InDel molecular marker combination and application thereof in heterologous cell therapy pharmacokinetic detection

Abstract

The invention discloses an InDel molecular marker combination and application of the InDel molecular marker combination in heterologous cell therapy pharmacokinetic detection. The InDel molecular marker combination is selected from one or more of 161 InDel loci on a human chromosome; relative to a reference genome hg19, position information and mutation types of the 161 InDel sites are shown in a table 1. The InDel molecular marker combination can be used for distinguishing donor cells and recipient cells for cell therapy, different donors and recipients are detected by adopting the InDel molecular marker combination, after a specific InDel site is selected, the mutation frequency of the InDel site of a sample (the recipients subjected to cell therapy) after medication is detected, and the mutation frequency of the InDel site is detected. According to the InDel site mutation frequency, the proportion of heterologous cells in the sample can be calculated, the problem that no mature analysis method exists in heterologous cell therapy pharmacokinetic research is effectively solved, and the InDel molecular marker combination can also distinguish donors and receptors with fewer sites.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and in particular relates to an InDel molecular marker combination and an application thereof in pharmacokinetic detection of heterologous cell therapy. Background Art

[0002] Immunotherapy has become the cornerstone of cancer treatment, and it includes a wide range of strategies aimed at releasing, directing, and enhancing the patient's own immune system through culture transfer. Among them, cell therapy is an important immunotherapy method. To implement cell therapy more widely, a key challenge is how to bypass the cumbersome procedures of finding HLA-matched healthy related or unrelated donors, collecting their cells for engineering, and infusing them into one patient. Although this tailored "one donor, one patient" strategy is feasible and has been proven to be effective, they have some limitations, including the long preparation cycle of autologous CAR-T, the inability to optimize the quality of patient T cells, which may lead to poor expansion yield, and the ineffective in vitro expansion of tumor-infiltrating leukocytes. [1] , the cost of surgery is high [2] These problems pose a serious challenge to autologous cell therapy. In contrast, allogeneic cell therapy uses T cells from donors. Since the donors do not undergo chemotherapy, the immune cells are more capable of eliminating cells carrying the target antigen. [3] . Allogeneic cell therapies can be delivered as “off-the-shelf” products, addressing the challenges of autologous cell therapies. Since HLA-A, -B, and -DR matching may negate graft-versus-host disease (GVHD), cell therapy products generated from selected individuals can be applied to a wider population. Therefore, cell banks can be generated from optimal T cell subsets from healthy individuals, reducing production costs while increasing applicability and effectiveness. Therefore, allogeneic cell therapies have the potential to break through the limitations of autologous cell therapies [4] .

[0003] The difficulty of the pharmacokinetics (PK) of “off-the-shelf” heterologous cell products lies in finding markers that distinguish between allogeneic and autologous cells. In preclinical testing of off-the-shelf cell therapy products, the difference between human genes and animal genes can be used to design unique primers specifically for human genes, thereby achieving PK testing through PCR. When human tumor models are used preclinically, the method will be affected by human tumor DNA. At the same time, this preclinical method is not applicable to clinical sample testing. Preclinical and clinical experiments based on this method require the development and verification of different methods, which will not only cause time delays, but also greatly increase economic costs.

[0004] In recent years, a new generation of polymorphic site insertion or deletion polymorphisms (InDel or DIP), that is, polymorphic genetic markers formed by the insertion or deletion of different types and sizes of DNA segments (4-22bp) in the genome, have been widely used in forensic evidence identification, paternity testing, and other medical tests. InDel markers have the following characteristics: (1) They are abundant in the human genome, second only to SNPs; (2) They have a similar natural mutation rate to SNPs (~2.3×10 -9 ); (3) InDel amplified fragments are shorter (less than 160bp), which is more suitable for detecting highly degraded and low-copy DNA templates, increasing the success rate of DNA degradation detection typing; (4) InDel sites have significant population and racial differences; (5) InDel detection can be performed using NGS technology, and the technology platform has strong universal applicability. Therefore, in recent years, it has gradually attracted the attention of domestic and foreign medical scholars. Summary of the invention

