NeoantigenicityJ.M. Sánchez Torres

H.U. Princesa, Madrid

Braun DA, et al. CCRes 2016

Multiple mechanisms influence the response to immune checkpoint blockade, including:

• High mutational and neoantigen load• Low intratumoral heterogeneity• Infiltration with a clonal T-cell population• Deficiencies in DNA repair machinery

Genomic correlates of response to immunotherapy

• Cancer arises as a result of the accumulation of DNA damage and genetic alterations, some of which can give rise to mutated, non-self peptides presented byHLA molecules and elicit T-cell responses

• Theses immunogenic mutated peptides orneoantigens are foreign in nature and displayexquisite specificity

• Consistently associated with antitumor T-cellreactivity and clinical efficacy of ICB

• Identification of personalized neoantigens remainschallenging

Aldous & Dong. Biol Med Ch 2018; Garcia-Garijo A, et al. Front Immunol 2019

Neoantigens

• Tumor-specific antigens

• Result of nonsynonymous mutations

• They are generally unique to a tumor

• Neoantigen load: number of neoantigenspotentially presented by the MHC class I

• The number of neoantigens on cancer cells directly correlates to immunotherapy success

• Highly mutated tumors are more prone to formneoantigens, but not all formed neoantigens are immunogenically relevant

Chabanon RM, et al. CCRes 2016; Braun DA, et al. CCRes 2016; Karpanen & Olweus, Frontiers Imm 2017

Braun DA, et al. CCRes 2016

Neoantigens

• Only a small fraction of all tumor somatic non-synonimous mutations identified represent bona fide neoantigens: mutated peptides that are processed, presented on the cell surface HLA molecules of cancer cells and are capable of triggering immune responses in patients

• CD8 T cells recognize antigens presented by MHC I or HLA by tumor cells

• Neoantigen recognition is an importantcomponent of the antitumor immune response

Capietto A-H, et al. Current Opinion in Immunology 2017; Sharma P & Allison JP. Science 2015; Garcia-Garijo A, et al. Frontiers Immunol 2019; LeDT, et al. NEJM 2015

Activation of T cells requires two signals: Interaction between TCR and antigen in the context of MHC (signal 1) plus CD28 costimulation (signal 2)

Neoantigens

1. Expression of antigenic proteins: Origin and abundance of the peptides

2. Antigen processing. The binding affinity of the peptidefor MHCI is determined by the sequence and thelength of peptides, and MHCI polymorphism.

3. Antigen presentation. The MHCI/peptide complexesare presented on the cell surface. The stability of theMHCI/peptide complex at the cell surface has beenassociated with immunogenicity.

4. TCR recognition. Specific CD8 T cells recognize theMHCI/peptide complexes throug their TCR

Peptide processing

Capietto A-H, et al. Current Opinion in Immunology 2017

• Several parameters impact the level of MHCI/peptide complexes:

Of all the possible peptides that can be generated from a protein, only a few have the adequate length and affinity for MHCI to form a stable complex that is transported to the cell surface for presentation to CD8 T cells

• The abundance of MHCI/peptide complexes displayed at the cell surface is critical for T cell activation

• Not all mutant peptides presented on MHCI elicit a T cell response

• The recognition of the mutant peptides as foreign is critical forimmunogenicity

• The degree of difference between the mutated peptide and itswild-type (self) peptide counterpart (its recognition as foreign bythe immune system) impacts the capacity of the mutatedneoantigen to elicit a T cell response

• The immunogenic potential of a mutation may vary with itsposition within the peptide

• T cell response depends on the stregth of the TCR-MHCI /peptidecomplex interaction

Neoantigen immunogenicity

Capietto A-H, et al. Current Opinion in Immunology 2017

• Most mutations are induced by carcinogens, UV or DNA repairdefects, and are unique to each individual tumor

• Defects in the DNA repair machinery are associated withimproved survival and durable clinical benefit from ICB

• Closely reflecting the DNA damage repair capacity of tumor cells and their intrinsic genomic instability, the mutational loadand its associate tumor-specific neoantigens appear as keypredictive paths to anticipate potential clinical benefits of ICB

• Tumors harboring deleterious mutations in the MMR , BER and NER pathways were found to carry a high number of candidateneoantigens and associated with clinical benefit from ICI

Neoantigens and DNA repair capacity

Chabanon RM, et al. CCRes 2016; Le DT, et al. NEJM 2015; Brown SD, et al. Genome Res 2014DNA repair defects and their association with anti-PD-(L)1 efficacy

