Method and System for Neoantigen Analysis

Abstract

A method for characterizing a target peptide through a detection approach such as mass spectrometry is provided, including: introducing at least one guard molecule to mix with the target peptide; and applying the detection approach for the characterization of the target peptide. Each guard molecule is configured to have similar characteristics as the target peptide, yet is still distinguishable therefrom by the detection approach, such as having a mass spectrometry-distinguishable different M/z value compared with the target peptide. The method can be used to characterize a neoantigen peptide through mass spectrometry, upstream of which the method can further include steps for tissue sample preparation, HLA molecules enrichment, elution, clean-up, and purification. Some or all of these steps can be configured to be executed in a substantially automatic manner with little or no manual intervention. A system for implementing the neoantigen analysis method is further provided.

Claims

1. A method for a characterization of a target peptide through a detection approach, the method comprising: (1) introducing at least one guard molecule to mix with the target peptide, wherein each of the at least one guard molecule is configured to have similar characteristics as the target peptide, and yet is further configured to be distinguishable from the target peptide by the detection approach; and (2) applying the detection approach for the characterization of the target peptide.

2. The method of claim 1, wherein the detection approach comprises mass spectrometry analysis, wherein: each of the at least one guard molecule is configured to have an M/z value that is distinguishable from the target peptide by the mass spectrometry analysis.

3. The method of claim 2, wherein the at least one guard molecule comprises a guard peptide.

4. The method of claim 3, wherein: the guard peptide has a same amino acid residue sequence as the target peptide; and at least one amino acid residue in the guard peptide is a heavy isotope-labeled amino acid.

5. The method of claim 3, wherein only one amino acid residue in the guard peptide differs from the target peptide.

6. The method of claim 3, wherein at least two amino acid residues in the guard peptide differ from the target peptide.

7. The method of claim 3, wherein the guard peptide has a scrambled sequence compared with the target peptide.

8. The method of claim 2, wherein the at least one guard molecule comprises a non-peptide compound.

9. The method of claim 2, wherein the target peptide is a neoantigen peptide.

10. The method of claim 2, wherein the neoantigen peptide is KRAS_Q61H, KRAS_Q61L, KRAS_Q61R, IDH2_R140Q, TP53_Y220C, TP53_R248W, TP53_R213L, KRAS_G12V_9mer, KRAS_G12V_10mer, KRAS_G12D_9mer, or KRAS_G12D_10mer.

11. The method of claim 9, wherein the neoantigen peptide is from a tissue sample obtained from a subject, the method further comprising a tissue sample preparation step prior to step (1), wherein the tissue preparation step comprises: providing the tissue sample, wherein the tissue sample is a frozen tissue sample; grinding the frozen tissue sample, under an impact of at least 8,000 psi, to thereby obtain a frozen single-cell tissue powder; and treating the frozen single-cell tissue powder before obtaining a treated tissue sample.

12. The method of claim 11, wherein: in the providing the tissue sample of the tissue preparation step, the tissue sample is snap-frozen in liquid nitrogen; and in the grinding the frozen tissue sample, the impact is approximately 10,000 psi.

13. The method of claim 11, wherein the treating the frozen single-cell tissue powder before obtaining a treated tissue sample comprises: lysis, sonication, and centrifugation, wherein the treated tissue sample is from a supernatant after the centrifugation.

14. The method of claim 13, further comprising, after the treating the frozen single-cell tissue powder before obtaining a treated tissue sample: performing an analysis over genomic DNA obtained from a pellet after the centrifugation.

15. The method of claim 11, further comprising a human leukocyte antigen (HLA) molecules enrichment step after the tissue sample preparation step and prior to step (1), wherein the HLA molecules enrichment step comprises: passing the treated tissue sample through an HLA enrichment column, wherein the HLA enrichment column comprises a matrix with anti-HLA antibodies immobilized thereon.

16. The method of claim 15, further comprising an elution step after the HLA molecules enrichment step, wherein the elusion step comprises: applying an elution buffer having a low pH to the HLA enrichment column to thereby obtain an eluate containing the neoantigen peptide, wherein the elution buffer comprises the at least one guard molecule.

