Nucleic acid molecule having binding affinity to a target molecule and a method for generating the same
20190367921 ยท 2019-12-05
Assignee
Inventors
- Florian Jarosch (Berlin, DE)
- Sven Klussmann (Berlin, DE)
- Simone Sell (Berlin, DE)
- Werner Purschke (Berlin, DE)
- Christian Maasch (Berlin, DE)
- Axel Vater (Berlin, DE)
- Kai Hohlig (Berlin, DE)
Cpc classification
G01N2400/10
PHYSICS
G01N2500/04
PHYSICS
C12N15/111
CHEMISTRY; METALLURGY
G01N33/5308
PHYSICS
C12N2320/13
CHEMISTRY; METALLURGY
C12N15/115
CHEMISTRY; METALLURGY
International classification
C12N15/115
CHEMISTRY; METALLURGY
C12N15/11
CHEMISTRY; METALLURGY
G01N33/53
PHYSICS
Abstract
The present invention is related to a method for generating a nucleic acid molecule capable of binding to a target molecule comprising the following steps: a) providing a reference nucleic acid molecule, wherein the reference nucleic acid molecule is capable of binding to the target molecule and wherein the reference nucleic acid molecule comprises a sequence of nucleotides, wherein the sequence of nucleotides comprises n nucleotides; b) preparing a first level derivative of the reference nucleic acid molecule, wherein the first level derivative of the reference nucleic acid molecule differs from the reference nucleic acid molecule at one nucleotide position, wherein the first level derivative is prepared by replacing the ribonucleotide at the one nucleotide position by a 2-deoxyribonucleotide in case the reference nucleic acid has a ribonucleotide at the nucleotide position and wherein the first level derivative is prepared by replacing the 2-deoxyribonucleotide at the one nucleotide position by a ribonucleotide in case the reference nucleic acid has a 2-deoxyribonucleotide at the nucleotide position and wherein the nucleotide position at which the replacement is made is the modified nucleotide position; and c) repeating step b) for each nucleotide position of the reference nucleic acid molecule, thus preparing a group of first level derivatives of the reference nucleic acid molecule, wherein the group of first level derivatives of the reference nucleic acid molecule consists of n first level derivatives, wherein each of the first level derivatives of the reference nucleic acid molecule differs from the reference nucleic acid molecule by a single nucleotide replacement and wherein each of the first level derivatives of the reference nucleic acid molecule has a single modified nucleotide position which is different from the single modified nucleotide of all of the single modified nucleotide positions of the other first level derivatives of the group of first level derivatives of the reference nucleic acid molecule.
Claims
1.-97. (canceled)
98. An L-nucleic acid molecule capable of binding to a target molecule by a mechanism other than base pairing obtainable by a method comprising the following steps: a) providing a reference L-nucleic acid molecule, wherein the reference L-nucleic acid molecule binds the target molecule, and wherein the reference L-nucleic acid molecule comprises a sequence of L-nucleotides, wherein the sequence of L-nucleotides comprises n L-nucleotides; b) preparing a first level derivative of the reference L-nucleic acid molecule, wherein the first level derivative of the reference L-nucleic acid molecule differs from the reference L-nucleic acid molecule at one nucleotide position; wherein the first level derivative is prepared by replacing 2-deoxyribonucleotide at the one nucleotide position by a ribonucleotide in case the reference L-nucleic acid molecule has a 2-deoxyribonucleotide at the one nucleotide position; wherein the first level derivative is prepared by replacing ribonucleotide at the one nucleotide position by a 2-deoxyribonucleotide in case the reference L-nucleic acid molecule has a ribonucleotide at the one nucleotide position; and wherein the nucleotide position at which the replacement is made is the modified nucleotide position; c) repeating step b) for each nucleotide position of the reference L-nucleic acid molecule, thereby preparing a group of first level derivatives of the reference L-nucleic acid molecule, wherein the group of first level derivatives of the reference L-nucleic acid molecule consists of n first level derivatives, wherein each of the first level derivatives of the reference L-nucleic acid molecule differs from the reference L-nucleic acid molecule by a single nucleotide replacement and wherein each of the first level derivatives of the reference L-nucleic acid molecule has a single modified nucleotide position which is different from the single modified nucleotide of all of the single modified nucleotide positions of the other first level derivatives of the group of first level derivatives of the reference L-nucleic acid molecule; d) determining a binding characteristic of each of the n first level derivatives of the reference L-nucleic acid molecule that binds the target molecule, wherein the binding characteristic comprises binding affinity of the first level derivative(s) of the reference L-nucleic acid molecule that binds the target molecule, wherein the binding affinity is expressed as KD value; and e) identifying first level derivative(s) of the reference L-nucleic acid molecule that binds the target molecule, comprising binding affinity that exceeds binding affinity of the reference L-nucleic acid molecule that binds the target molecule, thereby obtaining L-nucleic acid molecules that bind(s) the target molecule by a mechanism other than base pairing.
99. The L-nucleic acid molecule according to claim 98, wherein first level derivative(s) of the reference L-nucleic acid molecule that binds the target molecule, identified in step e) comprise a binding affinity that exceeds a predetermined threshold value.
100. The L-nucleic acid molecule according to claim 99, wherein the predetermined threshold value is Y with Y being the quotient of(binding affinity of the reference L-nucleic acid molecule)/(binding affinity of a first level derivative) and wherein Y>1, Y2, Y5 or Y10.
101. The L-nucleic acid molecule according to claim 98, wherein the L-nucleic acid molecule comprises at least one modification.
102. The L-nucleic acid molecule according to claim 101, wherein excretion rate of the L-nucleic acid molecule comprising a sequence of L-nucleotides and at least one modification group from an organism is decreased compared to an L-nucleic acid molecule consisting of the sequence of L-nucleotides.
103. The L-nucleic acid molecule according to 101, wherein the L-nucleic acid molecule comprising a sequence of L-nucleotides and at least one modification has an increased retention time in an organism compared to an L-nucleic acid molecule consisting of the sequence of L-nucleotides.
104. An L-nucleic acid molecule capable of binding to a target molecule, wherein the L-nucleic acid molecule has a binding affinity to the target molecule, wherein the binding affinity of the L-nucleic acid molecule to the target molecule is increased compared to the binding affinity of a reference L-nucleic acid molecule to the target molecule, wherein a) the L-nucleic acid molecule comprises a sequence of nucleotides and the reference L-nucleic acid molecule comprises a sequence of L-nucleotides, or b) the L-nucleic acid molecule comprises a sequence of L-nucleotides and at least one modification group and the reference L-nucleic acid molecule comprises a sequence of L-nucleotides and the at least one modification group, wherein the sequence of L-nucleotides of the L-nucleic acid molecule and the sequence of L-nucleotides of the reference L-nucleic acid molecule are at least partially identical with respect to the nucleobase moiety of the L-nucleotides but differ with respect to the sugar moiety of the L-nucleotides, wherein the sequence of L-nucleotides of the L-nucleic acid molecule consists of both L-ribonucleotides and 2-L-deoxyribonucleotides and wherein the sequence of L-nucleotides of the reference L-nucleic acid molecule consists of either L-ribonucleotides or 2-L-deoxyribonucleotides.
105. The L-nucleic acid molecule according to claim 104, wherein the L-nucleic acid molecule and/or the reference L-nucleic acid molecule are antagonists of an activity mediated by the target molecule.
106. The L-nucleic acid molecule according to claim 104, wherein excretion rate of the L-nucleic acid molecule comprising a sequence of L-nucleotides and at least one modification group from an organism is decreased compared to an L-nucleic acid molecule consisting of the sequence of L-nucleotides.
107. The L-nucleic acid molecule according to claim 104, wherein the L-nucleic acid molecule comprising a sequence of L-nucleotides and at least one modification has an increased retention time in an organism compared to an L-nucleic acid molecule consisting of the sequence of L-nucleotides.
108. The L-nucleic acid molecule according to claim 104, wherein the L-nucleic acid molecule comprises at least one modification.
109. The L-nucleic acid molecule according to claim 108, wherein excretion rate of the L-nucleic acid molecule comprising a sequence of L-nucleotides and at least one modification group from an organism is decreased compared to an L-nucleic acid molecule consisting of the sequence of L-nucleotides.
110. The L-nucleic acid molecule according to 108, wherein the L-nucleic acid molecule comprising a sequence of L-nucleotides and at least one modification has an increased retention time in an organism compared to an L-nucleic acid molecule consisting of the sequence of L-nucleotides.
111. The L-nucleic acid molecule according to claim 104, wherein the nucleic acid molecule comprises a method for the treatment and/or prevention of a disease.
112. The nucleic acid molecule according to of claim 104, wherein the nucleic acid molecule comprises a method for the diagnosis of a disease.
113. A pharmaceutical composition comprising the L-nucleic acid molecule according to claim 104 and a pharmaceutically acceptable carrier.
114. A method comprising administering to a subject in need of treatment the L-nucleic acid molecule according to claim 104 and a pharmaceutically acceptable carrier.
115. A method comprising exposing a sample of a subject suspected of comprising a condition to the L-nucleic acid molecule according to claim 104 and determining whether a complex is formed with said L-nucleic acid molecule.
Description
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EXAMPLE 1: NUCLEIC ACID MOLECULE HAVING INCREASED BINDING AFFINITY TO THE TARGET Molecule S1P
[0305] Starting from a nucleic acid molecule binding to S1P which was the result of a development process involving as a starting point the immediate screening product of the SELEX process, the method of the present invention was used in order to improve the binding affinity of the nucleic acid molecule to its target. In the instant case, the nucleic acid molecule binding to S1P was nucleic acid molecule L-S1P-215-F9-002.
[0306] Nucleic acid molecule L-S1P-215-F9-002 is a Spiegelmer, i.e. an L-nucleic acid molecule, which is capable of binding to S1P, has of a nucleotide sequence according to SEQ ID NO: 5 and consists of 44 ribonucleotides.
