KIR/HLA Genotyping (Hybrid Capture NGS)

KIR Genotyping by Hybrid-Capture NGS

Immunogenetic characterization of donors and starting materials for allogeneic / off-the-shelf CAR-NK and cell-therapy products. Using solution-phase targeted hybrid capture, all 13 KIR genes and HLA class I are enriched in a single reaction, sequenced on the Illumina short-read platform, and interpreted with the KIR-dedicated PING pipeline. One assay returns KIR gene content, copy number (CNV), allele-level genotypes (GL string), centromeric/telomeric haplotypes and the B-content score, together with HLA-A/B/C typing and KIR–HLA ligand pairing — providing molecular-level evidence for donor selection, immunogenetic characterization of the Master Cell Bank (MCB) / starting material, and IND/BLA regulatory submissions.

1. Background

Natural killer (NK) cells decide whether to kill in real time through a set of germline-encoded receptors; among them, the killer-cell immunoglobulin-like receptor (KIR) family recognizes HLA class I molecules and transmits inhibitory or activating signals. In allogeneic / off-the-shelf CAR-NK and cell therapy, the donor's KIR and HLA genotypes jointly shape NK alloreactivity and education, making them core variables for donor selection and product immunogenetic characterization. The KIR locus lies at chromosome 19q13.4, spans 100–350 kb, carries 4–20 genes per haplotype with more than 600 known alleles, and is one of the most structurally complex and polymorphic regions of the human genome.

Clinical evidence has translated this biology into practice. In haploidentical hematopoietic cell transplantation, a KIR-ligand mismatch in the donor-versus-recipient direction releases the corresponding NK subset from inhibition and produces alloreactivity, which is associated with reduced leukemia relapse (Ruggeri et al., 2002); selecting unrelated donors by the KIR B-haplotype “B-content score” improves outcomes in acute myeloid leukemia (AML), with a disease-specific effect (Cooley et al., 2010). On the cell-therapy side, cord-blood-derived anti-CD19 CAR-NK has shown encouraging response rates and a favorable safety profile in early-phase trials (Liu et al., 2020). Importantly, KIR (chromosome 19) and HLA class I (chromosome 6) are independent and unlinked — carrying a given inhibitory KIR (the “key”) is informative only when it is known whether its HLA ligand (the “lock”) is present. KIR must therefore be typed together with HLA class I; the associations above are correlative, disease-specific evidence and are not a prediction or guarantee of product efficacy.

KIR genotyping is challenging because the gene family is highly homologous and copy-number variable, with deletions, duplications and fusions. Gene-content-only methods (PCR-SSP/SSO) cannot provide copy number or allele-level information: for just two KIR genes, roughly 22.5% of individuals in the 1000 Genomes dataset carry a novel allele or a structural variant. Solution-phase targeted hybrid capture combined with short-read sequencing and a KIR-dedicated interpretation pipeline (PING) resolves gene content, copy number and allele-level genotypes within one system, and captures HLA class I in the same reaction — meeting the need for joint typing at the source (Norman et al., 2016; Marin et al., 2021).

From a regulatory standpoint, KIR/HLA genotyping is not a mandatory release attribute; it is a scientific input for donor selection and for immunogenetic characterization of the starting material / Master Cell Bank. This service can serve as supporting evidence for donor and starting-material genetic-background characterization within the frameworks of the FDA guidance “Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products” (2024), ICH Q5D (derivation and characterization of cell substrates), ICH Q2(R2) (validation of analytical procedures), and China's NMPA “Technical Guidelines for Research and Evaluation of Cell Therapy Products (Trial)” (2017) and CDE “Technical Guidelines for Pharmaceutical Research and Evaluation of Immune Cell Therapy Products (Trial)” (2022). The method can be qualified fit-for-purpose or fully validated per ICH Q2(R2) according to the development stage.

2. Technical Principle

This service is built on solution-phase targeted hybrid capture that simultaneously enriches the 13 KIR genes and HLA class I, followed by Illumina paired-end short-read sequencing and interpretation with the KIR-dedicated PING pipeline, returning KIR gene content, copy number and allele-level genotypes — together with HLA-A/B/C typing — from a single reaction. The core steps are as follows:

(1) Sample receipt and QC

Genomic DNA (or donor-derived cells) is received and assessed for concentration, purity and integrity to confirm it meets library-preparation and capture requirements.

(2) Library preparation and hybrid capture

Genomic DNA is fragmented, prepared into a dual-indexed library, hybridized in solution to biotinylated probes (covering the 13 KIR genes and HLA-A/B/C), separated from non-target DNA on streptavidin-coated magnetic beads, and enriched over two rounds to yield the target library.

(3) Illumina short-read sequencing

Paired-end sequencing is performed to the target coverage depth; the reference configuration is MiSeq v3 2×300 bp, and read length and platform can be adjusted with appropriate method validation.

