SERUM ALBUMIN-BINDING IMMUNOGLOBULIN VARIABLE DOMAINS

Abstract

The present invention relates to amino acid sequences binding to serum albumin. In particular, the present invention relates to improved immunoglobulin single variable domains (also referred to herein as “ISV's” or “ISVD's”), and more in particular improved heavy-chain immunoglobulin single variable domains, binding to serum albumin, as well as to proteins, polypeptides and other constructs, compounds, molecules or chemical entities that comprise such improved serum albumin binders.

Claims

1.-13. (canceled)

14. A nucleotide sequence or nucleic acid that encodes an immunoglobulin single variable domain that binds to serum albumin, wherein said immunoglobulin single variable domain comprises: a CDR1 according to Abm that is the following sequence: GFTFDTSSML (SEQ ID NO: 13); and a CDR2 according to Abm that is the following sequence: VIHQSGTPTY (SEQ ID NO: 14); and a CDR3 according to Abm that is the following sequence: FPSSRMKFDY (SEQ ID NO: 15); and having: an amino acid residue L or V at position 11; and an amino acid residue T, V or L at position 89; and an amino acid residue T, K or Q at position 110; and an amino acid residue S, K or Q at position 112; such that (i) position 89 is T; or (ii) position 89 is L and position 11 is V; or (iii) position 89 is L and position 110 is K or Q; or (iv) position 89 is L and position 112 is K or Q; or (v) position 89 is L and position 11 is V and position 110 is K or Q; or (vi) position 89 is L and position 11 is V and position 112 is K or Q; or (vii) position 11 is V and position 110 is K or Q; or (viii) position 11 is V and position 112 is K or Q; wherein the positions are according to Kabat numbering.

15. The nucleotide sequence or nucleic acid according to claim 14, wherein position 11 is V and: position 89 is L; or position 110 is K or Q; or position 112 is K or Q; or position 89 is L and position 110 is K or Q; or position 89 is L and position 112 is K or Q; wherein the positions are according to Kabat numbering.

16. The nucleotide sequence or nucleic acid according to claim 14, wherein position 89 is L and: position 11 is V; or position 110 is K or Q; or position 112 is K or Q; or position 11 is V and position 110 is K or Q; or position 11 is V and position 112 is K or Q; wherein the positions are according to Kabat numbering.

17. The nucleotide sequence or nucleic acid according to claim 14, wherein position 110 is K or Q and: position 11 is V; or position 89 is L; or position 11 is V and position 89 is L; wherein the positions are according to Kabat numbering.

18. The nucleotide sequence or nucleic acid according to claim 14, wherein position 112 is K or Q and: position 11 is V; or position 89 is L; or position 11 is V and position 89 is L; wherein the positions are according to Kabat numbering.

19. The nucleotide sequence or nucleic acid according to claim 14, wherein position 89 is T, wherein the position is according to Kabat numbering.

20. The nucleotide sequence or nucleic acid according to claim 14, wherein said immunoglobulin single variable domain comprises an amino acid sequence that is chosen from the amino acid sequences of SEQ ID NOs: 72 to 99.

21. The nucleotide sequence or nucleic acid according to claim 14, wherein said immunoglobulin single variable domain consists of an amino acid sequence that is chosen from the following amino acid sequences: SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, or SEQ ID NO: 99.

22. The nucleotide sequence or nucleic acid according to claim 14, wherein said immunoglobulin single variable domain consists of an amino acid sequence that is one of the following sequences: SEQ ID NO: 78 or SEQ ID NO: 92.

23. The nucleotide sequence or nucleic acid according to claim 14, wherein said immunoglobulin single variable domain further comprises a C-terminal extension (X).sub.n, in which n is 1 to 10; and each X is an amino acid residue that is independently chosen.

24. The nucleotide sequence or nucleic acid according to claim 23, wherein each X is independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

25. The nucleotide sequence or nucleic acid according to claim 14, wherein said immunoglobulin single variable domain further comprises a D at position 1 and/or an E1D mutation, wherein the position is according to Kabat numbering.