[0307] The binding characteristics of nucleic acid molecule L-S1P-215-F9-002 was determined by competitive Spiegelmer pull-down assay (as described in Example 9). Nucleic acid molecule L-S1P-215-F9-002 binds S1P with an affinity of 31.5 nM (
[0308] In order to improve the binding characteristics of nucleic acid molecule L-S1P-215-F9-002, derivatives of nucleic acid molecule L-S1P-215-F9-002 were synthesized. Said derivatives were L-nucleic acid molecules having the same sequence of nucleobasesguanine, cytosine, adenine, and uracil or thymine (in the case of a 2deoxyribonucleotide)as nucleic acid molecule L-S1P-215-F9-002, however, differed at a single position as to the sugar moiety of the nucleotide which was a 2-deoxyribonucleotide rather than a ribonucleotide. In accordance therewith, derivative 1 had a 2-deoxyribonucleoside at position 1 of the nucleotide sequence according to SEQ ID NO: 6, derivative 2 had a 2-deoxyribonucleotide at position 2 of the nucleotide sequence according to SEQ ID NO: 7, etc. Because nucleic acid molecule L-SIP-215-F9-002 consists of 44 nucleotides a total of 44 derivatives was synthesized in order to provide a complete set of all possible derivatives of nucleic acid molecule L-S1P-215-F9-002 carrying a single ribonucleotide to 2-deoxyribonucleotide substitution. Said complete set of derivatives is shown in
[0309] The binding affinity to S1P of each derivative of said complete set of derivatives of nucleic acid molecule L-S1P-215-F9-002 was determined using the competitive pull-down assay described in Example 9, and compared to the binding affinity of nucleic acid molecule L-S1P-215-F9-002 (
[0310] As may be taken from
[0311] As may be taken from said figures, derivatives L-S1P-215-F9-002-D01, L-S1P-215-F9-002-D11, L-S1P-215-F9-002-D19, L-S1P-215-F9-002-D21, L-S1P-215-F9-002-D22, L-S1P-215-F9-002-D32 which have a 2-deoxyribonucleotide at positions 1, 11, 19, 21, 22, and 32, respectively, belong to the first group of derivatives, i.e. derivatives of nucleic acid molecule L-S1P-215-F9-002 where substitution of a ribonucleotide by a 2-deoxyribonucletoide results in an improved binding affinity for S1P (
[0312] Derivatives L-S1P-215-F9-002-D05, L-S1P-215-F9-002-D12, L-S1P-215-F9-002-D13, L-S1P-215-F9-002-D14, L-S1P-215-F9-002-D15, L-S1P-215-F9-002-D16, L-S1P-215-F9-002-D39, L-S1P-215-F9-002-D40, L-S1P-215-F9-002-D41, L-S1P-215-F9-002-D42 and L-S1P-215-F9-002-D43 which have a 2-deoxyribonucleotide at positions 5, 12, 13 14, 15, 16, 39, 40, 41, 42 and 43, respectively, belong to the second group of derivatives, i.e. derivatives where substitution of a ribonucleotide by a 2-deoxyribonucletoide does not affect the binding affinity for S1P. Accordingly, positions 5, 12, 13, 14, 15, 16, 39, 40, 41, 42 and 43 are not suitable to confer improved binding affinity nor do they have negative impact on binding affinity to S1P compared to nucleic acid molecule L-S1P-215-F9-002 (
[0313] Finally, derivatives were obtained which resulted in reduced binding affinity or a profound loss of binding. These derivatives, namely L-S1P-215-F9-002-D02, L-S1P-215-F9-002-D03, L-S1P-215-F9-002-D04, L-S1P-215-F9-002-D06, L-S1P-215-F9-002-D07, L-S1P-215-F9-002-D08, L-S1P-215-F9-002-D09, L-S1P-215-F9-002-D10, L-S1P-215-F9-002-D17, L-S1P-215-F9-002-D18, L-S1P-215-F9-002-D20, L-S1P-215-F9-002-D23, L-S1P-215-F9-002-D24, L-S1P-215-F9-002-D25, L-S1P-215-F9-002-D26, L-S1P-215-F9-002-D27, L-S1P-215-F9-002-D28, L-S1P-215-F9-002-D29, L-S1P-215-F9-002-D30, L-S1P-215-F9-002-D31, L-S1P-215-F9-002-D33, L-S1P-215-F9-002-D34, L-S1P-215-F9-002-D35, L-S1P-215-F9-002-D36, L-S1P-215-F9-002-D37, L-S1P-215-F9-002-D38 and L-S1P-215-F9-002-D44 which have a 2-deoxyribonucletoide at position 2, 3, 4, 6, 7, 8, 9, 10, 17, 18, 20, 23, 24, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37, 38 and 44, respectively, belong to the third group of derivatives, i.e. derivatives of nucleic acid molecule L-S1P-215-F9-002 where substitution of a ribonucleotide by a 2-deoxyribonucletoide negatively affects the binding affinity to S1P (
[0314] In order to assess whether the binding affinity of the derivatives of nucleic acid molecule L-S1P-215-F9-002 can be further increased by introducing more than one substitution a group of further derivatives was generated (
[0315] Competitive Spiegelmer pull-down assays of these derivatives showed that combining ribonucleotide to 2-deoxyribonucleotide substitutions at multiple positions of the L-S1P-215-F9-002 Spiegelmer resulted in a further improvement of binding affinity for S1P. A Spiegelmer containing two substitutions, namely adenosine-5-phosphate to 2 deoxyadenosine-5-phosphate at position 19 and guanosine-5-phosphate to 2-deoxyguanosine-5-phosphate at position 21 (termed L-S1P-215-F9-002-D21-19) showed improved binding affinity compared to L-S1P-215-F9-002 and as well to L-S1P-215-F9-002-D21 containing a single substitution, namely guanosine-5-phosphate to 2-deoxyguanosine-5-phosphate at position 21 (
[0316] In order to prove and compare the functionality of Spiegelmers L-S1P-215-F9-002 and L-S1P-215-F9-002-D01-19-21-32 both nucleic acid molecules were synthesized comprising an amino-group at their 5-ends. To the amino-modified Spiegelmers a 40 kDa PEG-moiety was coupled leading to Spiegelmers 5-40 kDa-PEG-L-S1P-215-F9-002 and 5-40 kDa-PEG-L-S1P-215-F9-002-D01-19-21-32 (also referred to as NOX-S93). Synthesis and PEGylation of the Spiegelmer is described in Example 7.
[0317] An in vitro cell-culture assay (protocol see Example 11) confirmed that improved affinity to S1P translates into an enhanced inhibition of S1P function. 5-40 kDa-PEG-L-S1P-215-F9-002 and 5-40 kDa-PEG-L-S1P-215-F9-002-D01-19-21-32 (also referred to as NOX-S93) inhibited S1P-induced arrestin recruitment in a reporter cell line expressing human S1P-receptor EDG1 with IC.sub.50 values of 22.5 nM and 10.3 nM, respectively (
EXAMPLE 2: NUCLEIC ACID MOLECULE HAVING INCREASED BINDING AFFINITY TO THE TARGET MOLECULE HUMAN CGRP
[0318] Starting from a nucleic acid molecule binding to CGRP which was the result of a development process involving as a starting point the immediate screening product of the SELEX process, the method of the present invention was used in order to improve the binding affinity of the nucleic acid molecule to its target. In the instant case, the nucleic acid molecule binding to human CGRP was nucleic acid molecule 226-F2-001.
[0319] Nucleic acid molecule 226-F2-001 is a Spiegelmer, i.e. a L-nucleic acid molecule, which is capable of binding to human CGRP, has a nucleotide sequence according to SEQ ID NO: 55 and consists of 50 ribonucleotides.
[0320] The binding characteristics of nucleic acid molecule 226-F2-001 were determined by surface plasmon resonance measurement (as described in Example 8). Nucleic acid molecule 226-F2-001 binds human CGRP with an affinity of 2.6 nM (
[0321] In order to improve the binding characteristics of nucleic acid molecule 226-F2-001, derivatives of nucleic acid molecule 226-F2-001 were synthesized. Said derivatives were L-nucleic acid molecules having the same sequence of nucleobasesguanine, cytosine, adenine, and uracil or thymine (in the case of a 2deoxyribonucleotide)as nucleic acid molecule 226-F2-001, however, differed at a single position as to the sugar moiety of the nucleotides which was a 2-deoxyribonucleotide rather than a ribonucleotide. In accordance therewith, derivative 1 (termed 226-F2-001-D01) had a 2-deoxyribonucleoside at position 1 of the nucleotide sequence according to SEQ ID NO: 56, derivative 2 (termed 226-F2-001-D02) had a 2-deoxyribonucleotide at position 2 of the nucleotide sequence according to SEQ ID NO: 57, etc. Because nucleic acid molecule 226-F2-001 consisting of 50 nucleotides a total of 50 derivatives were synthesized in order to provide a complete set of all possible derivatives of nucleic acid molecule 226-F2-001 carrying a single ribonucleotide to 2-deoxyribonucleotide substitution. Said complete set of derivatives is shown in
[0322] The binding affinity to human CGRP of each derivative of said complete set of derivatives of nucleic acid molecule 226-F2-001 was determined by surface plasmon resonance measurement described in Example 8, and compared to the binding affinity of nucleic acid molecule 226-F2-001. From a set of at least 5 individually determined K.sub.D values of 226-F2-001 the mean value was calculated (mean+/ standard error). K.sub.D values of individual derivatives were determined and changes in affinity are given as x-fold improvement compared to mean K.sub.D of 226-F2-001, wherein the value of x-fold improvement is the quotient of the K.sub.D of 226-F2 001 and the derivative of 226-F2 001. The determined standard error indicates a cutting point for positive hits. The data of the x-fold improved affinities is indicated in
[0323] As may be taken from
[0324] As may be taken from said figures, derivatives 226-F2-001-D03, 226-F2-001-D05, 226-F2-001-D08, 226-F2-001-D09, 226-F2-001-D14, 226-F2-001-D16, 226-F2-001-D19, 226-F2-001-D22, 226-F2-001-D23, 226-F2-001-D24, 226-F2-001-D25, 226-F2-001-D26, 226-F2-001-D28, 226-F2-001-D30, 226-F2-001-D33, 226-F2-001-D34, 226-F2-001-D37, 226-F2-001-D39, 226-F2-001-D41, 226-F2-001-D42, 226-F2-001-D44, 226-F2-001-D45, 226-F2-001-D46, 226-F2-001-D47, 226-F2-001-D48, 226-F2-001-D49, 226-F2-001-D50 which have a 2-deoxyribonucleotide at position 03, 05, 08, 09, 14, 16, 19, 22, 23, 24, 25, 26, 28, 30, 33, 34, 37, 39, 41, 42, 44, 45, 46, 47, 48, 49, 50, respectively, belong to the first group of derivatives, i.e. derivatives of nucleic acid molecule 226-F2-001 where substitution of a ribonucleotide by a 2-deoxyribonucletoide results in an improved binding affinity for human CGRP (
[0325] Derivatives 226-F2-001-D04 and 226-F2-001-D27 which have a 2-deoxyribonucleotide at position 4 and 27, respectively, belong to the second group of derivatives, i.e. derivatives where the substitution of a ribonucleotide by a 2-deoxyribonucletoide does not affect the binding affinity for human CGRP. Accordingly, positions 4 and 27 are not suitable to confer improved binding affinity nor do they have a negative impact on binding affinity to human CGRP compared to nucleic acid molecule 226-F2-001 (
[0326] Finally, derivatives were obtained which resulted in reduced binding affinity or a profound loss of binding. These derivatives, namely 226-F2-001-D01, 226-F2-001-D02, 226-F2-001-D06, 226-F2-001-D07, 226-F2-001-D10, 226-F2-001-D11, 226-F2-001-D12, 226-F2-001-D13, 226-F2-001-D15, 226-F2-001-D17, 226-F2-001-D18, 226-F2-001-D20, 226-F2-001-D21, 226-F2-001-D29, 226-F2-001-D31, 226-F2-001-D32, 226-F2-001-D35, 226-F2-001-D36, 226-F2-001-D38, 226-F2-001-D40 and 226-F2-001-D43, which have a 2-deoxyribonucleotide at position 1, 2, 6, 7, 10, 11, 12, 13, 15, 17, 18, 20, 21, 29, 31, 32, 35, 36, 38, 40 and 43, respectively, belong to the third group of derivatives, i.e. derivatives of nucleic acid molecule 226-F2-001 where substitution of a ribonucleotide by a 2-deoxyribonucletoide negatively affects the binding affinity to human CGRP (
[0327] In order to assess whether the binding affinity of the derivatives of nucleic acid molecule 226-F2-001 can be further increased by introducing more than one substitution another derivative was generated. Said derivative started from the first group of derivatives where substitution of a ribonucleotide by a 2-deoxyribonucletoide resulted in an improved binding affinity for human CGRP. Starting from nucleic acid molecule 226-F2-001, the derivative had substitutions of ribonucleotides by 2-deoxyribonucleotides at the two positions 41 and 44 (referred to as 226-F2-001-D41/D44), i.e. those positions thatbesides position 19conferred the strongest improvement in binding affinity to nucleic acid molecule 226-F2-001.
