Cancer vaccines: Building a bridge over troubled waters

Abstract

Cancer vaccines aim to direct the immune system to eradicate cancer cells. Here we review the essential immunologic concepts underpinning natural immunity and highlight the multiple unique challenges faced by vaccines targeting cancer. Recent technological advances in mass spectrometry, neoantigen prediction, genetically and pharmacologically engineered mouse models, and single-cell omics have revealed new biology, which can help to bridge this divide. We particularly focus on translationally relevant aspects, such as antigen selection and delivery and the monitoring of human post-vaccination responses, and encourage more aggressive exploration of novel approaches.

Figure 1.. Natural immunity to an intracellular pathogen.

Infected and stressed host cells (top left) release PAMP/DAMPs, activating APCs, like DCs. Activated DCs mature, upregulating phagocytosis, antigen presentation and co-stimulatory molecules, and migrate to a draining lymph node (dLN); lymph drainage also transports local antigens to the dLN. The activated DCs present MHCII restricted peptides to CD4⁺ T cells, which can in turn license DCs (including via CD40L:CD40 signals; see Inset A) to cross-present antigen on MHCI and activate CD8⁺ T cells. Cytokines from DCs also shape CD4⁺ Helper T cell differentiation. DCs, via p:MHCI stimulation of TCR and co-stimulatory signals (Inset B) activate CD8 cells to respond, produce IL2 and IL-12 (for autocrine signaling) and differentiate into cytotoxic T lymphocytes (CTLs). CD4⁺ T cells potentiate CTL differentiation via IL2, maintain CTL effector function in viral infection via IL21 and induce CD8⁺ resident memory differentiation via IFNγ (Inset C). CD4⁺ T cells that recognize cognate p:MHCII on B cells, can provide co-stimulation (Inset D) to support affinity maturation, antibody class switching and plasma cell differentiation. Abbreviations: Pathogen- /Damage- associated molecular pattern (PAMP/DAMP), Dendritic cell (DC), T4 (CD4⁺ Helper T cell), T8 (CD8⁺ T cell), CTL (Cytotoxic T lymphocyte), B (B cell), p:MHC (peptide bound multi-histocompatibility complex), TCR (T Cell Receptor).

Figure 2:. Immunosuppression in the Tumor Immune Microenvironment (TIME).

The TIME is made up of malignant cells, stomal cells and immune cells, all of which can suppress tumor directed immunity by acting directly or indirectly on effector T cells (T_eff). Boxes depict select immunosuppressive mechanisms employed by these various cell types: cancer cells (Ca), regulatory T cells (T_reg), dendritic cells (DC), macrophages (MΦ) and myeloid derived suppressor cells (MDSC; (Veglia et al., 2018)), and cancer associated fibroblasts (CAF).

Figure 3:. Vaccines, a bridge to cure?

While there are many barriers to natural immune responses to cancer, we propose that focusing on four pillars (innate and adaptive immune cells, antigen targets, delivery strategies and co-therapies) will help vaccines become a bridge from cancer to immune-based cure.

Figure 4:. Neoantigens, our blind spots and their presentation.

(A) Potential neoantigen sources in cancer, adapted with modifications from Gupta et. al. (Gupta et al., 2021). Conventional bioinformatic techniques efficiently detect neoantigens derived from single nucleotide variations (SNVs) and insertions/deletions (Indels) (circled in blue). Largely beyond current clinical prediction techniques (red text) are neoantigens from some somatic tandem duplication and fusion transcripts, endogenous retroviruses, transcriptional variations (alternative splicing, exitrons and A-to-I editing), and translational abnormalities (unannotated NuORFs, defective translation products and peptides from alternative start sites). (B) and (C) depict antigen presentation pathways in MHCI and MHCII respectively. (B) For MHCI, the proteasome process endogenous proteins into short peptides, which the TAP complex transports into the endoplasmic reticulum. Immune cells can diversify the peptidome by, processing proteins from phagosomes to allow exogenous peptide cross-presentation (i.e. cDC1s), and/or by immunoproteasome processing, which provides different protease specificities via replacement of three constitutive proteasome subunits with alternative subunits (Murata et al., 2018). In the ER, peptides are further processed and chaperones/editors, including Tapasin, facilitate MHCI loading with stable 8–10 aa peptides, before transport to the cell surface where T cells survey them via TCR:p:MHCI interactions that generally require CD8αβ:MHCI interactions. (C) For MHCII, proteins are processed into smaller peptides in phagosomes, auto-phagosomes (products of autophagy) and lysosomes (Roche and Furuta, 2015), which eventually fuse with the late endosome. There proteolytic cleavage of the invariant chain, CLIP (which facilitates MHCII folding during translation and resides partially in its antigen binding grove), allows loading of 13–25 amino-acid peptides on to MHCII; of note, MHCII is more promiscuous in peptide binding and peptides are considerably longer than for MHCI. p:MHCII complexes can then be edited by HLA-DM, which selects for peptides that are more stably bound to MHCII, before being transported to the cell surface for surveillance by where CD4⁺ T cell TCRs. In contrast to the importance of CD8 in TCR:p:MHCI interactions, TCR:p:MHCII interactions are not as dependent on CD4 recognition of MHC.

