Mantle-cell lymphoma neoantigens are derived from immunoglobulins

This week Nature published new study that could explain why B cell-derived lymphomas are not easily rejected by body's immune system. It also highlights some of the obvious forgetfulness regarding basic immunology concepts.  

In this paper, research group from Stanford analyzed neo-antigenic burden in untreated mantle-cell lymphoma, a subtype of B-cell non-Hodgkin lymphoma. Rather than simply doing whole exome-sequencing of lymphoma DNA to identify tumor associated non-synonymous somatic mutations, the authors focused on direct proteomic analysis of cancer MHC ligands, epitopes, by liquid chromatography and tandem mass spectrometry (LC–MS/MS). 

Interestingly, though the authors found 13–175 non-synonymous somatic mutations per patient within genes known to mutate in mantle-cell lymphoma, such as TP53, CCND1, none of the mutated neo-epitopes from these genes were actually presented by MHC molecules. Only exception were neo-antigens derived from immunoglobulin (Ig) genes themselves. Surprise, surprise! 

As the authors wrote "presentation of variable-region peptides by MHC-II suggests that Ig neoantigen recognition by CD4 does not inhibit lymphoma development." Why that could be the case? As we know, when B cell responds to antigen it undergoes hypermutations within its Ig variable regions. In essence, every responding B cell generates neo-antigens within its Ig molecules. These are antibodies the body is using to defend against pathogens. Obviously Ig hypermutated B cells (and memory B cells or plasma cells derived from such B cells) are not ordinarily rejected by body's own immune system. It is strange that the authors did not mention this obvious contradiction.  

David Usharauli

PD-1 expression on tumor-infiltrating T cells does not correlate with antigen-specific response

Patients selection [stratification] and then monitoring for immunotherapy effectiveness is part of precision medicine. In humans, especially, when determination of cancer neo-antigens is not always feasible, clinical diagnostic tests are focused on surrogate markers to tell whether patient has a tumor antigen-specific immune response. More recently, PD-1 has become one of such surrogate markers. However it is not clear whether expression of PD-1 could truly correlate with T cell antigen-specific response.

New paper in Journal Immunology provided data to suggest that at least in mice tumor models PD-1 expression does not correlate with T cell antigen-specific response within tumor tissue.

For example, they showed that OT-I CD8 T cells would express PD-1 irrespective whether tumor expresses or not specific OVA antigen. In contrast, Nur77 (part of TCR signaling) expression correlated with tumor neo-antigen expression. 

This study indicate that diagnostic tests measuring PD-1 expression on tumor-infiltrating T cells may over-estimate tumor antigen-specific immune response and lead to unpredictable outcomes during antibody immunotherapy.

David Usharauli

Harnessing donor TCR specificity for cancer immunotherapy

Cancerous tissues harbor protein mutations that can be recognized by immune system as neoantigens. However, when tumor progresses it indicates that either (A) patient lacks T cells with adequate affinity to tumor neoantigens or (B) tumor environment actively suppresses immune response [or both]. 

For example, novel drug class of checkpoint inhibitors targeting CTLA4 and PD1/PD-L1 inhibitory circuits operating in T cells (Keytruda, Opdivo, Yervoy, Tecentriq) work on option B by modulating tumor suppressive micro-environment.

Another approach obviously would be an option A by using engineered T cells expressing tumor specific T cell receptors. Ideally, patient's own T cells can be expanded and re-infused back to attack tumor cells. But, more likely, patient will lack T cells with  adequate affinity to tumor neoantigens due to TCR editing. 

To overcome this limitation, new study published in journal Science suggested to use instead tumor-specific TCRs harvested from healthy donors. Here, the authors led by T cell expert Ton Schumacher, showed that HLA-matched healthy donors contain T cells with sufficient affinity and specificity to recipient's tumor neoantigens (of note, Ton Schumacher is also affiliated with biotech company Kite Pharma).

This study focused on HLA-A*02:01-restricted neoantigens from stage IV melanoma patients. Neoantigens were identified with whole-exome and RNA sequencing and selected for further analysis based on high predicted binding affinity to HLA-A*02:01. Autologous monocyte-derived dendritic cells transfected with mRNA encoding the candidate epitopes and cultured with healthy donor T cells. All 4 healthy donor  T cells specifically detected mutated tumor neoantigens with greater sensitivity.

Tumor neoantigen-specific T cell response was confirmed in epitope pulse experiment using WT or mut epitopes.

Furthermore, when donor T cell derived TCRs were re-introduced by gene transfer, resulting T cells were specific to patient's mut neoantigen and did not recognize, for example, 3rd party tumor cells.

In summary, this short but definitive study points to a growing and undeniable evidence in support for T cell based cancer immunotherapy. By incorporating donor derived TCR specificity this strategy vastly expands the reach of T cells immunotherapy. The challenge remains how to streamline this process (exome sequencing, MHC:peptide binding prediction, TCR identification, TCR transduction and re-infusion) to make it affordable for every cancer patient.  