17. The method of claim 16, further comprising a clean-up step after the elution step and prior to step (2), wherein the clean-up step comprises: passing the eluate through a trap column for at least one time to thereby trap the neoantigen peptide therewithin, wherein the trap column comprises a matrix capable of binding with the neoantigen peptide but having a lower or no binding affinity to impurities; and eluting the trap column to thereby obtain a cleaned eluate.

18. The method of claim 17, further comprising a purification step after the clean-up step and prior to step (2), wherein the purification step comprises: passing the cleaned eluate through a size exclusion column (SEC) for collecting a neoantigen peptide-containing fraction.

19. The method of claim 18, wherein at least two consecutive steps of the HLA molecules enrichment step, the elution step, the clean-up step, the purification step, and step (2) are substantially automatic.

20. The method of claim 19, wherein all steps of the HLA molecules enrichment step, the elution step, the clean-up step, the purification step and step (2) are substantially automatic.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0067] FIG. 1 illustrates a general process for the Valid-NEO pipeline according to certain embodiments of the disclosure;

[0068] FIG. 2A and FIG. 2B together illustrate a scheme of neoantigen isolation and purification in the Valid-NEO pipeline according to certain embodiments of the disclosure;

[0069] FIGS. 3A-3F together show a table summarizing the cancer types of the patients, the selected genetic mutation features of their tumors, and the possible neoantigen sequences flanking the highest prevalence mutation site on a cancer driver gene;

[0070] FIG. 4 shows a comparison result of the protein extraction efficiencies between UniCeller and three other traditional approaches;

[0071] FIG. 5 shows reproducibility evaluation results of the Valid-NEO pipeline analysis; and

[0072] FIG. 6 shows HPLC chromotograms for neoantigen purification through an SEC column.

DETAILED DESCRIPTION

[0073] In order to further describe the neoantigen analysis method and system as provided above, one specific embodiment (i.e. Embodiment 1) is provided below.

Embodiment 1

[0074] FIG. 1 illustrates a general process for the Valid-NEO pipeline to analyze a neoantigen peptide. Specifically, tumor tissue specimen from a subject is first harvested through biopsy or surgical resection, which is then processed, through tissue lysis, sonication and centrifuge, to obtain HLA molecules (in supernatant) and genomic DNA (in pellet). Patient specific mutations were revealed through genomic sequencing of the tumor DNA from the pellet by DEEPER-Seq pipeline (Wang et al., 2017). Mutation callings are made and potential sequences for neoantigens and neoantigen peptides (called “MaxRec sequences”) were established. Neoantigens are then isolated and purified from tissue lysate supernatant through a multi-step procedure which is illustrated in FIGS. 2A and 2B and described below, then neoantigens are directly detected and quantified through mass spectrometry.

[0075] FIGS. 2A and 2B illustrate a scheme of the multi-step process of neoantigen isolation and purification in the Valid-NEO pipeline according to certain embodiments of the disclosure. As illustrated in the two figures, there are mainly 5 steps in Valid-NEO system to obtain neoantigens, Step 1) HLA enrichment by antibody column with repeated loading; Step 2) Elution of HLA molecules and separation of neoantigens followed by trap column loading; Step 3) Neoantigen clean up and removal of HLA molecules; Step 4) Neoantigen elution and loading of SEC column; Step 5) Purification of neoantigens through SEC column.

[0076] In this embodiment of the multi-step process for neoantigen isolation and purification in the Valid-NEO pipeline, it is noted that a series of valves (see “Valves 1-5” in the figure), a series of pumps (see PUMPS 1-5), a series of DAD detectors (see “Detectors 1-3), and a Fraction Collector, are also included in the system. The schematic configuration and connection for each component in the system is also illustrated in FIGS. 2A and 2B.

