Antibodies specific for disease-associated antigens (DAA) expressed in non-malignant diseases reveal potential new tumor-associated antigens (TAA) for immunotherapy or immunoprevention.

Immune responses to a large number of mutated and non-mutated tumor antigens have been studied in an attempt to unravel the highly complex immune response to cancer. Better understanding of both the effectors and the targets of successful immunosurveillance can inform various immunotherapeutic approaches, which can strengthen or replace natural immunosurveillance that a tumor has managed to escape. In this review we highlight targets of antibodies generated in the context of diseases other than cancer, such as asthma, allergies, autoimmune disorders, inflammation and infections, where the antibody presence correlates either with an increased or a reduced lifetime risk of cancer. We focus on their target antigens, self-molecules abnormally expressed on diseased cells or cross-reactive with exogenous antigens and found on cancer cells as tumor associated antigens (TAA). We refer to them as disease-associated antigens (DAA). We review 4 distinct categories of antibodies according to their target DAA, their origin and their reported impact on cancer risk: natural antibodies, autoantibodies, long-term memory antibodies and allergy-associated antibodies. Increased understanding and focus on their specific targets could enable a more rational choice of antigens for both therapeutic and preventative cancer vaccines and other more effective and less toxic cancer immunotherapies.

The "Great Debate" at Immunotherapy Bridge 2021, December 1st-2nd, 2021.

As part of the 2021 Immunotherapy Bridge virtual congress (December 1-2, Naples, Italy), the Great Debate sessions featured experts who were assigned counter opposing views on four important questions in immunotherapy today. The first topic was whether oncolytic viruses or other specific immunomodulators were the more promising approach for intralesional therapy. The second was whether early surrogate endpoints, such as response rate or progression-free survival, correlate with long-term overall survival was considered. Thirdly, whether vaccines can transform cold into hot tumors was discussed and, finally, broad versus deep analytic profiling approaches to gain insights into immune-oncology development were compared. As with previous Bridge congresses, presenters were invited by the meeting Chairs and positions taken during the debates may not have reflected their respective personal view. In addition, the views summarised in this article are based on available evidence but may reflect personal interpretation of these data, clinical experience and subjective opinion of the speaker.

The "Great Debate" at Immunotherapy Bridge 2020, December 3rd, 2020.

As part of the 2020 Immunotherapy Bridge virtual congress (December 2nd-3rd, Italy), the Great Debate session featured counterpoint views from leading experts on three clinical questions in immunotherapy today. The first of these was whether antitumoral vaccination is still a treatment option. The second topic debated whether anti-programmed death (PD)-1/PD-ligand (L)1 blockade should be the backbone for immunotherapy combination. Finally, the use of innovative study designs and surrogate endpoints was considered from both an academic and industry perspective. For each topic, two experts presented the argument and counter-argument in support of two different points of view. As with previous Bridge congresses, the debates were assigned by meeting Chairs and positions taken by experts during the debates may not have necessarily reflected their respective personal view. The views summarised in this article are based on available evidence but may reflect personal interpretation of these data, clinical experience and subjective opinion of the speaker.

Colorectal cancer prevention: Immune modulation taking the stage.

Prevention or early detection is one of the most promising strategies against colorectal cancer (CRC), the second leading cause of cancer death in the US. Recent studies indicate that antitumor immunity plays a key role in CRC prevention. Accumulating evidence suggests that immunosurveillance represents a critical barrier that emerging tumor cells have to overcome in order to sustain the course of tumor development. Virtually all of the agents with cancer preventive activity have been shown to have an immune modulating effect. A number of immunoprevention studies aimed at triggering antitumor immune response against early lesions have been performed, some of which have shown promising results. Furthermore, the recent success of immune checkpoint blockade therapy reinforces the notion that cancers including CRC can be effectively intervened via immune modulation including immune normalization, and has stimulated various immune-based combination prevention studies. This review summarizes recent advances to help better harness the immune system in CRC prevention.

Altered Expression of the Epithelial Mucin MUC1 Accompanies Endoscopic Recurrence of Postoperative Crohn's Disease.

