Methods of monitoring immune responses

Patent metadata

Field	Value
Publication number	US-2016258951-A1
Application number	US-201415032820-A
Country	US
Kind code	A1
Filing date	Oct 28, 2014
Priority date	Oct 28, 2013
Publication date	Sep 8, 2016
Grant date	—

Abstract

The human HLA-A2:1g dimer molecule is a recombinant protein comprising a mouse IgG antibody fused with two human MHC Class I HLA-A2 molecules. Any peptide (usually 8-10 amino acids in length) can be loaded into the peptide-binding groove of the two HLA molecules, for example, by incubating a mixture of the dimer and peptide solution overnight. The resulting N peptide-specific HLA-A2:1g dimer mixture can then be added to PBMCs from peripheral blood samples in order to detect CD8 T lymphocytes which express T cell receptors that are capable of specifically interacting with and binding to the peptide HLA-A2:1g dimer molecules. The presence of such specific binding activity and interactions can then be detected by additional staining with fluorescence-conjugated antibodies.

First claim

1 . A method of monitoring an immune response to a test peptide, the method comprising:
(a) incubating peripheral blood mononuclear cells (PBMCs) obtained from a patient with a fusion protein comprising a mouse immunoglobulin fused to two human MEW class I HLA molecules, wherein the test peptide has been loaded into the peptide binding groove of the two human MEW class I HLA molecules; (b) quantitating the cytotoxic CD8 T cells specific for the test peptide; and (c) normalizing the result obtained in step (b) by subtracting a background value obtained by quantitating the cytotoxic CD8 T cells specific for a negative control peptide, wherein the negative control peptide is the E37 peptide having the amino acid sequence RIAWARTEL (SEQ ID NO:4).

2 . A method of monitoring an immune response to a test peptide, the method comprising:
(a) incubating peripheral blood mononuclear cells (PBMCs) obtained from a patient with a fusion protein comprising a mouse immunoglobulin fused to two human MEW class I HLA molecules, wherein the test peptide has been loaded into the peptide binding groove of the two human MEW class I HLA molecules; (b) incubating PBMCs obtained from the patient with the fusion protein, wherein a negative control peptide has been loaded into the peptide binding groove of the two human MHC class I HLA molecules, and wherein the negative control peptide is the E37 peptide having the amino acid sequence RIAWARTEL (SEQ ID NO:4); (c) quantitating the cytotoxic CD8 T cells specific for the test peptide; (d) quantitating the cytotoxic CD8 T cells specific for the negative control peptide to obtain a background value; (e) normalizing the result obtained in step (c) by subtracting the background value obtained in step (d).

3 . The method of claim 1 , wherein the background value is about 0.3%.

4 . The method of claim 1 , wherein the test peptide is a class I restricted Her2/neu derived peptide.

5 . The method of claim 4 , wherein the test peptide is E75, GP2, GP2′, or Her 577 .

6 . The method of claim 4 , wherein the test peptide is E75.

7 . The method of claim 1 , wherein the fusion protein comprises a mouse IgG fused to two human MHC class I HLA-A2 molecules.

8 . The method of claim 1 , wherein quantitating the cytotoxic T cells specific for the test peptide is achieved using immunofluorescent staining or fluorescent activated cell sorting (FACS).

9 . The method of claim 1 , further comprising a step before (a) of loading the test peptide into the peptide binding groove of the two human MHC class I HLA molecules of the fusion protein.

10 . The method of claim 9 , wherein the loading step comprises incubating the test peptide with the fusion protein overnight at 37° C.

11 . The method of claim 1 , wherein quantitating the cytotoxic T cells specific for the test peptide is expressed as a dimer index.

12 . The method of claim 1 , wherein the method is used to assess the clinical response of a HER2/neu derived peptide vaccine in a breast cancer patient, wherein the breast cancer patient is disease free following standard therapy.

13 . The method of claim 12 , wherein the HER2/neu derived peptide vaccine comprises E75 and GM-CSF or GP2 and GM-CSF.

14 . The method of claim 13 , wherein the cytotoxic CD8 T cells specific for the test peptide are quantified at baseline, after a primary vaccination, and at 6 months post primary vaccination.

15 . The method of claim 1 , wherein a more robust cytotoxic CD8 T cell response to the test peptide at 6 months post primary vaccination indicates that the patient is less likely to experience breast cancer recurrence or more likely to have a longer disease free survival.

16 . The method of claim 1 , wherein a low cytotoxic CD8 T cell response to the test peptide at baseline indicates that the patient is less likely to experience breast cancer recurrence or more likely to have a longer disease free survival.

17 . The method of claim 16 , wherein the patient has low to intermediate HER2 expression (IHC 1 or 2+ or FISH<2.2).

18 . A kit comprising a fusion protein comprising a mouse immunoglobulin fused to two human MHC class I HLA molecules and a negative control peptide, wherein the negative control peptide is an E37 peptide having the amino acid sequence RIAWARTEL (SEQ ID NO:4).

19 . The kit of claim 18 , further comprising a positive control peptide.

20 . The kit of claim 18 , further comprising a test peptide, wherein the test peptide is a class I restricted Her2/neu derived peptide.

21 . The kit of claim 20 , wherein the test peptide is E75, GP2, GP2′, or Her 577 .

22 . The kit of claim 18 , wherein the fusion protein comprises a mouse IgG fused to two human MHC class I HLA-A2 molecules.

23 . The kit of claim 18 , further comprising a labeled antibody that binds to the immunoglobulin of the fusion protein.

24 . The kit of claim 18 , wherein the E37 peptide is loaded into the peptide binding groove of the two human MHC class I HLA molecules of the fusion protein.

25 . The kit of claim 19 , wherein the test peptide is loaded into the peptide binding groove of the two human MHC class I HLA molecules of the fusion protein.

26 . The kit of claim 18 , further comprising a buffer for diluting one or more of the negative control peptide, positive control peptide, or test peptide.

Assignees

• Henry M Jackson Found Advancement Military Medicine Inc

Inventors

• Ponniah Sathibalan
• Peoples George E
• Storrer Catherine

Classifications

• G01N33/57515

  of the breast · CPC title

• G01N33/56966 Primary

  Animal cells · CPC title

• G01N2800/52

  Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis · CPC title

• G01N33/505 Primary

  involving T-cells · CPC title

• G01N33/6878

  in epitope analysis · CPC title