TRPV1 EPITOPES AND ANTIBODIES
20230322916 · 2023-10-12
Inventors
Cpc classification
C07K2317/76
CHEMISTRY; METALLURGY
C07K16/28
CHEMISTRY; METALLURGY
C07K2317/34
CHEMISTRY; METALLURGY
A61K47/6849
HUMAN NECESSITIES
A61K47/6811
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to antibodies that bind to TRPV1. The invention also relates to certain epitopes of the protein TRPV1. The invention also relates to immunoconjugates and compositions comprising such antibodies. The invention also provides methods of producing such antibodies. The invention further provides the use of such antibodies for therapeutic purposes, for example in the treatment of pain.
Claims
1. An antibody which binds to TRPV1, wherein said antibody binds to TRPV1 in the extracellular region of TRPV1 and wherein said antibody preferentially inhibits capsaicin-induced activation of TRPV1 as opposed to heat-induced activation of TRPV1.
2. The antibody of claim 1, wherein said antibody binds to an epitope of TRPV1 in the region of TRPV1 defined by amino acid residues 599-656 of TRPV1 (SEQ ID NO:1).
3. The antibody of claim 1, wherein said antibody binds to TRPV1 at an epitope that is in the region defined by amino acid residues 599-630, amino acid residues 599-606, amino acid residues 519-614, amino acid residues 599-622, amino acid residues 607-630, amino acid residues 615-630, amino acid residues 623-630, amino acid residues 631-643, amino acid residues 644-656 or amino acid residues 610-620 of TRPV1 (SEQ ID NO:1), or binds to an epitope of TRPV1 that is in the region of TRPV1 defined by amino acid residues 599-601 and residues 653-655 of TRPV1.
4. The antibody of claim 1, wherein said antibody binds to TRPV1 at an epitope that is in the region defined by amino acid residues 599-630.
5. The antibody of claim 1, wherein said antibody binds to an isolated peptide, said isolated peptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, or comprising a sequence that has 1, 2, or 3 amino acid substitutions or additions or deletions compared with said amino acid sequence.
6. The antibody of claim 1, wherein said antibody binds to an isolated peptide, said isolated peptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:18, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, or comprising a sequence that has 1, 2, or 3 amino acid substitutions or additions or deletions compared with said amino acid sequence.
7. The antibody of claim 1, wherein said antibody binds to an isolated peptide, said isolated peptide consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO:18, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, or consisting of a sequence that has 1, 2, or 3 amino acid substitutions or additions or deletions compared with said amino acid sequence.
8. The antibody of claim 1, wherein said antibody binds to an isolated peptide, said isolated peptide consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO:18, SEQ ID NO:16 and SEQ ID NO:17.
9. The antibody of claim 5, wherein said isolated peptides are linear peptides or cyclic peptides.
10. The antibody of claim 1, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:448, preferably SEQ ID NO: 449.
11. The antibody of claim 1, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein (i) said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:77, a VH CDR2 that has the amino acid sequence of SEQ ID NO:78 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:79, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:80, a VL CDR2 that has the amino acid sequence of SEQ ID NO:81 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:82, or sequences substantially homologous thereto; (ii) said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:41, a VH CDR2 that has the amino acid sequence of SEQ ID NO:42 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:43, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:44, a VL CDR2 that has the amino acid sequence of SEQ ID NO:45 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:46, or sequences substantially homologous thereto; (iii) said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:103, a VH CDR2 that has the amino acid sequence of SEQ ID NO:104 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:105, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:106, a VL CDR2 that has the amino acid sequence of SEQ ID NO:107 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:108, or sequences substantially homologous thereto; (iv) said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:123, a VH CDR2 that has the amino acid sequence of SEQ ID NO:124 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:125, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:126, a VL CDR2 that has the amino acid sequence of SEQ ID NO:127 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:128, or sequences substantially homologous thereto; (v) said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:143, a VH CDR2 that has the amino acid sequence of SEQ ID NO:144 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:145, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:146, a VL CDR2 that has the amino acid sequence of SEQ ID NO:147 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:148, or sequences substantially homologous thereto; (vi) said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:183, a VH CDR2 that has the amino acid sequence of SEQ ID NO:184 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:185, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:186, a VL CDR2 that has the amino acid sequence of SEQ ID NO:187 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:188, or sequences substantially homologous thereto; (vii) said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:203, a VH CDR2 that has the amino acid sequence of SEQ ID NO:204 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:205, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:206, a VL CDR2 that has the amino acid sequence of SEQ ID NO:207 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:208, or sequences substantially homologous thereto; (viii) said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:223, a VH CDR2 that has the amino acid sequence of SEQ ID NO:224 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:225, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:226, a VL CDR2 that has the amino acid sequence of SEQ ID NO:227 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:228, or sequences substantially homologous thereto; (ix) said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:283, a VH CDR2 that has the amino acid sequence of SEQ ID NO:284 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:285, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:286, a VL CDR2 that has the amino acid sequence of SEQ ID NO:287 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:288, or sequences substantially homologous thereto; (x) said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:303, a VH CDR2 that has the amino acid sequence of SEQ ID NO:304 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:305, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:306, a VL CDR2 that has the amino acid sequence of SEQ ID NO:307 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:308, or sequences substantially homologous thereto; (xi) said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:323, a VH CDR2 that has the amino acid sequence of SEQ ID NO:324 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:325, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:326, a VL CDR2 that has the amino acid sequence of SEQ ID NO:327 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:328, or sequences substantially homologous thereto; or (xii) said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:363, a VH CDR2 that has the amino acid sequence of SEQ ID NO:364 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:365, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:366, a VL CDR2 that has the amino acid sequence of SEQ ID NO:367 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:368, or sequences substantially homologous thereto, wherein said substantially homologous sequences are sequences containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequences, or said substantially homologous sequence is a sequence containing conservative amino acid substitutions of the given CDR sequences.
12. The antibody of claim 10, wherein said antibody comprises a VL CDR2, a VL CDR3, a VH CDR1, a VH CDR2 and a VH CDR3 that have amino acid sequences as defined together in any one of parts (i) to (xii) of claim 11.
13. The antibody of claim 1, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises: (a) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:77, (b) a VH CDR2 that has the amino acid sequence of SEQ ID NO:78, and (c) a VH CDR3 that has the amino acid sequence of SEQ ID NO:79; and wherein said light chain variable region comprises: (d) a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:80, (e) a VL CDR2 that has the amino acid sequence of SEQ ID NO:81, and (f) a VL CDR3 that has the amino acid sequence of SEQ ID NO:82.