BACKGROUND: MUC1-glycoprotein is expressed at low levels and in fully glycosylated form on epithelial cells. Inflammation causes MUC1 overexpression and hypoglycosylation. We hypothesized that overexpression of hypoglycosylated MUC1 would be found in postoperative Crohn's disease (CD) recurrence and could be considered an additional biomarker of recurrence severity. METHODS: We examined archived neo-terminal ileum biopsies from patients with prior ileocecal resection who had postoperative endoscopic assessment of CD recurrence and given a Rutgeerts ileal recurrence score. Consecutive tissue sections were stained using 2 different anti-MUC1 antibodies, HMPV that recognizes all forms of MUC1 and 4H5 that recognizes only inflammation-associated hypoglycosylated MUC1. RESULTS: A total of 71 postoperative CD patients were evaluated. There was significant increase in MUC1 expression of both glycosylated/normal (P<0.0001) and hypoglycosylated/abnormal (P<0.0001) forms in patients with severe endoscopic CD recurrence (i3+i4), ileal score i2, compared with patients in endoscopic remission (i0+i1). Results were similar regardless of anti-TNF-α use. Although MUC1 expression and Rutgeerts scores were in agreement when characterizing the majority of cases, there were a few exceptions where MUC1 expression was characteristic of more severe recurrence than implied by Rutgeerts score. CONCLUSIONS: MUC1 is overexpressed and hypoglycosylated in neo-terminal ileum tissue of patients with postoperative CD recurrence. Increased levels are associated with more severe endoscopic recurrence scores, and this is not influenced by anti-TNF-α use. Discrepancies found between Rutgeerts scores and MUC1 expression suggest that addition of MUC1 as a biomarker of severity of postoperative CD recurrence may improve categorization of recurrence status and consequently treatment decisions.

Myeloid derived suppressor cells in cancer, premalignancy and inflammation: A roadmap to cancer immunoprevention.

The ultimate success of any form of cancer therapy or cancer prevention depends on its ability to engage the power of the immune system to completely eliminate a growing tumor, lower the life-time tumor risk and establish long-term memory to prevent recurrence or future tumors. For that reason, all therapies but especially immunotherapies depend on the immune health (immunocompetence) of each treated individual. Cancer and chronic illnesses, combined with a usually more advanced age of cancer patients or those at risk for cancer are known to severely suppress multiple antitumor functions of the immune system. Understanding the critical mechanisms controlling and mediating immune suppression can lead to additional therapies to alleviate the effects of those mechanisms and improve the outcome of cancer therapy and prevention. We introduce and review here a highly immunosuppressive cell population found in cancer, precancer, and chronic inflammatory diseases, myeloid derived suppressor cells (MDSC). First described in the setting of advanced cancer, their presence and immunosuppressive activity has been seen more recently in early premalignant lesions and in chronic inflammatory diseases leading to cancer. We describe the detrimental effects of their presence on cancer immunotherapy, immunosurveillance and immunoprevention and review early attempts to develop drugs to eliminate them or reduce their negative impact.

A Believer's Overview of Cancer Immunosurveillance and Immunotherapy.

The field of tumor immunology has grown around the idea that one of the important roles of the immune system is to eliminate cancer. This idea was difficult to reconcile with the accepted notion that the immune system evolved to distinguish self from nonself and therefore tumors derived from self-tissues would not be recognized. Lack of appropriate animal models prevented experimental testing of cancer immunosurveillance. This changed with the realization that the immune system evolved to recognize danger and with the advent of mouse models deficient in one or more immune function, which showed predicted increases in susceptibility to cancer. Simultaneously, technical advances that enabled the study of the human immune system provided data for the existence of tumor-specific T cells and Abs and led to molecular identification of tumor Ags, fully validating the cancer immunosurveillance hypothesis. Immunotherapy designed to strengthen cancer immunosurveillance has achieved unprecedented clinical successes.

Immunobiology and immunosurveillance in patients with intraductal papillary mucinous neoplasms (IPMNs), premalignant precursors of pancreatic adenocarcinomas.