[0328] Surface plasmon resonance measurement of 226-F2-001-D41/44 showed that combining ribonucleotide to 2-deoxyribonucleotide substitutions at more than one position of 226-F2-001, namely cytidine-5-phosphate to 2-deoxycytidine-5-phosphate at position 41 and 44, resulted in a further improvement of binding affinity to human CGRP compared to derivatives containing a single substitution, i.e. 226-F2-001-D41 or 226-F2-001-D44 (
[0329] In order to prove and compare the functionality of Spiegelmers 226-F2-001 and 226-F2-001-D41 both nucleic acid molecules were synthesized comprising an amino-group at their 5-ends. To the amino-modified Spiegelmers a 40 kDa PEG-moiety was coupled leading to Spiegelmers 226-F2-001-540 kDa-PEG and 226-F2-001-D41-540 kDa-PEG (also referred to as NOX-L41). Synthesis and PEGylation of the Spiegelmer is described in Example 7.
[0330] An in vitro cell-culture assay (protocol see Example 12) confirmed functionality for both Spiegelmers by showing that they effectively inhibited human CGRP-induced cAMP production in a reporter cell line expressing human CGRP-receptor (
EXAMPLE 3: NUCLEIC ACID MOLECULE HAVING INCREASED BINDING AFFINITY TO THE TARGET MOLECULE HUMAN C5A
[0331] Starting from a nucleic acid molecule binding to C5a which was the result of a development process involving as a starting point the immediate screening product of the SELEX process, the method of the present invention was used in order to improve the binding affinity of the nucleic acid molecule to its target. In the instant case, the nucleic acid molecule binding to human C5a was nucleic acid molecule NOX-D19001.
[0332] Nucleic acid molecule NOX-D19001 is Spiegelmer, i.e. an L-nucleic acid molecule, which is capable of binding to human C5a, has of a nucleotide sequence according to SEQ ID NO: 107 and consists of 44 ribonucleotides.
[0333] The binding characteristics of nucleic acid molecule NOX-D19001 was determined by surface plasmon resonance measurement (as described in Example 8). Nucleic acid molecule NOX-D19001 binds human C5a with an affinity of 1.4 nM as also shown in
[0334] In order to improve the binding characteristics of nucleic acid molecule NOX-D19001, derivatives of nucleic acid molecule NOX-D19001 were synthesized. Said derivatives were L-nucleic acid molecules having the same sequence of nucleobasesguanine, cytosine, adenine, and uracilas nucleic acid molecule NOX-D19001, however, differed at a single position as to the sugar moiety of the nucleotides which was a 2-deoxyribonucleotide rather than a ribonucleotide. In accordance therewith, derivative 1 (termed NOX-D19001-D01) had a 2-deoxyribonucleoside at position 1 of the nucleotide sequence according to SEQ ID NO: 108 derivative 2 (termed NOX-D19001-D02) had a 2-deoxyribonucleotide at position 2 of the nucleotide sequence according to SEQ ID NO: 109 etc. Because of nucleic acid molecule NOX-D19001 consisting of 44 nucleotides a total of 44 derivatives were synthesized in order to provide a complete set of all possible derivatives of nucleic acid molecule meeting the above requirement of a single substitution of a ribonucleotide by a 2-deoxyribonucleotide. Said complete set of derivatives is shown in
[0335] The binding affinity to human C5a of each derivative of said complete set of derivatives of nucleic acid molecule NOX-D19001 was determined by surface plasmon resonance measurement described in Example 8, and compared to the binding affinity of nucleic acid molecule NOX-D19001. From a set of at least 5 individual determined K.sub.D values of NOX-D19001 the mean value was calculated (meanstandard error). K.sub.D values of individual derivatives were determined and changes in affinity are given as x-fold improvement compared to mean NOX-D19001, wherein the value of the x-fold improvement is the quotient of the K.sub.D of NOX-D19001 and the derivative of NOX-D19001. The determined standard error indicates a cutting point for positive hits. The data of the x-fold improved affinity is indicated in
[0336] As may be taken from
[0337] As may be taken from said figures, derivatives NOX-D19001-D01, NOX-D19001-D02, NOX-D19001-D09, NOX-D19001-D16, NOX-D19001-D17, NOX-D19001-D22, NOX-D19001-D25, NOX-D19001-D29, NOX-D19001-D30, NOX-D19001-D32, NOX-D19001-D40, NOX-D19001-D42, and NOX-D19001-D43 which have a 2-deoxyribonucleotide at positions 1, 2, 9, 16, 17, 22, 25, 29, 30, 32, 40, 42, and 43, respectively, belong to the first group of derivatives, i.e. derivatives where the substitution of a ribonucleotide by a 2-deoxyribonucletoide results in an improved binding affinity for human C5a (
[0338] Derivatives NOX-D19001-D03, NOX-D19001-D23, NOX-D19001-D26, NOX-D19001-D35, NOX-D19001-D38, NOX-D19001-D39 and NOX-D19001-D44 which have a 2-deoxyribonucleotide at positions 3, 23, 26, 35, 38, 39 and 44, respectively, belong to the second group of derivatives, i.e. derivatives where the substitution of a ribonucleotide by a 2-deoxyribonucleotide does not change or affect the binding affinity for human C5a. The positions 35 and 44 result in an improved binding affinity of NOX-D19001 for human C5a. Accordingly, positions 3, 23, 38, and 39 are not suitable to confer to nucleic acid molecule NOX-D19001 an improved binding affinity to human C5a, however, do not have a negative impact on binding affinity of nucleic acid molecule NOX-D19001 to human C5a either.
[0339] Finally, derivatives were obtained which resulted in reduced binding affinity or a profound loss of binding affinity. These derivatives, namely NOX-D19001-D04, NOX-D19001-D05, NOX-D19001-D06, NOX-D19001-D07, NOX-D19001-D08, NOX-D19001-D10, NOX-D19001-D11, NOX-D19001-D12, NOX-D19001-D13, NOX-D19001-D14, NOX-D19001-D15, NOX-D19001-D18, NOX-D19001-D19, NOX-D19001-D20, NOX-D19001-D21, NOX-D19001-D24, NOX-D19001-D27, NOX-D19001-D28, NOX-D19001-D31, NOX-D19001-D33, NOX-D19001-D34, NOX-D19001-D36, NOX-D19001-D38 and NOX-D19001-D41, accordingly, belong to the third group of derivatives, i.e. those derivatives of nucleic acid molecule NOX-D19001 where the substitution of a ribonucleotide by a 2-deoxyribonucletoidenegativelyaffects the binding affinity for human C5a. Accordingly, positions 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 24, 27, 28, 31, 33, 34, 36 and 41, have a negative impact on the binding affinity of nucleic acid molecule NOX-D19001 to human C5a.
[0340] In order to assess whether the binding affinity of the derivatives of nucleic acid molecule L-NOX-D19001 can be further increased by introducing more than one substitution a group of further derivatives was generated. Such group of further derivatives started from the above first group of derivatives comprising derivatives where the substitution of a ribonucleotide by a 2-deoxyribonucletoide resulted in an improved binding affinity for human C5a. Starting from nucleic acid molecule NOX-D19001, the derivatives of the further group of derivatives had a substitution of a ribonucleotide by a 2-deoxyribonucleotide at at least two of positions 9, 30, 32 and 40, i.e. those positions which have been proven to be suitable to confer to nucleic acid molecule NOX-D19001 an improved binding affinity to human C5a.
