DE112021001605T5 - BIOMARKER PANEL SPECIFIC FOR MYELOID-DERIVED SUPPRESSIVE CELLS - Google Patents

Abstract

The invention relates to the use of transcript variants of genes encoding surface proteins known to be found on the surface of granulocytic myeloid suppressor cells (gMDSC) in the characterization of MDSC populations (eMDSC, gMDSC and mMDSC- populations). The use of isoform proteins encoded by transcript variants found to be specific for MDSC as surface markers will also allow the development of new treatment approaches in chronic inflammation, autoimmune diseases and cancer.

Description

technical field

The invention relates to the use of transcript variants of genes encoding surface proteins known to be found on the surface of granulocytic myeloid-derived suppressor cells (gMDSC) for the characterization of MDSC populations (eMDSC-, gMDSC - and mMDSC populations). The use of isoform proteins encoded by transcript variants shown to be specific for MDSC as surface markers will also enable the development of new treatment approaches in chronic inflammation, autoimmune diseases and cancer.

State of the art

Although almost 20 years have passed since the discovery of myeloid-derived suppressor cells (MDSCs) in cancer patients, the functional importance of these cells in the immune system is still not understood. The results of the conducted studies demonstrate that the MDSC population plays a role in the negative regulation of the immune system in cancer and other diseases.

Common features of the MDSC populations are that they are myeloid-derived, that they are pathologically activated although immature, and that they have a remarkable ability to suppress T cells. Although researchers have defined different MDSC subgroups in different diseases, the studies conducted are often not universally accepted due to the methodological and technical differences in characterizing the identified MDSC populations. The fact that specific biomarkers for the MDSC population have not yet been discovered underscores the issue of trust in MDSC biology research.

Different combinations of the surface markers CD33, CD11b, IL-4Ra, CD14, CD15, CD16, CD66b, VEGFR1 and HLA-DR are often used for human MDSCs. While CD11b, CD33 and IL-4Ra are used to detect the general MDSC population, in addition to these three markers CD14-CD16 low VGFR1 +HLA-DR- G-MDSC and CD15+/- CD16-CD66b+/-VGFRI-HLA-DR apply low M-MDSC as subtypes.

Recent studies on multicolor immunophenotyping have shown that there are six different MDSC subtypes [MDSC1, CD14+IL-4Roc+; MDSC2, CD15+IL-4Ra+; MDSC3, Lin-(CD3, CD14, CD16, CD19, CD20, CD56)-HLA-DR-CD33+; MDSC4, CD14+HLA-DR-/low; MDSC5, CD11b+CD14-CD15+ and MDSC6, CD15+FSC low SSC high] in humans. In addition to these markers, there are publications stating that about 32 different surface molecules can be associated with the MDSC phenotype.

• • CD11b⁺ CD 14^- CD66b⁺ oder CD1 Ib⁺ CD 14^- CD15⁺ granulozytäre MDSC-Population (gMDSC),
• • CD11b⁺ CD14⁺ HLA-DR^-/low CD66b^- monozytäre MDSC-Population (mMDSC),
• • Lin^- (CD3, CD14, CD15, CD19, CD56) HLA-DR^- CD33⁺ MDSC-Population im Frühstadium (eMDSC).

Although several subtypes of myeloid regulatory cells have been identified in the studies to date, the myeloid regulatory cells can be divided into three major subtypes: granulocytic MDSC (or polymorphonuclear (PMN) MDSC), monocytic MDSC, and early-stage MDSC populations. The immunophenotypic classification used by researchers for these three major populations with a common consortium is as follows:

• • CD11b ⁺ CD 14 ^- CD66b ⁺ or CD1 Ib ⁺ CD 14 ^- CD15 ⁺ granulocytic MDSC population (gMDSC),
• • CD11b ⁺ CD14 ⁺ HLA-DR ^-/low CD66b ^- monocytic MDSC population (mMDSC),
• • Lin ^- (CD3, CD14, CD15, CD19, CD56) HLA-DR ^- CD33 ⁺ early stage MDSC population (eMDSC).

