Methods for characterizing loss of antigen presentation
This invention relates to methods for screening for a genotype for loss of antigen presentation via MHC class I in a subject and/or respectively detecting a subject's increased risk of resistance against immunotherapy such as against vaccination.
1. A method for screening for a genotype for loss of antigen presentation via major histocompatibility complex (MHC) class I, comprising: obtaining a sample containing nucleic acid from a human subject, and identifying a chromosomal deletion and inversion event in a chromosome 15, wherein the deletion and inversion event results in the loss of the beta-2-microglobulin (B2M) locus, and wherein the presence of the chromosomal deletion and inversion event indicates that the human subject possesses a genotype for loss of antigen presentation via MHC class I, wherein the identifying comprises (i) amplifying a nucleic acid sequence comprising the sequence set forth in SEQ ID NO: 9 and/or a sequence complementary to SEQ ID NO: 9 to generate an amplification product, (ii) contacting a probe to the amplification product, and (iii) detecting hybridization of the probe to the amplification product.
2. The method of claim 1 , wherein identifying a chromosomal deletion and inversion event in a chromosome 15 comprises detecting the presence or absence of a nucleic acid sequence comprising a sequence selected from the group consisting of:
(i) a sequence identifying a chromosomal deletion and inversion event in a chromosome 15, wherein the deletion and inversion event results in the loss of the beta-2-microglobulin (B2M) locus, and comprising the sequence set forth in SEQ ID NO: 9, the sequence set forth in SEQ ID NO: 9 surrounding the end point of the deletion, which is the start point of the inverted region,
(ii) the sequence set forth in SEQ ID NO: 17 or a portion thereof of at least 40 nucleotides comprising the sequence set forth in SEQ ID NO: 10,
(iii) the sequence set forth in SEQ ID NO: 21 or a portion thereof of at least 40 nucleotides comprising the sequence set forth in SEQ ID NO: 10,
(iv) a sequence complementary to the sequences set forth in any one of (i) to (iii), and
(v) a sequence which is at least 90% identical to the sequences set forth in any one of (i) to (iv), wherein detection of the nucleic acid sequence indicates the presence of a chromosomal deletion and inversion event in a chromosome 15 that is predictive of a genotype for loss of antigen presentation via MHC class I.
3. The method of claim 1 , wherein identifying a chromosomal deletion and inversion event in a chromosome 15 comprises amplifying a nucleic acid sequence comprising a sequence selected from the group consisting of:
(i) a sequence identifying a chromosomal deletion and inversion event in a chromosome 15, wherein the deletion and inversion event results in the loss of the beta-2-microglobulin (B2M) locus, and comprising the sequence set forth in SEQ ID NO: 9, the sequence set forth in SEQ ID NO: 9 surrounding the end point of the deletion, which is the start point of the inverted region,
(ii) the sequence set forth in SEQ ID NO: 17 or a portion thereof of at least 40 nucleotides comprising the sequence set forth in SEQ ID NO: 10,
(iii) the sequence set forth in SEQ ID NO: 21 or a portion thereof of at least 40 nucleotides comprising the sequence set forth in SEQ ID NO: 10,
(iv) a sequence complementary to the sequences set forth in any one of (i) to (iii), and
(v) a sequence which is at least 90% identical to the sequences set forth in any one of (i) to (iv).
4. The method of claim 3 , wherein the amplification is performed using a set of primers selected from: (a) a set comprising the sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16; (b) a set comprising the sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 19; and (c) a set comprising the sequences set forth in SEQ ID NO: 22 and SEQ ID NO: 23.
5. A method for screening for a genotype for loss of antigen presentation via MHC class I, by identifying a chromosomal deletion and inversion event in a chromosome 15 resulting in the loss of the beta-2-microglobulin (B2M) locus comprising: obtaining a sample containing nucleic acid from a subject, hybridizing to the nucleic acid a group of primers, amplifying the hybridized group of primers, and detecting the presence or absence of an amplification product, wherein detection of the amplification product indicates the presence of a chromosomal deletion and inversion event in a chromosome 15 resulting in the loss of the beta-2-microglobulin (B2M) locus that is predictive of a genotype for loss of antigen presentation via MHC class I;
wherein the group of primers is selected from: (a) the group of primers comprising the sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16; (b) the group of primers comprising the sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 19; and (c) the group of primers comprising the sequences set forth in SEQ ID NO: 22 and SEQ ID NO: 23.