14. The antibody of claim 1, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein (i) the light chain variable region has the amino acid sequence of SEQ ID NO:76, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:75, or a sequence having at least 80% sequence identity thereto; (ii) the light chain variable region has the amino acid sequence of SEQ ID NO:58, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:57, or a sequence having at least 80% sequence identity thereto; (iii) the light chain variable region has the amino acid sequence of SEQ ID NO:40, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:39, or a sequence having at least 80% sequence identity thereto; (iv) the light chain variable region has the amino acid sequence of SEQ ID NO:102, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:101, or a sequence having at least 80% sequence identity thereto; (v) the light chain variable region has the amino acid sequence of SEQ ID NO:122, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:121, or a sequence having at least 80% sequence identity thereto; (vi) the light chain variable region has the amino acid sequence of SEQ ID NO:142, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:141, or a sequence having at least 80% sequence identity thereto; (vii) the light chain variable region has the amino acid sequence of SEQ ID NO:162, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:161, or a sequence having at least 80% sequence identity thereto; (viii) the light chain variable region has the amino acid sequence of SEQ ID NO:182, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:181, or a sequence having at least 80% sequence identity thereto; (ix) the light chain variable region has the amino acid sequence of SEQ ID NO:202, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:201, or a sequence having at least 80% sequence identity thereto; (x) the light chain variable region has the amino acid sequence of SEQ ID NO:222, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:221, or a sequence having at least 80% sequence identity thereto; (xi) the light chain variable region has the amino acid sequence of SEQ ID NO:242, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:241, or a sequence having at least 80% sequence identity thereto; (xii) the light chain variable region has the amino acid sequence of SEQ ID NO:262, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:261, or a sequence having at least 80% sequence identity thereto; (xiii) the light chain variable region has the amino acid sequence of SEQ ID NO:282, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:281, or a sequence having at least 80% sequence identity thereto; (xiv) the light chain variable region has the amino acid sequence of SEQ ID NO:302, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:301, or a sequence having at least 80% sequence identity thereto; (xv) the light chain variable region has the amino acid sequence of SEQ ID NO:322, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:321, or a sequence having at least 80% sequence identity thereto; (xvi) the light chain variable region has the amino acid sequence of SEQ ID NO:362, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:361, or a sequence having at least 80% sequence identity thereto; (xvii) the light chain variable region has the amino acid sequence of SEQ ID NO:382, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:381, or a sequence having at least 80% sequence identity thereto; or (xviii) the light chain variable region has the amino acid sequence of SEQ ID NO:402, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:401, or a sequence having at least 80% sequence identity thereto.
15. The antibody of claim 1, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:343, a VH CDR2 that has the amino acid sequence of SEQ ID NO:344 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:345, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:346, a VL CDR2 that has the amino acid sequence of SEQ ID NO:347 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:348, or sequences substantially homologous thereto, wherein said substantially homologous sequences are sequences containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequences, or said substantially homologous sequences are sequences containing conservative amino acid substitutions of the given CDR sequences.
16. The antibody of claim 1, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein the light chain variable region has the amino acid sequence of SEQ ID NO:342, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:341, or a sequence having at least 80% sequence identity thereto.
17. The antibody of claim 1, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:423, a VH CDR2 that has the amino acid sequence of SEQ ID NO:424 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:425, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:426, a VL CDR2 that has the amino acid sequence of SEQ ID NO:427 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:428, or sequences substantially homologous thereto, wherein said substantially homologous sequences are sequences containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequences, or said substantially homologous sequences are sequences containing conservative amino acid substitutions of the given CDR sequences.
18. The antibody of claim 1, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein the light chain variable region has the amino acid sequence of SEQ ID NO:422, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:421, or a sequence having at least 80% sequence identity thereto.
19. The antibody of claim 1, wherein said antibody is a polyclonal antibody or a monoclonal antibody.
20. The antibody of claim 1, wherein said antibody is a whole antibody comprising an antibody constant region.
21. The antibody of claim 1, wherein said antibody is an IgG antibody.
22. The antibody of claim 1, wherein said antibody is an antigen binding fragment of an antibody.
23. A composition comprising an antibody of claim 1 and a diluent, carrier or excipient, preferably a pharmaceutically acceptable diluent, carrier or excipient.
24. A nucleic acid molecule comprising a nucleotide sequence that encodes an antibody of claim 1, or a set of nucleic acid molecules each comprising a nucleotide sequence, wherein said set of nucleic acid molecules together encode an antibody of claim 1.
25. A method of producing an antibody according to claim 1, comprising the steps of: (i) culturing a host cell comprising (i) one or more nucleic acid molecules encoding an antibody according to claim 1 or (ii) a set of nucleic acid molecules each comprising a nucleotide sequence, wherein said set of nucleic acid molecules together encode an antibody of claim 1, or (iii) one or more recombinant expression vectors comprising one or more of said nucleic acid molecules, under conditions suitable for the expression of the encoded antibody; and (ii) isolating or obtaining the antibody from the host cell or from the growth medium/supernatant.
26. (canceled)
27. (canceled)
28. A method of treating pain, said method comprising administering to a patient in need thereof a therapeutically effective amount of an antibody as defined in claim 1.
29. (canceled)
30. (canceled)
31. An isolated peptide, wherein said isolated peptide is as defined in claim 5.
32. A conjugate comprising an isolated peptide as defined in claim 5 and a peptide carrier.
33. The conjugate of claim 32, wherein said peptide carrier is keyhole limpet hemocyanin (KLH), ovalbumin (OVA) or bovine serum albumin (BSA).
Description
[1092] The invention will now be further described in the following non-limiting Example with reference to the following drawings:
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EXAMPLES
Example 1—Peptides and Antibody Generation and Testing
Materials and Methods
Chemicals
[1109] Cell culturing medium (DMEM/Ham's F12 with glutamine and Ham's F12), fetal bovine serum, and Accutase were purchased from PAA. Zeocin was purchased from Invitrogen.
[1110] The control antibody (Rabbit Gamma Globulin—RRID: AB_2532177—catalogue number 31887) was purchased from Thermo Fisher Scientific. Mavatrep and AMG517 were purchased from MedChemExpress. All other chemicals were purchased from Sigma. The following buffers were used; F: 140 mM NaCl, 5 mM KCl, 1 mM CaCl.sub.2, 1 mM MgCl.sub.2 10 mM HEPES, 10 mM D-glucose, pH 7.4, G: 120 mM KCl, 2 mM MgCl.sub.2, 10 mM HEPES, 10 mM EGTA, pH 7.2. H: 50 mM Tris, 100 mM NaCl, 10 mM MgCl.sub.2, 1 mM EDTA, 0.01% Triton X-100 and 1 mM DTT.
Cell Culture
[1111] Adherent Chinese hamster ovary (CHO) cells with a tetracycline regulated expression system (T-REx) of hTRPV1 (human TRPV1) were cultivated in medium (DMEM/F12 with glutamine) supplemented with 10% fetal bovine serum (FBS), Zeocin (350 μg/ml), and Blasticidin (5 μg/ml). 18-24 hours before use, the cells were incubated in medium supplemented with 10% FBS and Doxycycline (1 μg/ml) in order to induce hTRPV1 expression.
Antibody Development
[1112] For each epitope (peptide) used for antibody generation, synthetic peptides including an additional cysteine(s) residue(s), were synthesized and purified. These synthetic peptide sequences are set forth as SEQ ID NO:16 (OTV3), SEQ ID NO:17 (OTV4), SEQ ID NO:18 (OTV5), SEQ ID NO:19 (OTV6), SEQ ID NO:20 (OTV7), SEQ ID NO:21 (OTV8), SEQ ID NO:22 (OTV9), SEQ ID NO:23 (OTV10), SEQ ID NO:24 (OTV11), SEQ ID NO:25 (OTV12), SEQ ID NO:26 (OTV13), SEQ ID NO:27 (OTV14) and SEQ ID NO:28 (OTV15).
[1113] For the linear epitopes (peptides) (SEQ ID NO:16 (OTV3), SEQ ID NO:17 (OTV4), SEQ ID NO:19 (OTV6), SEQ ID NO:20 (OTV7), SEQ ID NO:21 (OTV8), SEQ ID NO:22 (OTV9), SEQ ID NO:23 (OTV10), SEQ ID NO:24 (OTV11), SEQ ID NO:25 (OTV12), SEQ ID NO:26 (OTV13)), the peptides (epitopes) were linked by the terminal cysteine residue to keyhole limpet hemocyanin (KLH). For the cyclic epitopes (peptides) (SEQ ID NO: 18 (OTV5), SEQ ID NO:27 (OTV14) and SEQ ID NO:28 (OTV15)) the peptides (epitopes) were then linked to keyhole limpet hemocyanin (KLH) via the propargyl group. The KLH-linked epitopes (peptides) were used to produce polyclonal antibodies by immunization of specific pathogen-free (SPF) rabbits following injection of the KLH linked peptides.