Figure 5:. Dendritic cells, PAMPs and DAMPs.

The diverse dendritic cell lineage expresses a broad array of pattern recognition receptors (PRR). PRRs include Toll like receptors (TLRs), which are present as transmembrane proteins to directly detect PAMPs externally (i.e. cell surface; TLR 1,2,4,5,6) or within endosomes (TLR 3,7,8,9; which focus on nucleic acids released from internalized pathogens) (Fitzgerald and Kagan, 2020); cell surface C-Lectin type Receptors (CLRs; (Geijtenbeek and Gringhuis, 2009)), which directly detect external PAMPs/DAMPs, and in the case of CLEC9A/DNRG1 and DEC205/CLEC13B, can facilitate phagocytosis of dead and dying cells. Intracellular PRRs, including cGAS/STING and RIG-I/MDA5, recognize intracellular nucleic acids. Nod-like receptors (NLR), including NLRP3, can participate in PAMP/DAMP sensing via interactions with caspases and the inflammasome. (Inset A) DC subsets most commonly considered in vaccine production, with lineage defining cell surface markers and transcription factors, differential PRR expression and unique functions. (Inset B) Select PAMPs/DAMPs, the PRRs that detect them, and mimics that are either commonly used as clinical vaccine adjuvants or are under pre-clinical testing. (Inset C) LPS and oxPAPC signaling via caspase-11 highlights differential processing of DAMP and PAMP signals, even via the same PRR pathway. LPS and oxPAPC both induce IL-1β production via caspase 11 and downstream NLRP3/atypical inflammasome signaling. While LPS stimulation leads to short lived, pyroptotic IL-1β-expressing DCs, OxPACP blocks pyroptosis, inducing a hyperactivated state and efficient CTL priming.

Figure 6:. Measuring success.

Ideally a cancer vaccine will demonstrate improved survival or progression free survival (A-i), although cancer vaccine trials are not usually powered to do so. Trials more typically focus on putative correlates of vaccine effectiveness via ex vivo analysis of T cells from the blood or tumor samples (A-ii) or analysis of tumor biopsies (A-iii). (A-ii) T cell assays include: ELISPOT, which enumerates T cell clones responsive to a defined peptide/epitope; flow cytometry based assays, which can identify antigen-specific T cells via p:MHC conjugates (e.g. tetramers), and define T cell function, activation and cytotoxic potential via cytokine, surface 41BB and CD107a exposure; bulk TCR sequencing can temporally track clonotype frequencies; single cell RNA and TCR sequencing (not broadly used in vaccine studies) may enable the linkage of clonotype changes to changes in cell state (e.g. effector, central memory, progenitor exhausted and exhausted). Linking TCR sequence to antigen specificity is not readily possible except through more extensive functional studies. (A-iii) Re-biopsy of tumor can provide evidence of vaccine effectiveness, for example, if genomic or expression analyses demonstrate loss of antigen presenting machinery or neoantigens, or decreased neoantigen expression. Newer spatial multiplexed IHC, in situ hybridization (Nanostring) and sequencing (Slide-seq), may show differences in T cell infiltration patterns (especially when paired with TCR sequencing methods). (B) Schema of epitope spreading. We propose that analysis of epitope spreading may best balance feasibility and sensitivity for vaccine efficacy. Epitope spreading is the concept that one antigen-specific immune response begets another: First, a vaccine primes a CD8⁺ T cell (purple T₈ cell recognizing a purple neoantigen; B-i). That vaccine specific CTL migrates to the tumor (B-ii), where it recognizes and destroys a cancer cell, releasing DAMPs that stimulate a DC maturation and phagocytoses tumor cell remnants, including non-vaccine targeted neoantigens (red). The DC migrates to a draining LN (B-iii), where it presents neoantigens to T cells. A CD8⁺ T cell (red) with specificity for a new neoantigen is de novo primed and expanded, returning to the tumor (B-iv). B-bottom, model ELISPOT data reflecting epitope spreading is shown, with vaccine induced neoantigen responses appearing after vaccine priming, but non-vaccine neoantigen responses from epitope spreading, appearing only later.