[0341] Surface plasmon resonance measurement of these representative example showed, as depicted in
[0342] Spiegelmers NOX-D19001-D09-30, NOX-D19001-D09-32, NOX-D19001-D09-40, NOX-D19001-D30-32, NOX-D19001-D30-40 and NOX-D19001-D32-40 all containing two substitutions, namely uridine-5-phosphate to 2-deoxy-uridine-5-phosphate at position 9/30, 9/32, 9/40, 30/32, 30/40 and 32/40 showed better binding affinity as the molecules containing one substitution, namely uridine-5-phosphate to 2-deoxy-uridine-5-phosphate at position 9, 30, 32 or 40 (
[0343] Spiegelmers NOX-D19001-D09-30-32, NOX-D19001-D09-30-40, NOX-D19001-D09-32-40 and NOX-D19001-D30-32-40 were synthesized to test whether three substitutions in the nucleic acid molecule NOX-D19001, namely uridine-5-phosphate to 2-deoxy-uridine-5-phosphate at position 9/30/32, 9/30/40, 9/32/40 and 30/32/40 led to further improved binding affinity in comparison to the NOX-D19001-D09-30, NOX-D19001-D09-32, NOX-D19001-D09-40, NOX-D19001-D30-32, NOX-D19001-D30-40 and NOX-D19001-D32-40 all containing two substitutions. Spiegelmers NOX-D19001-D09-30-40 and NOX-D19001-D09-32-40 all containing three substitutions, namely uridine-5-phosphate to 2-deoxy-uridine-5-phosphate at position 9/30/40 and 9/32/40 showed better binding affinity as the molecules containing two substitution, namely uridine-5-phosphate to 2-deoxy-uridine-5-phosphate at position 9, 30, 32 or 40 (
[0344] Combining the four positions 9, 30, 32 or 40 (nucleic acid molecule NOX-D19001-D09-30-32-40) for substitution, namely uridine-5-phosphate to 2-deoxy-uridine-5-phosphate at position 9/30/32/40 led to further improvement in comparison to the Spiegelmers NOX-D19001-D09-30-40 and NOX-D19001-D09-32-40 all containing three substitutions (
[0345] Substitution of an additional ribonucleotide by 2-deoxyribonucleotide at position 16 or 17 of NOX-D19001-D09-30-32-40 had a further positive effect on the binding affinity to human C5a (see NOX-D19001-D09-16-30-32-40 and see NOX-D19001-D09-17-30-32-40
[0346] In order to prove and compare the functionality of spiegelmers NOX-D19001 and NOX-D19001-6DNA both nucleic acid molecules were in an in vitro cell-culture assay (protocol see Example 14). As shown in
[0347] As shown before, the substitution of multiple ribonucleotides by 2-deoxyribonucleotides in the nucleic acid molecule NOX-D19-001 led to improved affinity to human C5a. However, such improvement can only be reached if the multiple substitutions are the result of single substitutions that already lead an improvement in binding to human C5a. The substitution of the ribonucleotide at position 7 by a 2-deoxyribonucleotides led to reduced affinity (see
EXAMPLE 4: DERIVATIVES OF NUCLEIC ACID MOLECULE NOX-G11STABI2 HAVING INCREASED BINDING AFFINITY TO THE TARGET MOLECULE GLUCAGON
[0348] Starting from a nucleic acid molecule binding to glucagon which was the result of a development process involving as a starting point the immediate screening product of the SELEX process, the method of the present invention was used in order to improve the binding affinity of the nucleic acid molecule to its target. In the instant case, the nucleic acid molecule binding to human glucagon was nucleic acid molecule NOX-G11stabi2.
[0349] Nucleic acid molecule NOX-G11stabi2 is a spiegelmer, i.e. an L-nucleic acid molecule, which is capable of binding to human glucagon, has of a nucleotide sequence according to SEQ ID NO: 172 and consists of 54 ribonucleotides.
[0350] The binding characteristics of nucleic acid molecule NOX-G11 stabi2 was determined by surface plasmon resonance measurement (as described in Example 8). Nucleic acid molecule NOX-G11 stabi2 binds human glucagon with an affinity of 67.1 nM as also shown in
[0351] In order to improve the binding characteristics of nucleic acid molecule NOX-G11 stabi2, derivatives of nucleic acid molecule NOX-G11 stabi2 were synthesized. Said derivatives were L-nucleic acid molecules having the same sequence of nucleobasesguanine, cytosine, adenine, and uracil or alternatively thymine (in the case of a 2deoxyribonucleotide)as nucleic acid molecule NOX-G11 stabi2, however, differed at a single position as to the sugar moiety of the nucleotides which was a 2-deoxyribonucleotide rather than a ribonucleotide. In accordance therewith, derivative 1 (termed NOX-G11-D01) had a 2-deoxyribonucleoside at position 1 of the nucleotide sequence according to SEQ ID NO: 172 derivative 2 (termed NOX-G11-D02) had a 2-deoxyribonucleotide at position 2 of the nucleotide sequence according to SEQ ID NO: 173 etc. Because of nucleic acid molecule NOX-G11 stabi2 consisting of 54 nucleotides a total of 54 derivatives were synthesized in order to provide a complete set of all possible derivatives of nucleic acid molecule meeting the above requirement of a single substitution of a 2-ribonucleotide by a 2-deoxyribonucleotide. Said complete set of derivatives is shown in
[0352] The binding affinity to human Glucagon of each derivative of said complete set of derivatives of nucleic acid molecule NOX-G11 stabi2 was determined by surface plasmon resonance measurement described in Example 8, and compared to the binding affinity of nucleic acid molecule NOX-G11 stabi2. From a set of at least 5 individual determined K.sub.D values of NOX-G11stabi2 the mean value was calculated (meanstandard error). K.sub.D values of individual derivatives were determined and changes in affinity are given as x-fold improvement compared to mean NOX-G11stabi2, wherein the value of the x-fold improvement is the quotient of the K.sub.D of NOX-G11stabi2 and the derivative of NOX-G11stabi2. The determined standard error indicates a cutting point for positive hits. The data of the x-fold improved affinity is indicated in
[0353] As may be taken from
[0354] As may be taken from said figures, derivatives NOX-G11-D01, NOX-G11-D02, NOX-G11-D03, NOX-G11-D04, NOX-G11-D05, NOX-G11-D06, NOX-G11-D07, NOX-G11-D08, NOX-G11-D09, NOX-G11-D10, NOX-G11-D12, NOX-G11-D13, NOX-G11-D14, NOX-G11-D15, NOX-G11-D16, NOX-G11-D18, NOX-G11-D19, NOX-G11-D20, NOX-G11-D21, NOX-G11-D22, NOX-G11-D23, NOX-G11-D24, NOX-G11-D25, NOX-G11-D26, NOX-G11-D27, NOX-G11-D28, NOX-G11-D29, NOX-G11-D30, NOX-G11-D32, NOX-G11-D36, NOX-G11-D38, NOX-G11-D44, NOX-G11-D46, NOX-G11-D48 and NOX-G11-D53 which have a 2-deoxyribonucleotide at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 36, 38, 44, 46, 48 and 53, respectively, belong to the first group of derivatives, i.e. derivatives where the substitution of a ribonucleotide by a 2-deoxyribonucletoide results in an improved binding affinity for human glucagon (
[0355] Derivatives NOX-G11-D11, NOX-G11-D17, NOX-G11-D31, NOX-G11-D33, NOX-G11-D34, NOX-G11-D35, NOX-G11-D39, NOX-G11-D40, NOX-G11-D43, NOX-G11-D45, NOX-G11-D50 and NOX-G11-D52, which have a 2-deoxyribonucleotide at positions 11, 17, 31, 33, 34, 35, 39, 40, 43, 45, 50 and 52, respectively, belong to the second group of derivatives, i.e. derivatives where the substitution of a ribonucleotide by a 2-deoxyribonucletoide does not change or affect the binding affinity for human glucagon. Accordingly, positions 11, 17, 31, 33, 34, 35, 39, 40, 43, 45, 50 and 52 are not suitable to confer to nucleic acid molecule NOX-G11stabi2 an improved binding affinity to human glucagon, however, do not have a negative impact on binding affinity of nucleic acid molecule NOX-G11stabi2 to human glucagon either.
[0356] Finally, derivatives were obtained which resulted in reduced binding affinity or a profound loss of binding affinity. These derivatives, namely NOX-G11-D37, NOX-G11-D41, NOX-G11-D42, NOX-G11-D47, NOX-G11-D49, NOX-G11-D51 and NOX-G11-D54, accordingly, belong to the third group of derivatives, i.e. those derivatives of nucleic acid molecule NOX-G11stabi2 where the substitution of a ribonucleotide by a 2-deoxyribonucletoidenegatively affects the binding affinity for human Glucagon. Accordingly, positions 37, 41, 42, 47, 49, 51 and 54, have a negative impact on the binding affinity of nucleic acid molecule NOX-G11stabi2 to human Glucagon.
EXAMPLE 5: DERIVATIVES OF NUCLEIC ACID MOLECULE 259-H6-002 HAVING INCREASED BINDING AFFINITY TO THE TARGET MOLECULE GLUCAGON
[0357] Starting from a nucleic acid molecule binding to glucagon which was the result of a development process involving as a starting point the immediate screening product of the SELEX process, the method of the present invention was used in order to improve the binding affinity of the nucleic acid molecule to its target. In the instant case, the nucleic acid molecule binding to human glucagon was nucleic acid molecule 259-H6-002.
[0358] Nucleic acid molecule 259-H6-002, a Spiegelmer, i.e. an L-nucleic acid molecule, which is capable of binding to human glucagon, has a nucleotide sequence according to SEQ ID NO: 287 and consists of 46 2-deoxyribonucleotides.
