Multiple Escape Mechanisms Research Articles

Abstract A039: Detection of immune cell glycosylation as an indicator of metabolic activity in the tumor tissue microenvironment using multimodal mass spectrometry imaging

Abstract Glycosylation on the cell surface is a major target and mediator of the immune response to cancers. It is expected that any immune cells present in these tissues will be actively engaged in responding to the presence of the tumor, however, multiple escape mechanisms used by the targets are known to suppress these immune responses. It follows that in this tumor microenvironment, any response, or non-response, to a cancer immunotherapeutic will also be mediated by glycans present in the target tissues and immune cells interacting with them. From a data archive of over 500 FFPE human tumor tissues assessed for N-glycan imaging MS analysis, a subset of tissues (prostate, colon, pancreas, lung) with notable intra- and peri-tumor immune cell clusters were re-evaluated for detection of N-glycans that co-localize to these regions. The metabolic premise for this is that the Warburg metabolites glucose and glutamine are both required for N-glycan biosynthesis and therefore active immune cells would have detectable glycan signatures. In most tumor types evaluated, there was minimal to no detection of N-glycans. This would be consistent with a tumor microenvironment deficient in metabolites due to the presence of the tumor, or other immunosuppressive mechanisms. A subset of the tissues did have glycan signatures associated with immune cell clusters, and the glycan structures present in each cluster were recorded. The same tissues were assessed by multiplexed MALDI-immunohistochemistry (IHC) to identify the immune cell types present in each cluster. Established method workflows were used for the N-glycan MALDI imaging and MALDI-IHC analyses. Previously characterized N-glycans and immune cell clusters (CD4, CD8, CD11b, CD163) in SARS-CoV2 infected autopsy lung tissues were used as positive controls for an active immune microenvironment. Distinct N-glycan species are associated with each immune cell type in these tissues. Detected N-glycans included high mannose structures, and a series of tri- and tetra-antennary structures with one fucose and a bisecting N-acetylglucosamine (GlcNAc). While the high mannose glycans were detected in other areas of the tissues, the bisecting branched N-glycans were distinctly enriched in the immune cell clusters. In these selected tissue subsets, immune cell clusters distal to tumor regions had readily detected high mannose N-glycans and tri-and tetra-antennary bisecting GlcNAc structure. In general, the immune cell clusters adjacent to the tumor region had minimal to no glycan expression relative to the more distal regions. We hypothesize that when N-glycans are detected by imaging mass spectrometry in tissue immune cell clusters that this represents tumors with more active immune functional states, and the lack of detection represents immune-suppressed tumors. Citation Format: Richard R Drake, Kameisha Radford, Caroline Kittrell, Kristin Wallace, Peggi M Angel. Detection of immune cell glycosylation as an indicator of metabolic activity in the tumor tissue microenvironment using multimodal mass spectrometry imaging [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A039.

Treatment Options in BRAF-mutant Metastatic Colorectal Cancer

Treatment of BRAF-mutant colorectal cancer (CRC) traditionally represents an unmet need, mainly due to its unfavourable prognostic outlook, limited options for targeted treatment and scarce benefit from epithelial growth-factor receptor (EGFR) inhibitors. Recently, the development of BRAF V600E inhibitors has expanded the therapeutic armamentarium, although exclusive targeting of BRAF has proved to be an unsuccessful strategy due to reactivation of the mitogen-activated protein kinase pathway through multiple escape mechanisms. Combination strategies that exploit simultaneous inhibition of BRAF, EGFR and/or mitogen-activated protein/extracellular signal-regulated kinase have achieved greater success, with the BEACON CRC trial providing the first evidence for an improvement in survival with a chemotherapy-free approach in pre-treated patients with CRC, leading to regulatory approval for the combination of encorafenib and cetuximab. Subsequent research efforts attempt to build on these foundations, exploring targeted maintenance strategies and conceivably moving the combination towards the first line of therapy soon, as well as laying the foundation for the use of liquid biopsy as a guidance tool in a precision oncology approach.

