# US 2019/0170742 A1 · Compositions, kits and methods for in vitro antigen presentation, assessing vaccine efficacy, and assessing immunotoxicity of biologics and drugs

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- Number: US 2019/0170742 A1
- Document ID: US20190170742A1
- Status: Published application
- Kind: A1
- Application number: 16/228,050
- Filed: 2018-12-20
- Published: 2019-06-06
- Earliest claimed: 2009-05-19
- Assignees: University of Miami
- Applicants: University of Miami
- Inventors: Pirouz Mohammad DAFTARIAN; Paolo SERAFINI; Vance Paul LEMMON; Angel KAIFER; Bonnie Beth BLOMBERG; Raquibul CHOWDHURY; Norma KENYON
- Web page: https://patentgrep.com/patent/US20190170742A1

## CPC

- A61K 48/0091: {Purification or manufacturing processes for gene therapy compositions}
- C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- G01N 33/505: {involving T-cells}
- G01N 33/54346: {Nanoparticles}
- G01N 33/56977: {HLA or MHC typing}
- G01N 33/6866: {Interferon}
- C12N 2810/85: mammalian
- G01N 2333/57: IFN-gamma
- G01N 2333/70539: MHC-molecules, e.g. HLA-molecules
- G01N 2500/04: Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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

Nanoparticle-based compositions, assays, kits, methods and platforms for delivering an antigen (peptides, proteins) or a nucleic acid encoding an antigen to professional APCs (PAPCs) result in the generation of autologous APCs that present a natural peptide repertoire of the antigen for use in assessing the efficacy of a vaccine (e.g., a cytotoxic T lymphocyte (CTL) response to a particular antigen) or other therapy or intervention (cell-based therapy, adjuvant therapy, etc.). The compositions, kits, assays and methods also can be used for delivering a drug or biologic or portion thereof to APCs for assessing the immunogenicity of drugs and biologics. The composition, kits, assays and methods involve the combined use of MHC targeting, universal DR binding peptides (e.g., PADRE, HA) with charged (e.g., positively-charged) highly branched polymeric dendrimers (e.g., PAMAM and other dendrimers) as vehicles for the targeted delivery of nucleic acids, peptides, biologics, drugs, or polypeptides to APCs, giving rise to a new nanoparticle-based method for assessing the immune response (CTL response) to a vaccination or other therapy or intervention, or for assessing the immunogenicity of a biologic or drug. Targeted delivery of nucleic acids, peptides, biologics, drugs, or polypeptides to APCs for effective expression and processing generates more physiologically relevant target antigens for evaluation of cell-mediated immune responses to vaccination, for example, and provides a low-cost approach for rapid generation of reagents and development of assay systems for more accurate profiling of immuno-logical responses to infection, immunization, and other therapies or interventions. Immunoevaluation kits using targeted nanoparticle-based antigen delivery are described herein.

## Claims

### Claim 1

A method of detecting an immune response against a vaccine, the method comprising the steps of:
a) preparing or providing a composition comprising a plurality of charged highly branched polymeric dendrimers each having conjugated thereto at least one universal DR binding peptide and at least one peptide or polypeptide antigen or a nucleic acid encoding the at least one antigen, wherein the at least one universal DR binding peptide and the nucleic acid or at least one peptide or polyeptide antigen are conjugated to the exterior surface of the plurality of charged highly branched polymeric dendrimers such that the at least one universal DR binding peptide specifically binds to professional antigen presenting cells (PAPCs),
wherein the vaccine comprises the antigen;
b) obtaining a first sample comprising PAPCs from the subject prior to vaccination of the subject;
c) dividing the first sample into a first portion of the first sample and a second portion of the first sample;
d) contacting the first portion of the first sample with the composition under incubation conditions such that the plurality of charged highly branched polymeric dendrimers are taken up by the PAPCs and such that the antigen is processed by the PAPCs and presented by the PAPCs in combination with MHC class II;
e) washing the first portion of the first sample and the second portion of the first sample;
f) combining the first portion of the first sample and the second portion of the first sample at two or more ratios, resulting in a first plurality of mixtures;
g) incubating the first plurality of mixtures for one or more hours;
h) examining the plurality of mixtures for the presence of at least one molecule or marker that is indicative of an immune response to the vaccine and determining the level of the at least one molecule or marker;
i) obtaining a second sample comprising PAPCs from the subject after the subject has been vaccinated;
j) dividing the second sample into a first portion of the second sample and a second portion of the second sample;
k) contacting the first portion of the second sample with the composition under incubation conditions such that the plurality of charged highly branched polymeric dendrimers are taken up by the PAPCs in the first portion of the second sample and such that the antigen is processed by the PAPCs in the first portion of the second sample and presented by the PAPCs in the first portion of the second sample in combination with MHC class II;
l) washing the first portion of the second sample and the second portion of the second sample;
m) combining the first portion of the second sample and the second portion of the second sample at two or more ratios, resulting in a second plurality of mixtures;
incubating the first plurality of mixtures for one or more hours;
n) examining the second plurality of mixtures for the presence of the at least one molecule or marker and determining the level of the at least one molecule or marker;
o) comparing the level of the at least one molecule or marker in the first plurality of mixtures with the level of the at least one molecule or marker in the second plurality of mixtures; and
p) correlating a higher level of the at least one molecular or marker in the second plurality of mixtures than in the first plurality of mixtures with an immune response to the vaccine.