Premalignant lesions for many cancers have been identified, and efforts are currently directed toward identification of antigens expressed on these lesions that would provide suitable targets for vaccines for cancer prevention. Intraductal papillary mucinous neoplasms (IPMNs) are premalignant pancreatic cysts of which a subset has the potential to progress to cancer. Currently, there are no validated predictive markers for progression to malignancy. We hypothesized that the presence or absence of immune surveillance of these lesions would be one such factor. Here we show that the tumor antigen MUC1, which is abnormally expressed on pancreatic cancer and is a target for cancer immunosurveillance, is also abnormally expressed on premalignant IPMN. We show that some IPMN patients make MUC1-specific IgG. Moreover, we show evidence of CD4 and CD8 T cell infiltration into IPMN areas of high dysplasia suggesting an ongoing immune response within the lesions. We also found, however, increased levels of circulating myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in some IPMN patients as well as evidence of T cell exhaustion. Further studies correlating immunosurveillance or immunosuppression with IPMN progression to malignancy will help define the immune response as a biomarker of risk, leading potentially to a vaccine to boost spontaneous immunity and prevent progression to cancer.

Novel mechanisms underlying the immediate and transient global tolerization of splenic dendritic cells after vaccination with a self-antigen.

Dendritic cells (DCs) are important orchestrators of the immune response, ensuring that immunity against pathogens is generated, whereas immunity against healthy tissues is prevented. Using the tumor Ag MUC1, we previously showed that i.v. immunization of MUC1 transgenic mice, but not wild-type, with a MUC1 peptide resulted in transient tolerization of all splenic DCs. These DCs did not upregulate costimulatory molecules and induced regulatory T cells rather than effector T cells. They were characterized by suppressed expression of a cohort of pancreatic enzymes not previously reported in DCs, which were upregulated in DCs presenting the same MUC1 peptide as a foreign Ag. In this article, we examined the self-antigen-tolerized DC phenotype, function, and mechanisms responsible for inducing or maintaining their tolerized state. Tolerized DCs share some characteristics with immature DCs, such as a less inflammatory cytokine/chemokine profile, deficient activation of NF-κB, and sustained expression of zDC and CCR2. However, tolerized DCs demonstrated a novel inducible expression of aldehyde dehydrogenase 1/2 and phospho-STAT3. Suppressed expression of one of the pancreatic enzymes, trypsin, in these DC impeded their ability to degrade extracellular matrix, thus affecting their motility. Suppressed metallopeptidases, reflected in low expression of carboxypeptidase B1, prevented optimal Ag-specific CD4(+) T cell proliferation suggesting their role in Ag processing. Tolerized DCs were not refractory to maturation after stimulation with a TLR3 agonist, demonstrating that this tolerized state is not terminally differentiated and that tolerized DCs can recover their ability to induce immunity to foreign Ags.

Antigen choice determines vaccine-induced generation of immunogenic versus tolerogenic dendritic cells that are marked by differential expression of pancreatic enzymes.

Dendritic cells (DC) elicit immunity to pathogens and tumors while simultaneously preserving tolerance to self. Efficacious cancer vaccines have been a challenge because they are based on tumor Ags, some of which are self-Ags and thus subject to self-tolerance. One such Ag is the tumor-associated mucin MUC1. Preclinical testing of MUC1 vaccines revealed existence of peripheral tolerance to MUC1 that compromises their efficacy. To identify mechanisms that act early postvaccination and might predict vaccine outcome, we immunized human MUC1 transgenic mice (MUC1.Tg) i.v. with a MUC1 peptide vaccine against which they generate weak immunity and wild-type (WT) mice that respond strongly to the same peptide. We analyzed differences in splenic DC phenotype and function between the two mouse strains at 24 and 72 h postvaccination and also performed unbiased total gene expression analysis of the spleen. Compared to WT, MUC1.Tg spleens had significantly fewer DC, and they exhibited significantly lower expression of costimulatory molecules, decreased motility, and preferential priming of Ag-specific Foxp3(+) regulatory T cells. This tolerogenic DC phenotype and function was marked by a new putative biomarker revealed by the microarray: a cohort of pancreatic enzymes (trypsin, carboxypeptidase, elastase, and others) not previously reported in DC. These enzymes were strongly upregulated in the splenic DC from vaccinated WT mice and suppressed in the splenic DC of vaccinated MUC1.Tg mice. Suppression of the enzymes was dependent on regulatory T cells and on signaling through the IL-10R and correlated with global downregulation of DC immunostimulatory phenotype and function.

Cancer vaccines: between the idea and the reality.