[0005] In order to solve the technical problems existing in the prior art, the present invention provides an InDel molecular marker combination and its application in the pharmacokinetic detection of allogeneic cell therapy. By utilizing the differences in InDel between people, the corresponding mutation sites are found for the donor, so as to distinguish the donor cells from the recipient cells. First, based on the InDel database, appropriate InDel is selected and InDel capture probe is designed to establish an InDel panel; secondly, based on NGS technology, a second-generation sequencing method for allogeneic cell PK is established.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] The first aspect of the present invention provides an InDel molecular marker combination, wherein the InDel molecular marker combination is selected from one or more of 161 InDel sites on human chromosomes; the position information and mutation types of the 161 InDel sites relative to the reference genome hg19 are as follows:

[0008]

[0009]

[0010]

[0011]

[0012] In the present invention, the reference genome may also be selected from the common reference genome in the art (eg, hg38), and the corresponding InDel molecular marker combination defined by the position information should also fall within the protection scope of the present invention.

[0013] In some embodiments of the present invention, the above-mentioned InDel sites are selected based on the public information of 2504 samples in the third phase of the 1000 Genomes Project (1KGP).

[0014] In some embodiments of the present invention, the InDel site selects biallelic genes as an alternative.

[0015] In some embodiments of the present invention, biallelic InDel polymorphic sites located in coding regions are removed to eliminate possible correlation between the selected sites and a certain type of disease as much as possible.

[0016] In some embodiments of the present invention, the mutation frequencies (AF) of the remaining biallelic polymorphic sites in the entire 2504 samples are calculated, and polymorphic sites with AF greater than 0.25 are retained.

[0017] In some embodiments of the present invention, polymorphic sites with a heterozygous frequency greater than 0.4 in the population are retained. In the present invention, heterozygosity refers to the inconsistency between the mutation from the mother and the mutation from the father of a polymorphic site, and the heterozygous frequency refers to the proportion of heterozygous samples in a population to the total number of samples.

[0018] In some embodiments of the present invention, InDel polymorphic sites with at least two base differences compared to the reference genome are retained.

[0019] In some embodiments of the present invention, polymorphic sites located in the repeat region or within 150 bp near the repeat region are removed. The repeat region information is obtained from rmsk.txt.gz in the UCSC (University of California, Santa Cruz) database.

[0020] In some embodiments of the present invention, possible pathogenic sites associated with a disease are removed according to the ClinVar database.

[0021] In some embodiments of the present invention, polymorphic sites with mutation lengths less than 5 bp are retained.

[0022] In some embodiments of the present invention, polymorphic sites with mutation frequencies between 0.25-0.75 in each ethnic group are retained.

[0023] In some embodiments of the present invention, mutation sites with a GC content within the range of 0.4-0.6 within 50 bp upstream and downstream of the polymorphic site are retained.

[0024] The second aspect of the present invention provides the use of the InDel molecular marker combination as described in the first aspect of the present invention in the preparation of a product for detecting the pharmacokinetics of allogeneic cell therapy, such as a kit or a chip.

[0025] The third aspect of the present invention provides a kit or chip for detecting the pharmacokinetics of allogeneic cell therapy, wherein the kit or chip comprises a reagent for detecting the InDel molecular marker combination as described in the first aspect of the present invention.

[0026] In some embodiments of the present invention, the reagent includes a probe combination for capturing the region where the InDel site is located.

[0027] In some embodiments of the present invention, the capture region of the probe combination is as follows:

[0028]

[0029]

[0030]

[0031]

[0032] In some embodiments of the present invention, the kit further comprises a reagent for extracting genomic DNA of a sample and a reagent for performing InDel detection based on NGS (Next Genome Sequencing) technology.

[0033] In some embodiments of the present invention, the reagents for InDel detection based on NGS technology include reagents used for DNA fragmentation of genomic DNA of the sample to be tested, preparation of DNA library and / or DNA library hybridization capture.

