# US 11,029,317 B2 · Compositions and methods for detecting anti-endothelial cell antibodies in allograft rejection

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,029,317 B2
- Document ID: US11029317B2
- Status: Granted
- Kind: B2
- Application number: 15/512,223
- Filed: 2015-09-18
- Published: 2017-09-28
- Granted: 2021-06-08
- Earliest claimed: 2014-09-18
- Assignees: The Johns Hopkins University; Immucor Gti Diagnostics, Inc.
- Applicants: Immucor Gti Diagnostics, Inc.; The Johns Hopkins University
- Inventors: Minnie M. Sarwal; Tara Sigdel; Annette M. Jackson
- Web page: https://patentgrep.com/patent/US11029317B2

## CPC

- G01N 33/6854: {Immunoglobulins}
- G01N 33/50: Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing (measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q)
- G01N 33/564: for pre-existing immune complex or autoimmune disease {, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9}
- G01N 33/6893: {related to diseases not provided for elsewhere}
- G01N 2800/245: Transplantation related diseases, e.g. graft versus host disease
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Provided herein are methods, compositions, and kits for diagnosing allograft rejection of organ transplants by identifying the presence of anti-endothelial cell antibodies. Such methods and compositions are independent of external confounders such as recipient age, transplant center, assay, cause of end-stage disease, co-morbidities, immunosuppression usage, and the like.

## Claims

### Claim 1

A method of treating an individual who has received an organ transplant, comprising:
(1) increasing the administration of a therapeutically effective amount of one or more immunosuppressive therapeutics if the individual's antibody expression profile indicates that the individual has an increased risk of developing allograft rejection (AR);
(2) maintaining the administration of a therapeutically effective amount of one or more immunosuppressive therapeutics if the individual's antibody expression profile indicates that the individual does not have an increased or decreased risk of developing AR; or
(3) decreasing the administration of a therapeutically effective amount of one or more immunosuppressive therapeutics if the individual's antibody expression profile indicates that the individual has a decreased risk of developing AR;
wherein the antibody expression profile comprises the level of at least four antibodies from a biological sample from the individual that form an affinity interaction with at least a portion or peptide fragment of at least four proteins selected from Table 2; and
wherein a similarity of the level of the at least four antibodies to the positive reference standard indicates increased risk of developing AR and a similarity of the measured level of the at least four antibodies to the negative reference standard indicates decreased risk of developing AR.

### Claim 2 (depends on 1)

The method of claim 1, wherein the biological sample is selected from the group consisting of whole blood, blood, serum, plasma, urine, mucus, saliva, cerebrospinal fluid, tissues, biopsies and combinations thereof.

### Claim 3 (depends on 2)

The method of claim 2, wherein the at least four antibodies are anti-endothelial cell antibodies.

### Claim 4 (depends on 3)

The method of claim 3, wherein the anti-endothelial cell antibodies form an affinity interaction with endoglin, Fms-like tyrosine kinase-3 (FLT3) ligand, EGF-like repeats and discoidin I-like domains 3 (EDIL3), and intercellular adhesion molecule 4 (ICAM4).

### Claim 5 (depends on 1)

The method of claim 1, further comprising isolating the at least four antibodies by incubating the biological sample with an antibody capture element.

### Claim 6 (depends on 5)

The method of claim 5, wherein the antibody capture element comprises an antibody specific antigen, a capture bead, and capture solid support.

### Claim 7 (depends on 6)

The method of claim 6, wherein the antibody capture element comprises an antibody specific antigen.

### Claim 8 (depends on 7)

The method of claim 7, wherein the antibody specific antigen is selected from the group consisting of: a full length protein, protein fragment, peptide, polypeptide, recombinant peptide, recombinant protein, and a polypeptide fragment.

### Claim 9 (depends on 8)

The method of claim 8, wherein the antibody specific antigen comprises at least a portion or peptide fragment of at least four proteins selected from Table 2.

### Claim 10 (depends on 9)

The method of claim 9, wherein the at least four proteins are endoglin, Fms-like tyrosine kinase-3 (FLT3) ligand, EGF-like repeats and discoidin I-like domains 3 (EDIL3) and intercellular adhesion molecule 4 (ICAM4).

### Claim 11 (depends on 7)

The method of claim 7, wherein the antibody specific antigen is labelled with an isolation tag selected from the group consisting of biotin, a GST tag, an amide group, and a streptavidin tag.

### Claim 12 (depends on 11)

The method of claim 11, wherein the isolation tag is a biotin.

### Claim 13 (depends on 5)

The method of claim 5, wherein the antibody capture element is isolated with a secondary capture element.

### Claim 14 (depends on 13)

The method of claim 13, wherein the secondary capture element is immobilized on a solid support comprising a bead or a plate.

### Claim 15 (depends on 13)

The method of claim 13, wherein the secondary capture element comprises streptavidin.

### Claim 16 (depends on 1)

The method of claim 1, further comprising measuring the level of the at least four antibodies by a detection element.

### Claim 17 (depends on 16)

The method of claim 16, wherein the detection element comprises a labelled secondary antibody.

### Claim 18 (depends on 1)

The method of claim 1, wherein the antibody expression profile further comprises the level of a donor specific HLA antibody.

### Claim 19 (depends on 18)

The method of claim 18, wherein the level of the donor specific HLA antibody is detected by a cell based assay or a solid-phase immunoassay.

### Claim 20 (depends on 19)

The method of claim 19, wherein the solid-phase immunoassay comprises an ELISA, and wherein the cell based assay comprises a cross-match assay.

### Claim 21 (depends on 1)

The method of claim 1, further comprising administering pulse solumedrol and increased baseline target levels of a therapeutically effective amount of one or more immunosuppressive therapeutics if the individual has an increased risk of developing AR.

### Claim 22 (depends on 21)

The method of claim 21, wherein the pulse solumedrol is administered at cumulative doses of about 30 to about 50 mg/kg in 3-6 doses.

### Claim 23 (depends on 1)

The method of claim 1, wherein the one or more immunosuppressive therapeutics are selected from: steroids, antibodies, calcineurin inhibitors, antiproliferatives, mammalian target of rapamycin (mTOR) inhibitors, and therapeutic plasmapheresis.

### Claim 24 (depends on 23)

The method of claim 23, wherein the steroids comprise prednisone, prednisolone, and methyl-prednisolone.

### Claim 25 (depends on 23)

The method of claim 23, wherein the antibodies comprise muromonab-CD3, antithymocyte immune globulin, daclizumab, basiliximab, Rituximab, cytomegalovirus-immune globulin, and immune globulin.

### Claim 26 (depends on 23)

The method of claim 23, wherein the calcineurin inhibitors comprise cyclosporine, and tacrolimus.

### Claim 27 (depends on 23)

The method of claim 23, wherein the antiproliferatives comprise mycophenolate mofetil, and azathioprine.

### Claim 28 (depends on 23)

The method of claim 23, wherein the mTOR inhibitors comprise rapamycin, and everolimus.

### Claim 29 (depends on 18)

The method of claim 18, wherein the presence of the donor specific HLA antibody indicates an increased risk for developing an AR.