In patent document no WO2016/196451A1 states that the lectin-type oxidized LDL receptor-1 (OLR-1) (LOX-1) protein, presented as a candidate marker specific for MDSC populations, is used to identify a subpopulation of MDSC called gMDSC or PMN-MDSC -Populations can be used. However, the utility of the ORL-1 protein as a gMDSC biomarker has not been adequately supported by other teams conducting studies in myeloid cells and has proved inconsistent due to differing results across studies. Since the gMDSC population heterogeneously expresses the ORR-1 protein in the tumor microenvironment, it was concluded that the ORR-1 protein is only expressed by a subset of the gMDSC population and it was also expressed by other polymorphonuclear cells. Because of this, it was determined that OLR-1 is not a specific marker for the MDSC subpopulation called gMDSC or PMN-MDSC.

In the patent specification no EP2619585B1 mentions the use of myeloid cell biomarkers in the identification, prediction and follow-up of renal cell carcinoma (RCC) and colorectal carcinoma (CRC). MDSC phenotype 5-specific markers (CD11b, CD14, CD15, CD80, CD83, CD86, and HLA-DR) are used to separate the MDSC-5 population from other populations using flow cytometry.

Another document, which mentions the markers used to label the MDSC populations and the strategies used, similarly mentions the markers to be used when separating the MDSC populations by flow cytometry. This paper provides an overview of the strategy that should be used in studies of MDSC biology, as specific markers for MDSC populations have not yet been discovered. As mentioned in the paper, it is concluded that functional, biochemical and molecular assays are necessary to validate MDSC populations, as is the use of the proposed marker combinations in flow cytometry.

It is known that the cell surface markers mentioned in the prior art are also transported in different amounts and/or transiently during normal differentiation, maturation or activation processes of myeloid cells. Therefore, in addition to the surface markers used in the identification of MDSCs, their immunosuppressive function almost always needs to be demonstrated as well. Considering the different human-to-human effects of inflammatory diseases such as cancer on the immune system, the importance of standardization studies to accurately and reliably identify these myeloid regulatory cells has been highlighted.

As a result of the differentiation, maturation and activation processes of immune cells, their biological behavior and gene expression profiles change. The precursor mRNA synthesized through gene expression is processed by various mechanisms to reach maturity that can encode a protein. At this stage, post-transcriptional mechanisms of “alternative splicing” come into play, and isoforms of different lengths are synthesized that do not carry some units of the protein in question. Another mechanism leading to the formation of different variants is the use of "alternative transcription start sites" when they are within the gene region.

Although it is known that there are functional differences between the isoforms of some proteins, information on the function and expression dynamics of the isoforms of most proteins is limited. There are few studies on the importance of alternative transcription mechanisms in myeloid cells. Furthermore, no information has been found in the literature on which transcript variants encode the surface markers of human MDSCs in the differentiation, maturation, and activation steps.

Summary of the Invention

In the context of the invention, transcript variants of surface molecules known to be present on the surface of gMDSC are first screened using a real-time PCR method in order to find biomarker candidates with a specificity that surface molecules with consistency or reliability problems can replace, which are often used to determine MDSCs.

It has been shown that protein isoforms encoded by transcript variants distinct from the variants examined can be used as new and unique molecules for monitoring and/or targeting of MDSCs. With the invention it was shown that CDID tv2, CD44 tv1 and CD44 tv2 variants can be biomarkers specific for eMDSC and/or mMDSC populations distributed within the monocyte population, while CD33 tv2, CDID tv1 -, CD16 tv1, CD16 tv2 and CDI6 tv3 transcript variants may be biomarkers specific for gMDSC populations.

The use of isoform proteins encoded by transcript variants found to be specific for MDSCs as surface markers will also allow the development of new treatment approaches in chronic inflammation, autoimmune diseases and cancer.

The greatest difference between the invention and other studies and patents is that it can be ensured that the MDSC populations can be recognized by a variant-specific marker. The invention is based on variant differences of markers specific for MDSC cells. As a brief explanation: there are, for example, 3 transcript variants of gene A: A1, A2 and A3; these 3 variants encode 3 protein isoforms of the A gene: AP1, AP2 and AP3; MDSC cells contain the encoded proteins of the same gene (AP1, AP2 and AP3 in our example) on their surface.