[0034] The third aspect of the present invention provides a method for detecting the pharmacokinetics of allogeneic cell therapy, the method comprising: using the InDel molecular marker combination as described in the first aspect of the present invention or the kit or chip as described in the second aspect of the present invention to process the sample to be tested to obtain a hybridized DNA library, and then performing high-throughput sequencing and analyzing the sequencing results.

[0035] In some embodiments of the invention, the method is for non-diagnostic purposes.

[0036] In some embodiments of the present invention, the method comprises the steps of:

[0037] (1) Extracting genomic DNA from human peripheral blood;

[0038] (2) fragmenting the genomic DNA and constructing a DNA library;

[0039] (3) hybridization capture of a DNA library using the probe combination or the kit or chip;

[0040] (4) Perform streptavidin magnetic bead capture, amplification, magnetic bead purification, and fragment distribution analysis quality control on the hybridized DNA library;

[0041] (5) Perform high-throughput sequencing and bioinformatics analysis on the DNA library obtained after quality control.

[0042] In some embodiments of the present invention, the bioinformatics analysis comprises the following steps:

[0043] 1) Align the sequencing reads to the human reference genome;

[0044] 2) Remove repetitive sequences generated during the amplification process;

[0045] 3) Detect InDel mutations and count the test results.

[0046] In some embodiments of the present invention, in step 1), the sequencing reads are aligned with the human reference genome using the bwa mem algorithm, the results are saved as BAM files, and the aligned BAM files are quality controlled; the human reference genome is preferably the reference genome hg38 or the reference genome hg19.

[0047] In some embodiments of the present invention, in step 2), the sequence processing software Picard is used to remove the repeated sequences generated during the PCR amplification process.

[0048] In some embodiments of the present invention, in step 3), the genome analysis toolkit GATK is used to detect InDel mutations.

[0049] In some embodiments of the present invention, the yield of the genomic DNA is not less than 5 ng, preferably not less than 10 ng.

[0050] In some embodiments of the present invention, the genomic DNA is subjected to DNA fragmentation by an ultrasonic shearing device;

[0051] Preferably, the parameters of the ultrasonic interrupter are: maximum incident power 75W, working coefficient 10%, the number of ultrasonic energy transfers during the ultrasonic wave acting on the sample is 200, and the processing time is 260sec.

[0052] In some embodiments of the present invention, the hybridization capture uses an overnight slow hybridization method (65° C. overnight).

[0053] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0054] The reagents and raw materials used in the present invention are commercially available.

[0055] The positive and progressive effects of the present invention are:

[0056] The present invention provides an InDel molecular marker combination for distinguishing cell therapy donor and recipient cells. Different recipients and donors can be distinguished at different InDel sites. The InDel molecular marker combination of the present invention is used to detect different donors and recipients. After selecting a specific InDel site, the mutation frequency of the InDel site of the sample after medication (receptor treated with cell therapy) is detected. According to the mutation frequency of the InDel site, the proportion of heterologous cells in the sample can be calculated, which effectively solves the problem of no mature analysis method for heterologous cell therapy pharmacokinetic research. Further, the InDel combination designed by the present invention can distinguish donors and recipients with relatively few sites; the InDel combination designed by the present invention can be widely used not only in East Asian populations, but also in other major populations in the world (for example, Africa, South Asia, Europe, and America). DETAILED DESCRIPTION

[0057] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0058] Example 1 Screening of InDel mutation sites

[0059] InDel mutation screening was performed based on the 2504 sample information published by the International 1000 Genomes Project (1KGP) phase 3. The specific steps are as follows:

[0060] 1. Select the polymorphic sites of biallelic genes according to the vcf file of 1KGP phase3;

[0061] 2. Download the V43 version of the genome annotation file (gtf file) from NCBI (National Center for Biotechnology Information), and screen the polypeptide sites located in the non-coding region according to the gtf file;

[0062] 3. Calculate the mutation frequency (AF) of the remaining biallelic polymorphic sites in all 2504 samples, and retain the polymorphic sites with AF greater than 0.25;

