Patent Application
20230266307
The Sequence Listing is submitted as an ASCII text file, named Sequence Listing.txt, created on May 28, 2023, 15,299, bytes, which is incorporated by reference herein.
The invention concerns a device for tumour antigen identification and a method for tumour antigen identification; a tumour antigen identified following use of said device and/or method; a pharmaceutical composition comprising said tumour antigen; a method of treating cancer using said device and/or said method; a method of stratifying patients for cancer treatment using said device and/or said method; a treatment regimen involving stratifying patients for cancer treatment using said device and/or method and then administering a cancer therapeutic; and a tumour antigen identified using said device and/or said method for use as a cancer vaccine or immunogenic agent or cancer therapy.
CD8+ T-cells have a key role in the detection and elimination of cells that present abnormal peptides on their surface as a result of pathogenic infection, such as viral infection, or malignant transformation. The cross-reactivity of the T-cell receptor (TCR) means T-cells can recognize an incredibly large variety of different target peptides. This phenomenon allows a relatively small number of T-cells to recognize multiple pMHC (peptide:Major Histocompatibility Complex) molecules that are representative of abnormal cells and so potentially health or life threatening.
However, an undesirable side effect of this mechanism is that an immune response directed against a pathogen might overcome the tolerance threshold for a highly homologous self-antigen, causing a deleterious off-target effect that is mediated by the cross-reactivity of T-cells; a process known as molecular mimicry. In the field of autoimmunity, the potentially deleterious consequences of such homology between self and pathogenic peptides is well-known, however, it has not been explored in cancer.
Indeed, it has been thought that the best prognostic markers for successful outcome in cancer immunotherapeutic treatment is a high tumor antigen mutational rate and abundant T-cell infiltration. This thinking is based upon the fact that a tumor that has a high number of mutations will have a higher chance of being recognized and eliminated by infiltrating T-cells.
Nevertheless, some studies have shown that the qualitative properties of tumor antigens might be more important than their quantity. Furthermore, it has been observed that anti-viral T-cells populate the tumor microenvironment [28], however whether their role is active or just bystander is still unclear.
Onco-immunology and immunotherapy have completely changed the way cancer is being treated over the last decade, particularly when using checkpoint inhibitors (ICIs) where outstanding clinical results have been seen but, unfortunately, only in a small number of patients. It is becoming clear that immunotherapy with ICIs will only fully work when targeting specific tumor antigens. However, at the moment there is not an easy and rapid method to identify these tumor antigens.
Herein, we hypothesized that tumors might present peptides that share a high degree of homology or affinity scoring with pathogen-derived peptides, in particular, viral peptides and this might enable pathogenically-generated, cross-reactive T-cells to recognize and kill tumor cells.
To this end, we have developed a bioinformatic device called HEX (Homology Evaluation of Xenopeptides) to automatically and very simply identify tumor antigens that are highly similar to viral peptides. Using this device, we have observed that anti-viral T-cell immunity, mediated by a peptide with high homology or affinity scoring with a cancer antigen, can also actively control tumor growth in both a prophylactic and a therapeutic setting. This observation demonstrates the cross-reactivity of activated T-cells against both homologous viral-derived peptides and tumor-derived peptides.
Subsequently, we have also found that a humoral response to cytomegalovirus (CMV) was able to stratify the response of melanoma patients' to checkpoint inhibitor therapy (anti-PD1). Indeed, peptides homologous with CMV and melanoma, identified through use of the device HEX, were found to foster Inf-g release in Pheripheral Blood Mononuclear Cells (PBMCs) from CMV seropositive melanoma patients.
According to a first aspect of the invention there is provided a device for tumour antigen identification comprising:
at least one flow-through channel containing a plurality of supports to which there is attached at least one molecule or at least one complex that has bound thereto at least one anti-MHC (Major Histocompatibility Complex) antibody or at least one anti-pan-Human Leukocyte Antigen (HLA) antibody whereby a pMHC (peptide:Major Histocompatibility Complex) in a sample flowing through said channel can be extracted from said sample using said at least one antibody.
Reference herein to a anti-pan-HLA antibody is to an antibody that recognises or has specificity for any of the different HLAs present in mammals, in particular humans.
More ideally still, said antibody recognises or has specificity for a particular HLA type such as MHC-I and selected from at least one of the following group: MHC class I A, B, and C.
Additionally or alternatively, said antibody recognises or has specificity for a particular HLA type such as MHC-II selected from at least one of the following group: MHC class II DP, DM, DO, DQ, and DR.
Yet more preferably still said antibody is anti-human.
In a preferred embodiment of the invention said molecule or complex is a complex of thiol and alkyl (“ene”) functional groups, ideally, involving the stoichiometric ratio of 1.5 to 1.0 within the range of 0.15:0.1-1500:1000 (tetrathiol: triallyl).
More ideally still, the fabrication of the device is based on UV-initiated photoreaction between the thiol and alkyl (“ene”) functional groups. In yet a further preferred embodiment of the invention the device is made by UV photopolymerization with biotin-PEG4-alkyne (Sigma, 764213). This is followed by reaction with avidin: streptavidin. Although, it is within the scope of the invention to attach the avidin to the biotic before attaching the biotic to the pillar and vice versa.
In still yet a further preferred embodiment said device is functionalised by reacting said antibody with said streptavidin. Ideally, more than one antibody is reacted with said streptavidin whereby each complex functionalised with streptavidin carries a plurality, such 2 or 3, of said antibodies such as a plurality of anti-pan-HLA antibodies.
In a further preferred embodiment of the invention the micropillar array was preconditioned with protein, such as bovine serum albumin (BSA) (ideally 100 μg/mL in 15 mM PBS, 10 min incubation) after streptavidin functionalization and before antibody binding.
In a more preferred aspect of the invention said supports comprise a plurality of micropillars, such as those present in a microfluidic device and, preferably, said pillars are arranged in an array within said device. Ideally, said micropillars are made of a same or similar composition to that which is used in conventional microfluidic devices e.g. for implementing immobilized enzyme reactions.
According to a further aspect of the invention there is provided a method for tumour antigen identification comprising:
Reference herein to homology/affinity includes reference to a comparison of sequence structures in part v) above using one of: homology/affinity as defined herein; or identity which is determined by the number of identical residues over a defined length in a given alignment; or similarity which is determined by the number of identical residues or conservative substitutions having similar physicochemical properties over a defined length in a given alignment.