6. The method of claim 5 , wherein the group of primers is selected from (a) the group of primers comprising the sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16.
7. The method of claim 5 , wherein the group of primers is selected from (b) the group of primers comprising the sequences set forth in SEQ ID NO: 18, and SEQ ID NO: 19.
8. The method of claim 7 , wherein the amplification product has a length of 80 to 100 bp, preferably 90 to 95 bp, more preferably about 94 bp.
9. The method of claim 7 , further comprising hybridizing to the nucleic acid a probe comprising the sequence set forth in SEQ ID NO: 20 or a sequence complementary to SEQ ID NO: 20.
10. The method of claim 5 , wherein the group of primers is selected from (c) the group of primers comprising the sequences set forth in SEQ ID NO: 22, and SEQ ID NO: 23.
11. The method of claim 10 , wherein the amplification product has a length of 80 to 95 bp, preferably 85 to 90 bp, more preferably about 88 bp.
12. The method of claim 10 , further comprising hybridizing to the nucleic acid a probe comprising the sequence set forth in SEQ ID NO: 24 or a sequence complementary to SEQ ID NO: 24.
13. The method of claim 1 , wherein the deletion starts after position chr15:44,962,085 and ends at position chr15:45,166,582.
14. The method of claim 1 , wherein the inversion comprises an inversion of the sequence downstream of the deleted region and follows the deletion.
15. The method of claim 1 , wherein the inverted region starts at position chr15:45,166,583 and ends at position chr15:45,304,626.
16. The method of claim 1 , wherein the sequence surrounding the end point of the deletion, which is the start point of the inverted region comprises the sequence set forth in SEQ ID NO: 9 or a sequence complementary to SEQ ID NO: 9.
17. The method of claim 1 , wherein identifying a chromosomal deletion and inversion event comprises performing polymerase chain reaction (PCR).
18. The method of claim 1 , wherein the loss of antigen presentation via MHC class I indicates resistance against immunotherapy.
19. The method of claim 18 , wherein the immunotherapy is tumor immunotherapy.
20. The method of claim 6 , wherein the amplification product has a length of 160 to 200 bp, preferably 170 to 190 bp, more preferably about 180 bp.
21. The method of claim 1 , wherein the amplifying step comprises amplifying a nucleic acid sequence comprising the sequence set forth in SEQ ID NO: 10 and/or a sequence complementary to SEQ ID NO: 10 to generate an amplification product.
22. A method for screening for a genotype for loss of antigen presentation via major histocompatibility complex (MHC) class I, comprising: obtaining a sample containing nucleic acid from a subject, and identifying a chromosomal deletion and inversion event in a chromosome 15, wherein the deletion and inversion event results in the loss of the beta-2-microglobulin (B2M) locus, and wherein the presence of the chromosomal deletion and inversion event indicates that the subject possesses a genotype for loss of antigen presentation via MHC class L wherein the identifying comprises (i) amplifying a nucleic acid sequence comprising the sequence set forth in SEO ID NO: 9 and/or a sequence complementary to SEO ID NO: 9 to generate an amplification product, (ii) contacting a probe to the amplification product, and (iii) detecting hybridization of the probe to the amplification product, wherein the amplifying step comprises amplifying a nucleic acid sequence comprising the sequence set forth in SEQ ID NO: 11 and/or a sequence complementary to SEQ ID NO: 11 to generate an amplification product.
23. A method for screening for a genotype for loss of antigen presentation via major histocompatibility complex (MHC) class I, comprising: obtaining a sample containing nucleic acid from a subject, and identifying a chromosomal deletion and inversion event in a chromosome 15, wherein the deletion and inversion event results in the loss of the beta-2-microglobulin (B2M) locus, and wherein the presence of the chromosomal deletion and inversion event indicates that the subject possesses a genotype for loss of antigen presentation via MHC class L wherein the identifying comprises i) amplifying a nucleic acid sequence comprising the sequence set forth in SEO ID NO: 9 and/or a sequence complementary to SEO ID NO: 9 to generate an amplification product, (ii) contacting a probe to the amplification product, and (iii) detecting hybridization of the probe to the amplification product, wherein the amplifying step comprises amplifying a nucleic acid sequence comprising the sequence set forth in SEQ ID NO: 12 and/or a sequence complementary to SEQ ID NO: 12 to generate an amplification product.