Le DT, et al. NEJM 2015

Tumors with MMR deficiency are more responsive to PD-1 blockade

• Patients with mCRC treated with pembrolizumab: MMR-deficient vs MMR-proficient

• MMR-deficient tumors have a high tumor mutation load by WES (mean somatic mutations: 1782 vs. 73, P=0.007)

• High mutation loads (high number of NSM) were associated with betteroutcomes and durable benefit from antibody-based ICB immunotherapy

HR 0.10 HR 0.22

Schumacher TN & Schreiber RD. Science 2015; Chabanon RM, et al. CCRes 2016; Rizvi NA, et al. Science 2015

Estimate of the neoantigen repertoire in human cancer

The presence of high tumor mutational burden can increase thelikelihood of neoantigens formation

The most mutated tumors may alsobe the most immunogenic ones

Mutational load and Neoantigen load

Formation of neoantigens

Only a small fraction of candidate neoantigens elicits spontaneous responses in the cancer patient’s autologous T-cell repertoire

Karpanen T & Orweus J. Frontiers Immunol 2017

Only about 1.2% of mutations with neoantigenic potential are spontaneously recognized in patients with melanoma, gastrointestinal, lung, and ovarian cancers

Schumacher TN & Schreiber RD. Science 2015; Garcia-Garijo A, et al. Frontiers Immunol 2019Cancer exome-based identification of neoantigens

WES/RNA sequencingand mass spectrometry analysis

Prediction algorithm to assessMHCI peptide immunogenicityand make feasible to track T cells

Identification of HLA-bindingpeptides

MHC-peptide multimer orfunctional assays

Identification of immune-relevant neoantigens

PFS in high and low NSM burdenNSM burden in p with DCB and NDB

Magnitude of HERC1 reactive CD8+ T cell response measured in peripheral bloodRizvi NA, et al. Science 2015

Neoantigen burden in p with DCB & NDB PFS in high and low neantigen burden

• ICI are demonstrating that adaptive immunity can be harnessed for the treatmentof cancer: ICI unleash a patient’s own T cells to kill tumors

• NSCLC treated with pembrolizumab (34 p)

• The quantity of neoantigens per tumor corelated with mutation burden

• Anti-PD-1 therapy can induce neoantigen specific T cell response and tm regression

• Efficacy correlated with:

• Molecular smoking signature

• Higher mutational burden

• Higher neoantigen burden

• DNA repair pathway mutation

Mutational landscape determines sensitivity to PD-1 blockade in NSCLC

Treatment of patients with T cells recognizing neoantigens

Rosenberg SA & Restifo NA. Science 2015

Aldous AR, et al. Bio Med Chem 2018; Pan R-Y, et al. J Immunol Research 2018

Personalized neoantigen peptide vaccine

Neoantigen peptide vaccine

Aldous AR, et al. Bio Med Chem 2018

Shared neoantigen vaccine

Personalized neoantigen vaccine

Tran E, et al. N Eng J Med 2016

Patient with metastatic colorectal cancer: Resection of lung mets

Cultures of Tumor Infiltrating Linfocites (TIL):TIL contained CD8+ T cells that specifically recognized mutant KRAS G12D

Infusion TIL composed by CD8+ T cells reactive to mutant KRAS G12D4

Objective regression of lung metastases

Direct evidence of the protective role of neoantigens: Neoantigen TIL therapy inducing regression of metastatic lesions

Progression of one lesion after therapy:

Resection of the lesion:Loss of expression of the HLA-C*08-02 class I MHC molecule(required for direct tumor recognition by the transferredKRAS G12D-specific T cells):

Immune evasion

The infusion of CD8+ cells targeting mutant KRAS mediated effective antitumorimmunotherapy against a cancer that expressed mutant KRAS G12D

Expansion of CD8+ T cells

Sahin U, et al. Nature 2017

-All patients developed T cell responses against vaccine neo-epitopes -Vaccine-induced T cell infiltration -Neo-epitope-specific killing of tumour cells were shown in post-vaccination resected mets-The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained PFS -Two of the five patients with metastatic disease experienced objective responses -A third patient developed a complete response to vaccination in combination with PD-1 blockade therapy.

RNA-based poly-neoepitope approach: Identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient

13 melanoma patients

T cells directed against mutant neo-epitopes drive cancer immunity

Chen DS & Mellman I. Immunity 2013

Neoantigens: conclusions

• The formation of neoantigens is only one of a number of essential conditions fora succesful immune attack of cancer cells (Schumacher and Schreiber, Science 2015)