(4) PING bioinformatics and interpretation

Using the single-copy framework gene KIR3DL3 as a normalizer, gene content and copy number are determined by clustering the relative ratios of uniquely aligned reads (PING_gc); a genotype-matched reference is then built and multi-round alignment yields allele-level genotypes (GL string) with assembled centromeric/telomeric haplotypes (PING_allele). HLA class I is typed from the same data, and the KIR–HLA ligand pairing and B-content score are reported.

Figure 1. Workflow of KIR/HLA genotyping by hybrid capture + PING.

3. Features and Advantages

KIR and HLA typed in one reaction: A single capture yields the 13 KIR genes and HLA-A/B/C together, intrinsically satisfying the “KIR must be read with HLA” prerequisite and avoiding cross-platform discrepancies.

Three resolution tiers in one assay: Gene content → copy number (CNV) → allele-level genotype plus centromeric/telomeric haplotype, all delivered from a single dataset.

Structural variation covered: Probes designed against complete KIR haplotypes detect deletions, duplications and fusions, and are more robust than alignment-only approaches.

High fidelity and scalable: Short-read sequencing offers high per-base accuracy; large sample numbers can be run in parallel per batch, suited to population/cohort studies and multi-batch characterization screening.

Standardized output: KIR/HLA genotypes are reported in the standard GL string format for cross-laboratory comparison and alignment with submission data packages.

4. Applications

Allogeneic / off-the-shelf CAR-NK: immunogenetic characterization of donors and starting materials.

Transplant donor selection: adding KIR to HLA typing to assess the B-content score and the direction of KIR-ligand mismatch (correlative, disease-specific evidence).

Starting material / Master Cell Bank (MCB) characterization: genetic-background documentation of donor-derived cells in support of release.

Population and cohort studies: large-scale association studies of KIR/HLA genotypes with disease or treatment outcome.

IND/BLA characterization: supporting evidence for donor selection and immunogenetic characterization of cell-therapy products.

5. Report and Deliverables

The report provides quantifiable, genotype-anchored and comparable immunogenetic evidence. Core contents include:

KIR gene content: presence/absence calls for the 13 KIR genes.

Copy number (CNV): per-gene KIR copy number normalized to KIR3DL3.

Allele-level genotype: in GL string format, with flags for novel alleles or novel SNP combinations.

Haplotype composition: centromeric/telomeric (Cen/Tel) and A/B haplotype composition.

B-content score: calculated from the number of Cen-B and Tel-B motifs (0–4).

KIR–HLA ligand pairing: assignment of HLA-C C1/C2, HLA-Bw4 and HLA-A3/A11 epitopes and the mismatch direction.

HLA class I typing: HLA-A/B/C alleles from the same capture.

Data delivery: full analysis report (PDF), KIR/HLA genotype list (GL string), variant and alignment files, and raw sequencing data.

Figure 2. Representative results of KIR gene content, copy number, allele-level genotype and KIR–HLA ligand.

6. Service Workflow

*Standard turnaround: approximately 20–30 working days; batch samples and add-on items are subject to the final plan.

7. Sample Requirements

*Detailed requirements follow the latest Sample Submission Guide; this service is not suitable for severely degraded samples. Please schedule and confirm the plan before shipping.

8. Technical Specifications

9. References

[1] Norman PJ, et al. Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing. Am J Hum Genet. 2016;99(2):375–391. doi:10.1016/j.ajhg.2016.06.023

[2] Marin WM, et al. High-throughput Interpretation of Killer-cell Immunoglobulin-like Receptor Short-read Sequencing Data with PING. PLoS Comput Biol. 2021;17(8):e1008904. doi:10.1371/journal.pcbi.1008904

[3] Cooley S, Weisdorf DJ, Guethlein LA, et al. Donor selection for natural killer cell receptor genes leads to superior survival after unrelated transplantation for acute myelogenous leukemia. Blood. 2010;116(14):2411–2419. doi:10.1182/blood-2010-05-283051

[4] Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science. 2002;295(5562):2097–2100. doi:10.1126/science.1068440

[5] Liu E, Marin D, Banerjee P, et al. Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors. N Engl J Med. 2020;382(6):545–553. doi:10.1056/NEJMoa1910607

[6] Milius RP, Mack SJ, Hollenbach JA, et al. Genotype List String: a grammar for describing HLA and KIR genotyping results in a text string. Tissue Antigens. 2013;82(2):106–112.

[7] ICH. Q2(R2): Validation of Analytical Procedures. Step 4 version, 2023.

[8] Robinson J, Halliwell JA, Hayhurst JD, et al. The IPD and IMGT/HLA database (IPD-KIR / IPD-HLA). Nucleic Acids Res. 2015;43:D423–D431.