[1114] The peptides were produced by solid phase peptide synthesis (SPPS) with capping step. Cyclization was done by oxidizing terminal cysteines, creating a disulphide bridge between peptide ends. Linear peptides were conjugated to KLH by coupling SH-group on cysteine to NH2-group on KLH. The cyclic peptides were conjugated by click chemistry using a propargyl group on the peptide and azide on KLH. Antibodies were purified using a Protein G column followed by affinity purification against the peptide.
[1115] The antibodies were affinity purified and subjected to an ELISA test. The ELISA test showed that the antibodies were able to bind to their respective peptides (i.e. the respective peptide used to immunize the rabbit to produce the relevant antibody) when the peptide was immobilized on a surface (data not shown).
[1116] Generation of both synthetic peptides and polyclonal antibodies were performed by Innovagen AB (Lund, Sweden).
Electrophysiology
Electrophysiological Patch Clamp Recordings—OTV4, OTV5 & OTV12
[1117] Whole cell recordings were performed using a microfluidic device for patch clamp recordings (Dynaflow, Cellectricon AB, Göteborg, Sweden) together with an Axopatch 200B (Molecular Devices, USA) patch clamp amplifier. The cells were adherent Chinese hamster ovary (CHO) cells, as described above. Bath and pipette (borosilicate glass capillaries, i.d. 0.86 mm; GC150F-7.5, Harvard Apparatus Ltd) solutions contained buffer F and G, respectively. The cells were clamped at −60 mV and the current signals were recorded with a sampling frequency of 10 kHz and low pass filtered at 2 kHz. The patch-clamp recordings were acquired using digital/analogue sampling (Axon Digidata 1550) and acquisition software (Clampex version 10.7, Molecular Devices). In experiments testing OTV4 antibody, OTV5 antibody, OTV12 antibody, AMG 517, Mavatrep and control antibody (ThermoFischer #31887), current amplitudes were measured by exposing cells to capsaicin, with and without antibody or small molecule (the small molecules are AMG 517 and Mavatrep). The cells were exposed to 100 nM capsaicin in buffer F for ˜20 s, followed by buffer F for 60 s, antibody (or small molecule) in buffer F for 60 s and then 100 nM capsaicin together with antibody (or small molecule) in buffer F for ˜20 s.
[1118] Measurements where the seal resistance shifted largely during treatment were excluded from analysis.
Electrophysiology Patch Clamp—Data Analysis—OTV4, OTV5 & OTV12
[1119] For all measurements, the recorded amplitude of the peak during stimulation with antibody+capsaicin was divided by the recorded amplitude of the peak during stimulation with capsaicin. Measurements were performed on cells from at least two different cell culture dishes. Each data point (n) represents a single cell. Statistical analysis was performed with one-way analysis of variance in combination with Dunnett's post-hoc test comparing each antibody to the control antibody. p<0.05 was considered as statistically significant. Normality was assessed using the Shapiro-Wilk test. Data is presented as mean±SEM.
Calcium Imaging Method for Assessing Inhibition of Capsaicin-Induced Activation of TRPV1
[1120] CHO cells expressing hTRPV1 were incubated with 4.4 μM of the calcium indicator Fluo-3 AM for 30 min at 37° C. and thereafter washed and then incubated with various concentrations of antibodies dissolved in PBS (OTV4 n=12, OTV7 n=12, OTV9 n=12, OTV12 n=11, OTV13 n=11, or control antibody n=12 (ThermoFischer #31887)) for 1 h, at room temperature. Calcium content within the cells was then monitored by measuring fluorescence intensity using a plate reader (CLARIOstar, BMG Labtech) before and after application of 1 μM capsaicin and 150 μM Ca.sup.2+ to the antibody solution covering the cells. The total calcium uptake caused by capsaicin in those samples that were prior incubated with antibodies was normalized against the calcium uptake caused by capsaicin activation (i.e. capsaicin+calcium) only (i.e. with no prior antibody incubation). Data is represented as mean±SEM. Statistical significance was determined using a Kruskal-Wallis test followed by Dunn's multiple comparison.
Imaging
Calcium Imaging of Heat Response
[1121] In order to measure the antibodies' effect on hTRPV1 heat response (42° C.) an optical heating system was used to deliver heat pulses to cells. The cells were adherent Chinese hamster ovary (CHO) cells, as described above. A commercial microfluidic device, the Biopen Prime (Fluicell AB), was used to deliver the antibodies to cells. Heat increases the open probability of hTRPV1 channels which causes an influx of calcium ions. This influx was measured using the calcium indicator Fluo-3. The experiments were performed in the same glass bottomed Petri dishes as the cells were grown in (50 mm uncoated, MatTek). All cell experiments including Fluo-3 AM incubation were performed using DMEM/F-12, HEPES cell culture medium without phenol red (Gibco). The “AM” ester is initially attached to the Fluo-3 indicator, which makes it cell permeable. Once added to the cell culture medium the Fluo-3 AM enters the cell and the “AM” ester part is cleaved off, leaving the Fluo-3 indicator inside the cell. The Fluo-3 indicator itself is non-cell permeable so it remains inside the cell.
Confocal Microscopy
[1122] Cells were imaged using a Bio-Rad MRC 1024 confocal unit fitted with a dual Calypso laser (Cobolt, Solna, Sweden) attached to a Nikon Diaphot 200 inverted microscope and a Nikon Plan Apo 20× dry objective (N.A. 0.75 Nikon, Tokyo, Japan). Excitation wavelengths used were 491 nm (Fluo-3) and the emitted light was collected through a 522 nM filter. Images were acquired for the full view of the 20× objective. The frame rate was one image per 7 sec and the pixel resolution 1024×1024.
Heat Probe
[1123] A laser heating system was used to locally increase the temperature to 42° C. around selected cells. The laser heating system was built in-house by Fluicell AB. This optical local heating system is based upon a CW 4W 1470-nm semiconductor diode laser (4PN-106, Seminex Corporation, USA) driven by a 20 A benchtop power source (ARO-4320, Arroyo Instruments). This delivers a localized beam to the sample (a group of cells in our case) through a 105 μm core, 0.22 NA, broadband optical fiber (M63L01, Thorlabs). The optical fiber is coupled to a 5 mm fiber optic cannula (CFMLC21L05, 105 μm, 0.22 NA, Thorlabs) so that it can be precisely positioned at any desired location in a Petri dish.
[1124] A narrow beam of 1470 nm radiation, exiting the tip of the optical fiber, induces local heating of the water within its path. The extent of heating is determined by the beam intensity which is modulated by the current setting of the laser, and the distance between the tip of the fiber and the sample. In this study, the current and distance are optimized to achieve a sample temperature of 42° C. The relationship between distance, applied current and temperature was calibrated using a previously described technique (Wegrzyn, I., et al. An optofluidic temperature probe. Sensors (Switzerland) (2013), 13(4), 4289-4302, doi:10.3390/s130404289), using a fluidic device (Biopen, Fluicell AB), probing the fluorescent responses.
Biopen
[1125] A Biopen Prime (Fluicell AB) was used to deliver antibodies to cells. The Biopen is a free-standing microfluidic device which can be readily positioned using micromanipulators such that the tip can be aligned adjacent to a selected group of cells in a Petri dish, to locally deliver a compound without contamination of the surrounding environment. The solutions to be delivered by the Biopen are loaded into wells on the Biopen to minimize compound consumption. The switching between solutions is controlled by dedicated software.