In a preferred method of the invention said library is of human pathogen-derived antigens and it comprises a curated library of known pathogen-derived antigens, ideally derived from pathogenic viruses, or more suitably the proteomes thereof, which viruses infect mammals, ideally humans, such as any one or more, or ideed any combination, of the following viruses:
Abyssoviridae; Ackermannviridae; Actantavirinae; Adenoviridae; Agantavirinae; Aglimvirinae; Alloherpesviridae; Alphaflexiviridae; Alphaherpesvirinae; Alphairidovirinae; Alphasatellitidae; Alphatetraviridae; Alvernaviridae; Amalgaviridae; Amnoonviridae; Ampullaviridae; Anelloviridae; Arenaviridae; Arquatrovirinae; Arteriviridae; Artoviridae; Ascoviridae; Asfarviridae; Aspiviridae; Astroviridae; Autographivirinae; Avsunviroidae; Avulavirinae; Bacilladnaviridae; Baculoviridae; Barnaviridae; Bastillevirinae; Bclasvirinae; Belpaoviridae; Benyviridae; Betaflexiviridae; Betaherpesvirinae; Betairidovirinae; Bicaudaviridae; Bidnaviridae; Birnaviridae; Bornaviridae; Botourmiaviridae; Brockvirinae; Bromoviridae; Bullavirinae; Caliciviridae; Calvusvirinae; Carmotetraviridae; Caulimoviridae; Ceronivirinae; Chebruvirinae; Chordopoxvirinae; Chrysoviridae; Chuviridae; Circoviridae; Clavaviridae; Closteroviridae; Comovirinae; Coronaviridae; Corticoviridae; Crocarterivirinae; Cruliviridae; Crustonivirinae; Cvivirinae; Cystoviridae; Dclasvirinae; Deltaflexiviridae; Densovirinae; Dicistroviridae; Endornaviridae; Entomopoxvirinae; Equarterivirinae; Eucampyvirinae; Euroniviridae; Filoviridae; Fimoviridae; Firstpapillomavirinae; Flaviviridae; Fuselloviridae; Gammaflexiviridae; Gammaherpesvirinae; Geminialphasatellitinae; Geminiviridae; Genomoviridae; Globuloviridae; Gokushovirinae; Guernseyvirinae; Guttaviridae; Hantaviridae; Hepadnaviridae; Hepeviridae; Herelleviridae; Heroarterivirinae; Herpesviridae; Hexponivirinae; Hypoviridae; Hytrosaviridae; lflaviridae; Inoviridae; Iridoviridae; Jasinkavirinae; Kitaviridae; Lavidaviridae; Leishbuviridae; Letovirinae; Leviviridae; Lipothrixviridae; Lispiviridae; Luteoviridae; Malacoherpesviridae; Mammantavirinae; Marnaviridae; Marseilleviridae; Matonaviridae; Mccleskeyvirinae Mclasvirinae; Medioniviridae; Medionivirinae; Megabirnaviridae; Mesoniviridae; Metaparamyxovirinae; Metaviridae; Microviridae; Mimiviridae; Mononiviridae; Mononivirinae; Mymonaviridae; Myoviridae; Mypoviridae; Nairoviridae; Nanoalphasatellitinae; Nanoviridae; Narnaviridae; Nclasvirinae; Nimaviridae; Nodaviridae; Nudiviridae; Nyamiviridae; Nymbaxtervirinae; Okanivirinae; Orthocoronavirinae; Orthomyxoviridae; Orthoparamyxovirinae; Orthoretrovirinae Ounavirinae; Ovaliviridae; Papillomaviridae; Paramyxoviridae; Partitiviridae; Parvoviridae; Parvovirinae; Pclasvirinae; Peduovirinae; Peribunyaviridae; Permutotetraviridae; Phasmaviridae; Phenuiviridae; Phycodnaviridae; Picobirnaviridae; Picornaviridae; Picovirinae; Piscanivirinae; Plasmaviridae; Pleolipoviridae; Pneumoviridae; Podoviridae; Polycipiviridae; Polydnaviridae; Polyomaviridae; Portogloboviridae; Pospiviroidae; Potyviridae; Poxviridae; Procedovirinae; Pseudoviridae; Qinviridae; Quadriviridae; Quinvirinae; Regressovirinae; Remotovirinae; Reoviridae; Repantavirinae; Retroviridae; Rhabdoviridae; Roniviridae; Rubulavirinae; Rudiviridae; Sarthroviridae; Secondpapillomavirinae; Secoviridae; Sedoreovirinae; Sepvirinae; Serpentovirinae; Simarterivirinae; Siphoviridae; Smacoviridae; Solemoviridae; Solinviviridae; Sphaerolipoviridae; Spinareovirinae; Spiraviridae; Spounavirinae; Spumaretrovirinae; Sunviridae; Tectiviridae; Tevenvirinae; Tiamatvirinae; Tobaniviridae; Togaviridae; Tolecusatellitidae; Tombusviridae; Torovirinae; Tospoviridae; Totiviridae; Tristromaviridae; Trivirinae; Tunavirinae; Tunicanivirinae; Turriviridae; Twortvirinae; Tymoviridae; Variarterivirinae; Vequintavirinae; Virgaviridae; Wupedeviridae; Xinmoviridae; Yueviridae; and Zealarterivirinae.
More preferably the pathogenic virus is a cytomegalovirus (CMV) or an Epstein-Barr virus (EBV) or, preferably, a Herpesvirus, a Poxvirus, a Hepadnavirus, an Influenza virus, a Coronavirus, a Hepatitis virus, a HIV, or a Bunyavirus, Most preferably said virus is non-oncolytic i.e. its replication is not specifically restricted to cancer cells..
In an alternative embodiment, said virus is oncolytic i.e. capable of infecting and killing cancer cells by selective replication in tumour versus normal cells.
In a preferred method said comparing said peptide of said bound pMHC (peptide:Major Histocompatibility Complex) with a library of pathogen-derived antigens involves a plurality of scorings (peptide affinity scoring and alignment scoring and similarity scoring and MHC-binding affinity scoring) to determine said homology/affinity or identity or similarity. Most preferably, said scoring is an homology/affinity scoring as described below.
In one embodiment, a matrix of rows (or columns) representing the amino acid position in the tumour peptide and columns (or rows) representing each of the 20 standard amino acids is generated and the amino acid positions of the tumour peptide are assigned the same high score and other positions are assigned the same low score as the pathogen-derived antigen/peptide. Tumour peptides with the highest degree of homology/affinity with a pathogen-derived antigen/peptide in terms of (in descending order of preference):
a) entire sequence structure (clearly a tumour peptide with 100% identity with a pathogen-derived peptide will recieve the highest score);
b) identity of key amino acids in hot-spot or key binding sites; and
c) the most number of of key amino acids in hot-spot or key binding sites will receive the highest scores.
Alignments are computed pairwise between peptides, in the query (pathogen-derived) set against the tumour set, or vice versa. For a given pair of peptides, their alignment is calculated by summing the distance scores between pairs of amino acids in the same position. Scoring is weighted to prioritize similarity between more central amino acids in the peptide.
Additionally, MHC class I binding affinity predictions are made. Methods for achieving same are known in the art such as using NetMHC (NetMHC4.0 or NetMHCpan4.1.) via the Application programming interface of IEDB (http://tools.iedb.org/main/tools-api/) and are then parsed and collated within the tool.
In an alternative embodiment of the invention, said comparing involves inputting lists of tumor peptides of 8 to 12 amino acides in length into the software. Firstly, the tool BLAST is employed to find Hits (similar sequences) in the library of pathogen-derived antigens. For this task the PAM30 tool is generally used, however both BLOSUM and PAM substitution matrices across several evolutionary distances are supported. Next, a pairwise refinement alignment using at least BLOSUM62 and, ideally also, a substitution matrix developed by Kim et al (described in BMC Bioinformatics. 2009;10:394. Published 2009 Nov. 30. doi:10.1186/1471-2105-10-394 Derivation of an amino acid similarity matrix for peptide: MHC binding and its application as a Bayesian prior. Kim Y, Sidney J, Pinilla C, Sette A, Peters B.) is performed in order to refine the alignment in a position-weighted fashon. A pair of peptides with high similarity in the TCR-interacting area (the central section of the peptides) receive a higher similarity score. Finally, the MHC-binding affinity prediction is produced for both the tumor and the viral cognate peptide using NetMHC4.0 or NetMHCpan4.1 as standalone command line tools. The results are ultimately automatically parsed and collated within the tool and a final score which summarize the above mentioned analysis is created.
According to a yet further aspect of the invention there is provided a cancer therapeutic or immunogenic agent or cancer vaccine comprising a tumour antigen identified using either, or both, the afore device and/or method.
According to a yet further aspect of the invention there is provided a tumour antigen for use in cancer therapy identified using either, or both, the afore device and/or method.