Commercially available antibodies recognize all protein isoforms of the A gene simultaneously, so the proteins AP1, AP2 and APS cannot be seen separately in our example. When AP1 is too elevated in the MDSC population and AP3 is too elevated in other cell groups, commercially available antibodies cannot detect it. Studies available in the prior art that do not make this distinction are able to separate cells that could be MDSC populations only by strategic approaches, by considering all isoforms of a protein equally. However, the "biomarker panel specific for myeloid-derived suppressive cells" according to the invention can characterize MDSC populations solely on the basis of surface proteins encoded by specific transcript variants.

character list

• 1 : Percent difference in the expression of transcript variants in colorectal cancer (CRC) compared to healthy subjects.
• 2 : Percent difference in expression of transcript variants in breast cancer (BC) compared to healthy subjects.
• 3 : Percent difference in expression of transcript variants in the gMDSC population compared to healthy and diseased PMN populations.
• 4 : Antigenicity score tables of candidate protein variants that can be used to determine the gMDSC population determined by the Kolaskar and Tongaonkar methods.

Elements/parts that define the invention

V1-7: Variant numbering

Detailed Description of the Invention

With the invention, the exon mRNA sequences and mature mRNA sequences of the gene sequences and transcript variants (tv) of a total of 45 surface molecules (CD33, CD14, CD16, CD1a, CD1d, CD44, EMR1(ADGRE1), PD-L1(CD274) , CD40, LGALS3, IL-IR1, IL-4R, IL6ST, ITGA6, lTGAL(CD11a) TFRC(CD71), FLT1(VEGFR1), CEACAM21, CD66a(CEACAMI), CD43, CD32, IL-6R, ITGA4, ITGAM( CD11b), ITGAX(CD11c), ITGAD(CD11d), CSFR1, CLEC10A(CD301), SELPLG(CD162), CD66d(CEACAM3), CD1b, CD1c, CD15, CD32, CD62L, CD13, CD66b5 C5AR1, VEGFR2, CD2, ITGA1 , ITGA2, ITGA3, ITGA5, and CD31) shown to be transported by MDSCs in various studies, determined from reference nucleotide sequences maintained in the United States National Center for Biotechnology Information (NCBI) - GenBank and Nucleotide; and ENSEMBL (European Bioinformatics Institute, Wellcome Trust Sanger Institute) are registered.

Since the main purpose of the invention is to identify new cell surface antigens that can be encoded by transcript variants for identification of MDSCs, it is considered the overriding criterion that the relevant transcript variants differ in the extracellular region of the protein.

• i. Wenigstens einer der Primer befindet sich an der Exon-Exon-Kreuzung,
• ii. Wenigstens 7 bp des 5'-Endes und wenigstens 4 bp des 3'-Endes des Primers, der an die Exon-Exon-Kreuzung angehängt werden soll, befinden sich auf den beiden Exons an der Kreuzung,
• iii. Die Primerlänge liegt zwischen 18 bis 22 bp,
• iv. Die Schmelztemperatur (Tm) des Primers liegt im Bereich von 57 bis 63 °C und die Schmelztemperaturdifferenz des Primerpaares beträgt höchstens 3 °C,
• v. Der GC-Anteil im Primer liegt im Bereich von 40 bis 60 %,
• vi. Es werden keine Sekundärstrukturen wie Haarnadel, Selbst-Dimer und Kreuz-Dimer gebildet,
• vii. Dinukleotid-Wiederholungen und gleiche Nukleotid-Wiederholungen kommen höchstens viermal vor,
• iix. Das 3'-Ende des Primers ist stabil und verursacht keine unspezifische Bindung,
• ix. Die Amplikonlänge liegt im Bereich von 70 bis 1000 (optimal 500 bp),
• x. Die Primer-Oligonukleotidsequenz bindet an den kodierenden Teil des Exons.
• xi. Primer binden nicht an genomische DNA,

The online tools Primer-BLAST (NCBI), OligoAnalyzer (Integrated DNA Technologies, USA) and offline FastPCR (Primer Digital, Finland) are used for primer design. The online tool Clustal Omega (The European Bioinformatics Institute, UK) is used for the analysis of sequence variants. The design algorithm for designing primers specific to variants can be used as follows:

• i. At least one of the primers is at the exon-exon junction,
• ii. At least 7 bp of the 5' end and at least 4 bp of the 3' end of the primer to be appended to the exon-exon junction is on the two exons at the junction,
• iii. The primer length is between 18 to 22 bp,
• IV. The melting temperature (Tm) of the primer is in the range of 57 to 63 °C and the melting temperature difference of the primer pair is at most 3 °C,
• v. The GC content in the primer is in the range of 40 to 60%,
• vi. Secondary structures such as hairpin, self-dimer and cross-dimer are not formed,
• vii. Dinucleotide repeats and same nucleotide repeats occur at most four times,
• iix. The 3' end of the primer is stable and does not cause non-specific binding,
• ix. The amplicon length is in the range of 70 to 1000 (optimal 500 bp),
• x. The primer oligonucleotide sequence binds to the coding part of the exon.
• xi. Primers do not bind to genomic DNA,

