CAMEL DERIVED POLYPEPTIDES BINDING HUMAN CD3, IDENTIFIED USING A NOVEL ONE-STEP METHOD

Abstract

The present invention relates to variable domains of camelid heavy-chain antibodies (VHH domains) directed against the human CD3 component of the T cell receptor and specifically to methods of use of said polypeptides for delivering prophylactic, therapeutic or diagnostic compounds to human T lymphocytes. It further describes a novel one-step method for the identification of phage displayed camel monoclonal antibodies.

Claims

1. An isolated variable domain of a camelid heavy-chain antibody (VHH domain) directed against CD3epslion (CD3) component of the T cell receptor, wherein said VHH domain comprises an amino acid sequence selected from the group consisting amino acid sequence SEQ ID NO: 1 to SEQ ID NO: 32.

2. A VHH domain according to claim 1, comprising a homo/hetero dimers of SEQ ID NO: 1 to SEQ ID NO: 32

3. A VHH domain according to claim 1, wherein it is obtainable by the method comprising the steps of: (a) immunizing a camelid with recombinant CD3, (b) isolating peripheral lymphocytes of the immunized camelid, obtaining the total RNA and synthesizing the corresponding cDNAs, (c) constructing a library of cDNA fragments encoding VHH domains, (d) transcribing the VHH domain-encoding cDNAs obtained in step (c) to mRNA using PCR, converting the mRNA to phage display format, and selecting the VHH domain by phage display, and (e) expressing the VHH domain in a vector.

4. An isolated polypeptide, comprising a VHH domain according to claim 1.

5. The polypeptide according to claim 4, wherein it contains at its C-terminus the amino acid sequence LEHHHHHH (SEQ ID NO: 42).

6. An isolated antibody or fragment thereof, comprising a VHH domain according to claim 1.

7. A therapeutic or diagnostic agent, comprising a VHH domain according to claim 1, linked, directly or indirectly, covalently or non-covalently to a substance of interest.

8. A therapeutic or diagnostic agent according to claim 7, wherein said substance of interest is a therapeutic or diagnostic compound selected from the group consisting of a peptide, an enzyme, a virus, a fluorophore, a heavy metal, a chemical entity and a radioisotope.

9. A therapeutic or diagnostic agent according to claim 8, wherein said substance of interest is a liposome or a polymeric entity comprising a therapeutic or a diagnostic compound selected from the group consisting of a peptide, an enzyme, a virus, a fluorophore, a heavy metal, a chemical entity and a radioisotope.

10. A therapeutic or diagnostic agent according to claim 8, wherein said therapeutic compound is selected from the group consisting of an anticancer compound, an immunosuppressant compound and an anti-inflammatory compound.

11. A therapeutic or diagnostic agent, comprising a polypeptide according to claim 4, linked, directly or indirectly, covalently or non-covalently to a substance of interest.

12. A therapeutic or diagnostic agent, comprising an antibody according to claim 6, linked, directly or indirectly, covalently or non-covalently to a substance of interest.

13. A kit comprising a VHH domain according to claim 1.

14. A kit comprising a polypeptide according to claim 4.

15. A kit comprising an antibody according to claim 6.

16. A kit comprising a diagnostic agent according to claim 7.

17. A pharmaceutical composition, comprising a therapeutic agent according to claim 7 and a pharmaceutically acceptable carrier.

18. A method of indicating the presence of a disorder mediated by infiltrating T lymphocytes comprising the steps of: a) contacting in vitro or ex vivo an appropriate biological sample with a VHH domain according to claim 1, b) determining the amount of CD3 in said biological sample, and c) comparing the amount determined in step (b) with a standard, a difference in amount indicating the presence of said disorder.

19. The method of claim 18, wherein said polypeptide contains at its C-terminus the amino acid sequence GQHHHHHH (SEQ ID NO: 33).

Description

BRIEF DESCRIPTION OF FIGURES AND TABLES

[0160] FIG. 1. The T-cell receptor complex with TCR- and TCR- chains (top), -chain accessory molecules (bottom) and CD3 (represented by CD3, CD3 and two CD3).

[0161] FIG. 2. Schematic representation of conventional and camelid antibodies and SDS/PAGE analysis in reducing and non-reducing condition of camelid antibodies.