According to a yet further aspect of the invention there is provided a tumour antigen for use in the manufacture of a medicament to treat cancer wherein said antigen is identified using either, or both, the afore device and/or method.
Most preferably said a cancer therapeutic or immunogenic agent or cancer vaccine or tumour antigen is any one or more of those listed in Tables 1-6.
According to a yet further aspect of the invention there is provided a method for treating cancer patients comprising:
identifying a tumour antigen using said device and/or method of the invention and then administering to an individual said tumour antigen or using said tumour antigen to expand a population of T-cells active against said tumour antigen and then administering said T-cells to a patient.
In a preferred embodiment said T cells are ex vivo T cells and/or cultured T cells, either allogeneic or autologous T cells.
According to a yet further aspect of the invention there is provided a pharmaceutical composition or immunogenic agent or vaccine comprising the tumour antigen of the invention and a pharmaceutically acceptable carrier, adjuvant, diluent or excipient.
Suitable pharmaceutical excipients are well known to those of skill in the art. Pharmaceutical compositions may be formulated for administration by any suitable route, for example intratumoral, intramuscular, intra-arterial, intravenous, intrapleural, intravesicular, intracavitary or peritoneal injection, buccal, nasal or bronchial (inhaled), transdermal or parenteral and may be prepared by any methods well known in the art of pharmacy.
The composition may be prepared by bringing into association the above tumour antigen with the carrier. In general, the formulations are prepared by uniformly and intimately bringing into association the tumour antigen with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. The invention extends to methods for preparing a pharmaceutical composition comprising bringing a tumour antigen as defined herein in conjunction or association with a pharmaceutically or veterinary acceptable carrier or vehicle.
According to a further aspect of the invention, there is provided a combination therapeutic for the treatment of cancer comprising: a tumour antigen identified using said device and/or method of the invention and at least one further cancer therapeutic.
Preferably, the further cancer therapeutic comprises most suitably cyclophosphamide as it downregulates T-regulatory cells. However, as will be appreciated by those skilled in the art, said further therapeutic can be any anti-cancer agent known in the art.
Preferably, the further cancer therapeutic comprises a checkpoint inhibitor (ICI).
The best characterized pathways for checkpoint inhibition are cytotoxic T-lymphocyte protein 4 (CTLA-4) pathway and programmed cell death protein 1 pathway (PD-1/PD-L1). Thus, the invention can be used in combination with at least one checkpoint modulator such as anti-CTLA-4, anti-PD1, or anti-PD-L1 molecules to counteract the immunosuppressive tumor environment and to cause a strong anti-immune response.
According to a yet further aspect of the invention there is provided a method of stratifying patients for checkpoint inhibitor cancer treatment comprising:
According to a yet further aspect or embodiment of the invention there is provided a method of stratifying patients for checkpoint inhibitor cancer treatment comprising: determining whether a patient is CMV-seropositive and where this is the case selecting the patient for treatment using an effective amount of at least a checkpoint inhibitor (ICI).
According to a yet further aspect of the invention there is provided a method of treating cancer comprising:
According to a yet further aspect or embodiment of the invention there is provided a method of treating cancer comprising:
determining whether a patient is CMV-seropositive and where this is the case administering an effective amount of at least a checkpoint inhibitor (ICI) to said patient.
According to a yet further aspect of the inevtion there is provided a method of stratifying patients for adenoviral cancer treatment comprising:
In a preferred embodiment said peptide is attached to said adenoviral vector using known technology such as that described in WO 2015/177098 and/or that contained herein, see PeptiCRAd preparation.
Reference herein to an “effective amount”, is one that is sufficient to achieve a desired biological effect, such as, cancer cell death.
It is understood that the effective dosage will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. Typically, the effective amount is determined by those administering the treatment.
Most preferably the cancer referred to herein includes any one or more of the following cancers: nasopharyngeal cancer, synovial cancer, hepatocellular cancer, renal cancer, cancer of connective tissues, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia/lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophagus cancer, gall bladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, parathyroid cancer, penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vagina cancer, vulva cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer.
In a particular preferred embodiment of the invention said sample is taken from a melanoma and said tumour antigen is homologous with a CMV antigen or peptide.
In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise”, or variations such as “comprises” or “comprising” is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.
All references, including any patent or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art.
Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.
Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.
Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.
Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
An embodiment of the present invention will now be described by way of example only with reference to the following wherein:
A schematic overview describing the new microchip methodology developed. Thiol-ene microchips incorporating free surface thiols are derivatized with biotin-PEG4-alkyne thiolene (Step1) and functionalized with a layer of streptavidin (Step2) after which a biotinylated pan-HLA antibody is immobilized on the micropillar surface (Step3) and cell lysate is loaded into the microchip (Step4). After adequate incubation time and washing steps, the HLA molecules are eluted by adding 7% acetic acid (Step5).
A) Binding efficacy of AlexaFluor 488-streptavidin on thiol-ene micropillars precoated with biotin-PEG4-alkyne at two different streptavidin incubation times (15 min and 1 h). B) The effect of streptavidin (nonfluorescent) concentration on the amount of immobilized biotinylated pan-HLA antibody quantitated through AlexaFluor 488-labeled secondary antibody. C) The effect of BSA incubation on the amount of immobilized biotinylated pan-HLA antibody quantitated through AlexaFluor 488-labeled secondary antibody. The efficiency of BSA in blocking nonspecific binding sites was assessed by preconditioning the micropillar array with BSA either before (BSA-panHLA) or after (panHLA-BSA) immobilization of the biotinylated pan-HLA antibody. D) The total amount of biotinylated pan-HLA antibody bound onto a single chip as a function of loading cycles. For each cycle, a fresh batch of the same (constant) pan-HLA antibody concentration was used. Significance was assessed by two-tailed unpaired Student's t-test, * p<0.05.
A: Scheme of the animal experiment: To assess if viral peptides similar to tumor peptides can impact tumor growth, 4 groups of C57BL6 mice were formed. A group of naïve mice was used as mock, the other three groups were each immunized with a different pool of viral peptides. The mice were immunized at 2 time points, 14 and 7 days, before the engraftment of the tumor.
B: After two weeks from the first immunization mice were injected subcutaneously with 3×105 murine melanoma B16-OVA cells. After the engraftment, tumor growth was followed by measuring with digital caliper every second day for nineteen days. P value was calculated using Two-Way ANOVA multiple comparison with Tukey's correction.
C: Mice were euthanized when the endpoint was reached. The splenocytes of the mice of each group were collected and pooled for an ELISpot assay. Each pool was then pulsed with the respective viral peptides (viral peptides homologous to TYR1, viral peptides homologous to TRP2, viral peptides homologous to GP100) to assess the response to the treatment. The dotted line shows the background produced by the negative control.
A: To assess the response toward the respective original tumor epitope, splenocytes from each group of immunization were pooled together and pulsed with the corresponding tumor peptide.
B: Comparison between the responses elicited by pulsing the splenocytes with the pooled viral peptides and the corresponding original tumor peptide.
C: Predicted affinity of the original tumor and respective similar viral pool of peptides to the murine MHC class I. 50 nM was considered as threshold to define peptides with “High-” and “Intermediate/Low Affinity” according to the IEDB guidelines.
D: Correlation between data from IFN-y response and predicted affinity.
E: Stratification of peptides based on their affinity and ability to stimulate IFN-y production. High affinity peptides (IC50 <50 nM) foster significantly higher production of INF-γ compared to Intermediate/Low affinity peptides (IC50 >50 nM). P value was calculated using t test with Mann Whitney correction. The range of p value was labeled with asterisks according to the following criteria: >0.05 (ns), ≤0.05 (*), ≤0.01 (**), ≤0.001 (***), ≤0.0001 (****).
