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There was a recent publication concerning use of IV administered RNA-lipoplexes with an adjusted electrical charge to encourage dendritic cells (DC) to pick up the RNA package encoding viral or mutant neo-antigens, for example, a sequence of melanoma tumor cell coded to RNA. The paper is at Systemic RNA delivery to dendritic cells

After reading that abstract and related papers (see Dendritic Cells Pulsed with RNA are Potent Antigen-presenting Cells In Vitro and In Vivo By David Boczkowski et al I came away with the concept that the RNA segment of encoded tumor protein was taken up by a DC and the DC translated that to a protein (antigen) that it presented with MHC of the appropriate class (class I I believe in these tests) to a naive T-cell, which would subsequently amplify and pursue and kill tumor cells with that antigen (by becoming CTL cytotoxic T cell leukocytes).

A quick look at my immunology text from NIH tells me that this mechanism (DC's bringing antigen to naive T-cells in lymphatic tissue) ensures "that rare antigen-specific T cells will encounter their specific antigen on an antigen-presenting cell surface."

My question is does this mean that the so-called "mutant neo-antigen" of a particular tumor in an individual must still find a T-cell receptive to that sequence (epitope) at least in part (say fractionally)? I guess I'm wondering whether there might be the case where a particularly diabolical tumor presented such novel sequences that no existing T-cell rare or otherwise could be receptive to a fraction of its sequence (even when a customized RNA delivery of a selected portion of the sequence was delivered via DC to the T-cell).

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  • $\begingroup$ After I posted this I looked at link and more or less answered my question. I gather I'm supposed to wait 24 hrs before answering my own question. Preview: Computer methods are used to identify sequences from a tumor mutanome that are likely to express non-synonymous protein present in a peptide presented on MHC molecules, so it is likely at least one mutated epitope would be immunogenic (T-cell druggable). $\endgroup$ – Dalton Bentley Jun 3 '16 at 22:30
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The OP (me) was unclear whether or not the new therapy had to ultimately present antigen specific to a particular naive T-cell. The answer is yes: Each T-cell is specific for a single antigenic determinant. An antigenic determinant is a small portion of an antigen, such as a certain sequence of amino acids in a protein, that the immune system (a T-cell receptor in this case) recognizes as non-self or altered self. The antigenic determinant is also known as an epitope. Typically an antigen has many different epitopes, each able to react with a specific antibody or T-cell receptor (when presented with MHC).

The confusion arose from the description of the RNA as encoding "mutant neo-antigens" (among other possibilities, e.g., viral or endogenous self-antigens). At first glance this seemed to imply that a tumor antigen constituting a novel polypeptide antigen ("mutant neo-antigen") was able to induce T-cell response (effector and memory T-cell responses) in the absence of any T-cells with a T-cell receptor specific to that neo-antigen. That would not be possible.

Through somatic gene rearrangement in development, T-cell receptors (TCR) have a wide range of antigen specificity, such that each T-cell has a unique receptor on its surface able to recognize foreign protein antigen associated with MHC molecules. Some estimate that human TCR and antibodies can respond specifically to 10 million different antigens.

Apparently the distinction is made between classical tumor-associated antigens that typically constitute autoantigens (which may appear frequently on the surface of cancer cells in conjunction with MHC and therefore generate some antibody response, possible ineffective) and neo-epitopes, the latter being the result of the estimated 25 – 30% of antigenic mutations (mutations not normally found in the genome) in tumors that could be made to induce immune response on the single- or poly-epitopic level. It appears that autoantigens are more modified normal protein that tend to appear somewhere in the disease process on a particular class of tumor than the result of entirely novel mutations. Since tumors usually have tens to hundreds of non-synonymous mutations, the mutanome of a tumor provides many possible targets for custom poly-epitope vaccines for each individual patient. Coding messenger RNA (mRNA), i.e., synthesizing mRNA to encode multiple transcripts for synthetic poly-epitopic nucleotide sequences, is an attractive antigen delivery format.

In order to locate a sequence that would be likely to act in this capacity (that is, to select tumor mutations likely to exhibit immunogenecity), an approach like the following serial selection has been suggested: (1) next generation sequencing (NGS) of an patient's individual tumor mutanome and patient genome (2) identify triplicate sequences present in the tumor mutanome but not in the patient genome, i.e., identify the mutations in the tumor that distinguish it from normal self (3) identify those mutations that are likely to code for a protein (4) identify those mutations that are likely to cause a non-synonymous protein change (5) identify those mutations meeting all the previous criteria that a predicted to be in a peptide presented on tumor MHC. An example quantifying this selection process is that of the murine B16F10 melanoma mutanome: The NGS exome profile (coding exons) predicted 12,842 mutations; 3570 of those predicted as somatic mutations; non-synonymous mutations 962; mutated peptide predicted to bind MHC 462; 50 of those selected for testing and of those 50, about a third of those tested strongly immunogenic when encoded to RNA and transfected to dendritic cells, i.e., inducing strong T-cell response. As a test of actual anti-tumor effect, mice were immunised with the encoded mutations identified and achieved complete tumor protection and survival in 40% of the group, compared with death of all control group animals.

Generalizing, a poly-epitopic vaccine formed from a tumor mutation should be selected to be specific to the tumor and not in the patient germline genome, to occur in a protein-coding transcript, to cause a change in protein sequence and that protein be expressed in tumor cells. In order for the mutation to induce a robust T-cell response, the mutation-containing epitope must be presented on the patient's MHC molecules in the dendritic cells during vaccination (and at least one T-cell must possess a receptor specific for one of the epitopes) and in the tumor cells for recognition (on the surface of the tumor cells). With the patient HLA haplotypte, MHC-binding epitopes can be predicted with computer algorithms. The immunogenecity of the computationally predicted epitopes can be tested in vitro with T-cell stimulation.

One of the challenges to RNA delivery has been degradation by ubiquitous extracellular RNAses. The study noted [Systemic RNA delivery to dendritic cells] solves that issue by protecting the RNA with lipid carriers to form RNA-lipoplexes (RNA-LPX). This has the additional adjuvant effect (the RNA-LPX) of triggering interferon-a (IFNa) release by plasmacytoid DCs and macrophages in a manner similar to early phases of viral infection.

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  • $\begingroup$ I would hesitate to say the liposomes "solved" the problem of protecting the RNA in circulation. It definitely helps though. mRNA delivery is a pain, but the study here probably benefited from liposome formulation's tendency to accumulate in liver and spleen. All of these nanoparticles tend to accumulate in those organs. $\endgroup$ – user137 Aug 4 '16 at 1:52
  • $\begingroup$ @user137 Good point. I just read Sahin's 2013 patent application at google.com/patents/WO2013143683A1?cl=en and that did make clear the difficulties you allude to, although Sahin's method of creating nanoparticle liposomes with electro-neutral or slightly negative charge apparently targets the spleen preferentially over other organs. This is the desired end if you want to get an RNA antigen payload to professional APCs without damaging other organs like the liver and lungs. Thanks for the clarification. $\endgroup$ – Dalton Bentley Aug 4 '16 at 22:06
  • $\begingroup$ @user137 ...and the Sahin neutral or slightly negative liposomes seem to have some of the advantages of PEGylation in decreasing the RES (reticuloendothelial system) clearance of lipoloplexes from blood, which may have been more of the central thrust of your comment, i.e., that nuclease degradation (protected by lipoplexes) is only one component of the problem of protecting the RNA in circulation (so I was indeed a bit optimistic earlier). $\endgroup$ – Dalton Bentley Aug 4 '16 at 22:33

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