26. A host or host cell containing and capable of expressing a nucleotide sequence or nucleic acid according to claim 14.

27. A method for preparing an immunoglobulin single variable domain comprising cultivating or maintaining the host or host cell according to claim 26 under conditions such that the host or host cell produces or expresses the immunoglobulin single variable domain.

28. The method of claim 27, further comprising isolating the immunoglobulin single variable domain.

29. A method for prevention and/or treatment of a disease or disorder using an immunoglobulin variable domain as encoded by the nucleotide sequence or nucleic acid according to claim 14.

Description

[0380] The invention will now be further described by means of the following non-limiting preferred aspects, examples and figures, in which:

[0381] FIG. 1 is a table listing some of the amino acid positions that will be specifically referred to herein and their numbering according to some alternative numbering systems (such as Aho and IMGT);

[0382] FIG. 2 shows an alignment of the Reference sequences referred to herein.

[0383] FIG. 3 lists the amino acid sequences referred to herein;

[0384] FIG. 4 shows two corresponding plots of data points obtained in Example 1 when 96 serum samples from human healthy subjects were tested for binding to Reference A and two representative variants of Reference A according to the invention (i.e. [Reference A+L11V+V89L+C-terminal alanine] and [Reference A+L11V+V89L+T110K+C-terminal alanine], respectively). Each dot represents the binding level for one of the 96 samples tested. The data points shown in the right hand panel and the left hand panel are the same; in the right hand panel the data points measured with each individual sample for each of the three compounds tested (i.e. Ref. A; Ref. A+L11V+V89L+114A; and Ref. A+L11V+V89L+T110K+114A) are connected by means of a line (as a result, the declination of the line gives an indication of the extent to which binding by pre-existing antibodies is reduced when the mutations of the invention and the C-terminal alanine are introduced);

[0385] FIG. 5 is a table listing the binding data (3 columns giving normalized PreAb binding levels (RU at 700) and 3 columns giving percentage of reduction in PreAb binding compared to the reference compound used, respectively) of the data points compiled in FIG. 4;

[0386] FIG. 6 shows two corresponding plots of data points obtained in Example 2 when 96 serum samples from human healthy subjects were tested for binding to Reference B and two representative variants of Reference B according to the invention (i.e. [Reference B+L11V+V89L+C-terminal alanine] and [Reference B+L11V+V89L+T110K+C-terminal alanine], respectively). Each dot represents the binding level for one of the 96 samples tested. The data points shown in the right hand panel and the left hand panel are the same; in the right hand panel the data points measured with each individual sample for each of the three compounds tested (i.e. Ref. B; Ref. B+L11V+V89L+114A; and Ref. B+L11V+V89L+T110K+114A) are connected by means of a line (as a result, the declination of the line gives an indication of the extent to which binding by pre-existing antibodies is reduced when the mutations of the invention and the C-terminal alanine are introduced);

[0387] FIG. 7 is a table listing the binding data (3 columns giving normalized PreAb binding levels (RU at 700) and 2 columns giving percentage of reduction in PreAb binding compared to the reference compound used, respectively) of the data points compiled in FIG. 6;

[0388] FIG. 8 shows two corresponding plots of data points obtained in Example 3 when 96 serum samples (66 from human healthy subjects and 30 from subjects assumed to contain pre-existing antibodies that can bind in the presence of a C-terminal alanine, including 13 samples from SLE patients) were tested for binding to Reference C, Reference D and two representative variants of Reference D according to the invention (i.e. [Reference D+L11V+V89L] and [Reference D+L11V+V89L+T1101(], respectively). Each dot represents the binding level for one of the 96 samples tested. The data points shown in the right hand panel and the left hand panel are the same; in the right hand panel the data points measured with each individual sample for each of the four compounds tested (i.e. Ref. C; Ref. D; Ref. D+L11V+V89L; and Ref. D+L11V+V89L+T110K) are connected by means of a line (as a result, the declination of the line gives an indication of the extent to which binding by pre-existing antibodies is reduced when the mutations of the invention and the C-terminal alanine are introduced);

[0389] FIG. 9 shows a plot of the data points obtained for four of the SLE samples that were tested in Example 3. The data points measured with for individual sample (i.e. “SLE 25”, “SLE 37”, “SLE39” and “SLE41”, respectively) are connected by means of a lines (as a result, the declination of each line gives an indication of the extent to which binding by pre-existing antibodies is reduced in each sample when the mutations of the invention are introduced);

[0390] FIG. 10 is a table listing the binding data (4 columns giving normalized PreAb binding levels (RU at 700) and 3 columns giving percentage of reduction in PreAb binding compared to the reference compounds used, respectively) of the data points compiled in FIG. 8.