[0063] 4. Heterozygosity refers to the mutation situation of a polymorphic site from the mother and the mutation situation from the father. When the mutation situation of the paternal and maternal sites at this site is inconsistent, the sample is a heterozygous site at this site. The heterozygous frequency of the remaining polymorphic sites in the population is calculated (that is, the number of samples with heterozygous sites at this site divided by the total number of samples), and the polymorphic sites with a heterozygous frequency greater than 0.4 are retained;

[0064] 5. Count the number of base differences between polymorphic site mutations and the reference genome, and retain polymorphic sites with at least two base differences;

[0065] 6. Remove polymorphic sites near the repeat region: According to the rmsk.txt.gz file in the UCSC (University of California, Santa Cruz) database (Repeat Masker was developed by Arian Smit et al. to screen scattered repeat sequences and low-complexity sequences in DNA sequences. The repeat sequences screened by Repeat Masker are stored in the UCSC database as rmsk.txt.gz files) remove polymorphic sites located in the repeat region and within 150 bp upstream and downstream of the repeat region;

[0066] 7. Remove possible pathogenic sites related to the disease: Download the ClinVar database, remove the possible pathogenic sites contained in the database and the polymorphic sites within 50 bp upstream and downstream of the possible pathogenic sites in the database;

[0067] 8. Count the lengths of the remaining polymorphic site InDel mutations, and retain polymorphic sites with mutation lengths less than 5 bp;

[0068] 9.1 The samples of KGP phase 3 were divided into different ethnic groups (AFR: African, AMR: Americas, EAS: East Asian, EUR: European, SAS: South Asian); the polymorphic sites with mutation frequencies between 0.25-0.75 in each ethnic group were retained;

[0069] 10. Count the GC content within 50 bp upstream and downstream of the remaining polymorphic sites, and retain mutation sites with GC content in the range of 0.4-0.6.

[0070] According to the above steps, 161 polymorphic sites were selected from the 1KGP database and are shown in Table 1, where the reference genome is hg19.

[0071] Table 1 InDel mutation site information

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[0073]

[0074]

[0075]

[0076] Example 2

[0077] The embodiment of the present invention uses the information of 2504 samples disclosed in the phase 3 of the International 1000 Genomes Project (1KGP) to verify the InDel mutation site. Specifically, the mutation files of the 2504 samples are respectively counted for the mutations of 161 InDel sites, and the mutation information of each sample at each site is shown in Table 3, wherein 0|0 in the mutation type represents that the site in the sample is captured by the probe but no mutation occurs; 0|1 represents that the site in the sample is a heterozygous mutation, with two genotypes of REF (reference genome) and ALT (mutation) (part of the bases are consistent with the REF base type, and part of the bases are consistent with the ALT base type); 1|1 represents that the site in the sample is a homozygous mutation, and the overall mutation type is consistent with the ALT base type; 1|2 represents that the site in the sample is a heterozygous mutation, with two genotypes of ALT1 and ALT2 (part of the base type is consistent with the ALT1 base type, and part of the base type is consistent with the ALT2 base type). As shown in Table 2 below, 2504 individuals can be completely distinguished by a combination of 161 InDel sites.

[0078] Table 2 Detection results of 161 InDel sites in 2504 samples of the International 1000 Genomes Project

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[0080]

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[0082]

[0083] *Only 10 samples are shown here

[0084] Example 3

[0085] The embodiment of the present invention provides a method for detecting PK of heterologous cells in cell therapy based on the second generation sequencing technology. In this embodiment, whole blood samples of 8 people are analyzed, and the operation steps specifically include:

[0086] 1. Design and synthesis of capture probes in the target region. Specifically, capture probes are designed for the regions where the 161 InDel sites are located. In this embodiment, 159 synthesized probes are used.

[0087] 2. Obtain sample DNA. Specifically, use human peripheral blood stored in sterile blood collection tubes containing anticoagulants (transported at 2-8°C and stored at -80°C) to extract genomic DNA ( Blood Genomic DNA Kit #EC101), the yield is not less than 10ng.

