences from 178 HLA-typed non-small cell lung cancer (NSCLC) patients, we have found up to 4,300 antigen specificity groups after applying stringent cutoffs. We sequenced TCR alpha/beta pairs from 15 patients with lung adenocarcinoma (n = 4,705). Results: We identified an antigen specificity group enriched in tumor compared to adjacent uninvolved lungs. Antigen screening of the T cell receptor belonging to this specificity group using an A02 yeast display libraries led to the identification of a dominant peptide after four rounds of enrichment. We functionally validated that the peptide derived from the protein TMEM161A stimulated Jurkat cells expressing the TCR alpha/beta receptor of interest. We show that full-length TMEM161A protein is processed and presented into a peptide that stimulates primary T cells expressing the TCR alpha/beta receptor. We observe that a peptide from Epstein-Barr virus (EBV) protein LMP2 also stimulated the same TCR alpha/beta receptor. We have show that TMEM161A RNA and protein are overexpressed in human lung cancer compared to adjacent uninvolved lungs. Conclusions: We have demonstrated a novel approach toward antigen discovery and identified a shared tumor antigen TMEM161A in human lung cancer.

Phase II study of nivolumab and relatlimab in advanced mismatch repair deficient (dMMR) cancers resistant to prior PD-(L)1 inhibition.

TPS839 Background: Cancers deficient in DNA mismatch repair (dMMR) are highly immunogenic tumors exhibiting high rates of response to immune checkpoint inhibitors targeting the programmed cell death-1 (PD-1)/PD-1 ligand (PD-L1) interaction. These tumors are characterized by high levels of microsatellite instability (MSI-H) and an exceptionally high tumor mutation burden, thought to underlie responsiveness to immunotherapy, with higher predicted immunogenicity of mutation-associated neoantigens. However, primary and acquired resistance are observed and diversity in responses is not fully explained by variations in mutation burden. Other immune checkpoints may be acting in parallel with PD-1/PD-L1. In particular, lymphocyte activation gene 3 (LAG3) mediates exhaustion of activated T cells and may have a role in resistance to PD-(L)1 inhibitors (PD-(L)1i); therefore, we hypothesized that the addition of LAG3 inhibitor (relatlimab) to the PD-1i nivolumab may overcome resistance in these tumors. Methods: Patients with advanced dMMR/MSI-H cancer who have progressive disease (by RECIST 1.1) during or within 6 months of PD-(L)1i containing therapy, and after at least 12 weeks of therapy, and meet other eligibility will be enrolled. All patients will receive nivolumab 480mg + relatlimab 160mg every 4 weeks until intolerance or progression, or up to a maximum of 2 years. The primary endpoint is objective response rate. Key secondary endpoints include safety, progression free and overall survival, and other response endpoints as measured by RECIST 1.1 and iRECIST criteria. Exploratory objectives will include analysis of the tumor microenvironment on biopsies obtained at baseline and on-treatment, analysis of T cell populations in the tumor and in the periphery and functional characterization of mutation-associated neoantigen-specific T cells. Studies of the microbiome will be conducted on stool and oral wash samples. Enrollment of 21 patients is planned. Clinical trial information: NCT03607890.

Pipeline for Discovering Neoepitopes Generated By Alternative Splicing in B-ALL

Pediatric B-cell acute lymphoblastic leukemias (B-ALL) are striking for their low mutational burdens. Ostensibly, this paucity of mutation-associated neoantigens could limit the development of novel immunotherapies. However, our lab has previously shown that globally aberrant splicing is a hallmark of B-ALL, as compared to normal pro-B cells. Here we hypothesize that alternative splicing represents a new source of targetable neo-epitopes, distinct from missense mutations. To test this hypothesis, we constructed a pipeline for neo-epitope discovery, validation, and therapeutic development. First, we obtained a very large RNA-seq dataset covering over 400 pediatric B-ALL samples from St. Jude Children's Research Hospital. For normal controls, RNA-seq was performed on 4 pro-B cell fractions from bone marrow donors. We then applied a bioinformatic splicing analysis, MAJIQ 2.0, to deeply mine the dataset for local splicing variants (LSVs) unique to B-ALL. To bypass MHC presentation barriers, we focused on LSVs mapping to exons encoding extracellular protein domains (ectoLSVs). This filtering yielded a list of 914 ectoLSVs in 430 genes. These ectoLSVs were further filtered for those that preserved opening reading frames, occurred with high prevalence, and demonstrated a large differential expression between B-ALL and normal controls. One such prominent event was the increased skipping in B-ALL of CD22 of exon 5-6 (Δex5-6). We validated this event at the RNA level with RT-PCR in 18 out of 18 primary B-ALL samples from CHOP Biobank. Using Nanopore-based long read RNA sequencing, we confirmed the Δex5-6 event exists within a larger translatable transcript. BALB/c mice were then immunized against a peptide containing the junction site of exon 4 and 7 to generate hybridomas; several monoclonal antibody (mAb) against CD22 Δex5-6 were successfully obtained. To validate the specificity of our mAbs, we used CRISPR to delete endogenous CD22 in OCI-Ly10 cells and reconstituted the cells with either CD22 Δex5-6 or full length CD22. The mAb demonstrated remarkable specificity for CD22 Δex5-6 and did not bind to the full-length isoform. We also demonstrated that at least one of these mAbs (clone 3A3) specifically binds to endogenously expressed CD22 Δex5-6 in multiple B-ALL cell lines by immunoprecipitation and Western blot analysis. Our next steps are to develop 3A3-based antibody-drug conjugates and chimeric antigen receptors for further therapeutic testing. In summary, our results validate that 1) our current discovery pipeline is able to identify targetable splicing-derived neo-epitopes, and that 2) antibodies with impressive specificity can be generated against such neo-epitopes. Furthermore, this new paradigm has the promise of increasing the repertoire of highly specific immunotherapy targets in B-ALL, despite its low mutation burden. Of note, this strategy could also be carried forward into therapeutic development for many other cancers beyond B-ALL. Disclosures No relevant conflicts of interest to declare.

Neoepitopes-based vaccines: challenges and perspectives

First generations of cancer vaccines using shared tumour antigens have been associated with disappointing clinical results. However, the paradigm shift introduced by immune checkpoint inhibitors has led to a renewed interest on anti-tumoural vaccination based on mutation-associated neoantigens. First clinical results are encouraging with some signs of clinical activity associated with induction of a specific immune response. In advanced or metastatic diseases, vaccination may either enhance the response to Programmed cell death 1 (PD-1/-L1) antagonists by increasing the number of effectors within the tumour or induce an anti-tumoural T-cell response in immunologically ‘cold’ tumours. There is also a strong rationale to use cancer vaccines in an adjuvant setting to induce a long-term control of the residual disease. Prediction of neoepitopes efficiently presented by Human Leukocyte Antigen (HLA) molecules remains a challenge, as well as identification of clonal neoantigens. Some mechanisms of resistance are already identified, such as tumour loss of neoepitopes-presenting HLA class I molecules. In this context, the role of CD4+ T cells induced by different cancer vaccines should be clarified. Finally, although studies have focused on mutated epitopes corresponding to single nucleotide variants, other neoantigens could be of strong interest such as those linked to tumour specific RNA-splicing abnormalities or associated with insertions-deletions.

Abstract NG01: Evolution of neoantigen landscape during immune checkpoint blockade in non-small cell lung cancer

Abstract NG01: Evolution of neoantigen landscape during immune checkpoint blockade in non-small cell lung cancer

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