David Usharauli

  

Melanoma patients harbor tumor mutation-specific PD1+ CD8 T cells in the peripheral blood

Steven Rosenberg's research group at National Institutes of Health (NIH) continues to define and refine condition for T cell-based cancer-specific immunotherapy. (for example, introduction of high-throughput personalized screening strategy capable of evaluating T cell reactivity to neo-antigens presented on all of the HLA restriction elements of the individual).

This time, their group showed that peripheral blood of melanoma patients harbors small but distinct PD1+ CD8 T cell population enriched in specificities for cancer associated neo-antigens.

If one compares CD8 T cells from PBMC v Tumor sites (TIL), blood derived T cells contains few PD1⁺/PD1^high CD8 T cells.

However, when the authors has expanded in vitro those sorted PD1⁺ CD8 T cells and co-cultured them with autologous dendritic cells expressing tumor neo-antigens (as tandem minigenes, TMG), they could identify circulating neoantigen-reactive CD8 T cells in three of the four melanoma patients evaluated.

Then the authors re-constructed blood PD1⁺/PD1^high CD8 T cell TCR specificity by (a) pairing the sequences encoding the two most-dominant TCR-α and TCR-β sequences, (b) cloning them into retroviral vectors and (c) transducing autologous PBMC. This TCR construct could [for example] detect neo-antigens derived from mutations in the genes MAGE family member A6 (MAGEA6).

Importantly, both PD1⁺/PD1^high CD8 T cells enriched from peripheral blood or T lymphocytes transduced with retroviruses expressing neo-antigen-specific TCRs could detect autologous tumor cell lines.

Finally, the authors found that blood and tumor site derived PD1⁺ CD8 T cell showed high degree of overlap in their TCR specificity [to tumor neo-antigens], suggesting that analysis of peripheral PD1⁺ CD8 T cells from cancer patients could reveal TCR specificities of tumor infiltrated lymphocytes.

In summary, this study is another evidence that cancer immunotherapy holds great promise in providing cancer antigen-tailored treatments. Identification of cancer neo-antigen specific T cells (TCRs) as shown in this study, would accelerate development of tumor-specific TCR constructs and could contribute in overcoming precursor limitation inherent to endogenous T cell clones.

David Usharauli

Almost every cancer patient harbors tumor-specific T cells

We know now that adaptive immune system (T and B cells) can detect single amino acid changes in mutated proteins. However, such efficiency of immune system was (and still) in odds with cancer development since tumors invariably express mutated proteins. So what's the deal?

Until recently in vitro detection of tumor-specific immune response was technically challenging. First, detection of mutated proteins or RNA was not easy task. Second, culture conditions for expansion and identification of viable tumor-infiltrated lymphocytes (TIL) specific for tumor antigens were not easy either. Today we have different situation. We can both detect and identify both tumor antigens and tumor antigen-specific TILs.

For example, new paper in Science showed that 9 out of 10 patients with GI tract cancers harbor tumor-specific T cells. However, out of 4 patients receiving adoptive transfer of in vitro expanded tumor-specific T cells, only 1 patient showed persistent anti-cancer response.

This study is kind of follow up from earlier study published in 2014. Here, for each cancer patient, the authors [led by Steven Rosenberg/NIH] first identified cancer mutations by rapid RNA sequencing and designed dozen of tandem minigene constructs containing mutated RNA sequences. These TMGs were then transfected into patient's autologous DCs and co-cultured with multiple TIL cultures harvested from metastatic tumors. This technique revealed presence of tumor-specific T cells (example below, TMG7, 14).

These tumor-specific TCRs were cancer antigen specific [showed no reactivity towards wild-type epitopes].

In 1 patient, the authors found TIL culture reactive to cancer driver gene KRAS (KRAS^G12D). The authors could identify HLA allele presenting mutated peptide (the authors even filed a patent for TCR directed to mutant KRAS. But the TCR itself is a product of nature and not patent eligible and TCR transduction into other T cells is by now considered "prior art". So I am not sure if they have a valid patent claim here. Another point is that it is safe to assume the authors already have earlier patents for transduction techniques and other TCRs. If so, then why they would need a new patent for just another TCR?).

however, the most relevant results are not shown in the paper but just discussed. The authors mentioned that 4 patients were treated with adoptively transferred T cells. However, only 1 patient receiving tumor-specific CD4 T cells showed persistent response and ongoing tumor regression, while other 3 patients who received tumor-specific CD8 T cells either do not show any response or showed only transient response. This suggest that unlike CD4 T cells, tumor-specific CD8 T cells may be not very efficient against solid tumors [lack of persistence in transferred hosts].

In summary, this article supports a notion that cancers do not go unnoticed by immune system. It appears that almost every cancer patient harbor tumor specific T cells that can be harvested, expanded and re-introduced back to patients to target tumors [alone or in combination of checkpoint inhibitors]. However, understanding biological difference between effectiveness of tumor-specific CD8 and CD4 T cells surely requires further research.  

David Usharauli