Calcium Imaging of Heat Response—Recording
[1126] 30 min before imaging the cell medium was changed to medium containing 36 μM Fluo-3-AM (F1242, Thermo-Fisher) and the samples were incubated for 30 min at RT and then washed and provided with fresh growth medium which contains Ca.sup.2+ (DMEM/F-12, HEPES cell culture medium without phenol red (Gibco)). The Biopen was positioned above a group of cells using a micromanipulator. The heat-probe was positioned 10 μm above the dish bottom and at approx. 100 μm distance from the Biopen outlet. To define which cells that are exposed to solution delivery from the Biopen, an initial pulse of Sulforhodamine B was delivered. After the Sulforhodamine B fluorescence declined the cells was optically heated for 7 s (42° C.) and the fluorescence response from Fluo-3 was measured (cell response, Peak #1). Subsequently, antibody solution (OTV4, OTV5, OTV12 antibody or control antibody (ThermoFischer #31887)) or small molecule solution (2 μM Mavatrep or 2 μM AMG-517) was delivered for 90 s and a second heat-pulse was applied during the last 7 s of application (cell response, Peak #2).
Data Analysis of Calcium Imaging of Heat Response
[1127] Data analyses were performed in Image J and GraphPad Prism software. The Sulforhodamine B pulse visualizes which cells that are reached by Biopen solution delivery and thereby defining which cell that will be included in the measurement. The fluorescence intensity of these cells was measured and averaged for each time point, to obtain an average curve for the cells stimulated in one experiment. The height of Peak #1 was measured to determine heat response without antibody present, and the height of Peak #2 was measured to determine heat response with antibody present. Peak #2 value was divided with Peak #1 to obtain a ratio. The ratio of the peaks for the OTV4, OTV5 and OTV12 antibodies was compared to the control antibody.
[1128] Statistical analysis was performed with one-way analysis of variance in combination with Dunnett's post-hoc test. p<0.05 was considered as statistically significant. Normality was assessed using Shapiro-Wilk test. Data is presented as mean±SEM. n equals the number of experiments containing on average 5-10 cells.
Results and Discussion
[1129] Moduli selective antagonistic antibodies of TRPV1, named antibodies OTV4, OTV5 & OTV12 were developed as described above (i.e. by immunizing rabbits with the stated KLH-linked OTV4, OTV5 and OTV12). These moduli selective antibodies are capable of preferentially inhibiting capsaicin activation of TRPV1 as opposed to heat activation of TRPV1, thus reducing or avoiding the heat-related side-effects that have been observed with previous small molecule antagonists.
[1130] The moduli selective effect of the OTV4, OTV5 and OTV12 antibodies was determined by comparing the degree of inhibition of capsaicin and heat activation of TRPV1, respectively. The activity profiles were compared to those of the small molecule antagonists Mavatrep (Manitpisitkul P, et al. Scand. J. Pain (2018), 18(2):151-164) and AMG-517 (Gavva, N. R. et al. Pain (2008), 136 (1-2), 202-210) (
[1131] During the patch-clamp experiments (which evaluated capsaicin-induced TRPV1 activation), cells were exposed to capsaicin followed by antibody (or small molecule) and finally capsaicin in the presence of antibody (or small molecule). During the fluorescence experiments (which evaluated heat-induced TRPV1 activation), cells were exposed to heat (42° C.) followed by antibody (or small molecule) and finally exposed to heat (42° C.) in the presence of antibody (or small molecule).
[1132] The level of heat and capsaicin inhibition by the antibodies can be found in
[1133] The OTV4 antibody elicited a 26.5% inhibition of capsaicin-induced TRPV1 activation at 533 nM and a −4.1% inhibition of temperature (heat)-induced TRPV1 activation at 2.7 μM (
[1134] The OTV5 antibody elicited a 62.4% inhibition of capsaicin-induced TRPV1 activation at 13.3 nM and a 10.0% inhibition of temperature (heat)-induced TRPV1 activation at 67 nM (
[1135] The OTV12 antibody elicited a 34.1% inhibition of capsaicin-induced TRPV1 activation at 13.3 nM and a −12.8% inhibition of temperature (heat)-induced TRPV1 activation at 67 nM (
[1136] Mavatrep elicited a 100% inhibition of capsaicin-induced TRPV1 activation at 100 nM and a 89.3% inhibition of temperature (heat)-induced TRPV1 activation at 100 nM (
[1137] AMG517 elicited a capsaicin inhibition of 100% inhibition of capsaicin-induced TRPV1 activation at 100 nM and a 88.8% inhibition of temperature (heat)-induced TRPV1 activation at 100 nM (
[1138] The control antibody elicited no inhibition of either capsaicin- or temperature-induced activation of TRPV1 (
[1139] All antibody concentrations evaluated can be seen in
[1140] In summary, all three antibodies (OTV4, OTV5 and OTV12 antibodies) demonstrated a moduli-selective activity profile by eliciting a higher inhibition of capsaicin activation compared to heat-activation even though evaluation of heat activation was performed at 5 times higher antibody concentration, with OTV5 having the highest difference. In contrast, both Mavatrep and AMG517 inhibited capsaicin and heat activation at close to equal levels at 100 nM. This clearly demonstrates that the OTV4, OTV5 and OTV12 antibodies are more than simple TRPV1 antagonists; they selectivity inhibit capsaicin induced activation of TRPV1 as opposed to heat-induced activation of TRPV1. The OTV4 and OTV12 antibodies, and especially the OTV5 antibody, are promising new candidates for TRPV1-targeted pain therapy and are currently pursued as drug candidates. These would not only be the first therapeutic anti-TRPV1 antibodies, they would also be the first therapeutic antibodies ever developed for an ion channel. The present data also establishes that epitopes on TRPV1 that correspond to (or correspond essentially to) the epitope (peptide) amino acid sequences used to generate the OTV4, OTV5 and OTV12 antibodies are useful epitopes to target (i.e. to generate antibodies against) in order to identify moduli selective antibodies as described herein.
[1141] In addition to containing data for the OTV4 and OTV12 polyclonal antibodies,
[1142] It is believed that the OTV7, OTV9 and OTV13 polyclonal antibodies preferentially inhibit capsaicin induced activation of TRPV1 as opposed to heat-induced activation of TRPV1. As described above, the inventors have also generated polyclonal antibodies OTV3, OTV6, OTV8, OTV10, OTV11, OTV13, OTV14 and OTV15. It is believed that these antibodies also preferentially inhibit capsaicin induced activation of TRPV1 as opposed to heat-induced activation of TRPV1.
[1143] It is evident from the above that the inventors have identified certain peptide sequences (epitope sequences) that correspond to (or correspond essentially to) epitopes on TRPV1 that are very useful to target with antibodies in order to achieve inhibition of TRPV1. In particular, the inventors have identified epitopes on TRPV1 that are very useful to target with antibodies in order to achieve preferential inhibition of capsaicin-induced activation of TRPV1 as opposed to heat-induced activation of TRPV1. Antibodies with such selectivity for inhibition of the capsaicin axis of TRPV1 will be therapeutically advantageous as avoiding (or reducing) concomitant inhibition of the heat axis of TRPV1 would avoid (or reduce) adverse effects such as hyperthermia or loss of heat sensation that are observed with other TRPV1 inhibitors (as discussed elsewhere herein).
Example 2—Generation and Testing of Monoclonal Antibodies (Antibodies Named OT-Ab1, OT-Ab2 and OT-Ab-3)
Materials and Methods
Production of Monoclonal Antibodies (Mouse IgG) Using the Hybridoma Technology
[1144] Mice were immunized with the cyclic peptide of SEQ ID NO:18 (OTV5) linked to keyhole limpet hemocyanin (KLH) (the KLH linked peptide is as described above in Example 1). Immune responses were evaluated with ELISA. After the immunization process mice were selected based on ELISA and/or FACS screening of serum, and spleen cells from mouse were extracted and fused with myeloma cells to produce hybridoma cells. Hybridomas were screened by ELISA and/or FACS to obtain positive clones (i.e. that produce antibody that binds to the target). After this screening, sub cloning of the selected hybridomas was performed and a further round of screening was performed using ELISA and/or FACS. Subcloned hybridomas were then used to produce monoclonal antibodies and the antibodies were purified. The binding properties of purified antibodies were tested using ELISA and/or FACS. Three monoclonal antibodies were identified, OT-Ab1, OT-Ab2 and OT-Ab3.