### Claim 2 (depends on 1)

The method of claim 1, wherein step d) of contacting the first portion of the first sample with the composition comprises adding mitomycin C for about 30 minutes.

### Claim 3 (depends on 1)

The method of claim 1, wherein the at least one molecule or marker that is indicative of an immune response to the vaccine comprises a cytokine.

### Claim 4 (depends on 3)

The method of claim 3, wherein the cytokine is IFN-γ.

### Claim 5 (depends on 1)

The method of claim 1, wherein the at least one molecule or marker that is indicative of an immune response to the vaccine comprises T cell activation or proliferation.

### Claim 6 (depends on 1)

The method of claim 1, wherein examining the first and second pluralities of mixtures for the presence of the at least one molecule or marker and determining the level of the at least one molecule or marker is performed using a cytokine assay or CTL assay.

### Claim 7 (depends on 1)

The method of claim 1, wherein the at least one universal DR binding peptide is a PADRE epitope.

### Claim 8 (depends on 7)

The method of claim 7, wherein the at least one universal DR binding peptide is two PADRE epitopes each having the amino acid sequence of SEQ ID NO: 1.

### Claim 9 (depends on 1)

The method of claim 1, wherein the at least one charged highly branched polymeric dendrimer is a PAMAM dendrimer.

### Claim 10

A method of detecting an immune response against a vaccine or other therapeutic intervention, the method comprising the steps of:
a) preparing or providing a first composition comprising a plurality of charged highly branched polymeric dendrimers each having conjugated thereto at least one universal DR binding peptide and at least one peptide or polypeptide antigen or a nucleic acid encoding the at least one antigen, wherein the at least one universal DR binding peptide and the nucleic acid or at least one peptide or polyeptide antigen are conjugated to the exterior surface of the plurality of charged highly branched polymeric dendrimers such that the at least one universal DR binding peptide specifically binds to professional antigen presenting cells (PAPCs),
wherein the vaccine or other therapeutic intervention comprises the antigen;
b) obtaining a first sample comprising PAPCs from the subject after the subject has been vaccinated;
c) dividing the first sample into at least a first portion and a second portion;
d) contacting the at least first portion with the first composition under incubation conditions such that the plurality of charged highly branched polymeric dendrimers are taken up by the PAPCs and such that the antigen is processed by the PAPCs and presented by the PAPCs in combination with MHC class II;
e) contacting the second portion with a second composition comprising a plurality of charged highly branched polymeric dendrimers each having conjugated thereto at least one universal DR binding peptide and at least one negative control peptide or polypeptide or a nucleic acid encoding the at least one negative control peptide or polypeptide, wherein the at least one universal DR binding peptide and the at least one control peptide or polyeptide antigen or nucleic acid encoding the at least one negative control peptide or polypeptide are conjugated to the exterior surface of the plurality of charged highly branched polymeric dendrimers such that the at least one universal DR binding peptide specifically binds to PAPCs;
f) examining the at least first portion contacted with the first composition for the presence of at least one molecule or marker that is indicative of an immune response to the vaccine or other therapeutic intervention, and determining the level of the at least one molecule or marker;
g) examining the at least second portion contacted with the second composition for the presence of the at least one molecule or marker, and determining the level of the at least one molecule or marker;
h) comparing the level of the at least one molecule or marker in the at least first portion contacted with the first composition with the level of the at least one molecule or marker in the at least second portion contacted with the second composition; and
i) correlating a higher level of the at least one molecular or marker in the at least first portion contacted with the first composition than in the at least second portion contacted with the second composition with an immune response to the vaccine.

### Claim 11 (depends on 10)

The method of claim 10, wherein the at least one control peptide or polyeptide antigen is albumin or luciferase.

### Claim 12 (depends on 10)

The method of claim 10, wherein the at least one molecule or marker that is indicative of an immune response to the vaccine or other therapeutic intervention comprises a cytokine.

### Claim 13 (depends on 12)

The method of claim 12, wherein the cytokine is IFN-γ.

### Claim 14 (depends on 10)

The method of claim 10, wherein the at least one molecule or marker that is indicative of an immune response to the vaccine or other therapeutic intervention comprises T cell activation or proliferation.

### Claim 15 (depends on 10)

The method of claim 10, wherein examining the at least first portion contacted with the first composition and the at least second portion contacted with the second composition for the presence of the at least one molecule or marker, and determining the level of the at least one molecule or marker in the at least first portion contacted with the first composition and the at least second portion contacted with the second composition is performed using a cytokine assay or CTL assay.

### Claim at position 16

16-26. (canceled)