A: Scheme of the animal experiment: 4 groups of C57BL mice were formed for each tumor cell line to be tested. Mice were subcutaneously injected either with B16-OVA or B16F10 cells at day 0. Once the tumor was palpable, mice were treated with saline solution (Mock group), uncoated adenovirus (Uncoated Virus group), adenovirus coated with pool of viral peptides homologous to TRP2 (Viral PeptiCRAd, VPC) or adenovirus coated with TRP2 peptide (TRP2 PeptiCRAd, TPC).
B: B16 OVA tumor individual growth. A threshold that defines the success of the therapy is identified by the median of all the tumor volumes of the last day.
C: B16 OVA tumor volume at the endpoint. The median of the tumor volume, showed as a dotted line, defines the success threshold of the therapy.
D: B16 OVA Contingency plot shows the number of responders per each group of treatment.
E: B16 F10 tumor individual growth. A threshold that defines the success of the therapy is identified by the median of all the tumor volumes of the last day.
F: B16 F10 Tumor volume at the endpoint. The median of the tumor volume, showed as dotted line, defines the success threshold of the therapy.
G: B16 OVA Contingency plot shows the number of responders per each group of treatment.
(C-F) P value was calculated using one-way ANOVA with Tukey's correction. The range of p value was labeled with asterisk according to the following criteria: >0.05(ns), ≤0.05 (*), ≤0.01 (**), ≤0.001 (***), ≤0.0001 (****).
(D-G) P value was calculated using Chi-square (and Fischer exact) test of the odds ratio. The range of p value was labeled with asterisk according to the following criteria: >0.05 (ns), ≤0.05 (*), ≤0.01 (**), ≤0.001 (***), ≤0.0001 (****).
The peptides in PeptiCRAd1 derived from HEX analysis. PeptiCRAd4 consisted of Valo-mD901 coated with gp70 423-431 (AH1-5).
Table 1. Peptides used for the animal experiment. Known melanoma tumor peptides (TRP2180-188, hGP10025-33 and TYR208-216) were analyzed with HEX. The best viral candidate peptides proposed by the software were selected and a pool composed by the best 4 xenopeptides per each original tumor epitope was tested in vivo.
Table 2. Peptides used for the ELISpot on patients' PBMCs. Known Melanoma-associated antigens were analyzed with HEX. The best human CMV-derived candidate peptide per each antigen was chosen to be tested in vitro.
Table 3. Comparative analysis between microchip-based Immuno Precipitation technology and the standard procedure. The table reports the total amount of antibody coated into the microchip-based IP technology and the standard procedure.
Table 4. Shows HEX output identified 13 tumour MHC restricted peptides with their counterpart pathogen-derived peptides.
Table 5. Table of peptides tested in the ELISPOT assay.
Table 6. Table of selected peptides for the in vivo PeptiCRAd assays shown in
The device of the invention (known as PeptiCHIP) is a flow-through construction containing thousands of pillars that are coated with a linker to which is attached the Biotin. Biotin is, in turn, connected to streptavidin which is bound with a HLA-specific capturing antibody, specifically, three molecules of HLA-specific capturing antibody.
Using conventional microfluidic manufacturing techniques, the fabrication of the PeptiCHIP is based on UV-initiated photoreaction between thiol and allyl (“ene”) functional groups in according to the following off-stoichiometric ratio of 150:100 (tetrathiol: triallyl).
Immediately after the afore fabrication, the PeptiCHIP is derivatized with the biotin-PEG4-alkyne (Sigma, 764213) by UV photopolymerization followed by reaction with avidin. This is undertaken as follows.
Step 1: We prepared a 1 mM biotin-PEG4-alkyne solution (stock of biotin-PEG4-alkyne in 10 mM in ethyleglycol). Wev tooke a small aliquot and added 1% (m/v) photoinitiator (Igracure® TPO-L, BASF), using 10% photoinitiator stock in methanol, thus, the final solution is 1 mM biotin+1% Lucirin in EG-MeOH 9:1*
We filled the chip with one volume of the solution of biotin-PEG4-alkyne and 1% Lucirin in EG:MeOH 9:1 (v/v), expose to UV for 1 min (using LED UV lamp λ=365 nm, I=15 mW/cm2).
We rinsed thoroughly first with methanol, then with milli-Q water, we flushed 1-2 mL of each solvent through the channel and stored it dry (if necessary).
STEP 2: After the derivation with biotin, the chip is functionalized with streptavidin. We prepared a stock of streptavidin of 0.01 mg/ml in PBS and added it to the CHIP for 15′ in the dark at room temperature. We washed 3 times with 200 ul of PBS.
We added BSA 100 ug/ml in 15 mM in PBS for 10′ at room temperature.
STEP 3: Reaction with anti-pan-HLA antibody. We added 25 ul of biotin anti-human HLA-A,B,C 1.6 ug/ul (Biolegend CAT. Number 311434) for 15′ at room temperature. We then washed three times with 200 ul of PBS and then the CHIP is ready for the immunoprecipitation of the MHC complex.
Tumor sample preparation: we detached the cells with EDTA 4 mM and washed once with PBS. Add 25 ul of Igepal 1% in PBS+ protease inhibitors. Centrfuge 500×g 10′+4° C. Cnetrifuge 20000×g 10′+4° C.
Immune-purification steps were carried out within a single microfluidic chip by adding a biotinylated pan-HLA antibody to a streptavidin-prefunctionalized solid support structure (i.e., the micropillar array) and then immobilizing the HLA onto the pan-HLA antibody coated solid surface.
In summary, off-stoichiometric thiol-enes (OSTEs) polymer based micropillar arrays were fabricated with a UV-replicamolding technique and biotinylated. Next, the biotinylated micropillars were functionalized with streptavidin and the biotinylated pan-HLA antibody was added, after which the cell lysate was loaded directly into the microfluidic chip to selectively trap the HLA-I complexes. After adequate washing, the trapped HLA-I complexes were eluted at room temperature by applying 7% acetic acid (
The efficiency of the streptavidin functionalization on micropillar arrays was examined with respect to two different incubation times (15 min and 1 h), with the help of fluorescent AlexaFluor488-streptavidin. The shorter incubation time was found sufficiently long for building the first streptavidin layer (
Finally, we sought to characterize the maximum amount of immobilized biotinylated pan-HLA antibody that can be bound onto a single chip by using the optimized protocol. This was evaluated through the use of multiple loading cycles of a new antibody batch of the same concentration (0.5 mg/mL) per a single microfluidic chip. In this case, the amount of the immobilized pan-HLA antibody was determined by comparing the pan-HLA antibody amount in the feed solution versus the output solution through an ELISA assay. It was observed that the amount of immobilized antibody increased almost linearly along with the number of loading cycles (
With the microchip setup, 1.74×1014 molecules of antibody could be immobilized and technically 4.5×106 cells could be investigated.
To assess whether the developed thiol-ene microchip could be exploited as a platform for antigen discovery, we immunopurified HLA peptides from the human B-cell lymphoblastoid cell line JY. The JY line has high expression of class I HLA and is homozygous for three alleles common in the human population (HLA-A*02:01, HLA-B*07:02 and HLA-C*07:02), and it has been extensively adopted for ligandome analysis. Consequently, the JY cell line was considered a suitable model for benchmarking the microchip-based antigen enrichment Immuno Precipitation technology.