[0077] As illustrated, each valve comprises a total of 6 ports (#1-6), each operably and controllably connected to an inlet or an outlet of other devices, such as the “Antibody Column” (i.e. HLA enrichment column), the “Trap Column”, the “SEC Column”, the pumps, DAD detectors, and the Fraction Collector. Each pump is configured to provide a driving force that drives the fluid to flow in the pipeline in a predetermined direction (as shown by the arrows in the figure), and each port is configured to open or close in a controlled manner based on the control signals that it receives. Each of the DAD detectors is configured to detect certain parameter of the fluid that it receives. A processor (not shown) is communicatively connected to each of the above components, and is configured, based on the detection signals transmitted from the DAD detectors, to control the coordinated working of each of the above components in a programed manner. For example, the processor may control the opening/closing status of each port of the valves, and may control the start or stop and flow rate of the each pump. As such, the coordinated working of each component of the system can realize an automatic sample processing, allowing the treated tissue sample (i.e. HLA/neoantigen-containing sample, or the “supernatant” in FIG. 1) to flow through the enrichment column, the trap column, and the SEC column in an automatic and controlled manner before running into the mass spectrometer for characterization.

[0078] Materials and Methods

[0079] Tumor Samples

[0080] Tumor samples from a total of 10 patients were obtained from BioIVT. This study was approved by the Institutional Review Boards for Human Research at Complete Omics Inc. and BioIVT, and complied with Health Insurance Portability and Accountability Act. Cancer types of the patients and selected genetic mutation features of their tumors are listed in the table shown in FIGS. 3A-3F. In the table, the amino acid residues in bold and underlined font represent the mutations of interest in a neoantigen peptide sequence.

[0081] Construction of Valid-NEO

[0082] Valid-NEO is an integrated system composed of five steps essential for neoantigen detection, including 1) Enrichment of HLA molecules, 2) Elution of neoantigens from antibody column, 3) Cleaning of neoantigens, 4) Elution of neoantigens from trap column, 5) Purification of neoantigens through SEC column. This integrated system is composed of a tandem series of HPLC systems, one mass spectrometer, and a set of optimized buffers including the MaxRec system.

[0083] HLA Molecule Extraction from Tissue Sample

[0084] Human tumor fresh frozen tissues were obtained from BioIVT (BioIVT, NY). 50 mg frozen tissue were wrapped in aluminum foil such that the tissue chunk was covered by at least four layers of aluminum foil. The wrapped tissue chunk was snap-frozen in liquid nitrogen. UniCeller (Complete Omics Inc, MD), an in-house built device designed to apply strong impact onto frozen tissue packs, was used to produce single-cell level powder from the tissue chunk, and this procedure can be repeated 5 times until the tissue chunk is completely ground into frozen single-cell powder. 1 mL NL buffer (Complete Omics Inc, MD) was added to the tissue powder and the tissue suspension was transferred into a protein lo-bind tube followed by five rounds of sonications through Bioruptor 300 (energy level 4.5, duty step 30 seconds, and delay step 59 seconds). The tissue lysate was incubated on ice for 1 hour, during which the suspension was pipetted up and down 20 times every 10 minutes, and one additional cycle of sonication was performed every 10 minutes. The tissue lysate was centrifuged at 4° C. for 30 minutes, and the clear supernatant was transferred to a new protein lo-bind tube. The supernatant containing HLA molecules was diluted with 4 volumes of NC buffer (Complete Omics Inc., MD), after which it was ready for HLA molecule isolation.

[0085] Online Enrichment of HLA Molecules Through Antibody-Column

[0086] Anti-HLA antibodies (clone W6/32) were immobilized on Protein A agarose beads (ThermoFisher Scientific, MA) through DMP (dimethyl pimelimidate)-based crosslinking reaction. 50 mL beads were then packed into an HLA enrichment column and flushed with 1 L NC buffer (Complete Omics Inc, MD). HLA-neoantigen suspension was filtered through a 0.22 μm filter, diluted with 4 volumes of the NC buffer and injected directly onto the HLA enrichment column. The flow-through was collected into a sample loop and re-injected onto the column. The injection was repeated for 4 more times, for a total of 5 passes of the suspension through the antibody column. During the repeated loadings, HLA molecules were depleted from the mobile phase and captured by the column, while the HLA-suspension was gradually diluted by NC buffer pushed into the system by the pump. The repeated loading ensured an efficient binding of the HLA molecules to the column and the sequential dilution of the sample with the mobile phase facilitates an improved cleaning efficiency and reduced nonspecific binding. The antibody column was then flushed with NC buffer at 1 mL/min for 20 minutes to remove unbound proteins and impurities (including salts and detergents).