Cloning and Sequencing of Mouse Hybridoma IgG
[1145] From mouse hybridomas, RNA was prepared from which cDNA was synthesized. Variable Light (VL) and Variable Heavy (VH) regions of cDNA were amplified and cloned into standard cloning vector separately. Identification of positive clones was done by colony PCR followed by gel electrophoresis. VL and VH DNA and amino acid sequences were obtained from positive clones.
[1146] Sequences of the antibodies OT-Ab1, OT-Ab2 and OT-Ab3 are set out in Tables C, B and A herein.
[1147] The IgG type of OT-Ab1 is IgG2b/kappa, OT-Ab2 is IgG1/kappa and OT-Ab3 is IgG1/kappa.
Determination of Binding of Antibodies to TRPV1-Expressing CHO Cells Using Fluorescence-Activated Cell Sorting (FACS)
[1148] For monoclonal antibody preparations, CHO-TRPV1 cells were incubated with 50 μl antibody preparation (serum, hybridoma supernatant or purified monoclonal antibody) for 30 min at 4° C., followed by 30 min incubation at 4° C. with 50 μl secondary antibody (anti-mouse IgG, conjugated to fluorescent probe). The negative control was secondary antibody only (anti-mouse IgG at 10 mg/mL in the 50 μL sample). Binding was assessed by measurement of fluorescence signal from cells and reported as mean fluorescence intensity (MFI). CHO-TRPV1 cells are adherent Chinese hamster ovary (CHO) cells with a tetracycline regulated expression system (T-REx) of hTRPV1 (human TRPV1) as described elsewhere. Doxycycline was used to induce expression of TRPV1 (denoted +TRPV1). Non-induced cells are denoted −TRPV1.
Measurement of Capsaicin-Induced Currents or NADA-Induced Currents by Patch Clamp
[1149] Whole cell recordings were performed using a microfluidic device for patch clamp recordings (Dynaflow, Cellectricon AB, Sweden) together with an Axopatch 200B (Molecular Devices, USA) patch clamp amplifier. The cells (in 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM D-glucose, pH 7.4) were clamped, using pipettes (i.d. 0.86 mm; GC150F-7.5, Harvard Apparatus Ltd; filled with 120 mM KCl, 2 mM MgCl2, 10 mM HEPES, 10 mM EGTA, pH 7.2, tip resistance 3-6 MOhm), at −60 mV and the current signals were recorded with a sampling frequency of 10 kHz and low pass filtered at 2 kHz. The patch-clamp recordings were acquired using digital/analogue sampling (Axon Digidata 1550) and acquisition software (Clampex version 10.7, Molecular Devices). Current amplitudes were measured by exposing cells to 100 nM capsaicin, 300 nM capsaicin or 1 μM NADA, with and without antibody. For experiments testing the effect of 100 nM capsaicin or 1 μM NADA, cells were first treated twice with capsaicin (or NADA) alone giving peak 1 and 2, then treated with antibody and then antibody together with capsaicin (or NADA) giving peak 3, followed by capsaicin (or NADA) alone giving peak 4. The effect is then calculated as (1−((peak 3)/((peak 2+peak 4)/2)))*100. This method of calculating effect has been described in the literature (Nikolaev, M. V. et al. TRPV1 activation power can switch an action mode for its polypeptide ligands. PLoS One 12, 1-16, 2017). For experiments testing the effect of 300 nM capsaicin cells were first treated twice with capsaicin alone giving peak 1 and 2, then treated with antibody or vehicle and then antibody or vehicle together with capsaicin giving peak 3, followed by capsaicin alone giving peak 4. The effect is then calculated as (1−((Peak3.sub.Ab/Peak2.sub.Ab)/(peak3.sub.veh/peak2.sub.veh)))*100.
Measurement of Heat-Induced Currents Using Ca.SUP.2+.-Imaging
[1150] In order to measure the antibodies' effect on hTRPV1 heat response an optical heating system was used to deliver heat pulses to cells (45° C.). A commercial microfluidic device, the Biopen Prime (Fluicell AB, Sweden), was used to deliver the antibodies to cells. Heat increases the open probability of hTRPV1 channels which causes an influx of calcium ions. This influx was measured using the calcium indicator Fluo-3. The experiments were performed in the same glass bottomed Petri dishes as the cells were cultured in (50 mm uncoated, MatTek). The cells were adherent TRPV1 expressing Chinese hamster ovary (CHO) cells, as described above. All cell experiments including Fluo-3 AM incubation were performed using buffer 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM D-glucose, pH 7.4. Cells were imaged using a Bio-Rad MRC 1024 confocal unit fitted with a dual Calypso laser (Cobolt, Solna, Sweden) attached to a Nikon Diaphot 200 inverted microscope and a Nikon Plan Apo 20× dry objective (N.A. 0.75 Nikon, Tokyo, Japan). In this example, the effect of the antibody was determined as follows: cells were first pulsed with heat, followed by cooldown, followed by administration of antibody or small molecule and a heat pulse in combination, followed by a cooldown. The small molecules Mavatrep or AG517 were applied directly to the bath solution using the same protocol as for the antibodies. The effect of the antibodies (or the small molecule inhibitors AMG517 or Mavatrep) on heat-induced activation of TRPV1 was assessed by determining the effect of the antibodies (or the small molecule inhibitors AMG517 or Mavatrep) on heat-induced influx of calcium ions into the cells.
[1151] The peak amplitudes represent the influx of calcium into the cells measured using the calcium indicator Fluo-3.
[1152] The ratio of the second to first peak amplitude was calculated for both antibody and vehicle. The percent of inhibition was calculated by comparing the aforementioned ratio for the antibody to the ratio for the vehicle. % inhibition is (1−((Peak2.sub.Ab/Peak1.sub.Ab)/(peak2.sub.veh/peak1.sub.veh)))*100.
Microscopy-Aided Determination of Antibody Binding to Live Cells
[1153] To demonstrate binding of OT-Ab1 to live cells by microscopy, live TRPV1-expressing CHO-cells were incubated for 1 hour with OT-Ab1, subsequently fixed in formaldehyde solution, stained with fluorescent secondary antibody and counterstained with Hoechst to visualize nuclei. Cells were observed with confocal microscopy equipped with fluorescence capability. Binding was assessed as fluorescence signal located at the cell membranes.
Results and Discussion
[1154] TRPV1 is an ion channel that is a member of the TRP-family that is considered to be very difficult to approach with antibodies due to its small extracellular region. Despite their irrefutable importance as drug targets, ion channels are significantly underexploited as antibody targets. In the present study, three modality-selective monoclonal antibodies (mAbs) were developed, that aside from being the first inhibitory mAbs developed against TRPV1, to our knowledge these are the most functionally complex antibodies developed against an ion channel. These antibodies preferentially inhibit capsaicin-induced activation of TRPV1 as opposed to heat-induced activation of TRPV1.