Hence, HLA-I complexes were immunoaffinity-purified using the thiol-ene microchips, functionalized with the amount of pan-HLA antibody as described above. Moreover, to determine the sensitivity of our approach, the protocol was challenged by using total cell numbers as low as 50×106, 10×106 and 1×106. The lysates were loaded into the microchips, and after adequate washing with PBS, the peptides were eluted with 7% acetic acid and analysed by tandem mass spectrometry. The entire workflow took an average, from the streptavidin functionalization to the elution of the tumour peptides, of <24 hours. A stringent false discovery rate threshold of 1% for peptide and protein identification was applied to generate data with high confidence. We were able to identify 5589, 2100 and 1804 unique peptides from 50×106, 10×106 and 1×106 cells, respectively (
As we sought to carefully analyse the ability of the microchip technology to enrich for natural HLA-I binders and to avoid potential co-eluting contaminants, we extensively characterized the eluted peptides. First, the eluted peptides from the JY cell line represented the typical length distribution of a ligandome data set, with 9mers as the most enriched peptide species (
Of the unique 9mers, 78%, 83% and 67% were predicted to be binders (described as binders in NetMHCpan4.0, applied rank 2% [24A-26A]) to either HLA-A*0201 or HLA-B*0702 alleles for 50×106, 10×106 and 1×106 cells, respectively (
Gibbs analysis was performed to deconvolute the consensus binding motifs of respective HLA-I alleles from the eluted 9mer peptides; these clustered in two distinct groups, with a preference for reduced amino acid complexity for residues at positions P2 and Ω, matching remarkably well with the known ones for HLA-A*0201 and HLA-B*0702 (
Next, in order to determine the role of the peptides identified, a gene ontology (GO) term enrichment analysis was performed on our list of 9mer binder source proteins. We observed an enrichment in nuclear and intracellular proteins, mainly those interacting with DNA, RNA or involved in catabolic activity. Finally, we set up an in vitro killing assay to further demonstrate the capacity of the microchip technology in isolating peptides in complex with HLA-I. To this end, a set of three peptides was selected from our JY data set to stimulate HLA-matched PBMCs; CD8+ T cells were purified from the PBMCs and adopted as effector cells in coculture with JY cells. To account for nonspecific cytotoxicity due to the effector cells per se, unstimulated PBMCs were used as a control. Real time cytolysis was then monitored. Interestingly, the CD8+ T cells pulsed with the peptides QLVDIIEKV (SEQ ID NO: 75; gene name PSME3) and KVLEYVIKV (SEQ ID NO: 76; gene name MAGEA1) showed about 10% specific cytolysis, whereas the CD8+ T cells pulsed with the peptide ILDKKVEKV (SEQ ID NO: 77; gene name HSP90AB3P) induced 15% specific cytolysis, indicating specific lysis in the presence of defined peptides.
To evaluate the validity of our HLA-I peptide lists identified by the microchip technology, we interrogated SysteMHC, a repository of the immunopetidomics data set generated by mass spectrometry. Among the unique 9mer binders identified in our data, 69%, 77% and 81% were also found in a previously published ligandome data set derived from the JY cell line (pride ID PXD000394) [3A] for 50×106, 10×106 and 1×106 cells, respectively (
Hence, these results demonstrated that the chip-based protocol can be exploited as a reliable Immuno Precipiation platform within the immunopeptidomic workflow.
Application of the sample to chip and elution of fractions for later analysis
We applied the sample through multiple cycles and incubated for 5 minutes (working at 4° C.). We washed 3 times with 200 ul of PBS and aspirated the last washing to empty the chip. We prepared a solution of 50% MeOH and 50% MilliQ. With the previous solution we prepared a 7% solution of Acetic Acid. We applied this solution to the CHIP and collected the fraction. The elution time was 5 minutes. On the same day, we purified the collected fractions. We prepared a SepPak cartridge for each tissue sample-for the HLA-1 peptides sample and labelled them. Using a syringe and the dedicated adaptor we washed the cartridge first once with 1 ml of 80% ACN in 0.1% TFA, then twice with 1 ml of 0.1% TFA. We load each of the biological samples on a SepPak cartridge. We passed them through slowly (speed of about 1 ml in 20 s).
We washed the cartridges twice with 1 ml of 0.1% TFA. We eluted the HLA binding peptides into a collection tube with 300 ul of 30% ACN in 0.1% TFA.
Homology(/affinity) Evaluation of Xenopeptides (HEX) is a novel in silico platform that compares similarity between tumor peptides (reference peptides) and pathogen-derived, such as viral, peptides (query peptides). It utilizes several metrics in order to expediate candidate peptide selection. This is done by incorporating both novel methods (peptide scoring and alignment scoring algorithm) and integrated pre-existing methods (MHC-I binding prediction). HEX comes with a number of precompiled databases of known proteins, such as proteins derived from viral pathogens and the human proteome (33).
The associated scoring matrix is generated ad hoc based on the amino acid composition of the reference peptide, as opposed to experimentally. In particular, in the matrix rows representing the amino acid position in the peptide and columns representing each of the 20 standard amino acids, amino acid positions of the reference peptide were assigned the same high score and other positions were assigned the same low score.
Alignments are computed pairwise between peptides in the query set against the reference set. For a given pair of peptides, their alignment is calculated by the summing the distance scores between pairs of amino acids in the same position. Scoring is weighted to prioritize similarity between more central amino acids in the peptide. HEX supports both BLOSUM and PAM substitution matrices across several evolutionary distances. Specifically, BLOSUM 62 was the matrix chosen for this study.
MHC class I binding affinity predictions are made using NetMHC [27] via the Application programming interface of IEDB (http://tools.iedb.org/main/tools-api/) and are then parsed and collated within the tool. The user can specify their desired scoring method or return a number of recommended results. Predictions for a number of human and murine MHC-I alleles are supported.
Users are able to select peptides by their own criteria or allow peptides to be selected by a random forest model. The random forest was trained on experimental outcomes of peptides chosen by the authors. Feature importance was determined by out-of-bag (OOB) increase in mean squared error (MSE) and cross-validated on an unseen sample of the peptides. HEX was developed as a web application using the R package Shiny and is accessible at https://picpl.arcca.cf.ac.uk/hex/app/ without user registration. The source code is available at https://github.com/whalleyt/hex.
In an alternative embodiment of the invention, HEX supports both BLOSUM and PAM substitution matrices across several evolutionary distances. Specifically, BLOSUM 62 was the matrix used in this study. HEX, first performs a BLAST search using PAM30 to find the Hits (similar sequences) in the reference library. Next, HEX performs a pairwise refinement alignment using both BLOSUM62 and a substitution matrix developed by Kim et al.
Ref: Kim Y, Sidney J, Pinilla C, Sette A, Peters B. Derivation of an amino acid similarity matrix for peptide: MHC binding and its application as a Bayesian prior. BMC Bioinformatics. 2009;10:394. Published 2009 Nov 30. doi:10.1186/1471-2105-10-394.
MHC class I binding affinity predictions are made using NetMHC4.0 (or NetMHCpan4.1) as a standalone command line tool and are then parsed and collated within the tool. The user can specify their desired scoring method or return a number of recommended results. Predictions for a number of human and murine MHC-I alleles are supported.
In total, 16 stage 4 metastatic melanoma patients were treated with anti-PD1 monoclonal antibody in the Helsinki University Central Hospital (HUCH) Comprehensive Cancer Center. Patients were randomly selected to receive either nivolumab (n=7) infusions every second week or pembrolizumab (n=9) infusions every third week. The study was approved by Helsinki University Central Hospital (HUCH) ethical committee (Dnro 115/13/03/02/15). Written informed consent was received from all patients and the study was conducted in accordance with the Declaration of Helsinki.