[0087] Online Elution of Neoantigen Peptides and Antibody Column Regeneration

[0088] Elution of the neoantigen peptides was performed with an increasing gradient (from 0 to 100% over a period of 5 minutes) of NE buffer (Complete Omics Inc, MD) through the column, followed by a constant flush with 100% NE buffer at 1 mL/min for 2 minutes. The antibody column was then neutralized by running an increasing gradient (from 0 to 100% over a period of 5 minutes) of NN buffer (Complete Omics Inc, MD), and followed by a 1 hour flushing with NC buffer at 1 mL/min. The eluted HLA molecules and neoantigen peptides were then subjected to further purifications.

[0089] Online Isolation and Purification of Neoantigen Peptides

[0090] HLA eluate containing neoantigen peptides was injected to pass through a trap column for a total of 5 times, followed by washing with 10 mL 0.1% formic acid. The cleaned peptides were eluted from the trap column through three cycles of acetonitrile gradients using mobile phase solvent A: 0.1% formic acid in water and mobile phase solvent B: 0.1% formic acid in acetonitrile. The gradient started from 0% solvent B and increased to 60% solvent B over 30 seconds, and then decreased to 0% solvent B over 30 seconds, and this 1-min gradient step was repeated three times at the follow rate of 1 mL/min followed by a high-speed flush at 2 mL/min with 100% solvent B for 1 minute. The follow through was collected 30 seconds after the initial gradient change took place and the collection was stopped 1 minute after the flushing step ended. A total of 4.5 mL of neoantigen peptide suspension was collected with an estimated 30% acetonitrile and 0.1% formic acid. The collected neoantigen suspension was directly loaded onto an SEC column packed with 1.7 μm particles with 125 Å pore size (Waters, Mass.). Before the analysis, NEO-SEC ladder (Complete Omics, MD) was spiked into the system to define the boundaries for collecting the neoantigens. Signature chromatography peaks were monitored to indicate the starting point (a peak representing 2000 Da) and the ending point (a peak representing 800 Da) for the collection. Flow-through containing the isolated neoantigen peptides was collected and subject to lyophilization before mass spectrometry analysis.

[0091] Mass Spectrometry Method Development

[0092] Heavy isotope labeled neoantigen peptides flanking gene mutations in patient cancer genomes were synthesized. Optimization of the detection parameters was performed with a two-step approach. Step 1) All possible ions (first to last) of each peptide were detected with a theoretical collision energy as well as two additional collision energies at 5 eV below and above the theoretical value (three collision energy values in total for each transition). The highest abundance transitions were selected for the next round of optimization. Step 2) High abundance transitions selected from previous step (>20 transitions for each charge status of the peptide target) were subject to a further optimization where for each transition 9 collision energy values were tested including the theoretical collision energy value as well as 4 steps of values below and above the theoretical value with a step-size of 2 eV. After two rounds of optimizations, detection parameters were manually curated to avoid false positive signals from co-detected impurities in the Valid-NEO matrix prepared from a reference human tumor sample, and an average of 8 to 10 transitions were selected as signature transitions for each target. Before and after each batch of analysis, Agilent 6495C Triple Quadrupole mass spectrometer was tuned using manufacturer's tuning mixture followed by MyProt-SRM Tuning Booster (Complete Omics, MD). Before each assay, to ensure the stable and consistent performance of the mass spectrometer throughout the entire study, MyProt-SRM Performance Standard (Complete Omics, MD), a mixture of standard peptides across a wide range of masses (M/z 100-1400) and a broad range of hydrophobicities, were analyzed. A system performance score was documented before every run.