[0359] The binding characteristics of nucleic acid molecule 259-H6-002 was determined by surface plasmon resonance measurement (as described in Example X). Nucleic acid molecule 259-H6-002 binds human glucagon with an affinity of 10.9 nM as also shown in
[0360] In order to improve the binding characteristics of nucleic acid molecule 259-H6-002, derivatives of nucleic acid molecule 259-H6-002 were synthesized. Said derivatives were L-nucleic acid molecules having the same sequence of nucleobasesguanine, cytosine, adenine, and uracilas nucleic acid molecule 259-H6-002, however, differed at a single position as to the sugar moiety of the nucleotides which was a 2-ribonucleotide rather than a desoxyribonucleotide. In accordance therewith, derivative 1 (termed 259-H6-002-R01) had a ribonucleoside at position 1 of the nucleotide sequence according to SEQ ID NO: 288 derivative 2 (termed 259-H6-002-R02) had a 2-ribonucleotide at position 2 of the nucleotide sequence according to SEQ ID NO: 289 etc. Because of nucleic acid molecule 259-H6-002 consisting of 46 nucleotides a total of 46 derivatives were synthesized in order to provide a complete set of all possible derivatives of nucleic acid molecule meeting the above requirement of a single substitution of a 2-deoxyribonucleotide by a ribonucleotide. Said complete set of derivatives is shown in
[0361] The binding affinity to human glucagon of each derivative of said complete set of derivatives of nucleic acid molecule 259-H6-002 was determined by surface plasmon resonance measurement described in Example 8, and compared to the binding affinity of nucleic acid molecule 259-H6-002, whereby the binding affinity to human glucagon of each derivative of said complete set of derivatives of nucleic acid molecule 259-H6-002 was determined by surface plasmon resonance measurement described in Example 8, and compared to the binding affinity of nucleic acid molecule 259-H6-002. From a set of at least 5 individual determined K.sub.D values of 259-H6-002 the mean value was calculated (mean+/standard error). K.sub.D values of individual derivatives were determined and changes in affinity are given as x-fold improvement compared to mean 259-H6-002, wherein the value of the x-fold improvement is the quotient of the K.sub.D of 259-H6-002 and the derivative of 259-H6-002. The determined standard error indicates a cutting point for positive hits. The data of the x-fold improved affinity is indicated in
[0362] As may be taken from
[0363] As may be taken from said figures, derivatives 259-H6-002-R8, 259-H6-002-R13, 259-H6-002-R22, 259-H6-002-R24, 259-H6-002-R30, 259-H6-002-R31, 259-116-002-R36, 259-H6-002-R38, 259-H6-002-R39, and 259-H6-002-R44, which have a ribonucleotide at positions 8, 13, 22, 24, 30, 31, 36, 38, 39, and 44, respectively, belong to the first group of derivatives, i.e. derivatives where the substitution of a 2-deoxyribonucleotide by a ribonucleotide results in an improved binding affinity for human glucagon (
[0364] Derivatives 259-H6-002-R04, 259-H6-R06, and 259-H6-R46 . . . which have a 2-ribonucleotide at positions 4, 6, and 46 respectively, belong to the second group of derivatives, i.e. derivatives where the substitution of a 2desoxyribonucleotide by a 2-ribonucleotide does not significantly change or affect the binding affinity for human glucagon. Accordingly, positions 4, 6, and 46 are not suitable to confer to nucleic acid molecule 259-H6-002 an improved binding affinity to human glucagon, however, do not have a negative impact on binding affinity of nucleic acid molecule 259-H6-002 to human glucagon either.
[0365] Derivatives 259-H6-R09 and 259-H6-R45 show a biphasic binding behaviour on the Biacore. Therefore the improvement factor were judged to be artificial and positions 9 and 45 were not further considered for improvement of binding affinity.
[0366] Finally, 31 derivatives were obtained which resulted in reduced binding affinity or a profound loss of binding affinity. These derivatives, namely 259-H6-002-R01, 259-H6-002-R02, 259-H6-002-R03, 259-H6-002-R05, 259-H6-002-R07, 259-H6-002-R10, 259-H6-002-R11, 259-H6-002-R12, 259-H6-002-R14, 259-H6-002-R15, 259-H6-002-R16, 259-H6-002-R17, 259-116-002-R18, 259-H6-002-R19, 259-H6-002-R20, 259-H6-002-R21, 259-H6-002-R23, 259-H6-002-R25, 259-H6-002-R26, 259-H6-002-R27, 259-H6-002-R28, 259-H6-002-R29, 259-H6-002-R32, 259-H6-002-R33, 259-H6-002-R34, 259-H6-002-R35, 259-H6-002-R37, 259-H6-002-R40, 259-H6-002-R41, 259-H6-002-R42, 259-H6-002-R43 accordingly, belong to the third group of derivatives, i.e. those derivatives of nucleic acid molecule 259-H6-002 where the substitution of a 2-deoxyribonucleotide by a ribonucleotidenegativelyaffects the binding affinity for human glucagon. Accordingly, positions 1, 2, 3, 5, 7, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 23, 25, 26, 27, 28, 29, 32, 33, 34, 35, 37, 40, 41, 42, and 43 have a negative impact on the binding affinity of nucleic acid molecule 259-H6-002 to human glucagon.
[0367] In order to assess whether the binding affinity of the derivatives of nucleic acid molecule L-259-H6-002 can be further increased by introducing more than one substitution a group of further derivatives was generated. Such group of further derivatives started from the above first group of derivatives comprising derivatives where the substitution of a 2-deoxyribonucleotide by a ribonucleotide resulted in an improved binding affinity for human glucagon. Starting from nucleic acid molecule 259-H6-002, the derivatives of the further group of derivatives had a substitution of a 2-deoxyribonucleotide by a ribonucleotide at at least two of positions 13, 24, 30 and 36, i.e. those positions which have been proven to be suitable to confer to nucleic acid molecule 259-H6-002 an improved binding affinity to human glucagon.
[0368] Surface plasmon resonance measurement of these representative example showed, as depicted in
[0369] Spiegelmers 259-H6-002-R13_R24 and 259-H6-002-R13_R36, all containing two substitutions, namely deoxyadenosine-5-phosphate to adenosine-5-phosphate at position 13, deoxyguanosine to guanosine-5-phosphate at position 24 and/or thymidine-5-phosphate to uridine-5-phosphate at position 36, showed better binding affinity as the molecules containing one substitution (
[0370] Spiegelmer 259-H6-002-R13_R24_R36 was synthesized to test whether three substitutions in the nucleic acid molecule 259-H6-002, namely deoxyadenosine-5-phosphate to adenosine-5-phosphate at position 13, deoxyguanosine-5-phosphate to guanosine-5-phosphate at position 24 and thymidine-5-phosphate to uridine-5-phosphate at position 36, led to further improved binding affinity in comparison to the spiegelmers 259-H6-002-R13_R24 and 259-H6-002-R13_R36, all containing two substitutions (
[0371] Combining the four positions 13, 24, 30 and 36 (nucleic acid molecule 259-H6-002-R13_R24_R30_R36) for substitution, namely deoxyadenosine-5-phosphate to adenosine-5-phosphate at position 13, deoxyguanosine-5-phosphate to guanosine-5-phosphate at position 24 and 30 and thymidine-5-phosphate to uridine-5-phosphate at position 36, led to a slight improvement in comparison to the Spiegelmer 259-H6-002-R13_R24_R36 containing three substitutions (
[0372] In order to prove and compare the functionality of Spiegelmers 259-H6-002, 259-H6-002-R13 and 259-H6-002-R13_R24_R36 all nucleic acid molecules were tested in an in vitro cell-culture assay (protocol see Example 14). As shown in
EXAMPLE 6: DERIVATIVES OF NUCLEIC ACID MOLECULE 257-E1-001 HAVING INCREASED BINDING AFFINITY TO THE TARGET MOLECULE GLUCAGON
[0373] Starting from a nucleic acid molecule binding to glucagon which was the result of a development process involving as a starting point the immediate screening product of the SELEX process, the method of the present invention was used in order to improve the binding affinity of the nucleic acid molecule to its target. In the instant case, the nucleic acid molecule binding to human glucagon was nucleic acid molecule 257-E1-001.
[0374] Nucleic acid molecule 257-E1-001 is a Spiegelmer, i.e. a L-nucleic acid molecule, which is capable of binding to human glucagon, has a nucleotide sequence according to SEQ ID NO: 27 and consists of 47 2-deoxyribonucleotides.
[0375] The binding characteristics of nucleic acid molecule 257-E1-001 were determined in the competitive pull-down assay format (as described in Example 10). Nucleic acid molecule 257-E1-001 binds human glucagon with an affinity of 186 nM as also shown in
[0376] In order to improve the binding characteristics of nucleic acid molecule 257-E1-001, derivatives of nucleic acid molecule 257-E1-001 were synthesized (as described in Example 7). Said derivatives were L-nucleic acid molecules having the same sequence of nucleobasesguanine, cytosine, adenine, and uracil or alternatively thymine (in the case of a 2deoxyribonucleotide)as nucleic acid molecule 257-E1-001, however, differed at a single position as to the sugar moiety of the nucleotides which was a ribonucleotide rather than a 2-deoxyribonucleotide. In accordance therewith, derivative 1 (termed 257-E1-R1-001) had a ribonucleoside at position 1 of the nucleotide sequence according to SEQ ID NO: 228, derivative 2 (termed 257-E1-R2-001) had a ribonucleotide at position 2 of the nucleotide sequence according to SEQ ID NO: 229 etc. Because of nucleic acid molecule 257-E1-001 consisting of 47 nucleotides a total of 47 derivatives was synthesized in order to provide a complete set of all possible derivatives of nucleic acid molecules meeting the above requirement of a single substitution of a 2-deoxyribonucleotide by a ribonucleotide. Said complete set of derivatives is shown in
[0377] The binding characteristics to human glucagon of each derivative of said complete set of derivatives of nucleic acid molecule 257-E1-001 was determined in competitive pull-down assays described in Example 10, and compared to the binding affinity of nucleic acid molecule 257-E1-001. As may be taken from
[0378] As may be taken from said figures, derivatives 257-E1-R9-001, 257-E1-R15-001, 257-E1-R18-001, 257-E1-R19-001, 257-E1-R29-001, and 257-E1-R30-001 which have a ribonucleotide at position 9, 15, 18, 19, 29, or 30, respectively, belong to the first group of derivatives, i.e. derivatives where the substitution of a 2-deoxyribonucleotide by a ribonucleotide results in an improved binding affinity for human glucagon (
[0379] Derivatives 257-E1-R26-001 and 257-E1-R46-001, which have a ribonucleotide at positions 26 and 46, respectively, belong to the second group of derivatives, i.e. derivatives where the substitution of a 2-deoxyribonucleotide by a ribonucleotide does not change or affect the binding affinity for human glucagon. Accordingly, positions 26 and 46 are not suitable to confer to nucleic acid molecule 257-E1-001 an improved binding affinity to human glucagon, however, do not have a negative impact on binding affinity of nucleic acid molecule 257-E1-001 to human glucagon either.