Blocking HIF to Enhance NK Cells: Hints for New Anti-Tumor Therapeutic Strategies?

Natural Killer (NK) cells are becoming an ever more promising tool to design new anti-tumor strategies. However, two major issues are still a challenge to obtain versatile and effective NK-based therapies: the way to maximize the persistency of powerful NK effectors in the patient, and the way to overcome the multiple escape mechanisms that keep away or suppress NK cells at the tumor site. In this regard, targeting the hypoxia-inducible factors (HIFs), which is important for both tumor progression and immune suppression, may be an opportunity. Especially, in the context of the ongoing studies focused on more effective NK-based therapeutic products.

Cell wall deficiency as an escape mechanism from phage infection.

The cell wall plays a central role in protecting bacteria from some environmental stresses, but not against all. In fact, in some cases, an elaborate cell envelope may even render the cell more vulnerable. For example, it contains molecules or complexes that bacteriophages recognize as the first step of host invasion, such as proteins and sugars, or cell appendages such as pili or flagella. In order to counteract phages, bacteria have evolved multiple escape mechanisms, such as restriction-modification, abortive infection, CRISPR/Cas systems or phage inhibitors. In this perspective review, we present the hypothesis that bacteria may have additional means to escape phage attack. Some bacteria are known to be able to shed their cell wall in response to environmental stresses, yielding cells that transiently lack a cell wall. In this wall-less state, the bacteria may be temporarily protected against phages, since they lack the essential entities that are necessary for phage binding and infection. Given that cell wall deficiency can be triggered by clinically administered antibiotics, phage escape could be an unwanted consequence that limits the use of phage therapy for treating stubborn infections.

Antigen presentation in cancer: insights into tumour immunogenicity and immune evasion.

Immune checkpoint blockade, which blocks inhibitory signals of T cell activation, has shown tremendous success in treating cancer, although success still remains limited to a fraction of patients. To date, clinically effective CD8+ T cell responses appear to target predominantly antigens derived from tumour-specific mutations that accumulate in cancer, also called neoantigens. Tumour antigens are displayed on the surface of cells by class I human leukocyte antigens (HLA-I). To elicit an effective antitumour response, antigen presentation has to be successful at two distinct events: first, cancer antigens have to be taken up by dendritic cells (DCs) and cross-presented for CD8+ T cell priming. Second, the antigens have to be directly presented by the tumour for recognition by primed CD8+ T cells and killing. Tumours exploit multiple escape mechanisms to evade immune recognition at both of these steps. Here, we review the tumour-derived factors modulating DC function, and we summarize evidence of immune evasion by means of quantitative modulation or qualitative alteration of the antigen repertoire presented on tumours. These mechanisms include modulation of antigen expression, HLA-I surface levels, alterations in the antigen processing and presentation machinery in tumour cells. Lastly, as complete abrogation of antigen presentation can lead to natural killer (NK) cell-mediated tumour killing, we also discuss how tumours can harbour antigen presentation defects and still evade NK cell recognition.

Multi Targeted CAR-T Cell Therapies for B-Cell Malignancies.

Chimeric antigen receptor (CAR) modified T cell therapy has revolutionized the treatment of relapsed and refractory hematological malignancies. Through targeting of the CD19 antigen on B cells durable remissions have been achieved in patients with B cell non-Hodgkin lymphoma and acute lymphoblastic lymphoma. Despite impressive responses, multiple escape mechanisms to evade CAR-T cell therapy have been identified, among which the most common is loss of the target antigen. In this review we will highlight outcomes to date with CD19 CAR-T cell therapy, describe the current limitations of single targeted CAR-T therapies, review identified tumor escape mechanisms, and lastly discuss novel strategies to overcome resistance via multi-targeted CAR-T cells.

Anti-Immune Strategies of Pathogenic Fungi.