Whether vaccines are designed to prepare the immune system for the encounter with a pathogen or with cancer, certain common challenges need to be faced, such as what antigen and what adjuvant to use, what type of immune response to generate and how to make it long lasting. Cancer, additionally, presents several unique hurdles. Cancer vaccines must overcome immune suppression exerted by the tumour, by previous therapy or by the effects of advanced age of the patient. If used for cancer prevention, vaccines must elicit effective long-term memory without the potential of causing autoimmunity. This article addresses the common and the unique challenges to cancer vaccines and the progress that has been made in meeting them. Considering how refractory cancer has been to standard therapy, efforts to achieve immune control of this disease are well justified.

Vaccines based on abnormal self-antigens as tumor-associated antigens: immune regulation.

Abnormal expression of "self" antigens on tumors compared with normal cells provides opportunities and challenges for development of cancer vaccines. We review recent work in pre-clinical transgenic mouse models and in clinical trials that has elucidated multiple regulatory mechanisms that interfere with the induction of effective immunity. We discuss these as being either part of the normal function of the immune system or being driven by the tumor microenvironment. Collectively this work shows that it is possible to design vaccines based on tumor-associated antigens and elicit effective immunity against abnormal expression of these antigens on tumors without causing autoimmunity.

Variables affecting CA15.3 tumor antigen expression and antibodies against it in female National Health and Nutritional Survey (NHANES) participants.

BACKGROUND: Cancers of ductal origin often express glycoprotein mucin 1 (MUC1), also known as CA15.3, with higher levels leading to poorer prognosis. Conversely, anti-MUC1 antibodies develop in some patients leading to better prognosis. We sought to identify epidemiologic factors associated with CA15.3 antigen or antibody levels. METHODS: Levels of CA15.3 antigen and anti-CA15.3 IgG antibodies were measured in archived sera from 2302 mostly healthy women from the National Health and Nutritional Survey (NHANES); and epidemiologic predictors of their levels were examined using multivariate and correlational analyses. RESULTS: Among racial groups, Black women had the highest level of CA15.3 antigen and lowest levels of antibodies. Increasing BMI and current smoking were associated with low anti-CA15.3 antibody levels. Low CA15.3 antigen levels were seen in oral contraceptive (OC) users and high levels in women who were pregnant or lactating at the time of blood collection, with the latter group also having high antibody levels. Past reproductive events associated with high antigen levels included: later age at menarche, having given birth, and history of endometriosis. Lower antigen levels were seen with increasing duration of OC use. Anti-CA15.3 antibody levels decreased with an increasing estimated number of ovulatory years. CONCLUSION: Key determinants of CA.15.3 antigen or antibody levels include: race, BMI, smoking, later menarche, childbirth, number of ovulatory cycles, and endometriosis. IMPACT: This study supports the premise that known epidemiologic factors affecting risk for or survival after MUC1-expressing cancers may, at least partially, operate through their association with CA15.3 antigen or antibody levels.

Advances and challenges in cancer immunoprevention and immune interception.

Invasive cancers typically evade immune surveillance through profound local and systemic immunosuppression, preventing their elimination or control. Targeting immune interventions to prevent or intercept premalignant lesions, before significant immune dysregulation has occurred, may be a more successful strategy. The field of cancer immune interception and prevention is nascent, and the scientific community has been slow to embrace this potentially most rational approach to reducing the global burden of cancer. This may change due to recent promising advances in cancer immunoprevention including the use of vaccines for the prevention of viral cancers, the use of cancer-associated antigen vaccines in the setting of precancers, and the development of cancer-preventative vaccines for high-risk individuals who are healthy but carry cancer-associated heritable genetic mutations. Furthermore, there is increasing recognition of the importance of cancer prevention and interception by national cancer organizations. The National Cancer Institute (NCI) recently released the National Cancer Plan, which includes cancer prevention among the top priorities of the institute. The NCI's Division of Cancer Prevention has been introducing new funding opportunities for scientists with an interest in the field of cancer prevention: The Cancer Prevention-Interception Targeted Agent Discovery Program and The Cancer Immunoprevention Network. Moreover, the Human Tumor Atlas Network is spearheading the development of a precancer atlas to better understand the biology of pre-invasive changes, including the tissue microenvironment and the underlying genetics that drive carcinogenesis. These data will inform the development of novel immunoprevention/immuno-interception strategies. International cancer foundations have also started recognizing immunoprevention and immune interception with the American Association for Cancer Research, Cancer Research UK and the Society for Immunotherapy of Cancer each implementing programming focused on this area. This review will present recent advances, opportunities, and challenges in the emerging field of cancer immune prevention and immune interception.