As a result of the analyses, 17 of the 48 genes were found to lack transcript diversity (having a single coding mRNA), and these genes were excluded from the targets. The CDIE gene was excluded from the analysis because it is rarely found on the cell surface and is usually located in intracellular organelles. Primer-oligonucleotide pairs were designed to distinguish 108 different variants of the remaining 31 MDSC-related genes. Their sequences, product lengths (size), and GenBank reference numbers are listed in Table 1.

The optimal reaction conditions for the oligonucleotide pairs are determined by conventional PCR. 5 of the variants optimized with conventional PCR could not be fully investigated due to the lack of positive control samples (testis, brain, pancreas and embryonic tissue, etc.). They are ITGAL tv2, LGALS3 tv3, ITGA6tv2, ILIRI tv3 and CD16 tv5.

In the context of the invention, 70 transcripts of 19 genes are evaluated, which differ primarily in their sequences that code for extracellular protein regions. These 70 transcripts that differing in the extracellular protein region are tested by conventional PCR analysis using cDNA samples derived from the total myeloid cell population isolated from colon cancer patients and healthy subjects. The resulting PCR products are visualized by gel electrophoresis.

Variants that are not expressed or only expressed to a very low extent in both groups were excluded from the panel according to the invention by analyzing variants obtained from the transcript pools of the entire myeloid cell population of colorectal cancer patients and healthy subjects.

For 47 transcript variants (D40 tv1, tv2, tv3; CD44 tv1, tv2, tv3, tv4, tv5, tv6, tv7 ve tv8; CD274 tv1, tv2, tv4; ADGRE1 tv2 ve tv5; CD1D tv1 ve tv2; ILIRI tv2,1, tv6, tv7, tv8 ve v9; IL4R tv1, tv4 ve tv5; TFRC tv2; CEACAMI(CD66a) tv1, tv2, tv4 ve tv6; CEACAM21 tv1, tv2 ve tv4; FLT1 tv2 ve tv4; IL6ST tv1, tv3; IT GAL.tv3 ve tv1, CD16 tv1, tv2, tv3, tv4; CD33 tv2 and tv3), which differ both in their sequences encoding extracellular protein regions and in their expression levels from healthy subjects based on conventional PCR data, comparisons are made with the real-time PCR performed ( 1 ). 1 Figure 12 shows the graph normalizing the neutrophil (PMN) and monocytic populations of colorectal cancer patients with the neutrophil and monocytic populations of healthy individuals. The percentage difference in neutrophil and monocyte population is given for all variants shown in the diagram. The parts without bar charts correspond to the value "0". For this comparison, cDNAs derived primarily from monocytes and PMN cells obtained from colorectal cancer patients and healthy subjects are used.

25 (CD16 tv1, tv2, tv3, tv4; CDID tv1, tv2; CD33 tv2; CD40 tv2, tv3; CD44 tv1, tv2, tv6, tv7, tv8; CE AC AM 1 (CD66a) tv4, tv6; CEACAM21 tv1, tv2 , tv4; FLT1 tv2, tv4; IL1R1 tv6; IL6ST tv2, ADGREI tv5; and IL4R tv4) of the 47 transcript variants differing in the colorectal cancer and healthy transcript pools are identified by real-time PCR with cDNA from the total monocyte and PMN Cell populations of breast cancer evaluated ( 2 ). 2 Figure 12 shows the graph in which the neutrophil and monocyte populations of breast cancer patients are normalized with the neutrophil and monocyte populations of healthy subjects. For the PMN population of breast cancer patients, the variants are CD16 tv1, tv2, tv3 and tv4, CDId tv1, CDId tv2, CD33 tv2, CD40 tv2 and tv3, CD44 tv6, tv7 and tv8, CD66a tv4 and tv6, CEACAM21 tv1, tv2 and tv4, FLT1 tv2 and tv4, IL1R1 tv6 and IL6ST tv2 shown; for the monocyte population, the variants CDID tv2, FLT1 tv2, CD44 tv1 and tv2, ADGRE1 tv5 and IL4R tv4 are shown. Only CDId tv2 is shown in both monocyte and PMN samples. Since there is no difference in the expression of FLT1 tv4 for PMN, this corresponds to the value "0".