Pathogenic fungi have developed many strategies to evade the host immune system. Multiple escape mechanisms appear to function together to inhibit attack by the various stages of both the adaptive and the innate immune response. Thus, after entering the host, such pathogens fight to overcome the immune system to allow their survival, colonization and spread to different sites of infection. Consequently, the establishment of a successful infectious process is closely related to the ability of the pathogen to modulate attack by the immune system. Most strategies employed to subvert or exploit the immune system are shared among different species of fungi. In this review, we summarize the main strategies employed for immune evasion by some of the major pathogenic fungi.

Multiplexed screening of natural humoral immunity identifies antibodies at fine specificity for complex and dynamic viral targets

Viral entry targets with therapeutic neutralizing potential are subject to multiple escape mechanisms, including antigenic drift, immune dominance of functionally irrelevant epitopes, and subtle variations in host cell mechanisms. A surprising finding of recent years is that potent neutralizing antibodies to viral epitopes independent of strain exist, but are poorly represented across the diverse human population. Identifying these antibodies and understanding the biology mediating the specific immune response is thus difficult. An effective strategy for meeting this challenge is to incorporate multiplexed antigen screening into a high throughput survey of the memory B cell repertoire from immune individuals. We used this approach to discover suites of cross-clade antibodies directed to conformational epitopes in the stalk region of the influenza A hemagglutinin (HA) protein and to select high-affinity anti-peptide antibodies to the glycoprotein B (gB) of human cytomegalovirus. In each case, our screens revealed a restricted VH and VL germline usage, including published and previously unidentified gene families. The in vivo evolution of paratope specificity with optimal neutralizing activity was understandable after correlating biological activities with kinetic binding and epitope recognition. Iterative feedback between antigen probe design based on structure and function information with high throughput multiplexed screening demonstrated a generally applicable strategy for efficient identification of safe, native, finely tuned antibodies with the potential for high genetic barriers to viral escape.

Coexistence of multiple escape mechanisms in a BRAFV600E-mutated cutaneous melanoma treated with vemurafenib.

9014 Background: Mechanisms of acquired resistance to vemurafenib are the subject of intense research. Here we report, for the first time, two coexisting yet mutually exclusive mechanisms of escape in a melanoma patient who presented an excellent clinical response following reintroduction of vemurafenib. Methods: To investigate the mechanisms of resistance to vemurafenib, we performed whole-exome sequencing using the Illumina technology of a pre-treatment paraffin-embedded lymph node metastasis (Pre) and of two progressing subcutaneous snap-frozen metastases of the right arm (PV1 and PV2). The tumor samples, along with germline controls, were sequenced at high coverage (mean range 268x-356x). Results: We identified 107 exonic somatic Single Nucleotide Variants (SNVs) in Pre, 139 SNVs in PV1, and 127 SNVs in PV2, generating a set of 202 different SNVs, 82 of which were common to all 3 samples. The non-synonymous to synonymous ratios were higher for PV1 (1.82) than for PV2 (1.46), and lower for Pre (1.31). C>T transitions, largely predominated in the samples, indicating light-induced damage. Two independent NRAS escape mutations (Q61K and Q61R) where observed in PV1, whereas appearance of a BRAF splice variant (lack of exon 4-10) was present in PV2. Most importantly, these 2 escape mechanisms were mutually exclusive, i.e. no BRAF splice variant was observed by PCR in PV1 and no NRAS mutation found in PV2. Conclusions: Our results clearly demonstrate that multiple molecular escape mechanisms can be both coexistent and mutually exclusive. These findings have clinical implications: firstly, local treatment of isolated progressing lesions and continuation of vemurafenib could be supported by the fact that the resistance mechanisms are not necessarily shared. This approach is currently being tested within clinical trials with preliminary results that seem to support this hypothesis. Secondly, the coexistence of divergent escapes within the same patient strongly argues that single biopsy analysis at progression might not reflect the molecular complexity of tumor progression, and therefore might not be sufficient to guide selection of second-line optimal combination therapy.