EXPERIMENTAL PART

[0391] The human samples used in the Experimental Part below were either obtained from commercial sources or from human volunteers (after all required consents and approvals were obtained) and were used in according with the applicable legal and regulatory requirements (including but not limited to those regarding medical secret and patient privacy)

[0392] In the Examples below, unless explicitly indicated otherwise, the binding of pre-existing antibodies that are present in the samples used (i.e. from healthy volunteers, rheumatoid arthritis (RA) patients and SLE patients) to the Nanobodies tested was determined using ProteOn as follows: Nanobodies were captured either on serum albumin or via a FLAGS tag using monoclonal anti-FLAG M2.

[0393] In case of binding of pre-existing antibodies on Nanobodies captured on human serum albumin (HSA) was evaluated using the ProteOn XPR36 (Bio-Rad Laboratories, Inc.). PBS/Tween (phosphate buffered saline, pH7.4, 0.005% Tween20) was used as running buffer and the experiments were performed at 25° C. The ligand lanes of a ProteOn GLC Sensor Chip were activated with EDC/NHS (flow rate 30 μl/min) and HSA was injected at 10 μg/ml in ProteOn Acetate buffer pH4.5 (flow rate 100 μl/min) to render immobilization levels of approximately 3200 RU. After immobilization, surfaces were deactivated with ethanolamine HCl (flow rate 30 μl/min). Nanobodies were injected for 2 minutes at 45 μl/min over the HSA surface to render a Nanobody capture level of approximately 200 RU. The samples containing pre-existing antibodies were centrifuged for 2 minutes at 14,000 rpm and supernatant was diluted 1:10 in PBS-Tween20 (0.005%) before being injected for 2 minutes at 45 μl/min followed by a subsequent 400 seconds dissociation step. After each cycle (i.e. before a new Nanobody capture and blood sample injection step) the HSA surfaces were regenerated with a 2 minute injection of HCl (100 mM) at 45 μl/min. Sensorgram processing and data analysis was performed with ProteOn Manager 3.1.0 (Bio-Rad Laboratories, Inc.). Sensorgrams showing pre-existing antibody binding were obtained after double referencing by subtracting 1) Nanobody-HSA dissociation and 2) non-specific binding to reference ligand lane. Binding levels of pre-existing antibodies were determined by setting report points at 125 seconds (5 seconds after end of association). Percentage reduction in pre-existing antibody binding was calculated relative to the binding levels at 125 seconds of a reference Nanobody.