Peripheral blood samples (3 ml EDTA blood, 50 ml Heparin blood) were collected from three time-points; before initiation of treatment, after one and three months of treatment. From these the plasma was separated by centrifuging and then stored at -70° C. The CMV and EBV IgG levels were measured from thawed EDTA plasma samples using VIDAS CMV IgG (BioMérieux, Marcy-I'Etoile, France) and Siemens Enzygnost Anti-EBV/IgG kits (Siemens Healthcare Diagnostics, Marburg, Germany). The immunoglobulins (IgA, IgM, IgG) from thawed Heparin plasma were measured in the central laboratory of the Helsinki University Central Hospital (HUSLAB).
the murine melanoma cell line B16-F10 was purchased from the American Type Culture Collection (ATCC; Manassas, Va., USA). Cells were cultured in RPMI (Gibco, Thermo Fisher Scientific, US) with 10% fetal bovine serum (FBS) (Life Technologies), 1% Glutamax (Gibco, Thermo Fisher Scientific, US), and 1% Penicillin and Streptomycin (Gibco, Thermo Fisher Scientific, US) at 37° C./5% CO2.
The cell line B16-OVA, a mouse melanoma cell line modified to constitutively express chicken Ovalbumin (OVA), was kindly provided by Prof. Richard Vile (Mayo Clinic, Rochester, Minn., USA). These cells were cultured in RPMI Low glucose (Gibco, Thermo Fisher Scientific, US) with 10% FBS (Gibco, Thermo Fisher Scientific, US), 1% Glutamax, 1% Penicillin and Streptomycin (Gibco, Thermo Fisher Scientific, US) and 1% Geneticin (Gibco, Thermo Fisher Scientific, US) at 37° C./5% CO2.
All cells were tested for mycoplasma contamination with a commercial detection kit (Lonza-Basel, Switzerland). Isolated human PBMCs were frozen in FBS supplemented with 10% DMSO and then maintained in liquid nitrogen until use. Cryopreserved PBMCs were thawed and rested overnight at 37° C./5% CO2 in complete RPMI medium supplemented with 10% FBS, 1% Glutamax, 1% penicillin-streptomycin over night before plating them for ELISPOT.
All the peptides used in this study were purchased from Zhejiang Ontores Biotechnologies Co. (Zhejiang, China) 5 mg >90% purity. The sequences of all the peptides used in this study are found in Table 1 and 2.
All PeptiCRAd complexes described in this work were prepared by mixing Adenoviruses and polyK-tailed peptides according to the following protocol: 1×109 vp(viral particles) were mixed with 20 ug poly-K tailed peptides (resuspended in water); after vortexing, the mixture was incubated at room temperature for 15 min; PBS was added, after the incubation, up to the injection volume (50 uL/mouse), successively, the solution was vortexed again and used for assays or animal injections. For the TRP2-PeptiCRAd, 1×109 vp were mixed with 20ug of 6K-TRP2180-188 peptide, while the Viral-PeptiCRAd was prepared using 1×109 vp mixed with 5 ug of each viral 6K-peptide homologous for TRP2280-188.
New PeptiCRAds were prepared before each experiment using fresh reagents. All dilutions of virus and peptides required before incubation for PeptiCRAd preparation, were performed in sterile PBS or water. The PeptiCRAds were then diluted in the buffer required by the assay.
Viruses were generated, propagated, and characterized as elsewhere described [20].
All animal experiments were reviewed and approved by the Experimental Animal Committee of the University of Helsinki and the Provincial Government of Southern Finland.
All the experiments were carried using C57BL/6JOlaHsd mice obtained from Scanbur (Karlslunde, Denmark).
For the immunization experiment 8- to 9-week-old immune competent female C57BL/6J mice were divided in 4 groups. N=3 mice were used as mock group, n=7 mice were used to form each of the three different treatment groups. Each treatment group was vaccinated with a different group of xenopeptides. Mice were vaccinated twice and injections were performed at one-week interval (day 0 & 7) at the base of the tail with 40 ug of peptides and 40ug of adjuvant (VacciGrade poly(I:C)—Invivogen) in a final injectable volume of 100 ul. Naïve mice (PBS injected) were used as Mock group. At day fourteen mice were injected with 3105 B16-OVA cells on the right flank and tumor growth was followed until endpoint was reached.
For the treatment of established tumors, we tested 2 different tumor cell lines: B16-OVA cells and the more aggressive B16F10 cells. 3*105 B16-OVA cells and 1*105 B16-F10 were injected subcutaneously on the right flank of 8- to 9-week-old immune competent female C57BL/6J mice. Successively, these mice were randomly divided in 4 groups of 7-8 mice for each tumor cell line. A mock group was treated with PBS; a second group was treated with uncoated Adenovirus; a third group was treated with Adenovirus coated with TRP2180-188 (TRP2—PeptiCRAd); the last group was treated with Adenovirus coated with TRP2-homologous viral peptides (Viral—PeptiCRAd).
Mice were intratumorally treated twice and injections were performed at two days interval (day 10 & 12 from tumor engraftment) and tumor growth was followed until endpoint was reached. The median of the tumor volume measurement of the last day identifies the therapeutic success threshold showed as a dotted line. Mice that at the end point showed a tumor volume below the threshold were considered responders, while mice above it were considered as non-responders.
Tumor growth was followed with a digital caliper measuring two dimensions of the tumor. Successively, the volume was mathematically calculated according to the following formula:
((long side)×(short side)2)/2
In all experiments, tumors were measured every second or third day until the tumor size reached the maximum allowed, and mice were then sacrificed and spleens collected.
To assess the amount of active antigen specific T-cells, interferon-γ (IFN-γ) secretion was measured by ELISPOT assay from IMMUNOSPOT (CTL, Ohio USA) for the murine IFN-γ and MABTECH (Mabtech AB, Nacka Strand, Sweden) for the human IFN-γ.
Fresh mice splenocytes collected at the end point of the experiment have been used. The procedure was carried out according to the manufacturer instructions. In brief, for murine IFN-γ, 3*105 of splenocytes/well were plated at day 0. Cells were stimulated with 2 ug/well of peptides. After 3 days of incubation at 37° C./5% CO2, plates were developed according to the kit's protocol.
For human IFN-γ ELISPOT, human PBMCs were thawed and let rest over-night at 37° C./5% CO2 in complete medium. The following day, 3×105 PBMCs/well were plated and stimulated with 2 ug/well of peptides. After 48 h of incubation at 37° C./5% CO2 the plates were developed according to the manufacturer's protocol. Plates were sent to CTL-Europe GmbH to be analyzed.
The EBV-transformed human lymphoblastoid B-cell line JY (ECACC HLA-type collection, Sigma Aldrich) was cultured in RPMI 1640 (GIBCO, Invitrogen, Carlsbad, Calif., USA) supplemented with 1% GlutaMAX (GIBCO, Invitrogen, Carlsbad, Calif., USA) and 10% heat inactivated foetal bovine serum (HI-FBS, GIBCO, Invitrogen, Carlsbad, Calif., USA).
Streptavidin (Streptomyces avidinii, affinity purified, lyophilized from 10 mm potassium phosphate, ≥13 U/mg protein) was purchased from Sigma-Aldrich (Saint Louis, Missouri, USA).
Biotin-conjugated anti-human HLA-A, B, C clone w6/32 was purchased from Biolegend (San Diego, Calif., USA) for analysis.
The following peptides were purchased from Ontores Biotechnologies Co., Ltd., were used throughout the study: KVLEYVIKV (SEQ ID NO: 75; gene name MAGE A1), ILDKKVEKV (SEQ ID NO: 76; gene name HSP90), and QLVDIIEKV (SEQ ID NO: 77; gene name PSME3).