[0093] Pre-Conditioning the System to Ensure Highest Sensitivity

[0094] In order to achieve the highest sensitivity for the assay, a strategy is developed to ensure a minimal sample loss by pre-conditioning and co-processing in the system with peptides that are “similar” to the ones being detected. The peptides used to ensure the maximal recovery of the assay are called MaxRec peptides. A MaxRec prediction algorithm was created to generate MaxRec peptide sequences based on the sequences, hydrophobicity and detectability (signal strengths detected in mass spectrometer) of the target peptides desired to be detected from the pipeline. MaxRec peptide sequences used in this study were shown in Table 1, where the amino acid residues in bold and underlined font represent the mutations of interest (i.e. target mutations), and the amino acid residues in italics font represent the altered residues used in the MaxRec peptides. All MaxRec peptides were synthesized at a high purity (>99.9%). A buffer system containing MaxRec peptides at the concentration of 100 femtomole/μL was injected into the Valid-NEO pipeline before each assay. MaxRec peptides passed through the pipeline at much higher concentrations than what would presumably be observed from the target peptides in clinical samples. Before clinical sample injection, the Valid-NEO pipeline was flushed with NC buffer for 30 minutes to deplete excessive unbound MaxRec peptides.

TABLE-US-00001 TABLE 1 MaxRec peptides used in this study Neo-antigen peptide ID sequence MaxRec peptides KRAS_Q61H ILDTAGHEEY ILDTAGH EY I DTAGHEEY ILD AGHEEY (SEQ ID NO. 496) (SEQ ID NO. 507) (SEQ ID NO. 518) (SEQ ID NO. 529) KRAS_Q61L ILDTAGLEEY ILDTAGL EY I DTAGLEEY ILD AGLEEY (SEQ ID NO. 497) (SEQ ID NO. 508) (SEQ ID NO. 519) (SEQ ID NO. 530) KRAS_Q61R ILDTAGREEY ILDTAGRE Y I DTAGREEY ILD AGREEY (SEQ ID NO. 498) (SEQ ID NO. 509) (SEQ ID NO. 520) (SEQ ID NO. 531) IDH2_R140Q SPNGTIQNIL SPN TIQNIL SPNGTIQNI SPNGT QNIL (SEQ ID NO. 499) (SEQ ID NO. 510) (SEQ ID NO. 521) (SEQ ID NO. 532) TP53_Y220C VVPCEPPEV VVPCEPP V VVPCEPPE V PCEPPEV (SEQ ID NO. 500) (SEQ ID NO. 511) (SEQ ID NO. 522) (SEQ ID NO. 533) TP53_R248W SSCMGGMNWR SSCM GMNWR S CMGGMNWR SSCMGGM WR (SEQ ID NO. 501) (SEQ ID NO. 512) (SEQ ID NO. 523) (SEQ ID NO. 534) TP53_R213L YLDDRNTFL YL DRNTFL YLDDRNTF YLDDRN FL (SEQ ID NO. 502) (SEQ ID NO. 513) (SEQ ID NO. 524) (SEQ ID NO. 535) KRAS_G12V_9mer VVGAVGVGK VVGAVG GK VVGAV VGK VVG VGVGK (SEQ ID NO. 503) (SEQ ID NO. 514) (SEQ ID NO. 525) (SEQ ID NO. 536) KRAS_G12V_10mer VVVGAVGVGK VVVGAVG GK VVVGAV VGK VVVG VGVGK (SEQ ID NO. 504) (SEQ ID NO. 515) (SEQ ID NO. 526) (SEQ ID NO. 537) KRAS_G12D_9mer VVGADGVGK VVGADG GK VVGAD VGK VVG DGVGK (SEQ ID NO. 505) (SEQ ID NO. 516) (SEQ ID NO. 527) (SEQ ID NO. 538) KRAS_G12D_10mer VVVGADGVGK VVVGADG GK VVVGAD VGK VVVG DGVGK (SEQ ID NO. 506) (SEQ ID NO. 517) (SEQ ID NO. 528) (SEQ ID NO. 539)

[0095] Data Deposition

[0096] The data reported in this article have been deposited via ProteomeXchange in

[0097] PeptideAtlas SRM Experiment Library (PASSEL) (identifier PASS01588).