[1155] Three examples of mouse monoclonal antibodies against human TRPV1, OT-Ab1, OT-Ab2 and OT-Ab3, were produced using the hybridoma technology. Specificity of binding to hTRPV1 was determined using FACS, employing a CHO cell line with inducible hTRPV1 expression. OT-Ab1, OT-Ab2 and OT-Ab3 bound to hTRPV1-expressing cells but not to non-hTRPV1-expressing cells indicating strongly specific binding to hTRPV1 (
[1156] OT-Ab1, OT-Ab2 and OT-Ab3 were evaluated for effect on hTRPV1 activation in several different ways. The patch clamp method was used to measure the antibodies' inhibitory activity on hTRPV1 capsaicin responses in hTRPV1-expressing CHO cells. One aspect tested was the propensity to inhibit capsaicin-induced currents at capsaicin concentrations of 100 nM or 300 nM. OT-Ab1, OT-Ab2 and OT-Ab3 inhibited capsaicin-induced currents (
[1157] The patch clamp method was also used to measure OT-Ab1 inhibitory activity on hTRPV1 NADA responses in hTRPV1-expressing CHO cells. Dose-dependent inhibition of hTRPV1 NADA-induced currents by antibody OT-Ab1 was observed at 1 μM NADA and two concentrations of OT-Ab1, and the relationship between dose and effect was statistically significant (
[1158] Ca.sup.2+-imaging was used to measure the inhibitory activity of OT-Ab1, OT-Ab2 and OT-Ab3 on hTRPV1 heat response in hTRPV1-expressing CHO cells. Small-molecule antagonists AMG517 and Mavatrep inhibited heat-induced Ca.sup.2+ uptake by around 75% in this example, whereas OT-Ab1, OT-Ab2 and OT-Ab3 did not inhibit heat-induced Ca.sup.2+ uptake (
[1159] The ability of OT-Ab1 to bind to TRPV1 on live cells was also assessed by fluorescence microscopy, and OT-Ab1 was found to bind to live TRPV1 expressing cells, binding at the cell membranes (data not shown)
[1160] The ability to inhibit activation of hTRPV1 by either capsaicin or NADA but not inhibit activation of hTRPV1 by heat indicates that these antibodies show modality-selective pharmacology, which is a highly desired property for a clinically acceptable and useful therapeutic agent.
[1161] In the present study, modality-selective monoclonal antibodies targeting hTRPV1, a clinically important target that has failed using small molecule approaches, have been developed. These antibodies are promising new candidates for TRPV1-targeted pain therapy and are currently pursued as drug candidates.
Example 3—Generation and Testing of Monoclonal Antibodies (Antibodies Named 32C8-1, 33C9-1, 34C11-1, 40B10-1, 41B5-1, 43D6-1, 44E8-1, 46B7-1, 46D9-1, 12C9-1, 12G6-1, 15D8-1, 16F1-1, 17E11-1, 17E9-1 and 18E10-1)
Materials and Methods
Production of Monoclonal Antibodies (Mouse IgG) Using the Hybridoma Technology
[1162] Mice were immunized with the linear peptide of SEQ ID NO:16 (Peptide OTV3) linked to keyhole limpet hemocyanin or were immunized with the linear peptide of SEQ ID NO:17 (Peptide OTV4) linked to keyhole limpet hemocyanin (KLH) (the KLH linked peptide is as described above in Example 1). Immune responses were evaluated with ELISA. After the immunization process mice were selected based on ELISA and/or FACS screening of serum, and spleen cells from mouse were extracted and fused with myeloma cells to produce hybridoma cells. Hybridomas were screened by ELISA and/or FACS to obtain positive clones (i.e. that produce antibody that binds to the target). After this screening, sub cloning of the selected hybridomas was performed and a further round of screening was performed using ELISA and/or FACS. Subcloned hybridomas were then used to produce monoclonal antibodies and the antibodies were purified. The binding properties of purified antibodies were tested using ELISA and/or FACS. 16 monoclonal antibodies were identified, 32C8-1, 33C9-1, 34C11-1, 40B10-1, 41B5-1, 43D6-1, 44E8-1, 46B7-1, 46D9-1, 12C9-1, 12G6-1, 15D8-1, 16F1-1, 17E11-1, 17E9-1 and 18E10-1. The peptide (immunogen) used to generate the 32C8-1, 33C9-1, 34C11-1, 40B10-1, 41B5-1, 43D6-1, 44E8-1, 46B7-1 and 46D9-1 antibodies was the peptide of SEQ ID NO:16 (OTV3), and as mentioned above this was linked to keyhole limpet hemocyanin. The peptide (immunogen) used to generate the 12C9-1, 12G6-1, 15D8-1, 16F1-1, 17E11-1, 17E9-1 and 18E10-1 antibodies was the peptide of SEQ ID NO:17 (OTV4), and as mentioned above this was linked to keyhole limpet hemocyanin. The suffix “−1” in each of the antibody names is merely indicative that each of these antibodies is a daughter clone of from a respective parental hybridoma (the parental hybridomas have the same names without the “−1” suffix, for example 32C8 is the parental hybridoma of the 32C8-1 antibody (daughter clone)).
[1163] For OTV4-based and OTV5-based purified antibodies the storage buffer was PBS (2.7 mM KCl, 1.5 mM KH.sub.2PO.sub.4, 137 mM NaCl, 8 mM Na.sub.2HPO.sub.4, pH 7.2).
[1164] For OTV3-based purified antibodies the storage buffer was PBS-Tween (0.02% Tween 80, 2.7 mM KCl, 1.5 mM KH.sub.2PO.sub.4, 137 mM NaCl, 8 mM Na.sub.2HPO.sub.4, pH 7.2).
Cloning and Sequencing of Mouse Hybridoma IgG
[1165] From mouse hybridomas, RNA was prepared from which cDNA was synthesized. Variable Light (VL) and Variable Heavy (VH) regions of cDNA were amplified and cloned into standard cloning vector separately. Identification of positive clones was done by colony PCR followed by gel electrophoresis. VL and VH DNA and amino acid sequences were obtained from positive clones.
[1166] Sequences of the antibodies 32C8-1, 33C9-1, 34C11-1, 40B10-1, 41B5-1, 43D6-1, 44E8-1, 46B7-1, 46D9-1, 12C9-1, 12G6-1, 15D8-1, 16F1-1, 17E11-1, 17E9-1 and 18E10-1 are set out in Tables E-T herein.
[1167] The IgG type of 15D8-1, 17E11-1, and 12G6-1 is IgG1/kappa. The IgG type of 16F1-1 is IgG2a/kappa. The IgG type of 17E9-1 and 12C9-1 is type IgG2b/kappa. The IgG type of 18E10-1 is IgG2c/kappa.
Cell Culture
[1168] Adherent Chinese hamster ovary (CHO) cells with a tetracycline regulated expression system (T-REx) of hTRPV1 (human TRPV1) were cultivated in medium (DMEM/F12 with glutamine) supplemented with 10% fetal bovine serum (FBS), Zeocin (350 μg/ml), and Blasticidin (5 μg/ml). 18-24 hours before use, the cells were incubated in medium supplemented with 10% FBS and Doxycycline (1 μg/ml) in order to induce hTRPV1 expression.
[1169] HEK293 cells and CHO S cells were grown under standard cell culture conditions.
Determination of Binding of Antibodies to TRPV1-Expressing CHO Cells Using Fluorescence-Activated Cell Sorting (FACS)
[1170] For monoclonal antibody preparations for FACS analysis, CHO TRPV1 cells, HEK 293 cells or CHO S cells were incubated with 50 μl antibody preparation (serum, hybridoma supernatant or purified monoclonal antibody or control) for 40 min at room temperature, followed by 30 min incubation at room temperature with 100 μl secondary antibody (anti-mouse IgG, conjugated to fluorescent probe). Binding was assessed by measurement of fluorescence signal from cells and reported as mean fluorescence intensity (MFI). CHO TRPV1 cells are adherent Chinese hamster ovary (CHO) cells with a tetracycline regulated expression system (T-REx) of hTRPV1 (human TRPV1) as described elsewhere. Doxycycline was used to induce expression of TRPV1. CHO S cells and HEK 293 cells were used as negative control cells. CHO S cells are a high-density suspension-adapted cell type used as a tool for high level expression of recombinant protein, with little or no basal expression of TRPV1. HEK 293 cells are human embryonic kidney 293 cells widely used in cell biology which have little or no basal expression of TRPV1.