Additionally, the following peptides were purchased from Chempeptide (Shangai, China):
VIMDALKSSY (SEQ ID NO: 78; gene name NNMT), FLAEGGGVR (SEQ ID NO: 79; gene name FGA) and EVAQPGPSNR (SEQ ID NO: 80; gene name HSPG2).
The ovarian tumour biopsy was collected from a patient with ovarian metastatic tumour (high grade serous), who signed an informed consent, under the studies approved by the Research Ethics Committee of the Northern Savo Hospital District with the approval number 350/2020. The samples were chopped in small pieces and treated with a digestion buffer containing collagenase type D (Roche) 1 mg/ml, Hyaluronidase (Sigma Aldrich) 100 μg/ml and DNase I (Roche) 1 mg/ml for 1 h at 37° C. The cell suspension was sequentially passed through a 500 μm and 300 μm cell strainer (pluriSelect) to obtain single cells.
Patient tissue samples for organoid cultures were obtained from the DEDUCER study (Development of Diagnostics and Treatment of Urological Cancers) at Helsinki University Central Hospital with approval number HUS/71/2017, 26.04.2017, ethical committee approval number 15.03.2017 Dnro 154/13/03/02/2016, and patient consent. The kidney sample was obtained from a nephrectomy of an adult male with a clear cell renal cell carcinoma (ccRCC, pTNM stage pT3a G2). The benign kidney tissue sample was used for the experiments. The carcinoma urothelial (bladder cancer, high grade, gradus III, 1×1 cm) was obtained from adult female, and the cancer tissue sample was used for the organoid culture.
Cells were isolated from the original tissue instantly after surgery by dissociating the tissue into small pieces and treating it with collagenase (40 units/ml) for 2-4 h. Benign and cancer cells of the kidney of a clear cell renal cell carcinoma patient cells were grown as organoids in F-medium [3:1 (v/v) of F-12 nutrient mixture (Ham)-DMEM (Invitrogen), 5% FBS, 8.4 ng/mL cholera toxin (Sigma), 0.4 μg/mL hydrocortisone (Sigma), 10 ng/mL epidermal growth factor (Corning), 24 μg/mL adenine (Sigma), 5 μg/mL insulin (Sigma), 10 μM ROCK inhibitor (Y-27632, Enzo Life Sciences, Lausen, Switzerland) and 1% penicillin-streptomycin with 10% Matrigel (Corning). The bladder tumour-derived organoids were grown in hepatocyte calcium medium (Corning)[15A] supplemented with 5% CSFBS (Thermo Fisher Scientific), 10 μM Y-27632 RHO inhibitor (Sigma), 10 ng/mL epidermal growth factor (Corning), 1% GlutaMAX (Gibco), 1% penicillin-streptomycin and 10% Matrigel. 6×106 cells were collected by centrifugation, washed in PBS to remove Matrigel and snap frozen before analyses.
The clinical HLA typing of tumour samples (ccRCC and bladder) was performed by the European Federation for Immunogenetics (EFI) -accredited HLA laboratory of the Finnish Red Cross Blood Service. Allele determination of three classical HLA-I genes HLA-A, -B and -C was performed by targeted PCR based next generation sequence (NGS) technique according to the protocol provided by the manufacturer (NGSgo® Workflow, GenDx, Utrecht, The Netherlands).
The allele assignment at 4-field resolution level was implemented by NGSengine Version: 2.11.0.11444 (GenDx, Utrecht, The Netherlands) using IPD IMGT/HLA database Release 3.33.0, https://www.ebi.ac.uk/ipd/imgt/hla/.
The following antibodies were used to analyse the cell surface expression of HLA-A2 and HLA-A, B, and C: PE-conjugated anti-human HLA-A2 (clone BB7.2, BioLegend 343306 San Diego, Calif., USA), PE-conjugated anti-human HLA-A, B, and C (clone W6/32, BioLegend 311406, San Diego, Calif., USA), and Human TruStain FcX block (BioLegend B247182, San Diego, Calif., USA).
The data were acquired using a BDLSR Fortessa Flow Cytometer. Flow cytometric analysis of renal cell carcinoma and bladder tumour-derived organoids was performed using a BD Accuri 6 plus (BD Biosciences) and analysed with FlowJo software (Tree Star, Ashland, Oreg., USA).
In order to study whether molecular mimicry between viruses and tumor could impact on tumor growth we needed to identify peptides sharing a high degree of homology/affinity. However, a tool to facilitate the identification of relevant target, to this scope, is lacking. Therefore, we developed HEX (Homology Evaluation of Xenopeptides), a device that compares the input sequences to a database of pathogen-derived antigens/peptides or protein sequences and selects highly homologous candidate pairs of peptides based on at least two of the three following criteria: 1) A B-score that corresponds to the likelihood of the peptides being recognized by a given TCR; 2) A positional weighted alignment score to prioritize the similarity in the area of interaction with the TCR; 3) the prediction of the MHC class I binding affinity (
We started from three melanoma-associated antigens, that have been applied successfully in a number of vaccination studies: TRP2180-188 (tyrosinase-related protein 2), GP10025-33 (aka PMEL; premelanosome protein) and TYR1208-216 (tyrosinase 1). TYR1208-216 is not predicted to be a binder of the murine MHC, and thus, was considered as an “irrelevant target”. With HEX we identified viral peptides that shared a high degree of homology/affinity with the input tumor epitopes. Pools of 4 viral-derived peptides per each original tumor epitope (Table 1) were chosen to be further evaluated in vivo.
To assess whether the viral-derived peptides would influence the tumor growth, we decided to mimic the viral infection by immunizing C57BL6 mice with selected viral peptide-pools followed by the tumor engraftment (
To further investigate the contribution of the selected viral peptides in the reduction of tumor growth, we collected the splenocytes of the mice at the endpoint for ELISpot assay (
We further investigated the reactivity of these splenocytes from the viral pre-immunized mice towards their respective cognate tumor antigen (
We have shown that the molecular mimicry between viral and tumor antigens can affect the tumor growth in preimmunized mice. Next, we wanted to assess whether the molecular mimicry directed-response could have an impact on already established tumors in naïve mice. To this end, mice were implanted with B16OVA tumors, or more aggressive and immunosuppressive B16F10 tumors and, subsequently, treated with the previously developed vaccine platform PeptiCRAd (20) to mimic a viral infection (
We selected a pool of melanoma-associated proteins [21] that were compared to the CMV proteome using HEX generating a list of tumor peptides with high similarity to CMV (Table 2). Tumor peptides and their CMV counterpart were used in ELISPOT assay showing that in a responder patient, PBMCs always responded to both viral and their counterpart tumor antigens, suggesting that the CMV infection had expanded viral T cell clones that could attack and kill tumor cells making seropositive patients more prone to react towards melanoma specific epitopes similar to CMV (
We challenged the platform for the investigation of a scarce tumour biopsy. Thus, an ovarian metastatic tumour (high grade serous) was collected from the patient and four pieces were derived from the tumoral border (S1, S2, S3 and S4); the central part of the tumour was collected as well (S5). Next, the samples were weighed and as summarized in
Consistently with a typical ligandome profile, metabolic processes were enriched in all the samples examined. Additionally, the analysis revealed an increase in skin development pathway proteins in line with the epithelial nature of the ovarian serous tumour here analyzed. Altogether, the results highlighted the feasibility of exploiting the developed microfluidic-chip platform to analyse scarce tumour biopsy and obtain personalized antigenic peptidse for tumour treatment.
The microchip-based protocol reveals the immunopeptidome landscape in patient-derived organoids
The microchip technology was challenged with as few as 6×106 cells from patient-derived organoids (PDOs). We selected two patients from an on-going precision medicine study for urological cancers, a nephrectomy sample containing both benign and cancer tissue from clear cell renal cell carcinoma (ccRCC) patient and 1×1 cm sample from bladder cancer patient, were further processed as 3D primary organoid cultures.