[0380] Finally, derivatives were obtained which resulted in reduced binding affinity or a profound loss of binding affinity. These derivatives, namely 257-E1-R1-001, 257-E1-R2-001, 257-E1-R3-001, 257-E4-R1-001, 257-E5-R1-001, 257-E1-R6-001, 257-E1-R7-001, 257-E1-R8-001, 257-E1-R10-001, 257-E1-R11-001, 257-E1-R12-001, 257-E1-R13-001, 257-E1-R14-001, 257-E1-R16-001, 257-E1-R17-001, 257-E1-R20-001, 257-E1-R21-001, 257-E1-R22-001, 257-E1-R23-001, 257-E1-R24-001, 257-E1-R25-001, 257-E1-R27-001, 257-E1-R28-001, 257-E1-R31-001, 257-E1-R32-001, 257-E1-R33-001, 257-E1-R34-001, 257-E1-R35-001, 257-E1-R36-001, 257-E1-R37-001, 257-E1-R38-001, 257-E1-R39-001, 257-E1-R40-001, 257-E1-R41-001, 257-E1-R42-001, 257-E1-R43-001, 257-E1-R44-001, 257-E1-R45-001, 257-E1-R47-001, accordingly, belong to the third group of derivatives, i.e. those derivatives of nucleic acid molecule 257-E1-001 where the substitution of a 2-deoxyribonucleotide by a ribonucleotidenegativelyaffects the binding affinity for human glucagon. Accordingly, positions 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 16, 17, 20, 21, 22, 23, 24, 25, 27, 28, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 47, have a negative impact on the binding affinity of nucleic acid molecule 257-E1-001 to human glucagon.
[0381] In order to assess whether the binding affinity of the derivatives of nucleic acid molecule 257-E1-001 can be further increased by introducing more than one substitution a group of further derivatives was generated. Such group of further derivatives started from the above first group of derivatives comprising derivatives where the substitution of a 2-deoxyribonucleotide by a ribonucleotide resulted in an improved binding affinity for human glucagon. Starting from nucleic acid molecule 257-E1-001, the derivatives of the further group of derivatives had a substitution of a 2-deoxyribonucleotide by a ribonucleotide at least at two of positions 9, 15, 18, 19, 29, and 30, i.e. those positions which have been proven to be suitable to confer to nucleic acid molecule 257-E1-001 an improved binding affinity to human glucagon.
[0382] In competitive pull-down assays representative examples showed, as depicted in
[0383] Spiegelmers 257-E1-R15/29-001 and 257-E1-R29/30-001, both containing two substitutions, namely 2-deoxyguanosine-5-phosphate to guanosine-5-phosphate at position 15, 2-deoxyadenosine-5-phosphate to adenosine-5-phosphate at position 29 and/or 2-deoxyadenosine-5-phosphate to adenosine-5-phosphate at position 30, showed better binding affinity as the molecules containing one substitution (
[0384] Spiegelmers 257-E1-R15/29/30-001 and 257-E1-R18/29/30-001 were synthesized to test whether three substitutions in the nucleic acid molecule 257-E1-001 led to a further improved binding affinity compared to Spiegelmers 257-E1-R15/29-001 and 257-E1-R29/30-001, all containing two substitutions. Namely, the substitutions of 2-deoxyguanosine-5-phosphate to guanosine-5-phosphate at position 15, 2-deoxyadenosine-5-phosphate to adenosine-5-phosphate at position 29, and 2-deoxyadenosine-5-phosphate to adenosine-5-phosphate at position 30 led to further improved binding affinity compared to the Spiegelmer 257-E1-R29/30-001. However, the binding affinity of the three-fold substituted Spiegelmer 257-E1-R15/29/30-001 to glucagon is comparable to that of Spiegelmer 257-E1-R15/29-001, containing two substitutions. The substitution of 2-deoxyguanosine-5-phosphate to guanosine-5-phosphate at position 18 instead of the 2-deoxyguanosine-5-phosphate to guanosine-5-phosphate substitution at position 15 in Spiegelmer 257-E1-R18/29/30-001 led to an increased binding affinity compared to 257-E1-R29/30-001, but to a decreased affinity compared to 257-E1-R15/29/30 (
[0385] Combining the four positions 15, 18, 29 and 30 (nucleic acid molecule 257-E1-R15/18/29/30-001) for substitution, namely 2-deoxyguanosine-5-phosphate to guanosine-5-phosphate at position 15, 2-deoxyguanosine-5-phosphate to guanosine-5-phosphate at position 18, 2-deoxyadenosine-5-phosphate to adenosine-5-phosphate at position 29, and 2-deoxyadenosine-5-phosphate to adenosine-5-phosphate at position 30, did not lead to further improvement in comparison to the Spiegelmer 257-E1-R15/29/30-001 containing three substitutions (
[0386] Surprisingly, the combination of six 2deoxyribonucleotide to ribonucleotide substitutions at positions 9, 15, 18, 19, 29, 30 (Spiegelmer 257-E1-R9/15/18/19/29/30-001=257-E1-6xR-001), namely 2-deoxyguanosine-5-phosphate to guanosine-5-phosphate at position 9, 2-deoxyguanosine-5-phosphate to guanosine-5-phosphate at position 15, 2-deoxyguanosine-5-phosphate to guanosine-5-phosphate at position 18, 2-deoxyguanosine-5-phosphate to guanosine-5-phosphate at position 19, 2-deoxyadenosine-5-phosphate to adenosine-5-phosphate at position 29, and 2-deoxyadenosine-5-phosphate to adenosine-5-phosphate at position 30, led to a further improved binding affinity to glucagon compared to the Spiegelmers 257-E1-R15/29-001 and 257-E1-R15/18/29/30-001, containing two and four substitutions, respectively (
[0387] In order to assess whether the binding affinity to glucagon of the Spiegelmer 257-E1-6xR-001, containing six 2-deoxyribonucleotide to ribonucleotide substitutions, can be further increased by the exchange of thymidine-5-phosphate with 5-methyl-uridine-5-phosphate instead of with uridine-5-phosphate, derivatives of nucleic acid molecule 257-E1-6xR-001 were synthesized. Said derivatives contain an additional, seventh substitution of thymidine-5-phosphates to 5-methyl-uridine-5-phosphate and are shown in
[0388] Finally, the binding affinity of Spiegelmer 257-E1-7xR-023 to glucagon was improved by a factor of 43 in comparison to the SELEX derived, unmodified Spiegelmer 257-E1-001.
EXAMPLE 7: SYNTHESIS AND DERIVATIZATION OF SPIEGELMERS
Small Scale Synthesis
[0389] Spiegelmers (L-RNA nucleic acids or L-DNA modified L-RNA nucleic acids) were produced by solid-phase synthesis with an ABI 394 synthesizer (Applied Biosystems, Foster City, Calif., USA) using 2TBDMS RNA and DNA phosphoramidite chemistry with standard exocyclic amine protecting groups (Damha and Ogilvie, 1993). For the RNA part of the oligonucleotide rA(N-Bz)-, rC(N-Ac)-, rG(N-ibu)-, and rU-phosphoramidites in the
Large Scale Synthesis Plus Modification
[0390] Spiegelmers were produced by solid-phase synthesis with an ktaPilot100 synthesizer (GE Healthcare, Freiburg) using 2TBDMS RNA and DNA phosphoramidite chemistry with standard exocyclic amine protecting groups (Damha and Ogilvie, 1993).
Pegylation of Spiegelmers
[0391] In order to prolong the Spiegelmer's plasma residence time in vivo, a 40 kDa polyethylene glycol (PEG) moiety was covalently coupled at the 5-end of the spiegelmers.
[0392] For PEGylation (for technical details of the method for PEGylation see European patent application EP 1 306 382), the purified 5-amino modified Spiegelmer was dissolved in a mixture of H.sub.2O (2.5 ml), DMF (5 ml), and buffer A (5 ml; prepared by mixing citric acidH.sub.2O [7 g], boric acid [3.54 g], phosphoric acid [2.26 ml], and 1 M NaOH [343 ml] and adding water to a final volume of 11; pH=8.4 was adjusted with 1 M HCl).
[0393] The pH of the Spiegelmer solution was brought to 8.4 with 1 M NaOH. Then, 40 kDa PEG-NHS ester (Jenkem Technology, Allen, Tex., USA) was added at 37 C. every 30 min in six portions of 0.25 equivalents until a maximal yield of 75 to 85% was reached. The pH of the reaction mixture was kept at 8-8.5 with 1 M NaOH during addition of the PEG-NHS ester.
[0394] The reaction mixture was blended with 4 ml urea solution (8 M), and 4 ml buffer B (0.1 M triethylammonium acetate in H.sub.2O) and heated to 95 C. for 15 min. The PEGylated Spiegelmer was then purified by RP-HPLC with Source 15RPC medium (Amersham), using an acetonitrile gradient (buffer B; buffer C: 0.1 M triethylammonium acetate in acetonitrile). Excess PEG eluted at 5% buffer C, PEGylated Spiegelmer at 10-15% buffer C. Product fractions with a purity of >95% (as assessed by HPLC) were combined and mixed with 40 ml 3 M NaOAC. The PEGylated Spiegelmer was desalted by tangential-flow filtration (5 K regenerated cellulose membrane, Millipore, Bedford Mass.).
EXAMPLE 8: BIACORE MEASUREMENT
Biacore Assay Setup
[0395] The Biacore 2000 instrument (Biacore AB, Sweden) was set to a constant temperature of 37 C. The instrument was cleaned using the DESORB method before the start of each experiment/immobilization of a new chip: After docking a maintenance chip, the instrument was consecutively primed with desorb solution 1 (0.5% sodium dodecyl sulphate, SDS), desorb solution 2 (50 mM glycine, pH 9.5) and HBS-EP pH 7.4 buffer. Finally, the system was primed with HBS-EP pH 7.4 buffer. All reagents were purchased from GE Healthcare unless otherwise indicated.
Target Immobilization
[0396] The target immobilization procedure was established individually for each target. Examples for the targets described herein are listed below:
Immobilization of Biotinylated Human L-Glucagon
[0397] The immobilization buffer was HBS-EP pH 7.4 buffer. Synthetic biotinylated human L-glucagon (glucagon1-29-AEEAc-AEEAc-biotin, custom synthesis by BACHEM, Switzerland) was immobilized on a carboxymethylated dextran-coated sensor chip (CM5, GE Healthcare) which had been prepared by covalent immobilization of soluble neutravidin (Sigma Aldrich, Germany) using a 1:1 mixture of 0.4 M EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in H.sub.2O) and 0.1 M NHS (N-hydroxysuccinimide in H.sub.2O). The reference flow cell on the same sensor chip was blocked with biotin.
Immobilization of Human L-C5a
[0398] The immobilization buffer was HBS-EP pH 7.4 buffer. Recombinant human L-C5a (Sigma Aldrich) in 10 mM NaOAc pH 5.5 was immobilized by amine coupling on a carboxymethylated dextran-coated sensor chip (CM5, GE Healthcare) which had been activated using a 1:1 mixture of 0.4 M EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in H.sub.2O) and 0.1 M NHS (N-hydroxysuccinimide in H.sub.2O).