Cancer immunotherapy: Progress and challenges in the clinical setting

Cancer immunologists have made enormous efforts to prove that innate and adaptive immune cells recognize tumor cells and induce tumor rejection in experimental animal models. These observations provided the rationale to study the role of the immune system in the control of tumor growth in cancer patients and to develop immunotherapeutic strategies to treat patients with cancers. Several lines of evidence suggest that anti-tumor immune responses may correlate with better clinical outcome in patients with cancers. Among them is the correlation between the presence of tumor-infiltrating T cells and the improved clinical outcome for patients with solid tumors [1–3]. In addition, a number of immunotherapies, such as high-dose IL-2 [4] and TA-specific monoclonal antibodies (mAb) [5], have provided long-term clinical benefits to a minority of cancer patients. Most importantly, the recent approval by the US FDA of the mAb ipilimumab directed against the co-inhibitory molecule CTLA-4 for patients with unresectable or metastatic melanoma represents a major breakthrough for mAb-based therapies in oncology practice [6]. The successful outcome of the randomized phase III clinical trial with ipilumumab has provided the much-needed incontrovertible clinical evidence that in humans, as in experimental animal models, the host’s immune system can control tumor growth. Furthermore, it has infused a considerable amount of optimism among tumor immunologists and clinical oncologists about the clinical potential of immunotherapy for the treatment of advanced cancers. However, there are also many examples of spontaneous or vaccine-induced TA-specific T- and B-cell immune responses that do not correlate with improved clinical status [7–9]. This discrepancy between immune and clinical responses underlines the need to better dissect the molecular and cellular events leading to tumor rejection in humans. Such an endeavor has greatly benefited from the molecular identification of TA expressed by human tumor cells, which are recognized by T cells and antibodies [5, 10, 11]. As a result, TA-specific immunotherapies have been implemented in clinical trials with molecularly defined cancer vaccines, TA-specific mAb and adoptive transfer of TA-specific T cells. Novel generations of cancer vaccines with molecularly defined TAs and potent adjuvants like toll-like receptor ligands appear to stimulate strong TA-specific T-cell responses but have shown evidence of clinical benefits in only a minority of patients with advanced cancer [7, 8, 12]. The adoptive transfer of TA-specific T cells remains technically challenging and the promising data obtained in terms of objective clinical responses and durability of responses from small monocentric clinical trials will need to be further confirmed in large multicenter clinical trials [13]. TA-specific mAb are clinically effective in a number of hematological malignancies and solid tumors and are routinely used in the clinic [5]. We now have a better understanding of the multiple mechanisms of tumor-induced immune escape, which are likely to cause the failure of the spontaneous or vaccine-induced immune responses to promote tumor regression in humans. In the tumor microenvironment, a number of negative regulators dampens anti-tumor immune responses and/or their therapeutic efficacy, including the production of cytokines (like TGF-β or IL-10), suppressive cells (regulatory T cells, myelosuppressive dendritic cells), defective antigen presentation by tumor cells (HLA or tumor antigen loss, antigen processing machinery defects), amino-acid catabolizing enzymes (indoleamine-2-3dioxygenase, arginase) and co-inhibitory pathways (like CTLA-4/CD28, PD-1/PD-L1) [14–17]. As a consequence, a number of therapies to specifically target these pathways are being developed to enhance TA-specific immune responses and to increase the likelihood of clinical benefits. In this article, commissioned to recognize National Cancer Survivors Day (the first Sunday in June each year, 5 June in 2011, see www.ncsdf.org), we will comment on the successes of immunotherapy of cancer in the clinical setting. In addition, we will discuss the challenges to optimize the use of cancer immunotherapies in the clinic.