[0088] 3. DNA fragmentation. Specifically, the genomic DNA was fragmented to 200 bp using an ultrasonic fragmentor (Covaris M220). The model of the fragmentation tube was microTUBE-50. The parameters used were as follows: peak incident power 75 W, duty factor 10%, number of cycles per burst of ultrasonic energy transferred during the ultrasonic action on the sample 200, and treatment time 260 sec.

[0089] 4. DNA library preparation. Specifically, according to the instructions of SureSelect XT HS2 DNA Reagent Kit, the DNA fragments were end-repaired / A-added and connected to adapters using SureSelect XT HS2 DNA Reagent Kit (#G9981A), and small fragments in the adapter-connected samples were removed using purification magnetic beads (AMPure XP Reagent #A63881) (hereinafter referred to as magnetic bead purification). The purified products were amplified and magnetically purified to obtain a purified DNA library.

[0090] 5. DNA library hybridization capture. Specifically, according to the instructions of SureSelect XT HS2 Target Enrichment Kit, Overnight Hybridization (#G9957A), the InDel probe Panel of the present invention (probes synthesized by Agilent Technologies Inc.) was used to hybridize the DNA library overnight, and the hybridized DNA library was captured with streptavidin magnetic beads, amplified, purified by magnetic beads, and subjected to quality control of fragment distribution analysis (Qsep 100).

[0091] 6. Sequencing on a machine. Specifically, a second-generation sequencer (MGISEQ 2000) is used to perform high-throughput sequencing on the library obtained in step 5 to obtain sequencing data.

[0092] 7. Bioinformatics analysis: specifically, quality control, comparison and variation detection analysis of offline data.

[0093] 1) Align the sequencing reads to the human reference genome (hg38, where the hg19 coordinates and hg38 coordinates can be converted to each other) using the bwa mem algorithm in the alignment software BWA (version: 0.7.17), and save the results as bam files. Perform quality control on the aligned bam files, check the alignment rate, coverage and other indicators to ensure that the data quality meets the standards.

[0094] 2) Removal of duplicate sequences: The sequence processing software Picard (version: 2.23.9) was used to remove duplicate sequences (PCR duplicate reads) generated during the PCR amplification process.

[0095] 3) Mutation detection: The genome analysis toolkit GATK (version: 4.1.9.0) was used to detect insertion and deletion (InDel) mutations at 161 InDel sites.

[0096] 4) Statistics of InDels detection results of 8 samples. Statistics of 161 InDel variation results detected by the GATK HaplotypeCaller algorithm for 8 samples (Table 3). As can be seen from the table below, the 8 individuals can be completely distinguished, and any two individuals can be distinguished by one probe site in the InDel combination. If downstream connection is required for qPCR (real-time fluorescence quantitative PCR) for DNA-level quantification, or ddPCR (droplet digital PCR) for cell-level quantification, primer probe design capture can be performed based on a single probe site.

[0097] Table 3 Detection results of 161 InDel sites in 8 samples

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[0099]

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[0101]

[0102] In summary, the InDel detection kit (containing 159 probes) designed based on the above 161 InDel sites can cover all sites and effectively distinguish different individuals based on the second-generation sequencing technology, helping to solve problems such as multi-individual sample detection, such as heterologous cell drug detection.

[0103] The above embodiments are merely descriptions of preferred embodiments of the present invention, and are not intended to limit the present invention. Various modifications and improvements made by those skilled in the art to the technical solution of the present invention without departing from the spirit and scope of the present invention shall still be covered by the claims of the present invention.

[0104] References

[0105] 1. Ando M, Nakauchi H. 'Off-the-shelf' immunotherapy with iPSC-derived rejuvenated cytotoxic T lymphocytes. Exp Hematol. 2017Mar; 47:2-12. doi:10.1016 / j.exphem.2016.10.009. Epub 2016Nov 5. PMID: 27826124.