[1171] A negative control was secondary antibody only (anti-mouse IgG at 10 mg/mL in the 50 μL sample). Another negative control, of non-specific binding, was mouse IgG. Another control was unstained CHO TRPV1 cells, which are cells not treated with anything and thus provide a measure of background fluorescence.
Measurement of Capsaicin-Induced Currents by Patch Clamp
[1172] Whole cell recordings were performed using a microfluidic device for patch clamp recordings (Dynaflow, Cellectricon AB, Sweden) together with an Axopatch 200B (Molecular Devices, USA) patch clamp amplifier. The cells (in Buffer A: 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM D-glucose, pH 7.4) were clamped, using pipettes (filled with Buffer B: 120 mM KCl, 2 mM MgCl2, 10 mM HEPES, 10 mM EGTA, pH 7.2), at −60 mV and the current signals were recorded with a sampling frequency of 10 kHz and low pass filtered at 2 kHz. The patch-clamp recordings were acquired using digital/analogue sampling (Axon Digidata 1550) and acquisition software (Clampex version 10.7, Molecular Devices).
[1173] Current amplitudes were measured by exposing cells to 100 nM capsaicin, with and without antibody. The cells were exposed to 100 nM capsaicin in buffer A for 20 s, followed by buffer A for 60 s, antibody in buffer A for 60 s and then 100 nM capsaicin together with antibody in buffer A for 20 s.
[1174] Data was analysed as follows: for all measurements, the amplitude of the peak during stimulation with antibody+capsaicin was divided by the amplitude of the peak during stimulation with capsaicin only. The obtained value was multiplied with 100 to obtain the cell response during antibody+capsaicin stimulation as a percentage of the control response (capsaicin only).
Measurement of Heat-Induced Currents Using Ca.SUP.2+.-Imaging
[1175] In order to measure the antibodies' effect on hTRPV1 heat response an optical heating system was used to deliver heat pulses to cells (45° C.). A commercial microfluidic device, the Biopen Prime (Fluicell AB, Sweden), was used to deliver the antibodies to cells. Heat increases the open probability of hTRPV1 channels which causes an influx of calcium ions. This influx was measured using the calcium indicator Fluo-3. The experiments were performed in the same glass bottomed Petri dishes as the cells were cultured in (50 mm uncoated, MatTek). The cells were adherent TRPV1 expressing Chinese hamster ovary (CHO) cells, as described above. All cell experiments including Fluo-3 AM incubation were performed using HEPES buffer (140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM D-glucose, pH 7.4). Cells were imaged using a Bio-Rad MRC 1024 confocal unit fitted with a dual Calypso laser (Cobolt, Solna, Sweden) attached to a Nikon Diaphot 200 inverted microscope and a Nikon Plan Apo 20× dry objective (N.A. 0.75 Nikon, Tokyo, Japan). In this example, the effect of the antibody was determined as follows: cells were first pulsed with heat, followed by cooldown, followed by administration of antibody or small molecule and a heat pulse in combination, followed by a cooldown. The effect of the antibodies on heat-induced activation of TRPV1 was assessed by determining the effect of the antibodies on heat-induced influx of calcium ions into the cells.
[1176] The peak amplitudes represent the influx of calcium into the cells measured using the calcium indicator Fluo-3.
[1177] The ratio of the second to first peak amplitude was calculated for both antibody and vehicle. The percent of inhibition was calculated by comparing the aforementioned ratio for the antibody to the ratio for the vehicle. % inhibition is (1−((Peak2.sub.Ab/Peak1.sub.Ab)/(peak2.sub.veh/peak1.sub.veh)))*100.
Measurement of Capsaicin-Induced Activity Using Fluorescence Imaging Plate Reader (FLIPR)
[1178] CHO cells with inducible TRPV1 expression were cultured in black, clear bottom 96-well microplates (Corning ltd.) and TRPV1 expression was induced. Cells were loaded with Fluo-3 AM Calcium indicator (Thermo Fisher, cat. no. F1242) by incubating the cells with 4 μM Fluo-3 in HEPES buffer (140 nM NaCl, 5 mM KCl, 1 mM CaCl.sub.2, 1 mM MgCl.sub.2, 10 mM HEPES, 10 mM D-glucose, pH 7.4) for 30 minutes at room temperature. After subsequent washing of cells with HEPES buffer to remove extracellular Fluo-3, serially diluted antibody was added to wells. Cells were incubated in the presence of antibody for 4 minutes at room temperature. Fluorescence measurements were made using a ClarioSTAR microplate reader (BMG Labtech) with excitation at 483 nm (bandwidth 14 nm) and emission at 530 (bandwidth 30 nm). Baseline fluorescence intensity was first measured, then a fixed amount of capsaicin (in HEPES buffer) was added to each well (to give a concentration of capsaicin that had been previously established as representing the EC50 value of capsaicin for the batch of cells under study, typically such a concentration was in the lower nM range e.g. 10 nM) and a second fluorescence intensity measurement was performed after a certain time (within minutes). EC50 is the concentration of a substance that gives half-maximal response of a biological process, in this case TRPV1-mediated Ca.sup.2+ entry into cells. Data is presented as the Fluorescence rate, which is calculated as fluorescence at a certain time after addition of capsaicin minus the fluorescence measured before capsaicin addition. The IC50 values for the tested antibodies was calculated. The IC50 value is the half maximal inhibitory concentration of a substance for a biological process under study, in this case capsaicin-induced cellular TRPV1-mediated Ca.sup.2+ influx.
Results and Discussion
[1179] In the study described in the present example, 16 mouse monoclonal antibodies against human TRPV1, 32C8-1, 33C9-1, 34C11-1, 40B10-1, 41B5-1, 43D6-1, 44E8-1, 46B7-1, 46D9-1, 12C9-1, 12G6-1, 15D8-1, 16F1-1, 17E11-1, 17E9-1, 18E10-1, were produced using the hybridoma technology. Specificity of binding to hTRPV1 was determined using FACS, employing a CHO cell line with inducible hTRPV1 expression. Hybridoma supernatant preparations from parental hybridomas from which the 32C8-1, 33C9-1, 34C11-1, 40B10-1, 41B5-1, 43D6-1, 44E8-1, 46B7-1 and 46D9-1 antibodies were derived bound to hTRPV1-expressing cells but not to non-hTRPV1-expressing cells indicating strongly specific binding to hTRPV1 (
[1180] Further testing of certain of the antibodies is described below.
[1181] 15D8-1, 16F1-1, 17E9-1 and 17E11-1 were evaluated for their effect on hTRPV1 activation in several different ways. The patch clamp method was used to measure the antibodies' inhibitory activity on hTRPV1 capsaicin responses in hTRPV1-expressing CHO cells. One aspect tested was the propensity to inhibit capsaicin-induced currents at capsaicin concentrations of 100 nM. Antibodies 15D8-1 (at 130 nM), 16F1-1 (at 253 nM), 17E9-1 (at 83 nM), and 17E11-1 (at 110 nM) inhibited capsaicin-induced currents at 100 nM capsaicin and therefore are antagonists of the capsaicin axis of TRPV1 (
[1182] We used FLIPR to determine the capacity to inhibit TRPV1-mediated capsaicin-induced Ca2+ uptake in hTRPV1-expressing CHO cells by antibodies 15D8-1, 16F1-1, 17E9-1, 17E11-1, 41B5-1, and 46B7-1. Efficacy of inhibition can be expressed by the IC50 value, which is the half maximal inhibitory concentration of a substance for the biological process under study, in this case cellular TRPV1-mediated Ca2+ influx. All these antibodies were inhibitory in this regard. From the data we calculated approximate IC50 values of 70 nM for 15D8-1, 170 nM for 41B5-1, 200 nM for 46B7-1, 215 nM for 16F1-1, 230 nM for 17E11-1, and 400 nM for 17E9-1 (
[1183] The antibodies 44E8-1, 40B10-1, 43D6-1 were also tested for their ability to inhibit the capsaicin axis of TRPV1, and each of these antibodies showed an inhibitory effect on the capsaicin axis (data not shown), i.e. showed inhibition of capsaicin-induced activation of TRPV1.