107 Escape from Neutralizing Antibody in Early Subtype C HIV-1 Infection

HIV-1 subtype C viruses circulate predominantly in India and sub-Saharan Africa, accounting for most global infections. We previously demonstrated that 9 out of 11 subtype C infected seroconvertors followed in a Zambian cohort rapidly developed high titer autologous neutralizing antibody (Nab) against the infecting virus. Here we characterized the Nab sensitivity of individual envelope (Env) glycoproteins that evolved during early infection in four of these seroconvertors. Env genes were PCR amplified from longitudinal patient PBMC DNA and plasma samples using single genome analysis, cloned into an expression vector, and screened for biological function using a pseudovirus assay. Neutralization sensitivity of each Env pseudovirus was evaluated against autologous plasma from longitudinal time points to identify variants that had escaped from contemporaneous Nab. The molecular mechanism of Nab escape was then investigated in detail in two subjects by replacing Env domains of the initial, Nab sensitive virus with those derived from 6 to 10 escape variants from the same patient. The monophyletic founder virus in the newly infected subjects was rapidly replaced by a succession of variants that were resistant to contemporaneous Nab but were subsequently neutralized by a de novo antibody response. In one patient, the virus used multiple escape mechanisms, even at a single time point. One of these escape pathways required sequence changes in both gp120 and the gp41 ectodomain, while another involved only the V3-V5 region of gp120. The V1V2 domain, however, did not contribute to neutralization escape in this subject. In contrast, in the second patient, the V1V2 domain played a major role in resistance in every Env analyzed. The changes in V1V2 conferred early and complete resistance against a monoclonal antibody isolated from the PBMC of this patient four years later, but changes outside of V1V2 were required to escape from the polyclonal antibody response. Our data demonstrate that subtype C viruses use multiple mechanisms to escape from potent autologous Nab, even within a single patient. These findings highlight the need to define and understand potential escape mechanisms in strategies to induce protective Nab.

Human CD4+ T Lymphocytes Recognize a Vascular Endothelial Growth Factor Receptor-2–Derived Epitope in Association with HLA-DR

Given the multiple escape mechanisms of tumor cells, immunotherapy targeting tumor-dependent stroma may be an effective cancer treatment strategy. Animal models indicate that inducing immunity to tumor endothelia engenders potent antitumor effects without significant pathology. Recently, the first human tumor endothelial antigen vascular endothelial growth factor receptor-2 (VEGFR-2) recognized by HLA class I-restricted CD8(+) T cells has been characterized. In this study, we sought to investigate specific recognition of this molecule by human CD4(+) T cells. To identify HLA-DR-restricted antigenic peptides on VEGFR-2 recognized by CD4(+) T cells of healthy donors and cancer patients. Nine candidate VEGFR-2 peptides with high binding probability to six common HLA-DRB1 alleles were synthesized using the SYFPEITHI algorithm. One 15-mer peptide (EKRFVPDGNRISWDS), mapping to the 167-181 region of VEGFR-2, stimulated CD4(+) T cells in association with several HLA-DR alleles, including DR4 and DR7. Importantly, the epitope could be naturally processed and presented both by HLA-DR-matched antigen-expressing proliferating endothelial cells and by dendritic cells loaded with the native antigen. Furthermore, circulating VEGFR-2-specific CD4(+) T cells were detected in 4 of 10 healthy donors and 12 of 40 cancer patients even after single-round peptide stimulation in short-term culture. Patient's T cells could recognize antigen-expressing proliferating endothelial cells in a HLA-DR-restricted fashion. These findings indicate an important role for the 167-181 region of VEGFR-2 in the stimulation of CD4(+) T cell responses to VEGFR-2 protein, and may be instrumental both for the development and monitoring of upcoming antitumor vessel vaccines against different cancers based on VEGFR-2 immunogens.