In the first phase, given the data obtained from breast cancer patients after colorectal cancer, the transcript variants CD 16 tv1, tv2, tv3, tv4, CDID tv1, tv2 and CD33 tv2 were examined in gMDSC populations. At this stage, the gMDSC population purified from breast and colorectal cancer patients, whose suppressive cell properties were confirmed by functional experiments, was tested for the mentioned 7 variants ( 3 ). 3 Figure 12 shows the graph in which the gMDSC population of breast cancer and colorectal cancer patients is normalized by (a) PMN cells from healthy subjects and (b) PMN cells from patients. Both diagrams show the percentage difference values of the variants CD33 tv2, CDId tv1 and tv2, CD16 tv1, tv2, tv3 and tv4 of the gMDSC population.

The analysis and the graphical results in the figures show that the variants CD33 tv2, CDID tv1, CD16 tv1, CD16 tv2 and CD16 tv3 are lower in the gMDSC population compared to the patient's PMN cells at the transcription level. Of these variants, CD33 tv2 and CDID tv1 were higher at transcript level in the gMDSC population, while variants CD16 tv1, CD16 tv2, and CD16 tv3 were at lower levels compared to healthy subjects.

When the equivalent of the differences found at the transcript level is analyzed at the protein level using bioinformatics tools, CD33 tv2 protein 12-19, CD16 tv1 and tv2 protein 117, CD16 tv3 protein 20-21, CDID tv1 differ -protein 203-295 in amino acid positions. CD33 tv2 protein 12-19, CD16 tv1 and tv2 1-17, CD16 tv3 protein 20-21, CD1d tv1 protein differ in amino acid sequences at positions 203-295.

When antigenic analyzes of the protein variants mentioned are carried out, peptide sequences are determined which show antigenicity at different positions of the CD33 tv2, CDID tv1, CD 16 tv1 and CD 16 tv2 proteins ( 4 ). 4 shows the antigenicity scores of the protein sequences CD33 tv2, CDID tv1, CD16 tv1 and tv2 and CD16 tv3. The positional regions of CD33 tv2 protein 12-19, CD16 tv1 and tv2 1-17, CD16 tv3 protein 20-21 and CDID tv1 protein 203-295 with amino acid differences are shown in red in the diagrams. The horizontal red line indicates the threshold.

It is noted that the same protein is obtained by translation of the CD16 tv1 and tv2 variants, the difference between the variants is due to the 5'UTR and the peptide region showing antigenicity is in the signal sequence. The CDId tv1 protein has a peptide region of 93 amino acids and amino acid sequences that show high antigenicity in this region, making the CDId tv1 protein the strongest candidate for characterizing gMDSC.

On the other hand, the CD33 tv2 protein has a region of 4 amino acids, part of which remains in the signal sequence region, but which is located in the N-terminal region after the signal sequence is cut off and has antigenicity and is very difficult for the development of antibodies use is.

It appears that the extracellular region of the CD33 tv1 and CD33 tv3 proteins can be used to detect the CD33 tv2 protein on the cell surface. Because the extracellular region of the CD33 tv1 and CD33 tv3 proteins is much longer, these proteins have extracellular peptide regions that are not present in the CD33 tv2 protein. Therefore, by developing antibodies that recognize all CD33 protein variants (tv1, tv2, tv3) and the CD33 tv1 and CD33 tv3 proteins, it will be possible to recognize cells carrying the CD33 tv2 protein variant.

The COST BM1404 Action cannot provide sufficient information and technical support for mMDSC and eMDSC populations, therefore gMDSC populations can be assessed within whole monocytes. Within the scope of the invention, it was shown by comparing the total monocyte transcripts from healthy volunteers with the total monocyte transcripts from breast cancer and colon cancer patients that the variants CDID tv2, CD44 tv1 and CD44 tv2 are specific biomarkers for eMDSC and/or mMDSC Populations distributed in the monocyte population.

• • als spezifische Biomarker für eMDSC- und/oder mMDSC-Populationen CDId tv2, CD44 tv1 und CD44 tv2 Varianten,
• • als Biomarker spezifisch für gMDSC-Populationen CD33 tv2, CDId tv1, CD16 tv1, CD16 tv2 und CD16 tv3 Transkriptvarianten.