Cellular and transcriptional profiles of peripheral blood mononuclear cells pre-vaccination predict immune response to preventative MUC1 vaccine.

A single arm trial (NCT007773097) and a double-blind, placebo controlled randomized trial ( NCT02134925 ) were conducted in individuals with a history of advanced colonic adenoma to test the safety and immunogenicity of the MUC1 tumor antigen vaccine and its potential to prevent new adenomas. These were the first two trials of a non-viral cancer vaccine administered in the absence of cancer. The vaccine was safe and strongly immunogenic in 43% (NCT007773097) and 25% ( NCT02134925 ) of participants. The lack of response in a significant number of participants suggested, for the first time, that even in a premalignant setting, the immune system may have already been exposed to some level of suppression previously reported only in cancer. Single-cell RNA-sequencing (scRNA-seq) on banked pre-vaccination peripheral blood mononuclear cells (PBMCs) from 16 immune responders and 16 non-responders identified specific cell types, genes, and pathways of a productive vaccine response. Responders had a significantly higher percentage of CD4+ naive T cells pre-vaccination, but a significantly lower percentage of CD8+ T effector memory (TEM) cells and CD16+ monocytes. Differential gene expression (DGE) and transcription factor inference analysis showed a higher level of expression of T cell activation genes, such as Fos and Jun, in CD4+ naive T cells, and pathway analysis showed enriched signaling activity in responders. Furthermore, Bayesian network analysis suggested that these genes were mechanistically connected to response. Our analyses identified several immune mechanisms and candidate biomarkers to be further validated as predictors of immune responses to a preventative cancer vaccine that could facilitate selection of individuals likely to benefit from a vaccine or be used to improve vaccine responses.

Characterization of latently infected EBV+ antibody-secreting B cells isolated from ovarian tumors and malignant ascites.

Introduction: Intra-tumoral B cells mediate a plethora of immune effector mechanisms with key roles in anti-tumor immunity and serve as positive prognostic indicators in a variety of solid tumor types, including epithelial ovarian cancer (EOC). Several aspects of intra-tumoral B cells remain unclear, such as their state of activation, antigenic repertoires, and capacity to mature into plasma cells. Methods: B lymphocytes were isolated from primary EOC tissue and malignant ascites and were maintained in cell culture medium. The stably maintained cell lines were profiled with flow cytometry and B cell receptor sequencing. Secreted antibodies were tested with a human proteome array comprising more than 21,000 proteins, followed by ELISA for validation. Originating tumor samples were used for spatial profiling with chip cytometry. Results: Antibody-secreting B lymphocytes were isolated from the ovarian tumor microenvironment (TME) of four different EOC patients. The highly clonal cell populations underwent spontaneous immortalization in vitro, were stably maintained in an antibody-secreting state, and showed presence of Epstein-Barr viral (EBV) proteins. All originating tumors had high frequency of tumor-infiltrating B cells, present as lymphoid aggregates, or tertiary lymphoid structures. The antigens recognized by three of the four cell lines are coil-coil domain containing protein 155 (CCDC155), growth factor receptor-bound protein 2 (GRB2), and pyruvate dehydrogenase phosphatase2 (PDP2), respectively. Anti-CCDC155 circulating IgG antibodies were detected in 9 of 20 (45%) of EOC patients' sera. Tissue analyses with multiparameter chip cytometry shows that the antibodies secreted by these novel human B cell lines engage their cognate antigens on tumor cells. Discussion: These studies demonstrate that within the tumor-infiltrating lymphocyte population in EOC resides a low frequency population of antibody-secreting B cells that have been naturally exposed to EBV. Once stably maintained, these novel cell lines offer unique opportunities for future studies on intratumor B cell biology and new target antigen recognition, and for studies on EBV latency and/or viral reactivation in the TME of non-EBV related solid tumors such as the EOC.

Pre-vaccination transcriptomic profiles of immune responders to the MUC1 peptide vaccine for colon cancer prevention.