Applying the developed microchip technology and a stringent false discovery rate threshold of 1% for peptide and protein identification, we were able to identify a total of 576 and 2089 unique peptides in ccRCC and bladder PDOs respectively (
To demonstrate that technology can be exploited for the rapid development of therapeutic cancer vaccines, we set up a killing assay. We focused our analysis on ccRCC samples. We used transcriptomics levels to select putative tumour antigens using PBMC and healthy kidney tissue as reference sets. Next, PBMCs from healthy volunteers were pulsed with the selected peptides, and CD8+ T cells isolated from those cells were used in the assay. T cells pulsed with the peptide EVAQPGPSNR (gene name HSPG2) showed about 10% specific cytolysis.
Finally, we sought to investigate the recall T cells response in the ccRCC patient. To this end, unfractionated PBMCs derived from the patient were in vitro stimulated with the peptide EVAQPGPSNR whereas unstimulated PBMCs were adopted as control. The derived CD8+T cells were then added to the ccRCC PDO showing about a 7% increase in killing activity compared to the control group.
The list of peptides was analysed with HEX. First, the software prioritized the peptides that were concurrent strong binders (cut off IC50 range 50 nM-500 nM according to NetMHC4.0) and that showed higher weighted alignment score (cut off 0.8-1 normalized weighted alignment score). This latter focuses the peptide's similarity in the area of interaction that most likely will engage the TCR of CD8+ T cells, in order to induce mediated immune response; the resultant peptides are then further categorized based on their overall percentage of identity to pathogen-derived antigens/peptides and IC50 binding affinity score. The ultimate output consisted in thirteen peptides with their counterpart pathogenic peptides (Table 4).
To determine the peptide immunogenicity, mice were pre-immunized with subcutaneous injection of each single peptide in presence of an adjuvant Poly(I:C); a group of mice was injected either with Poly(I:C) alone or saline as control as well. The splenocytes from those mice were harvested and tested for IFNy production upon specific stimuli (Table 5) in an ELISpot assay.
We then sought to verify whether the candidate peptides could be exploited as cancer vaccine for treatment of established tumors. To this end, we adopted PeptiCRAd, our previously developed cancer vaccine platform, that combines oncolytic adenoviruses with capsid attached (via a poly-lysine linker) poly-lysine modified peptides. The adenovirus used here is VALO-mD901 i.e., genetically modified to express murine OX40L and CD40L and previously shown to elicit tumor growth control and systemic antitumor response in murine melanoma model. Therefore, Balb/c mice were subcutaneously injected with the syngeneic tumor model CT26 in the left and right flank. (Day 0,
HEX is a bioinformatic tool that analyzes peptide sequences and compares them to a curated database of pathogen-derived viral proteomes looking for sequences with high similarity.
Since presented peptides are mainly involved in the interaction with the anchor residues of the MHC [25], our software returns an alignment score that is positionally weighted in order to prioritize the similarities occurring in the central section of the peptide, that is the position which is most involved in the interaction with the TCR [26]. Furthermore, in order to increase the chances that our target is a presented epitope, we ranked the resulting peptides according to the binding affinity for the chosen MHCs using, in one example, the IEDB NetMHC prediction tool API [27]; or NetMHC4.0 or NetMHCpan4.1b as stand alone tools.
We used HEX to identify viral-peptides homologous to three extensively characterized tumor associated antigens.
We found that preimmunization with our selected viral peptides pools, simulated an anti-viral immune status prior to the establishment of the tumor and efficiently slowed down the growth of subcutaneously injected melanoma tumor cells in mice indicating that the pre-exposure to viral-derived peptides can affect the tumor growth.
Next, we observed that the tumor-homologous viral-derived peptides have a similar effect on already established tumors when administrated during tumor progression, indicating that a viral infection occurring during the tumor progression could still impact on its growth.
Taken together our results suggest that the molecular mimicry between viral- and tumor-derived antigens is able to exert a control on the tumor growth even without relying on the pre-exposure or pre-acquired immunity.
Novel ICPIs have significantly improved patient survival in several solid tumors, especially in metastatic melanoma, when compared to the other commonly used therapies such as radiation and chemotherapies. However, despite the improved survival and efficient response rates, it is yet known why some patients do not benefit from ICPI-therapies. Our results indicated that patients with high titer of CMV-specific IgG levels had significantly longer progression free survival.
We observed that PBMCs, from patients with high anti-CMV-IgG titer, reacted with melanoma antigens similar to CMV peptides. This data indicates CMV-seropositivity contributes to a melanoma-specific T-cell immunity, and thus provide a clinical advantage for these patients undergoing ICPI therapy.
Here we show that viral infections could have an impact on the tumor growth and clearance. Additionally, we show the cross-reactivity of cytotoxic T-cells against viral-and homologous tumor-derived antigens selected using HEX. Based on our results we conclude that the molecular mimicry phenomenon could be exploited in the future for the development of novel therapies or, when accompanied with immunotherapies, potentially enhance the antitumor immune effect achieved by these therapies.
Moreover, designing effective tumour rejection and protection strategies requires the reliable identification of tumour peptides binding to HLA-I. The direct identification of peptides from the HLA-I complex still represents the best method. Nevertheless, the immunopeptidomics workflow is relatively complex and thus represents a major bottleneck in the antigen discovery process. Currently, the inability to analyse immunopeptidomes from a small amount of biological materials (e.g., tissue needle biopsy), the sample throughput, the cost and the affinity matrix adopted (which is both laborious and expensive to produce) in the conventional platform have been depicted as the main technical challenges to address.
In this work, we addressed several technical issues hindering the ligandome research, with main focus on the limited availability of material to analyse, on the cost of consumables and on prolonged protocols.
By exploiting the well-characterized biotin-streptavidin interaction to immobilize biotinylated pan-HLA antibody on streptavidin-functionalized surfaces, we were able to replace the conventional technology, based on affinity matrices prepared via cross-linking reactions with a microchip platform. The limitations posed by the paucity of the material (e.g., needle biopsy) inspired the work towards the implementation of a microfluidic protocol. In this work, we employed a custom microchip protocol involving a thiol-ene polymer-based micropillar array as the solid support for further biofunctionalization that enabled performing the entire IP procedure on a single microfluidic chip.
The validation of the identified peptides in an in vitro killing assay confirmed that the peptides identified when working the invention were actually presented on the JY cell surface, as they were killed in a specific CD8+ T cell-dependent fashion. The peptide ILDKKVEKV (SEQ ID NO: 3) found in our data set elicited the higher percentage of specific cytolysis.
Our approach thus offers an unexplored tool for immune-affinity purification.
20. C. Capasso, M. Hirvinen, M. Garofalo, D. Romaniuk, L. Kuryk, T. Sarvela, A. Vitale, M. Antopolsky, A. Magarkar, T. Viitala, T. Suutari, A. Bunker, M. Yliperttula, A. Urtti, V. Cerullo, Oncolytic adenoviruses coated with MHC-I tumor epitopes increase the antitumor immunity and efficacy against melanoma, Oncoimmunology 5, e1105429 (2016).
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| Number | Date | Country | Kind |
|---|---|---|---|
| 2006760.9 | May 2020 | GB | national |
This is the U.S. National Stage of International Application No. PCT/EP2021/061981, filed May 6, 2021, which was published in English under PCT Article 21(2), which claims the benefit of GB Application No. 2006760.9, filed May 7, 2020. The PCT application is herein incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/061981 | 5/6/2021 | WO |