Immobilization of Human L-Alpha-CGRP
[0399] The immobilization buffer was HBS-EP pH 7.4 buffer. Synthetic human L-CGRP (Bachem) in 10 mM NaOAc pH 5.5 was immobilized by amine coupling on a carboxymethylated dextran-coated sensor chip (CM5, GE Healthcare) which had been activated using a 1:1 mixture of 0.4 M EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in H.sub.2O) and 0.1 M NHS (N-hydroxysuccinimide in H.sub.2O).
Identification of Spiegelmer Derivatives with Improved Binding Affinity
[0400] Binding analysis and kinetic parameter assessment of the individual single-position modified spiegelmer derivatives was performed by injecting Spiegelmer at a concentration of 1 M at a device temperature of 37. Before and after the total injection series, as well as every tenth injection an injection of blank running buffer and of a Spiegelmer reference were injected to monitor sensor chip decay, due to the regeneration procedure or/and limited peptide stability on the sensor chip surface.
[0401] From at least 5 individually determined K.sub.d values of the parent (all-DNA or all-RNA) spiegelmer the mean value was calculated (meanstandard error). K.sub.d values of individual derivatives were determined and changes in affinity are given as x-fold improvement compared to the mean of the parent molecule, wherein the value of the x-fold improvement is the quotient of the K.sub.D of the parent molecule and the derivative of the parent molecule. The determined standard error indicates a cutting point for positive hits.
[0402] Data analysis and calculation of dissociation constants (K.sub.d) was done with the BLAevaluation 3.1.1 software (BIACORE AB, Uppsala, Sweden) and Prism 5.0 (GraphPad) software for calculation of mean values and standard errors.
[0403] Derivatives of the parent molecule that showed improved binding properties in respect of the target recognition (association constant k.sub.a) or/and Spiegelmer target complex stability (dissociation constant k.sub.d) resulting in an overall improved affinity (dissociation constant K.sub.d) were characterized by measuring detailed binding kinetics (injection of a concentration series of the respective Spiegelmer).
Detailed Kinetic Evaluation of Selected Derivatives of the Parent Molecule
[0404] Kinetic parameters and dissociation constants were evaluated by a series of Spiegelmer injections at concentrations of 2,000-1,000-500-200-125-62.5-31.3-15.6 (2x)-7.8-3.9-1.95-0.98-0.48-0.24-0.12-0 nM diluted in running buffer, starting with the lowest concentration. In all experiments, the analysis was performed at 37 C. using the Kinject command defining an association time of 240 to 360 seconds and a dissociation time of 240 to 360 seconds at a flow of 30 l/min. The assay was double referenced, whereas Flow Cell 1 (FC1) served as (blocked) surface control (bulk contribution of each Spiegelmer concentration) and a series of buffer injections without analyte determined the bulk contribution of the buffer itself. At least one Spiegelmer concentration was injected twice to monitor the regeneration efficiency and chip integrity during the experiments. Data analysis and calculation of dissociation constants (K.sub.d) was done with the BIAevaluation 3.1.1 software (BIACORE AB)
Combination of Identified Exchange Positions that Lead to Improved Binding
[0405] Finally two or more of the positive single positions for substitions were combined and the resulting sequences were again studied with detailed binding kinetics.
[0406] The results of the Biacore measurements are described in Examples 2 to 5.
EXAMPLE 9: COMPETITIVE SPIEGELMER PULL-DOWN ASSAY OF S1P SPIEGELMERS
[0407] Affinity constants of SU binding spiegelmers were determined by competitive pull-down assays. In order to allow for radioactive labeling of the spiegelmer by T4 polynucleotide kinase two guanosine residues in the D-configuration were added to the 5-end of the L-S1P-215-F9-002 spiegelmer. Unlabeled spiegelmers were then tested for their ability to compete with 300-600 pM radiolabeled spiegelmer L-S1P-215-F9-002-5diD-G for binding to a constant amount of biotinylated D-e-S1P, i.e. decreasing the binding signal according to the binding affinity of the non-labeled spiegelmer to D-e-S1P. D-e-S1P was used at a concentration of 8 nM resulting in a final binding of approximately 10% of radiolabeled spiegelmer L-S1P-215-F9-002-5diD-G in the absence of competitor spiegelmers. Assays were performed in 250 l selection buffer (20 mM Tris-HCl pH 7.4; 150 mM NaCl; 5 mM KCl; 1 mM MgCl.sub.2; 1 mM CaCl.sub.2; 0.1% [w/vol] Tween-20; 4 mg/ml bovine serum albumin; 10 g/ml Yeast-RNA) for 3-4 hours at 37 C. Biotinylated D-e-S1P and complexes of spiegelmer and biotinylated S1P were immobilized on 5 l Neutravidin Ultralink Plus beads (Pierce Biotechnology, Rockford, USA) which had been pre-equilibrated with selection buffer before addition to the binding reactions. Beads were kept in suspension for 30 min at 37 C. in a thermomixer. After removal of supernatants and appropriate washing, immobilized radioactivity was quantified in a scintillation counter. The percentage of binding or normalized percentage of bound radiolabeled spiegelmer L-S1P-215-F9-002-5diD-G was plotted against the corresponding concentration of competitor spiegelmer. Dissociation constants were obtained using GraphPad Prism software. The same assay format was used for comparative ranking of a set of different spiegelmers. In this case competitor spiegelmers were used at a single concentration as indicated.
EXAMPLE 10: DETERMINATION OF BINDING AFFINITY TO GLUCAGON (PULL-DOWN ASSAY)
[0408] For binding analysis to glucagon the glucagon binding nucleic acid molecules were synthesized as spiegelmers consisting of L-nucleotides. The binding analysis of spieglmers was done with biotinylated human L-glucagon consisting of L-amino acids.
Direct Pull-Down Assay
[0409] Two additional adenosinresidues in the D-configuration at the Spiegelmer's 5-end enabled 5-phosphate labeleling by T4 polynucleotide kinase (Invitrogen, Karlsruhe, Germany) using [-.sup.32P]-labeled ATP (Hartmann Analytic, Braunschweig, Germany). The specific radioactivity of labeled nucleic acids was 200,000-800,000 cpm/pmol. After de- and renaturation (1 94 C., ice/H.sub.2O) labeled nucleic acids were incubated at 100-700 pM concentration at 37 C. in selection buffer (20 mM Tris-HCl pH 7.4; 137 mM NaCl; 5 mM KCl; 1 mM MgCl.sub.2; 1 mM CaCl.sub.2; 0.1% [w/vol] Tween-20; 0.1% [w/vol] CHAPS) together with varying amounts of biotinylated human D- or L-glucagon, respectively, for 2-6 hours in order to reach equilibrium at low concentrations. Selection buffer was supplemented with 100 g/ml human serum albumin (Sigma-Aldrich, Steinheim, Germany), and 10 g/ml yeast RNA (Ambion, Austin, USA) in order to prevent unspecific adsorption of binding partners to surfaces of used plasticware or to the immobilization matrix. The concentration range of biotinylated L-glucagon for Spiegelmer binding was set from 0.32 nM to 5 M; total reaction volume was 50 Biotinylated glucagon and complexes of nucleic acids and biotinylated glucagon were immobilized on 4 l High Capacity Neutravidin Agarose particles (Thermo Scientific, Rockford, USA) which had been preequilibrated with selection buffer. Particles were kept in suspension for 20 min at the respective temperature in a thermomixer. Immobilized radioactivity was quantitated in a scintillation counter after removal the supernatant and appropriate washing. The percentage of binding was plotted against the concentration of biotinylated glucagon and dissociation constants were obtained by using software algorithms (GRAFIT; Erithacus Software; Surrey U.K.) assuming a 1:1 stoichiometry.
Competitive Pull-Down Assay for Ranking of Glucagon Binding Nucleic Acids
[0410] In order to compare the binding of different Spiegelmers to glucagon a competitive ranking assay was performed. For this purpose either the most affine spiegelmer available was radioactively labeled (see above) and served as reference for glucagon binding spiegelmers, respectively. After de- and renaturation the labeled nucleic acids were incubated at 37 C. with biotinylated glucagon in 50 or 100 l selection buffer at conditions that resulted in around 5-10% binding to the biotinylated glucagon after immobilization on 1.5 l High Capacity Neutravidin Agarose particles (Thermo Scientific, Rockford, USA) and washing without competition. An excess of de- and renatured non-labeled spiegelmer variants was added at different concentrations together with the labeled reference spiegelmer to parallel binding reactions. De- and renatured non-labeled Spiegelmer derivatives were applied at concentrations of 1, 10, and 100 nM together with the reference Spiegelmer in parallel binding reactions. The nucleic acids to be tested competed with the reference nucleic acid for target binding, thus decreasing the binding signal in dependence of their binding characteristics. The aptamer or Spiegelmer, respectively that was found most active in this assay could then serve as a new reference for comparative analysis of other glucagon binding nucleic acid molecules.
Competitive Pull-Down Assay for Determination of Affinity
[0411] In addition to comparative ranking experiments the competitive pull-down assay was also performed to determine the affinity constants of glucagon binding nucleic acids. For this purpose either a L-glucagon binding Spiegelmer was radioactively labeled and served as reference as described above. After de- and renaturation the labeled reference nucleic acid and a set of 5-fold dilutions ranging e.g. from 0.128 to 2000 nM of competitor molecules were incubated with a constant amount of biotinylated glucagon in 0.1 or 0.2 ml selection buffer at 37 C. for 2-4 hours. The chosen protein concentration should cause final binding of approximately 5-10% of the radiolabeled reference molecule at the lowest competitor concentration. In order to measure the binding constants of derivative nucleic acid sequences an excess of the appropriate de- and renatured non-labeled Spiegelmer variants served as competitors, whereas for Spiegelmers unmodified as well as PEGylated forms were tested. In another assay approach non-biotinylated glucagon at different concentrations competed against the biotinylated glucagon for Spiegelmer binding. After immobilization of biotinylated glucagon and the bound nucleic acids on 1.5 l High Capacity Neutravidin Agarose matrix, washing and scintillation counting (see above), the normalized percentage of bound radiolabeled Spiegelmer was plotted against the corresponding concentration of competitor molecules. The resulting dissociation constant was calculated employing the GraFit Software.