[0162] FIG. 3. Time course detection of CD3 specific antibody titers in serum of the immunized camel. Human recombinant CD80 was used as a negative control.

[0163] FIG. 4. Amplification and cloning of the VHH encoding cDNAs from the immunized camel.

[0164] FIG. 5. Overview of selection procedure used to identify CD3-binding VHHs.

[0165] FIG. 6. One-step enrichment for CD3 binding VHHs.

[0166] FIG. 7. Amino acid alignment of the CDR regions of 20 clones identified by the epitope specific elution selection procedure (SEQ ID NOS 43-102, respectively, in order of appearance)).

[0167] FIG. 8. ELISA assay on solid-phase immobilized CD3 of 4 unique CD3 specific clones identified via the epitope specific elution selection procedure. 2 VHH clones randomly picked from a preimmune library were used as a control.

[0168] FIG. 9. Pull down assay on CD3 of 4 unique CD3 specific clones identified via the epitope specific elution selection procedure. 2 VHH clones randomly picked from a preimmune library were used as a control.

[0169] FIG. 10. Western Blot. Cell lysates were prepared from Jurkat cells in lysis buffer (150 mM NaCl, 20 mM Hepes, pH 7.4, 1% Triton X-100, 10% glycerol and a mixture of protease inhibitors). Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membrane and incubated with 4 CD3-specific VHH or 2 VHH randomly picked from a preimmune library primary antibodies followed by anti-HA and horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences). The blot was developed with SuperSignal West substrate (Pierce) and exposed to film as shown in the Figure.

[0170] Table I Amino acid sequence listing of the peptides of the present invention.

[0171] FIG. 11. Table II. The extracellular portion of the CD3 antigen expressed on human T cells used to raise an antibody response in camels (SEQ. ID. NO: 103).

QUOTED PATENTS

[0172]

