Production of Hybrid Peptides by Antigen Presenting Cells

Abstract

This disclosure describes production of hybrid peptides, including hybrid insulin peptides (HIPs), by antigen presenting cells (APCs).

Claims

1. A method of preparing hybrid peptides in antigen presenting cells comprising: a. encapsulating at least two synthetic peptides or a protein within nanocarriers, b. incubating the nanocarriers with an antigen presenting cell (APC), wherein incubation results in the fusion of two or more peptides or portions of one protein to produce a hybrid peptide.

2. The method of claim 1, wherein all or part of the hybrid peptide is presented on the APC surface.

3. The method of claim 1, wherein the hybrid peptide comprises two or more fragments of the same peptide or protein.

4. The method of claim 1, wherein the hybrid peptide comprises two or more fragments of different peptides or proteins.

5. The method of claim 1, wherein at least one peptide or a protein is insulin, a fragment of insulin, a precursor of insulin, or a fragment of a precursor of insulin, and wherein a hybrid insulin peptide (HIP) is formed.

6. The method of claim 1, wherein the nanocarrier is 200-500 nm in diameter.

7. The method of claim 1, wherein the antigen presenting cell is a monocyte, dendritic cell, macrophage, B cell, or T cell.

8. (canceled)

9. (canceled)

10. A method of treating a patient with an immune disorder comprising administering one or more proteosome inhibitor, wherein the administering leads to inhibiting hybrid peptide production by APCs.

11. The method of claim 10, wherein inhibiting hybrid peptide production leads to a decrease in T-cell activation.

12. A method of evaluating an immune response in a sample comprising an APC comprising: a. encapsulating at least two synthetic peptides or a protein within nanocarriers; b. incubating the nanocarriers with the sample, wherein incubation results in the fusion of two or more peptides or portions of a protein to produce an APC presenting one or more hybrid peptides on the APC surface; and c. measuring an immune response to one or more hybrid peptides on the APC surface with a reporter assay.

13. The method of claim 12, wherein the reporter assay measures T-cell activation.

14. The method of claim 13, wherein the reporter assay uses hybridomas expressing CD4.

15. The method of claim 12, wherein the reporter assay measures interferon gamma levels.

16. The method of claim 12, wherein the reporter assay measures interleukin-10 levels.

17. The method of claim 12, wherein the hybrid peptide is a hybrid insulin peptide comprising insulin, a fragment of insulin, a precursor of insulin, or a fragment of a precursor of insulin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 shows hybrid peptide production by antigen presenting cells (APCs). Synthetic peptides or proteins are encapsulated within nanocarriers, and these nanocarriers are then fed to APCs. The APCs can then form hybrid peptides from the peptides or proteins within the nanocarriers. The nanocarrier may allow for preparation of hybrid peptides by APCs by holding different peptides or proteins in close proximity. Hybrid peptides may be generated from two copies of the same protein (intramolecular spliced peptides) or from two different proteins (intermolecular spliced peptides). The leftward slashes and rightward slashes in the intramolecular spliced peptides indicate that amino acid sequence from two separate copies of the same peptides (for example, two copies of insulin) were fused into the hybrid peptide. An intermolecular spliced peptide can be generated from copies of two different peptides (for example, insulin and a different peptide). These hybrid peptides can exist in an APC together with linear proteins (i.e., non-hybrid peptides, which are not spliced).

[0025] FIG. 2 shows intermolecular BDC2.5 HIP formation in murine APCs.

[0026] FIG. 3 shows that APCs stimulate HIP11 reactive T cells. HIP11 is a C-peptide/C-peptide HIP. Human PBMCs were stained with anti-CD3 antibody and magnetically sorted to deplete T cells. Pseudogranules were added to flowthrough and co-cultured with T-cell hybridoma cells (as described in Mann et al., Front Immunol 9 (11): 633 (2020)). The CD4 hybridoma cells have a ZsGreen reporter that can be activated (linked to NFAT), and the cells are HLA-DQ8 specific and reactive for HIP11. Flow cytometry analysis of TCR reporter was performed to measure T-cell stimulation.

[0027] FIGS. 4A-4F shows results with a variety of different APCs, including B cells (A), APCs without B cells (B), T cells (C), monocytes (D), dendritic cells (E), and macrophages (F).

[0028] FIG. 5 shows inhibition of HIP11 production (as measured by interferon gamma levels) by various proteosome inhibitors.

[0029] FIGS. 6A and 6B show inhibition of BDC2.5 recognized peptide production (as measured by interferon gamma levels) by various proteosome inhibitors at higher (A) and lower (B) concentrations.

DESCRIPTION OF THE SEQUENCES

[0030] Table 1 provides a listing of certain sequences referenced herein. Hyphenation indicates hybrid peptide junction. Bold sequences represent C-peptide components of HIPs. Italicized sequences represent N-terminal sequences of natural cleavage products.