[0098] Results

[0099] To maximize the recovery of HLA molecules from tumor tissue samples, it is critical to homogenize the frozen tissue into single-cell powder rapidly without thawing the sample. For this purpose, an equipment, called the “UniCeller”, was developed, which is capable of applying a strong impact (˜10,000 psi) to frozen tissue chunks. Tissue powder was produced through UniCeller and was then quickly dissolved in Neoantigen Lysis (NL) buffer (Materials and Methods), followed by repeated pipetting and programmed sonication (Materials and Methods). Through this procedure, it was shown that nearly 100% of the HLA molecules from the tissue sample was able to be extracted, which represents a greater recovery efficiency than when using traditional approaches including Dounce Homogenizer, Probe Sonicator and Bead Ruptor (see FIG. 4, which shows a comparison results of the recovery efficiencies between UniCeller and three other traditional approaches Specifically for the experiment, the same amount (50 mg) of tumor tissue was processed through different approaches, including using Dounce Homogenizer, Probe Sonicator, Bead Ruptor, and UniCeller, for extracting HLA complexes. W6/32 antibody was used for the blot). The higher yield observed when using the UniCeller suggests that HLA molecule, as a protein complex predominantly located on the cell surface, may be vulnerable to temperature change and harsh mechanical force in liquid suspension during extraction. In addition, few to none HLA molecules were left in the pellet from the UniCeller group, indicating that a higher extraction efficiency can be achieved when a strong mechanical impact is applied to rapidly generate single-cell level dry tissue powder, followed immediately by a moderate but repeated HLA extraction in lysis buffer.

[0100] The pellet obtained from the UniCeller tissue lysate was processed to extract genomic DNA (see FIG. 1). Single-stranded exomic regions of a selected panel of cancer driver genes were captured with proprietary dual RNA probes and sequenced through DEEPER-Seq pipeline (Wang et al., 2017). A mutation calling was made only when the point mutation is observed as a complementary pair of residues on both DNA strands came from the same DNA duplex molecule as previously described (Wang et al., 2017). Nine tumor samples bearing hotspot mutations in highly frequently mutated cancer driver genes K-Ras and TP53, as well as the slightly lower frequently mutated driver gene IDH2 were selected for a further evaluation of potential neoantigen presentations.

[0101] Antibody-column based affinity chromatography is more efficient and cost-effective than conventional immunoprecipitation and was thus adopted in Valid-NEO pipeline for enriching HLA molecules (Moser & Hage, 2010). To achieve a high enrichment efficiency, an antibody-conjugated column was packed with a 20-fold excess of antibodies (50 mg antibody) relative to the amount needed to enrich HLA molecules from a typical sample (50-100 mg wet tissue with ≥50% tumor mass), in addition repeated sample loadings were performed to ensure the binding between antibodies and HLA molecules in Neoantigen Capture (NC) buffer (see FIGS. 2A and 2B, Materials and Methods). Through elution from the column and incubation in acidic Neoantigen Elution (NE) buffer (Materials and Methods), HLA complexes were eluted, and neoantigen peptides were dissociated from HLA molecules (see FIGS. 2A and 2B). The HLA enrichment column was then regenerated by Neoantigen Neutralization (NN) buffer. The column can be used for at least 30 enrichment and elution procedure with no significant loss in performance observed for the neoantigens analyzed in this study (see FIG. 5, which shows reproducibility evaluation results of the Valid-NEO pipeline analysis. Specifically for the experiment, 10 tumor samples were processed through the same NeoTrue Valid-NEO pipeline to evaluate their endogenous neoantigen presentations. For each neoantigen, three replicates NeoTrue Valid-NEO assays were performed. There were 9 NeoTrue Valid-NEO assays for different neoantigens between replicate 1 and 2, as well as between replicate 2 and 3 for the detection of each neoantigen).