[0106] 2.Torikai H,Cooper LJ.Translational Implications for Off-the-shelfImmune Cells Expressing Chimeric Antigen Receptors.Mol Ther.2016Aug;24(7):1178-86.doi:10.1038 / mt.2016.106.Epub 2016May 16. PMID: 27203439; PMCID: PMC5088751.

[0107] 3. Qasim W. Allogeneic CAR T cell therapies for leukemia. Am J Hematol. (2019) 94:S50–S4.10.1002 / ajh.25399

[0108] 4.Gatla H,Uth N,Levinson Y,Navaei A,Sargent A,Ramaswamy S,FriedrichBen-Nun I.Enabling Allogeneic T Cell-Based Therapies:Scalable Stirred-TankBioreactor Mediated Manufacturing.Front Med Technol.2022May 30;4:850565.doi:10.3389 / fmedt.2022.850565.PMID:35707712;PMCID:PMC9189297.

Claims

1. An InDel molecular marker combination, characterized in that: The InDel molecular marker combination is selected from one or more of the 161 InDel sites on human chromosomes; the position information and mutation types of the 161 InDel sites relative to the reference genome hg19 are as follows:

2. Use of the InDel molecular marker combination as claimed in claim 1 in the preparation of a product for detecting the pharmacokinetics of heterologous cell therapy.

3. A kit or chip for detecting the pharmacokinetics of heterologous cell therapy, characterized in that: The kit or chip comprises a reagent for detecting the InDel molecular marker combination as claimed in claim 1; Preferably, the reagent comprises a probe combination for capturing the region where the InDel site is located; Further preferably, the capture region of the probe combination is as follows:

4. The kit or chip according to claim 3, characterized in that: The kit also includes reagents for extracting sample genomic DNA and reagents for InDel detection based on NGS technology.

5. The kit or chip according to claim 4, characterized in that: The reagents for InDel detection based on NGS technology include reagents for DNA fragmentation of genomic DNA of the sample to be tested, preparation of DNA library and / or DNA library hybridization capture.

6. A method for detecting the pharmacokinetics of heterologous cell therapy, characterized in that: The method comprises: using the InDel molecular marker combination as claimed in claim 1 or the kit or chip as claimed in any one of claims 3 to 5 to process the sample to be tested to obtain a hybridized DNA library, and then performing high-throughput sequencing and analyzing the sequencing results; Preferably, the method is for non-diagnostic purposes.

7. The method according to claim 6, characterized in that The method comprises the following steps: (1) Extracting genomic DNA from human peripheral blood; (2) fragmenting the genomic DNA and constructing a DNA library; (3) hybridization capture of a DNA library using the probe combination or the kit or chip; (4) Perform streptavidin magnetic bead capture, amplification, magnetic bead purification, and fragment distribution analysis quality control on the hybridized DNA library; (5) Perform high-throughput sequencing and bioinformatics analysis on the DNA library obtained after quality control.

8. The method according to claim 7, characterized in that The bioinformatics analysis comprises the following steps: 1) Align the sequencing reads to the human reference genome; 2) Remove repetitive sequences generated during the amplification process; 3) Detect InDel mutations and count the test results.

9. The method according to claim 8, characterized in that In step 1), the sequencing reads are aligned with the human reference genome using the bwa mem algorithm, the results are saved as BAM files, and the aligned BAM files are quality controlled; the human reference genome is preferably the reference genome hg38 or the reference genome hg19; And / or, in step 2), using sequence processing software Picard to remove repeated sequences generated during PCR amplification; And / or, in step 3), using the genome analysis toolkit GATK to detect InDel mutations.

10. The method according to claim 7 or 8, characterized in that The yield of the genomic DNA is not less than 5 ng, preferably not less than 10 ng; and / or, fragmenting the genomic DNA using an ultrasonic fragmentation instrument; Preferably, the parameters of the ultrasonic interrupter are: maximum incident power 75W, working coefficient 10%, the number of ultrasonic energy transfers during the ultrasonic wave acting on the sample is 200, and the processing time is 260 seconds.

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