Rational Design of AIDS Vaccines

Development of an effective vaccine against HIV has posed significant challenges to the scientific community. Lack of knowledge about the molecular pathogenesis of this disease, and the absence of naturally occurring, protective immune responses, which is attributed in large part to the diversity of the viral envelope and multiple escape mechanisms, have made it difficult to design successful candidates. Recent studies of the envelope have suggested that it is possible to enhance immunogenicity of the envelope by improving the breadth of the neutralizing antibody response and by stimulating cell-mediated immunity. This relies on an understanding of the structure of HIV Env and the use of site-specific mutation to create novel immunogens. Based on structural and functional data, our lab has developed DNA and adenoviral vectors which express envelope proteins containing alterations to regions in the variable loop to better control tropism and improve immunogenicity. These modified HIV genetic vaccine candidates have been tested in combination with Gag, Pol, and Nef immunogens, and shown to improve the potency of cellular and humoral immune responses in primates. An initial Phase I trial (VRC 004) dose escalation study to test a DNA vaccine composed of a 4-plasmid combination of clade B gag/pol/nef with clades A, B, and C envelope was recently completed in 50 healthy adults in the U.S. CD4+ and CD8+ T cell responses were detected in the majority of vaccinees using IFN-(ELISpot) and flow cytometric detection of intracellular IL-2 or IFN-γ. HIV-specific antibody response was detected in about one third of vaccinees. Combination modality regimens using a DNA vaccine prime followed by a viral vector boost have shown promise in nonhuman primate models of HIV infection. Phase I clinical trials to test the safety and immunogenicity of an adenoviral vaccine expressing proteins similar to the DNA, and a DNA prime, adenoviral boost regimen are in progress. The initial adenoviral vector vaccine uses an Ad5 serotype vector. However, pre-existing immunity can mitigate the efficacy, so alternate serotypes and modifications to the adenoviral fiber regions are being explored. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

HLA-G Proteins in Cancer: Do They Provide Tumor Cells with an Escape Mechanism?

Convincing clinical evidence indicates that the limited success of T-cell-based immunotherapy of malignant diseases is caused, at least in part, by the ability of malignant cells to escape from immune recognition and destruction. Among the multiple escape mechanisms identified, a major role is played by changes in the expression and/or function of HLA antigens expressed by tumor cells, because they may markedly affect tumor cell-host's immune system interactions. In this article, we review the data about the aberrant expression of the nonclassical HLA class I antigen HLA-G by tumor cells. Furthermore, we discuss the possible reasons for the conflicting information in the literature about HLA-G antigen expression by malignant cells. Lastly, in light of the well-documented immunotolerant function of HLA-G, we discuss the potential role of these antigens in the escape of tumor cells from immune recognition and destruction and in the clinical course of malignant diseases.

Immune selection of hot-spot beta 2-microglobulin gene mutations, HLA-A2 allospecificity loss, and antigen-processing machinery component down-regulation in melanoma cells derived from recurrent metastases following immunotherapy.

Scanty information is available about the mechanisms underlying HLA class I Ag abnormalities in malignant cells exposed to strong T cell-mediated selective pressure. In this study, we have characterized the molecular defects underlying HLA class I Ag loss in five melanoma cell lines derived from recurrent metastases following initial clinical responses to T cell-based immunotherapy. Point mutations in the translation initiation codon (ATG-->ATA) and in codon 31 (TCA-->TGA) of the beta(2)-microglobulin (beta(2)m) gene were identified in the melanoma cell lines 1074MEL and 1174MEL, respectively. A hot-spot CT dinucleotide deletion within codon 13-15 was found in the melanoma cell lines 1106MEL, 1180MEL, and 1259MEL. Reconstitution of beta(2)m expression restored HLA class I Ag expression in the five melanoma cell lines; however, the HLA-A and HLA-B,-C gene products were differentially expressed by 1074MEL, 1106MEL, and 1259MEL cells. In addition, in 1259MEL cells, the Ag-processing machinery components calnexin, calreticulin, and low m.w. polypeptide 10 are down-regulated, and HLA-A2 Ags are selectively lost because of a single cytosine deletion in the HLA-A2 gene exon 4. Our results in conjunction with those in the literature suggest the emergence of a preferential beta(2)m gene mutation in melanoma cells following strong T cell-mediated immune selection. Furthermore, the presence of multiple HLA class I Ag defects within a tumor cell population may reflect the accumulation of multiple escape mechanisms developed by melanoma cells to avoid distinct sequential T cell-mediated selective events.