[0102] HLA molecules and other large proteins were separated from neoantigen peptides by a trap column packed with C18 small pore spherical silica particles (diameter 100 Å). Neoantigens (molecular weight around 1.5 kDa) are significantly smaller than HLA molecules (molecular weight around 41 kDa), and will enter the pores therefore be efficiently retained by the C18 matrix that are predominately located inside the pores. The majority of HLA molecules and other large proteins are not efficiently retained by the column. Neoantigens bound to the trap column were then cleaned with 0.1% formic acid to remove HLA molecules and other impurities (see FIGS. 2A and 2B). Neoantigens were then eluted into a suspension composed of 30% acetonitrile 0.1% formic acid. The suspension was spiked with NEO-SEC ladder (Complete Omics Inc, MD), containing two sets of peptides with signature molecular weights at 2,000 and 800 Da (Materials and Methods), and subjected to fractionation through a size exclusion column (short as SEC or SEC column hereinafter) (see FIGS. 2A and 2B). During elution, absorbance at the wavelength of 280 nm was constantly measured by a diode array detector (i.e. DAD). Neoantigen fraction started to be collected after a signature peak at 2,000 Da was observed, and the collection stopped before an 800 Da signature peak was observed (see FIG. 6, which shows HPLC chromotograms for neoantigen purification through an SEC column. Specifically, neoantigen samples were loaded to SEC column together with NEO-SEC ladders. Two signature peaks were observed at 2000 Da and 800 Da, which marked the boundaries for neoantigen-containing fractions). The flow-through between the two signature peaks was collected and lyophilized. The neoantigen sample was then subjected to Valid-NEO mass spectrometry analysis with pre-optimized conditions (Materials and Methods).

[0103] To further improve the recovery ratio and the sensitivity of the pipeline, a neoantigen recovery system, called “Maximum Recovery (MaxRec) System”, was developed. A key element in MaxRec is a set of peptides with slightly different sequences (varying by only 1 or 2 amino acids) from the target peptides, and they are resuspended in the MaxRec system at a much higher abundance than the endogenous neoantigens. MaxRec peptides are designed to mimic the physical characteristics of the target peptides, so as to saturate the nonspecific binding opportunities in the system, thereby minimizing the loss of the target peptides due to such nonspecific interactions. Though sharing similar physical characteristics as target peptides, MaxRec peptides are chemically different, and can be easily distinguished from neoantigen targets based on the high resolution of modern mass spectrometers (see FIGS. 2A and 2B). The Triple Quadrupole Mass Spectrometer in Valid-NEO system is set up to be completely blind to MaxRec peptides and the presence of MaxRec system will not reduce the sensitivity of the platform, which is a feature of such type of instrument. The Valid-NEO neoantigen isolation system was pre-conditioned and spiked in the samples with the MaxRec peptides (see Table 1 above). Through MaxRec system, a significantly improved performance was able to be achieved in the detection of all neoantigen peptides in this study with an average increase in sensitivity by 27 folds (ranging from 2 to 67 folds) (the results are shown in Table 2 below).

TABLE-US-00002 TABLE 2 Neoantigen quantification through different approaches Valid-NEO Valid-NEO (w/MaxRec) (w/o copy number MaxRec) detected per tumor MANA-SRM Detected Detected abundance cell Detected Ratio Ratio to Ratio to (unit: atto (assuming Neoantigen to Standards Standards Standards mole) 50 M cells) KRAS_Q61H non-detectable ± 0.022 ± 0.006 1.045 ± 0.058 522.5 6.3 N/A KRAS_Q61L 0.027 ± 0.002 0.053 ± 0.004 0.729 ± 0.037 364.5 4.4 KRAS_Q61R non-detectable ±  0.04 ± 0.004 1.495 ± 0.156 747.5 9.0 N/A IDH2_R140Q non-detectable ± 0.015 ± 0.003 1.019 ± 0.057 509.5 6.1 N/A TP53_R175H 0.042 ± 0.004 0.266 ± 0.011 1.006 ± 0.045 503 6.1 TP53_Y220C non-detectable ± 0.054 ± 0.002 1.354 ± 0.014 677 8.2 N/A TP53_R248W  0.011 ± 0.00006 0.086 ± 0.034 0.173 ± 0.031 86.5 1.0 TP53_R213L non-detectable ± 0.373 ± 0.057 0.662 ± 0.03  331 4.0 N/A KRAS_G12V_ non-detectable ± 0.146 ± 0.022 5.458 ± 1.206 2729 32.9   9 mer N/A KRAS_G12V_ non-detectable ± 0.141 ± 0.024 6.381 ± 1.693 3190.5 38.4  10 mer N/A KRAS_G12D_ 0.0719 ± 0.012  0.145 ± 0.067 2.933 ± 1.227 1466.5 17.7   9 mer KRAS_G12D_ 0.113 ± 0.012 0.244 ± 0.104 6.518 ± 3.748 3259 39.3  10 mer