EXAMPLE 11: INHIBITION OF -ARRESTIN RECRUITMENT INDUCED BY S1P VIA EDG1 RECEPTOR BY S1P-BINDING SPIEGELMERS
[0412] PathHunter eXpress EDG-1 CHO-K1 -arrestin GPCR cells (DiscoverX) were seeded at 110.sup.4 cells per well in a white 96 well-plate with clear bottom (Greiner) and cultivated for 48 h at 37 C. and 5% CO.sub.2 in 100 l Culture Medium (DiscoverX). Stimulation solutions (D-e-S1P+various concentrations of Spiegelmer) are made up as 11 concentrated solutions in HBSS (Gibco) supplemented with 1 mg/ml BSA and 20 mM HEPES, mixed thoroughly and incubated at 37 C. for 30 min. 10 l stimulation solution were added per well (triplicates) and cells were incubated for 90 min at 37 C. and 5% CO.sub.2.
[0413] Upon receptor activation by D-e-S1P, the interaction of activated EDG1 with -arrestin leads to -galactosidase enzyme fragment complementation.
[0414] For quantification of -galactosidase activity 55 l Working Detection Reagent Solution (DiscoverX) were added and incubated for 90 min at room temperature. Luminescence was subsequently measured in a Fluostar Optima multidetection plate reader (BMG).
[0415] To show the efficacy of anti-S1P-spiegelmers, cells were stimulated with 10 nM D-e-S1P or D-e-S1P preincubated with various amounts of spiegelmers. The results show the percentage of luminescence signal normalized to the signal obtained without addition of spiegelmer. Mean valuesSD from triplicate cultures are shown.
EXAMPLE 12: INHIBITION OF ALPHACGRP-INDUCED CAMP PRODUCTION IN HUMAN NEUROBLASTOMA CELLS
[0416] Biological efficacy of CGRP-binding Spiegelmers was analysed as follows.
[0417] SK-N-MC human neuroblastoma cells (DSMZ, Braunschweig) were seeded at 510e4 cells/well in a flat-bottomed 96-well plate (Greiner) and cultivated for 48 h at 37 C. and 5% CO.sub.2 in 100 l in DMEM (1000 mg/L glucose, Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (FCS), 4 mM L-alanyl-L-glutamine (GLUTAMAX), 50 units/ml Penicillin and 50 g/ml Streptomycin.
[0418] Stimulation solutions (1 nM human or rat L-alphaCGRP (Bachem)+increasing concentrations of Spiegelmer) were prepared as triplicates in HBSS (Gibco) supplemented with 1 mg/ml BSA and 20 mM HEPES using v-bottomed 0.2 ml low profile 96-well plates and incubated at 37 C. for 60 min in total. Blank values (no L-alphaCGRP, no Spiegelmer) and control values (1 nM L-alphaCGRP, no Spiegelmer) were included as triplicates. 20 min prior to stimulation 1 mM phosphodiesterase inhibitor 3-Isobutyl-1-methylxanthine (IBMX, Sigma; 50 mM stock in DMSO diluted in HBSS/BSA/HEPES) was added to the cells and the stimulation solutions. For stimulation, cell culture medium was removed from the cells and substituted by 100 l pre-incubated stimulation solution. Cell were stimulated for 30 min at 37 C., 5% CO.sub.2. After removal of stimulation solutions cells were lysed by addition of 50 l/well assay/lysis buffer (Applied Biosystems, Tropix cAMP-Screen System kit) for 30 min at 37 C.
[0419] The amount of cAMP produced per well was subsequently measured using the Tropix cAMP-Screen ELISA System kit (Applied Biosystems) according to manufacturer's instructions. Briefly, a standard curve is prepared in assay/lysis buffer ranging from 6 nmol to 0.6 pmol cAMP/well. Cell lysates diluted in assay/lysis buffer and standard curves are added to microplates precoated with goat anti-rabbit IgG. cAMP alkaline phosphatase conjugate and anti-cAMP antibody are added to the samples and incubated for 60 min at room temperature. Subsequently, plates are washed and chemiluminescent substrate is added. After 30 min chemiluminescence is measured in a FLUOstar OPTIMA plate reader unit (BMG Labtech). The cAMP-Screen ELISA system is a competitive immunoassay format. Thus, light signal intensity is inversely proportional to the cAMP level in the sample or standard preparation.
[0420] This assay system was used to test Spiegelmers within the scope of Examples 1 and 7 described herein. The result is illustrated in
EXAMPLE 13: DETERMINATION OF INHIBITORY CONCENTRATION IN A CHEMOTAXIS ASSAY
[0421] Generation of a cell line expressing the human receptor for C5a A stably transfected cell line expressing the human receptor for C5a was generated by transfecting BA/F3 mouse pro B cells with a plasmid coding for the human C5a receptor (NCBI accession NM_001736 in pcDNA3.1+). Cells expressing C5aR were selected by treatment with geneticin and tested for expression with RT-PCR and for functionality with chemotaxis assay.
Chemotaxis Assay
[0422] The day before the experiment, cells are seeded in a new flask at 0.310.sup.6/ml. For the experiment, cells were centrifuged, washed once in HBH (HBSS, containing 1 mg/ml bovine serum albumin and 20 mM HEPES) and resuspended at 1.3310.sup.6 cells/ml. 75 l of this suspension were added to the upper compartments of a 96 well Corning Transwell plate with 5 m pores (Costar Corning, #3388; NY, USA). In the lower compartments recombinant human C5a (SEQ.ID. 50) or mouse C5a (SEQ.ID. 54) was pre-incubated together with Spiegelmers in various concentrations in 235 l HBH at 37 C. for 20 to 30 min prior to addition of cells. Cells were allowed to migrate at 37 C. for 3 hours. Thereafter the insert plate (upper compartments) was removed and 30 l of 440 M resazurin (Sigma, Deisenhofen, Germany) in phosphate buffered saline was added to the lower compartments. After incubation at 37 C. for 2.5 hours, fluorescence was measured at an excitation wavelength of 544 nm and an emission wavelength of 590 nm.
[0423] Fluorescence values are corrected for background fluorescence (no C5a in well) and plotted against Spiegelmer concentration. The IC.sub.50 values are determined with non-linear regression (4 parameter fit) using GraphPad Prism. Alternatively, the value for the sample without Spiegelmer (C5a only) is set 100% and the values for the samples with Spiegelmer are calculated as per cent of this. The per cent-values are plotted against Spiegelmer concentration and the IC.sub.50-values are determined as described above.
Determination of the Half-Maximal Effective Concentration (EC.SUB.50.) for Human and Mouse C5a
[0424] After 3 hours migration of BA/F3/huC5aR cells towards various human C5a or mouse C5a concentrations, dose-response curves for human and mouse C5a were obtained, indicating half effective concentrations (EC.sub.50) of 0.1 nM for huC5a and 0.3 nM for mC5a. For the experiments on inhibition of chemotaxis by Spiegelmers 0.1 nM human C5a and 0.3 nM mouse C5a were used.
EXAMPLE 14: INHIBITION OF GLUCAGON-INDUCED CAMP PRODUCTION BY GLUCAGON-BINDING SPIEGELMERS
[0425] A stably transfected cell line expressing the human receptor for glucagon was generated by cloning the sequence coding for the human glucagon receptor (NCBI accession NM_000160) into the pCR3.1 vector (Invitrogen). CHO cells adapted to growth in serum-free medium (UltraCHO, Lonza) were transfected with the glucagon receptor plasmid and stably transfected cells were selected by treatment with geneticin.
[0426] For an inhibition experiment CHO cells expressing the glucagon receptor were plated on a 96 well plate (cell culture treated, flat bottom) at a density of 4-610.sup.4/well and cultivated overnight at 37 C. 5% CO.sub.2 in UltraCHO medium containing 100 units/ml penicillin, 100 pg/ml streptomycin and 0.5 mg/ml geneticin. 20 min before stimulation a solution of 3-isobutyl-1-methylxanthine (IBMX) was added to a final concentration of 1 mM.
[0427] The stimulation solutions (glucagon+various concentrations of Spiegelmers) were made up in Hank's balanced salt solution (HBSS)+1 mg/ml BSA and were incubated for 30 min at 37 C. Shortly before addition to the cells, IBMX was added to a final concentration of 1 mM. For stimulation, the medium was removed from the cells and the stimulation solutions (glucagon+Spiegelmer) were added. After incubation for 30 min at 37 C. the solutions were removed and the cells were lysed in lysis-buffer which is a component of the cAMP-Screen System kit (Applied Biosystems). This kit was used for determination of the cAMP content following the supplier's instructions.
REFERENCES
[0428] The complete bibliographic data of the documents recited herein are, if not indicated to the contrary, as follows, whereby the disclosure of said references is incorporated herein by reference. [0429] Altschul S. F., Gish W., et al. (1990) Basic local alignment search tool. J Mol Biol. 215(3):403-10. [0430] Altschul S. F., Madden T. L., et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25(17):3389-402. [0431] Damha M J, Ogilvie K K. (1993) Oligoribonucleotide synthesis. The silyl-phosphoramidite method. Methods Mol Biol. 20:81-114 [0432] Klussmann S. (2006). The Aptamer HandbookFunctional Oligonucleotides and their Applications. Edited by S. Klussmann. WILEY-VCH, Weinheim, Germany, ISBN 3-527-31059-2 [0433] Kusser W. (2000) Chemically modified nucleic acid aptamers for in vitro selections: evolving evolution. J Biotechnol 74(1): 27-38. [0434] Mairal T., Ozalp V. C., Lozano Sanchez P., et al. (2008) Aptamers: molecular tools for analytical applications. Anal Bioanal Chem. 390(4):989-1007 [0435] McGinnis S., Madden T. L. et al. (2004) BLAST: at the core of a powerful and diverse set of sequence analysis tools. Nucleic Acids Res. 32 (Web Server issue):W20-5. [0436] Needleman and Wunsch (1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol. 48(3):443-53. [0437] Pearson and Lipman (1988) Improved tools for biological sequence comparison. Proc. Nat'l. Acad. Sci. USA 85: 2444 [0438] Smith and Waterman (1981), Adv. Appl. Math. 2: 482 [0439] Venkatesan N., Kim S. J., et al. (2003) Novel phosphoramidite building blocks in synthesis and applications toward modified oligonucleotides. Curr Med Chem 10(19): 1973-91 [0440] Wincott F, DiRenzo A, et al. (1995). Synthesis, deprotection, analysis and purification of RNA and ribozymes. Nucleic Acids Res.; 23(14):2677-84.
[0441] The features of the present invention disclosed in the specification, the claims and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.