The Clonotypic BCR IG of CLL Patients Contain Predicted T-Cell Class I Epitopes with Shared Structural Properties
Authors: Vardi Anna , Agathangelidis Andreas , Gkagkaridou Sofia , Schaap-Johansen Anna-Lisa , Karipidou Maria , Boukla Anna , Fylaktou Asimina , Stavroyianni Niki , Iskas Michail , Anagnostopoulos Achilles , Reker Hadrup Sine , Chatzidimitriou Anastasia , Marcatili Paolo , Stamatopoulos Kostas 
Published: November 2021

Abstract
Targeted therapies have revolutionized the treatment of chronic lymphocytic leukemia (CLL) with remarkable overall response rates. Against that, however, CLL remains incurable, indicating a need for novel strategies towards disease control and eventual eradication, including reinvigoration of anti-tumor immune responses. T cells in CLL display an oligoclonal profile and appear selected by restricted antigens, with recent evidence suggesting that the selecting epitopes may lie within the clonotypic B-cell receptor immunoglobulins (BcR IG). Should this prove to be the case, such neoepitopes could be exploited as idiotypic targets for cellular therapy or for peptide vaccine design, aiming to augment response to current treatments.
Here we performed ad hoc prediction of putative T-cell class I neoepitopes contained within the clonotypic BcR IGs of CLL patients, with an intended bias towards major stereotyped CLL subsets. We selected 27 patients to represent the full spectrum of CLL: (i) with mutated IGHV genes (M-CLL, n=5), (ii) with unmutated IGHV genes (U-CLL, n=5), (iii) assigned to major stereotyped subsets (subset #1, n=7; subset #2, n=5; subset #4, n=5). RT-PCR was performed for the heavy (H) and the light (K/L) IG chains using subgroup-specific Leader primers for the IGHV/IGK/LV gene and universal primers annealing to the constant domain (IGHG, IGHM, IGKC, IGLC), in order to produce the full-length V-(D)-J gene rearrangement sequence, plus the start of the constant domain. PCR products were subjected to direct double-strand Sanger sequencing with a quality-optimized protocol. The amino acid sequences were subsequently parsed in peptides and subjected to NetMHCpan. The rank score was calculated, considering the 4-digit HLA-A and -B typing for each individual patient. High- and medium-binding peptides (rank score &lt;2%) were selected. Exact matches to germline and/or proteome databases were excluded.
Overall, 1,007 predicted neoepitopes were identified. All patients had predicted CD8 + T-cell epitopes within the clonotypic BcR IG, either in the heavy chain (26/27 pts, n=632 epitopes) or the light chain (26/27 pts, n=375 epitopes). The majority of the peptides resulted from somatic hypermutations (SHMs) across the IGHV gene outside the complementarity-determining region 3 (CDR3; n=538, 53.4%). With the exception of few peptides located within the FR4 region (n=11, 0.1%), the remaining (n=458, 45.5%) involved (at least part of) the CDR3, which is particularly relevant given its small length (9-27 aa) within the full sequence (331-660 aa). There was no statistically significant difference in the rank score of peptides involving the CDR3 vs. all others. Peptide clustering assigned most of the predicted neoepitopes (970/1007, 96.3%) in 54 clusters of similar length and amino acid composition. Also, it revealed similar or identical predicted neoepitopes among different patients (30 clusters of two, 10 clusters of three, 8 clusters of four, 4 clusters of five, 1 cluster of six and 1 cluster of eight). Importantly, these clusters involved: (i) shared CDR3 patterns in patients assigned to the same stereotyped subset, but also (ii) subset-specific recurrent SHMs across the rearranged IGHV gene, e.g. G-to-E SHM at position 28 in the VH CDR1 of subset #4, a recurrent SHM in this subset. Also of note, the two most highly populated clusters involved peptides within the VL CDR3, and were biased towards specific subsets; the cluster of eight patients contained 4 patients assigned to subset #1 and the cluster of six patients included 4 patients assigned to subset #2.
In conclusion, in silico prediction identified a significant number of putative T-cell class I neoepitopes contained within the clonotypic BcR IG of CLL patients. The majority of these neoepitopes can be assigned to clusters based on amino acid similarity and are shared among different patients. Many of them culminate from subset-specific ('stereotyped') CDR3 patterns or recurrent SHMs, suggesting that the targeted SHM which shapes the CLL BcR IG repertoire may produce immunogenic CD8 + T-cell epitopes. Their actual immunogenicity has to be tested in ex vivo studies, currently underway by our group.

Disclosures
Anagnostopoulos: Abbvie: Other: clinical trials; Sanofi: Other: clinical trials ; Ocopeptides: Other: clinical trials ; GSK: Other: clinical trials; Incyte: Other: clinical trials ; Takeda: Other: clinical trials ; Amgen: Other: clinical trials ; Janssen: Other: clinical trials; novartis: Other: clinical trials; Celgene: Other: clinical trials; Roche: Other: clinical trials; Astellas: Other: clinical trials . Chatzidimitriou: Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.