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TABLE-US-00002 TABLEI SEQ.ID1 MAESGGGSVQTGGSLRLSCAYTASSVCMAW FRQAPGKEREGVAVTREGLTKTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYNYWGQGTQVTV SEQ.ID2 MAESGGGSVQTGGSLRLSCAYTASSVCMAW FRQAPGKEREGVAVTREGLTKTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYDYWGQGTQVTV SEQ.ID3 MAESGGGSVQTGGSLRLSCAYTASSVCMAW FRQAPGKEREGVAVTREGLTQTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYNYWGQGTQVTV SEQ.ID4 MAESGGGSVQTGGSLRLSCAYTASSVCMAW FRQAPGKEREGVAVTREGLTQTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYDYWGQGTQVTV SEQ.ID5 MAESGGGSVQTGGSLRLSCAYTASSVCMAW FRQAPGKEREGVAVTREGLPKTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYNYWGQGTQVTV SEQ.ID6 MAESGGGSVQTGGSLRLSCAYTASSVCMAW FRQAPGKEREGVAVTREGLPKTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYDYWGQGTQVTV SEQ.ID7 MAESGGGSVQTGGSLRLSCAYTASSVCMAW FRQAPGKEREGVAVTREGLPQTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYNYWGQGTQVTV SEQ.ID8 MAESGGGSVQTGGSLRLSCAYTASSVCMAW FRQAPGKEREGVAVTREGLPQTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYDYWGQGTQVTV SEQ.ID9 MAESGGGSVQTGGSLRLSCAYTASSVCMAW FRQAPGKEREGVAVTRDGLTKTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYNYWGQGTQVTV SEQ.ID10 MAESGGGSVQTGGSLRLSCAYTASSVCMAW FRQAPGKEREGVAVTRDGLTKTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYDYWGQGTQVTV SEQ.ID11 MAESGGGSVQTGGSLRLSCAYTASSVCMAW FRQAPGKEREGVAVTRDGLTQTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYNYWGQGTQVTV SEQ.ID12 MAESGGGSVQTGGSLRLSCAYTASSVCMAW FRQAPGKEREGVAVTRDGLTQTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYDYWGQGTQVTV SEQ.ID13 MAESGGGSVQTGGSLRLSCAYTASSVCMAW FRQAPGKEREGVAVTRDGLPKTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYNYWGQGTQVTV SEQ.ID14 MAESGGGSVQTGGSLRLSCAYTASSVCMAW FRQAPGKEREGVAVTRDGLPKTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYDYWGQGTQVTV SEQ.ID15 MAESGGGSVQTGGSLRLSCAYTASSVCMAW FRQAPGKEREGVAVTRDGLPQTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYNYWGQGTQVTV SEQ.ID16 MAESGGGSVQTGGSLRLSCAYTASSVCMAW FRQAPGKEREGVAVTRDGLPQTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYDYWGQGTQVTV SEQ.ID17 MAESGGGSVQTGGSLRLSCAYTASSLCMAW FRQAPGKEREGVAVTREGLTKTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYNYWGQGTQVTV SEQ.ID18 MAESGGGSVQTGGSLRLSCAYTASSLCMAW FRQAPGKEREGVAVTREGLTKTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYDYWGQGTQVTV SEQ.ID19 MAESGGGSVQTGGSLRLSCAYTASSLCMAW FRQAPGKEREGVAVTREGLTQTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYNYWGQGTQVTV SEQ.ID20 MAESGGGSVQTGGSLRLSCAYTASSLCMAW FRQAPGKEREGVAVTREGLTQTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYDYWGQGTQVTV SEQ.ID21 MAESGGGSVQTGGSLRLSCAYTASSLCMAW FRQAPGKEREGVAVTREGLPKTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYNYWGQGTQVTV SEQ.ID22 MAESGGGSVQTGGSLRLSCAYTASSLCMAW FRQAPGKEREGVAVTREGLPKTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYDYWGQGTQVTV SEQ.ID23 MAESGGGSVQTGGSLRLSCAYTASSLCMAW FRQAPGKEREGVAVTREGLPQTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYNYWGQGTQVTV SEQ.ID24 MAESGGGSVQTGGSLRLSCAYTASSLCMAW FRQAPGKEREGVAVTREGLPQTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYDYWGQGTQVTV SEQ.ID25 MAESGGGSVQTGGSLRLSCAYTASSLCMAW FRQAPGKEREGVAVTRDGLTKTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYNYWGQGTQVTV SEQ.ID26 MAESGGGSVQTGGSLRLSCAYTASSLCMAW FRQAPGKEREGVAVTRDGLTKTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYDYWGQGTQVTV SEQ.ID27 MAESGGGSVQTGGSLRLSCAYTASSLCMAW FRQAPGKEREGVAVTRDGLTQTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYNYWGQGTQVTV SEQ.ID28 MAESGGGSVQTGGSLRLSCAYTASSLCMAW FRQAPGKEREGVAVTRDGLTQTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYDYWGQGTQVTV SEQ.ID29 MAESGGGSVQTGGSLRLSCAYTASSLCMAW FRQAPGKEREGVAVTRDGLPKTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYNYWGQGTQVTV SEQ.ID30 MAESGGGSVQTGGSLRLSCAYTASSLCMAW FRQAPGKEREGVAVTRDGLPKTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYDYWGQGTQVTV SEQ.ID31 MAESGGGSVQTGGSLRLSCAYTASSLCMAW FRQAPGKEREGVAVTRDGLPQTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYNYWGQGTQVTV SEQ.ID32 MAESGGGSVQTGGSLRLSCAYTASSLCMAW FRQAPGKEREGVAVTRDGLPQTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYDYWGQGTQVTV SEQ.ID33 GQHHHHHH SEQ.ID34 MAESGGGSVQTGGSLRLSCAYTASSVCMAW FRQAPGKEREGVAVTREGLTKTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYNYWGQGTQVTV GQHHHHHH SEQ.ID35 MAESGGGSVQTGGSLRLSCAYTASSVCMAW FRQAPGKEREGVAVTREGLTKTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYDYWGQGTQVTV GQHHHHHH SEQ.ID36 MAESGGGSVQTGGSLRLSCAYTASSVCMAW FRQAPGKEREGVAVTREGLTQTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYC AARPTSPCTVDGELLASTYNYWGQGTQVTV GQHHHHHH CALL001: SEQ.ID37 5-GTCCTGGCTGCTCTTCTACAAGG-3 CALL002: SEQ.ID38 5-GGTACGTGCTGTTGAACTGTTCC-3 VHH-Back: SEQ.ID39 5-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3 VHH-For: SEQ.ID40 5-CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3