TABLE-US-00001 TABLE1 DescriptionoftheSequences SEQ ID Description Sequences NO BDC2.5HIP: LQTLAL-WSRMD 1 C-Peptide/ ChrA-WE14 ChromograninA WSRMDQLAKELTAE 2 WE14peptide C-Peptide MALWMRFLPLLALLFLWESHPT 3 peptide QAFVKQHLCGSHLVEALYLVCG (mouse) ERGFFYTPMSRREVEDPQVAQL ELGGGPGAGDLQTLALEVAQQK RGIVDQCCTSICSLYQLENYCN HumanHIP11 SLQPLAL-EAEDLQV 4 C-peptide EAEDLQVGQVELGGGPGAGSLQ 5 peptide PLALEGSLQ (human) C-terminal SLQPLAL 6 sequencefrom HIP11 N-terminal EAEDLQV 7 sequencefrom HIP11 HIP1 GQVELGG-WSKMDQLA 8 HIP2 GQVELGG-LEGQEEEE 9 HIP3 GQVELGGG-EAEDLQV 10 HIP4 GQVELGGG-GIVEQCC 11 HIP5 GQVELGGG-TPIESHQ 12 HIP6 GQVELGGG-NAVEVLK 13 HIP7 GQVELGGG-FLGEGHH 14 HIP8 GQVELGGG-SSPETLI 15 HIP9 SLQPLAL-WSKMDQL 16 HIP10 SLQPLAL-LEGQEEE 17 HIP12 SLQPLAL-GIVEQCC 18 HIP13 SLQPLAL-TPIESHQ 19 HIP14 SLQPLAL-NAVEVLK 20 HIP15 SLQPLAL-FLGEGHH 21 HIP16 SLQPLAL-SSPETLI 22 insB: SHLVEALYLVCGER 23 9-23 insB: SHLVEALYLVCGEE 24 9-23R22E ins.sub.64-79 GQVELGGGPGAGSLQP 25 ins.sub.75-90 GSLQPLALEGSLQKRG 26 ChgA.sub.334-349 KEWEDSKRWSKMDQLA 27 ChgA.sub.350-365 KELTAEKRLEGQEEEE 28 Ins.sub.49-64 FYTPKTRREAEDLQVG 29 Ins.sub.82-97 LEGSLQKRGIVEQCCT 30 IAPP.sub.15-30 VALNHLKATPIESHQV 31 IAPP.sub.66-81 GSNTYGKRNAVEVLKR 32 ScG1.sub.432-447 SDTREEKRFLGEGHHR 33 NP-Y.sub.60-75 TRQRYGKRSSPETLIS 34 Insulinspecific EAEDLQVGQVELGGGPGAGS 35 peptide Insulinspecific GSLQPLALEGSLQ 36 peptide Insulinspecific GPGAGSLQPLALEGSLQ 37 peptide Insulinspecific EAEDLQVGQVELGGGPGAGS 38 peptide Insulinspecific EAEDLQVGQVELGGGPGAGS 39 peptide LQ Insulinspecific GGGPGAGSLQPLALEGSLQ 40 peptide

[0031] A number of human HIP sequences have been presented in Baker et al. Diabetes 68 (9): 1830-1840 (2019), as shown in the Table 2 below.

TABLE-US-00002 TABLE2 AdditionalrepresentativehumanHIPS SEQ SEQ ID ID HIPS NO PeptideSequence* B-Chain NO PeptideSequence HIP1 8 GQVELGG-WSKMDQLA insB: 23 SHLVEALYLVCGER 9-23 HIP2 9 GQVELGG-LEGQEEEE insB: 24 SHLVEALYLVCGEE 9-23R22E HIP3 10 GQVELGGG-EAEDLQV HIP4 11 GQVELGGG-GIVEQCC Left PeptideSequence** control peptides HIP5 12 GQVELGGG-TPIESHQ ins.sub.64-79 25 GQVELGGGPGAGSLQP HIP6 13 GQVELGGG-NAVEVLK ins.sub.75-90 26 GSLQPLALEGSLQKRG HIP7 14 GQVELGGG-FLGEGHH HIP8 15 GQVELGGG-SSPETLI Right PeptideSequence control peptides HIP9 16 SLQPLAL-WSKMDQL ChgA.sub.334-349 27 KEWEDSKRWSKMDQLA HIP10 17 SLQPLAL-LEGQEEE ChgA.sub.350-365 28 KELTAEKRLEGQEEEE HIP11 5 SLQPLAL-EAEDLQV Ins.sub.49-64 29 FYTPKTRREAEDLQVG HIP12 18 SLQPLAL-GIVEQCC Ins.sub.82-97 30 LEGSLQKRGIVEQCCT HIP13 19 SLQPLAL-TPIESHQ IAPP.sub.15-30 31 VALNHLKATPIESHQV HIP14 20 SLQPLAL-NAVEVLK IAPP.sub.66-81 32 GSNTYGKRNAVEVLKR HIP15 21 SLQPLAL-FLGEGHH SCG1.sub.432-447 33 SDTREEKRFLGEGHHR HIP16 22 SLQPLAL-SSPETLI NP-Y.sub.60-75 34 TRQRYGKRSSPETLIS *Hyphenation indicates hybrid peptide junction. **Bold sequences represent C-peptide components of HIPs. ***Italicized sequences represent N-terminal sequences of natural cleavage products.

[0032] Mass spectrometry data are shown in Table 3.

TABLE-US-00003 TABLE3 Massspectrometrydata SampleID Reference PeptideSequences Positive HIP11 SLQPLAL-EAEDLQV Control (SEQIDNO:4) (HIP11 peptide) HIP11 HIP11 SLQPLAL-EAEDLQV Nanocarrier (SEQIDNO:4) C-Peptide HIP11 SLQPLAL-EAEDLQV Nanocarrier (SEQIDNO:4) Insulin EAEDLQVGQVELGGGPGAGS specific (SEQIDNO:35) Peptides GSLQPLALEGSLQ (SEQIDNO:36) GPGAGSLQPLALEGSLQ (SEQIDNO:37) PPI HIP11 SLQPLAL-EAEDLQV Nanocarrier (SEQIDNO:4) Insulin EAEDLQVGQVELGGGPGAGS specific (SEQIDNO:38) Peptides EAEDLQVGQVELGGGPGAGSLQ (SEQIDNO:39) GGGPGAGSLQPLALEGSLQ (SEQIDNO:40)

EQUIVALENTS

[0033] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0034] As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., +/5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.