Self-antigens abnormally expressed on tumors, such as MUC1, have been targeted by therapeutic cancer vaccines. We recently assessed in two clinical trials in a preventative setting whether immunity induced with a MUC1 peptide vaccine could reduce high colon cancer risk in individuals with a history of premalignant colon adenomas. In both trials, there were immune responders and non-responders to the vaccine. Here we used PBMC pre-vaccination and 2 weeks after the first vaccine of responders and non-responders selected from both trials to identify early biomarkers of immune response involved in long-term memory generation and prevention of adenoma recurrence. We performed flow cytometry, phosflow, and differential gene expression analyses on PBMCs collected from MUC1 vaccine responders and non-responders pre-vaccination and two weeks after the first of three vaccine doses. MUC1 vaccine responders had higher frequencies of CD4 cells pre-vaccination, increased expression of CD40L on CD8 and CD4 T-cells, and a greater increase in ICOS expression on CD8 T-cells. Differential gene expression analysis revealed that iCOSL, PI3K AKT MTOR, and B-cell signaling pathways are activated early in response to the MUC1 vaccine. We identified six specific transcripts involved in elevated antigen presentation, B-cell activation, and NF-kB1 activation that were directly linked to finding antibody response at week 12. Finally, a model using these transcripts was able to predict non-responders with accuracy. These findings suggest that individuals who can be predicted to respond to the MUC1 vaccine, and potentially other vaccines, have greater readiness in all immune compartments to present and respond to antigens. Predictive biomarkers of MUC1 vaccine response may lead to more effective vaccines tailored to individuals with high risk for cancer but with varying immune fitness.

Human mucin MUC1 RNA undergoes different types of alternative splicing resulting in multiple isoforms.

MUC1 is a transmembrane mucin with important functions in normal and transformed cells, carried out by the extracellular domain or the cytoplasmic tail. A characteristic feature of the MUC1 extracellular domain is the variable number of tandem repeats (VNTR) region. Alternative splicing may regulate MUC1 expression and possibly function. We developed an RT-PCR method for efficient isolation of MUC1 mRNA isoforms that allowed us to evaluate the extent of alternative splicing of MUC1 and elucidate some of the rules that govern this process. We cloned and analyzed 21, 24, and 36 isoforms from human tumor cell lines HeLa, MCF7, and Jurkat, respectively, and 16 from normal activated human T cells. Among the 78 MUC1 isoforms we isolated, 76 are new and different cells showed varied MUC1 expression patterns. The VNTR region of exon 2 was recognized as an intron with a fixed 5' splice site but variable 3' splice sites. We also report that the 3506 A/G SNP in exon 2 can regulate 3' splice sites selection in intron 1 and produce different MUC1 short isoform proteins. Furthermore, the SNP A to G mutation was also observed in vivo, during de novo tumor formation in MUC1(+/-)Kras(G12D/+)Pten(loxP/loxP) mice. No specific functions have been associated with previously reported short isoforms. We now report that one new G SNP-associated isoform MUC1/Y-LSP, but not the A SNP-associated isoform MUC1/Y, inhibits tumor growth in immunocompetent but not immunocompromised mice.

Post-translational covalent assembly of CAR and synNotch receptors for programmable antigen targeting.

Chimeric antigen receptors (CARs) and synthetic Notch (synNotch) receptors are engineered cell-surface receptors that sense a target antigen and respond by activating T cell receptor signaling or a customized gene program, respectively. Here, to expand the targeting capabilities of these receptors, we develop "universal" receptor systems for which receptor specificity can be directed post-translationally via covalent attachment of a co-administered antibody bearing a benzylguanine (BG) motif. A SNAPtag self-labeling enzyme is genetically fused to the receptor and reacts with BG-conjugated antibodies for covalent assembly, programming antigen recognition. We demonstrate that activation of SNAP-CAR and SNAP-synNotch receptors can be successfully targeted by clinically relevant BG-conjugated antibodies, including anti-tumor activity of SNAP-CAR T cells in vivo in a human tumor xenograft mouse model. Finally, we develop a mathematical model to better define the parameters affecting universal receptor signaling. SNAP receptors provide a powerful strategy to post-translationally reprogram the targeting specificity of engineered cells.