Further evidence for this result is the mirroring of the eMDSC and mMDSC populations in the total monocyte population in functional experiments. It is observed that the capacity to produce nitric oxide increases and the expression of arginase, cyclooxygenase and inhibitor molecules is higher in the total monocyte population of patients compared to healthy subjects. Consequently, protein isoforms encoded by the following can be used with the invention to detect MDSC populations

• • as specific biomarkers for eMDSC and/or mMDSC populations CDID tv2, CD44 tv1 and CD44 tv2 variants,
• • as a biomarker specific for gMDSC populations CD33 tv2, CDID tv1, CD16 tv1, CD16 tv2 and CD16 tv3 transcript variants.

SEQUENCE LOG

• <110> Hacettepe Universitesi
• <120> BIOMARKER PANEL SPECIFIC TO MYEOLID-DERIVED SUPPRESIVE CELLS
• <130> P21-0119
• <150> TR2020/03833 <151> 2020-03-12
• <160> 31
• <170> BiSSAP 1.3.6
• <210> 1 <211> 20 <212> DNA <213> Artificial sequence
• <220> <223> CD1d-tv1 sense primer
• <400> 1
  
• <210> 2 <211> 24 <212> DNA <213> Artificial sequence
• <220> <223> CD1d-tv1 antisense primer
• <400> 2
  
• <210> 3 <211> 21 <212> DNA
• <213> Artificial sequence
• <220> <223> CD33 tv2 sense primer
• <400> 3
  
• <210> 4 <211> 20 <212> DNA <213> Artificial sequence
• <220> <223> CD33 tv2 antisense primer
• <400> 4
  
• <210> 5 <211> 20 <212> DNA <213> Artificial sequence
• <220> <223> CD16 tv1 sense primer
• <400> 5
  
• <210> 6 <211> 23 <212> DNA <213> Artificial sequence
• <220> <223> CD16 tv1 antisense primer
• <400> 6
  
• <210> 7
• <211> 20 <212> DNA <213> Artificial sequence
• <220> <223> CD16 tv2 sense primer
• <400> 7
  
• <210> 8 <211> 19 <212> DNA <213> Artificial sequence
• <220> <223> CD16 tv2 antisense primer
• <400> 8
  
• <210> 9 <211> 20 <212> DNA <213> Artificial sequence
• <220> <223> CD16 tv3 sense primer
• <400> 9
  
• <210> 10 <211> 19 <212> DNA <213> Artificial sequence
• <220> <223> CD16 tv3 antisense primer
• <400> 10
  
• <210> 11 <211> 24 <212> DNA <213> Artificial sequence
• <220> <223> CD1d tv2 sense primer
• <400> 11
  
• <210> 12 <211> 24 <212> DNA <213> Artificial sequence
• <220> <223> CD1d-tv2 antisense primer
• <400> 12
  
• <210> 13 <211> 20 <212> DNA <213> Artificial sequence
• <220> <223> CD44 tv1 sense primer
• <400> 13
  
• <210> 14 <211> 22 <212> DNA <213> Artificial sequence
• <220> <223> CD44-tv1 antisense primer
• <400> 14
  
• <210> 15 <211> 21 <212> DNA <213> Artificial sequence
• <220> <223> CD44 tv2 sense primer
• <400> 15
  
• <210> 16 <211> 21 <212> DNA <213> Artificial sequence
• <220> <223> CD44 tv2 antisense primer
• <400> 16
  
• <210> 17 <211> 3795 <212> RNA <213> Homo sapiens
• <220> <223> CD1d transcript variant 1 mRNA
• <400> 17
  
  
  
  
• <210> 18 <211> 335 <212> PRT <213> Homo sapiens
• <220> <223> CD1d transcript variant 1 amino acid sequence
• <400> 18
  
  
• <210> 19 <211> 1081 <212> RNA <213> Homo sapiens
• <220> <223> CD33 transcript variant 2 mRNA
• <400> 19
  
  
• <210> 20 <211> 237 <212> PRT <213> Homo sapiens
• <220> <223> CD33 transcript variant 2 amino acid sequence
• <400> 20
  
  
• <210> 21 <211> 2103 <212> RNA <213> Homo sapiens
• <220> <223> CD16 transcript variant 1 mRNA
• <400> 21
  
  
  
• <210> 22 <211> 233 <212> PRT <213> Homo sapiens
• <220> <223> CD16 transcript variant 2 amino acid sequence
• <400> 22
  