[1184] Ca.sup.2+-imaging was used to measure the inhibitory activity of 15D8-1, 17E11-1, 41B5-1 and 46B7-1 on hTRPV1 heat response in hTRPV1-expressing CHO cells (
[1185] When compared to a vehicle control 15D8-1, 17E11-1 and 41B5-1 did not inhibit heat-induced Ca.sup.2+ uptake in a statistically significant manner. In other words, TRPV1 heat-induced activity remained at approximately 100% (where 100% is that of the vehicle) after antibody treatment (
[1186] The data described in this Example also shows that the antibody 46B7-1 preferentially inhibits capsaicin-induced activation of TRPV1 as opposed to heat-induced activation of TRPV1.
[1187] The ability to preferentially inhibit activation of hTRPV1 by capsaicin as opposed to activation of hTRPV1 by heat indicates a modality-selective pharmacology, which is a highly desired property for a clinically acceptable and useful therapeutic agent.
[1188] In the present study, modality-selective monoclonal antibodies targeting hTRPV1, a clinically important target that has failed using small molecule approaches, have been developed. These antibodies are promising new candidates for TRPV1-targeted pain therapy and are currently pursued as drug candidates.
Example 4—Generation of a Monoclonal Antibody (Antibody Named R4P1-C1)
Materials and Methods
Production of a Monoclonal Antibody (IgG) Using the Phage Display Technology
[1189] The antigen mixture for phage display consisted of 3 different peptides (OTV3, OTV4, OTV5) each synthesized and conjugated to 3 different carriers via N-terminal Cys (BSA, OVA+KLH). Thus there were nine different conjugates in total (i.e. OTV3+BSA, OTV3+OVA, OTV3+KLH, OTV4+BSA, OTV4+OVA, OTV4+KLH, OTV5+BSA, OTV5+OVA and OTV5+KLH). BSA is an abbreviation for bovine serum albumin. OVA is an abbreviation for ovalbumin. KLH is an abbreviation for keyhole limpet hemocyanin.
[1190] The peptide sequences were as follows:
TABLE-US-00046 OTV3: (SEQ ID NO: 16) CIEDGKNDSLPSESTSHRWRGPASRPPDSSYNS OTV4: (SEQ ID NO: 17) CIEDGKNDSLPSESTSHRWRGPACRPPDSSYNS OTV5: (SEQ ID NO: 18) (Pra-)CIEDGKNDSLPSESTSHRWRGPASRPPDSSYNSC(-CONH2)
[1191] The OTV3 and OTV4 peptides used are linear peptides. The OTV5 peptide used is a cyclic peptide.
Biopanning
[1192] Tubes were coated with antigen mixture, then washed, blocked, and washed again. A phage library depleted by exposure to carriers (BSA, OVA+KLH) was then added followed by incubation. The tubes were then washed again and phage binders were eluted with Glycine HCl followed by neutralization. Eluted phages were added to E. Coli culture for amplification, and the amplified phage was collected for a further round of biopanning. After the last biopanning, the polyclonal phage was tested by ELISA.
Polyclonal Phage ELISA
[1193] ELISA plates were coated with antigens conjugated to carriers or carriers alone (control), followed by washing, blocking, washing and addition of amplified eluted phages from the biopanning step. After additional washing, bound phage was detected using anti-phage-HRP antibody.
Monoclonal Phage ELISA
[1194] Single E. coli clones were picked randomly selected during the biopanning step and cultured. Supernatants containing phage were prepared and purified. ELISA plates were coated with antigens conjugated to carriers or carriers alone (control), followed by washing, blocking, washing and addition of purified phage. After additional washing, bound phage was detected using anti-phage-HRP antibody.
Gene Synthesis & Sub-Cloning
[1195] A vector construction (plasmid) was prepared as follows. The cDNA of the variable heavy (VH) and the variable light (VL) sequence from phage clone R4P1-C1 that was identified via phage display were chemically synthesized with optimization for mammalian expression in CHO cells, then sub-cloned into a mammalian cell expression vector in order to obtain the full-length sequences of the heavy (HC) and light (LC) chains of human IgG1.
[1196] Sequences of the antibody R4P1-C1 are set out in Table U herein.
[1197] The IgG type of the R4P1-C1 antibody made in this example is IgG1/kappa.
Expression & Purification of Monoclonal Antibody
[1198] Plasmid DNA was transiently co-transfected into CHO cells. Culture medium was collected after 14 days and recombinant antibodies were then purified on a Protein A/G column into PBS buffer (pH 7.5). Purity was assessed by both reduced and non-reduced SDS-PAGE with Coomassie blue staining.
Results and Discussion
[1199] This example describes the production of a monoclonal antibody by phage display. The phage library used in this case was a commercially available human naïve LiAb-Fab library (high diversity of 2×10E.sup.10 variants). After 4 rounds of biopanning and ELISA tests, 80 clones were selected and 5 different sequences were identified within this group of phage clones, represented by R4P1-C1, #1, #2, #3, and #4. Each of these phage clones were tested by ELISA for binding to each of the individual peptides OTV3, OTV4, and OTV5 each coupled to either BSA, OVA or KLH (a total of 9 peptide-conjugates). This is an important confirmation step to ensure clone specificity because due to their nature phages can sometimes bind unspecifically. In the ELISA tests (monoclonal phage ELISA tests) one phage Fab clone, R4P1-C1, bound specifically and strongly to all three of the peptides (all nine of the peptide-carrier conjugates), whereas the other phage Fab clones appeared to be non-specific binders (see Table GG below, all phages were tested at the same concentration).
TABLE-US-00047 TABLE GG #1 #2 R4P1-C1 #3 #4 1 2 1 2 1 2 1 2 1 2 OTV3-KLH 0.41 0.55 0.41 0.56 3.13 2.94 1.02 1.15 1.65 1.61 OTV4-KLH 0.60 0.68 0.38 0.40 2.81 2.51 0.78 0.88 1.06 0.91 OTV5-KLH 0.94 0.46 0.65 0.58 2.74 2.58 1.51 1.41 1.07 1.08 OTV3-OVA 0.80 0.89 1.03 1.16 2.85 2.72 1.70 1.66 2.19 2.09 OTV4-OVA 0.86 0.85 0.77 0.94 2.84 2.78 1.06 0.91 2.22 1.98 OTV5-OVA 0.62 0.73 0.66 0.61 2.61 2.49 2.06 1.99 2.22 1.80 OTV3-BSA 0.68 0.62 0.47 0.49 2.87 2.65 2.04 2.21 1.89 2.10 OTV4-BSA 0.56 0.52 0.67 0.82 2.95 2.80 2.32 2.11 1.52 1.34 OTV5-BSA 0.53 0.50 0.68 0.81 2.63 2.56 2.25 2.12 1.83 1.45 NC1 0.70 0.64 0.82 1.07 0.06 0.06 2.17 1.91 2.21 2.00 NC2 0.79 0.84 0.39 0.34 0.05 0.04 1.66 2.19 1.84 2.00 NC1, Negative control = coating with mix of 3 carriers alone (BSA + OVA + KLH) NC2, Negative control = coating with buffer only