[0104] It has been shown that almost all MHC class I associated neoantigens have a length between 8 to 12 amino acids (Sarkizova et al., 2020). For each sample, all potential neoantigen sequences flanking the highest prevalence mutation site on a cancer driver gene (a maximum of 50 possible neoantigen peptides for each missense mutation site) can be directly assayed for in a massively parallel manner without any prediction thus preventing uncertainties (see the table shown in FIGS. 3A-3F). Using Valid-NEO pipeline, nine fresh frozen tumor samples was analyzed to detect and quantify each patient's personalized neoantigens and compared their relative performance between MANA-SRM and the current integrated Valid-NEO pipeline (see Table 2). The patients with KRAS mutations at Q61 site have on average 6.6 copies of the neoantigen presented on each tumor cell surface, and the neoantigens flanking G12 site has an average of 32.1 copies presented per tumor cell. The presentation of TP53 neoantigens are low, ranging from 1 to 8 copies per cell, similar to IDH2 mutations with 6.1 copies of neoantigens presented per cell. Each assay was performed for three times, and the reproducibility of the pipeline was thoroughly evaluated (see Table 2, FIG. 5).

[0105] Discussion

[0106] Traditionally, cytotoxic chemotherapies have been the mainstay therapeutic agent for cancers, regardless of a given patient's individual genetic background of the disease (Bonadonna & Valagussa, 1983; Chan et al., 2012; Savage et al., 2009; Yagoda & Petrylak, 1993). While cytotoxic chemotherapies are still the first line treatment for many cancers, further molecular characterization of cancers has facilitated the development of small molecules or antibody-based agents that can treat a sub-population of the patients who are sharing the same genetic basis of their diseases (Sawyers, 2004; Scaltriti & Baselga, 2006; Sharkey & Goldenberg, 2006). With the development of next-generation sequencing (NGS), it is evident that each individual's cancer has its own genetic profile with varying degrees of overlaps in cancer driver gene mutations among patients (Bagnyukova et al., 2010; Cancer Genome Atlas Research et al., 2013; Chin et al., 2011; Vogelstein et al., 2013). In recent years, highly personalized cancer therapeutic approaches have achieved success through targeting a patient's specific neoantigens, offering hope with regards to the generalizability of such highly personalized treatments (Ott et al., 2017; Sahin et al., 2017). To reveal the neoantigen sequences needed for such personalized cancer therapeutics, algorithm-based or artificial intelligence (AI)-based predictions are often the choice when direct observation is impossible, but such predictions have been proven to be unreliable for clinical applications (Jurtz et al., 2017; Wang et al., 2019). Neoantigens can also be determined through co-culturing tumor cells with autologous T cells, followed by tetramer staining or peptide-pulsing assays, however these functional assays are technically difficult and time consuming, therefore cannot be readily adopted in clinical settings (Danilova et al., 2018; Lu et al., 2014). In Valid-NEO, no prediction is needed, and no cell culture is performed. Additionally, while the neoantigens evaluated in this study are all presented in the context of class I major histocompatibility complexes (MHC I), a similar concept can be readily applied to class II MHC as previously described (Wang et al., 2019).

[0107] Valid-NEO is the only pipeline developed so far to directly validate neoantigens from clinical samples in a sensitive, rapid and reproducible manner, and it helps pave the way for truly personalized cancer therapeutics.

REFERENCES

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