  
• <210> 23 <211> 2148 <212> RNA <213> Homo sapiens
• <220> <223> CD16 transcript variant 2 mRNA
• <400> 23
  
  
• <210> 24 <211> 2100 <212> RNA <213> Homo sapiens
• <220> <223> CD16 transcript variant 3 mRNA
• <400> 24
  
  
• <210> 25 <211> 232 <212> PRT <213> Homo sapiens
• <220> <223> CD16 transcript variant 3 amino acid sequence
• <400> 25
  
  
• <210> 26 <211> 3213 <212> DNA <213> Homo sapiens
• <220> <223> CD1d transcript variant 2 mRNA
• <400> 26
  
  
  
• <210> 27 <211> 242 <212> PRT <213> Homo sapiens
• <220> <223> CD1d transcript variant 2 amino acid sequence
• <400> 27
  
• <210> 28 <211> 5431 <212> RNA <213> Homo sapiens
• <220> <223> CD44 transcript variant 1 mRNA
• <400> 28
  
  
  
  
• <210> 29 <211> 742 <212> PRT <213> Homo sapiens
• <220> <223> CD44 transcript variant 1 amino acid sequence
• <400> 29
  
  
• <210> 30 <211> 5302 <212> RNA <213> Homo sapiens
• <220> <223> CD44 transcript variant 2 mRNA
• <400> 30
  
  
  
  
• <210> 31 <211> 699 <212> PRT <213> Homo sapiens
• <220><223> CD44 transcript variant 2 amino acid sequence
• <400> 31
  
  

QUOTES INCLUDED IN DESCRIPTION

This list of documents cited by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Patent Literature Cited

• WO 2016196451 A1 [0007]
• EP 2619585 B1 [0008]

Claims (3)

A method for detecting populations of myeloid-derived suppressive cells (MDSC), characterized in that the proteins encoded by the transcript variants are used to detect early-stage MDSC or monocytic MDSC or granulocytic MDSC populations. procedure after claim 1 , characterized in that if the MDSC population is granulocytic MDSC, (i) the protein represented by the amino acid sequence SEQUENCE ID NO 18 encoded by the CDID tv1 transcript variant represented by the nucleotide sequence SEQUENCE ID NO 17 replicates by the primer pair represented by SEQUENCE ID NO 1 and 2, (ii) the protein represented by the amino acid sequence SEQUENCE ID NO 20 encoded by the CD33-TV2 transcript variant represented by the nucleotide sequence SEQUENCE ID NO 19 replicates by the primer pair represented by SEQUENCE ID NO 3 and 4, (iii) the protein represented by the amino acid sequence SEQUENCE ID NO 22 encoded by the CD16 tv1 transcript variant represented by the nucleotide sequence SEQUENCE ID NO 21 replicated by the pair of primers represented by SEQUENCE ID NOS 5 and 6 is used, (iv) the protein represented by the amino acid sequence SEQUENCE ID NO 22 encoded by the CD16 tv2 transcript variant represented by the nucleotide sequence SEQUENCE ID NO 23 replicated by the primer pair represented by SEQUENCE ID NOS 7 and 8, (v) the protein represented by the amino acid sequence SEQUENCE ID NO 25 encoded by the CD16 tv3 - Transcript variant represented by the nucleotide sequence SEQUENCE ID NO 24 replicated by the primer pair represented by SEQUENCE ID NO 9 and 10 is used. procedure after claim 1 , characterized in that if the MDSC population is early-stage MDSC or monocytic MDSC, (i) the protein represented by the amino acid sequence SEQUENCE ID NO 27 encoded by the CDId tv2 transcript variant represented by the nucleotide sequence SEQUENCE ID NO 26 replicated by the primer pair represented by the nucleotide sequence SEQUENCE ID NOS 11 and 12, (ii) the protein represented by the amino acid sequence SEQUENCE ID NO 29 encoded by the CD44 tv1 transcript variant represented by the nucleotide sequence SEQUENCE ID NO 28 replicated by the primer pair represented by the nucleotide sequences SEQUENCE ID NOS 13 and 14, (iii) the protein represented by the amino acid sequence SEQUENCE ID NO 31 encoded by the CD44 tv2 variant represented by the nucleotide sequence SEQUENCE ID NO 30 replicated by the primer pair represented by the nucleotide sequences SEQUENCE ID NO 15 and 16 is used.

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