[0394] In case of binding of pre-existing antibodies on FLAG-tagged Nanobodies captured on monoclonal anti-FLAG M2 (Sigma) was evaluated using the ProteOn XPR36 (Bio-Rad Laboratories, Inc.). PBS/Tween (phosphate buffered saline, pH7.4, 0.005% Tween20) was used as running buffer and the experiments were performed at 25° C. The ligand lanes of a ProteOn GLC Sensor Chip were activated with EDC/NHS (flow rate 30 μl/min) and anti-FLAG M2 mAb was injected at 10 μg/ml in ProteOn Acetate buffer pH4.5 (flow rate 100 μl/min) to render immobilization levels of approximately 4000 RU. After immobilization, surfaces were deactivated with ethanolamine HCl (flow rate 30 μl/min). Nanobodies were injected for 2 minutes at 45 μl/min over the anti-FLAG M2 surface to render a Nanobody capture level of approximately 100 RU. To reduce non-specific binding of the blood samples to the anti-FLAG M2 surface 100 nM 3×FLAG peptide (Sigma) was added to the blood samples. The samples containing pre-existing antibodies were centrifuged for 2 minutes at 14,000 rpm and supernatant was diluted 1:10 in PBS-Tween20 (0.005%) before being injected for 2 minutes at 45 μl/min followed by a subsequent 600 seconds dissociation step. After each cycle (i.e. before a new Nanobody capture and blood sample injection step) the anti-FLAG M2 surfaces were regenerated with a 10 seconds injection of Glycine pH1.5 (10 mM) at 150 μl/min. Sensorgram processing and data analysis was performed with ProteOn Manager 3.1.0 (Bio-Rad Laboratories, Inc.). Sensorgrams showing pre-existing antibody binding were obtained after double referencing by subtracting 1) Nanobody-anti-FLAG M2 dissociation and 2) non-specific binding to reference ligand lane. Binding levels of pre-existing antibodies were determined by setting report points at 125 seconds (5 seconds after end of association). Percentage reduction in pre-existing antibody binding was calculated relative to the binding levels at 125 seconds of a reference Nanobody.

Example 1: Introducing the Mutations of the Invention in Reference a (SEQ ID NO: 1) Leads to a Reduction in Binding by Pre-Existing Antibodies

[0395] Reference A (SEQ ID NO: 1) and two representative examples of the improved variants of Reference A carrying the mutations according to the invention (SEQ ID NOs: 37 and 38, both with alanine-extension and tested with an N-terminal HIS6-FLAG3 tag, see SEQ ID NO:100) were tested for binding by pre-existing antibodies that are present in the samples from 96 serum samples from healthy human volunteers. The compounds were captured using the FLAG-tag and binding was measured using ProteOn according to the protocol given in the preamble to this Experimental Part.

[0396] The results are shown in FIG. 4. FIG. 5 lists the results for each of the samples that forms one of the data points in FIG. 4.

[0397] It can be seen that for most of the 96 samples tested, introducing the mutations according to the invention leads to a reduction in pre-existing antibody binding, with the degree of reduction generally being dependent on the level to which the pre-existing antibodies in each sample were capable of binding to Reference A.

Example 2: Introducing the Mutations of the Invention in Reference B (SEQ ID NO: 2) Leads to a Reduction in Binding by Pre-Existing Antibodies

[0398] Reference B (SEQ ID NO:2) and two representative examples of the improved variants of Reference B carrying the mutations according to the invention (SEQ ID NOs: 65 and 66, both with alanine-extension and tested with an N-terminal HIS6-FLAG3 tag, see SEQ ID NO:100) were tested for binding by pre-existing antibodies that are present in the samples from 96 serum samples from healthy human volunteers. The compounds were captured using the FLAG-tag and binding was measured using ProteOn according to the protocol given in the preamble to this Experimental Part.

[0399] The results are shown in FIG. 6. FIG. 7 lists the results for each of the samples that forms one of the data points in FIG. 6.

[0400] Similar to Example 1, it can be seen that for most of the 96 samples tested, introducing the mutations according to the invention leads to a reduction in pre-existing antibody binding, with the degree of reduction generally being dependent on the level to which the pre-existing antibodies in each sample were capable of binding to Reference B.

Example 3: Introducing the Mutations of the Invention in Reference C (SEQ ID NO: 3) and Reference D (SEQ ID NO: 4) Leads to a Reduction in Binding by Pre-Existing Antibodies

[0401] Reference C (SEQ ID NO: 3), Reference D (SEQ ID NO: 4) and two representative examples of the improved variants of Reference C and Reference D carrying the mutations according to the invention (SEQ ID NOs: 93 and 94, both with an alanine-extension as present in Reference D and tested with an N-terminal HIS6-FLAG3 tag, see SEQ ID NO:100) were tested for binding by pre-existing antibodies that are present in the samples from 66 serum samples from healthy human volunteers and 30 samples assumed to contain pre-existing antibodies capable of binding even when a C-terminal alanine is present (of which 13 were from SLE patients). The compounds were captured on human serum albumin and binding was measured using ProteOn according to the protocol given in the preamble to this Experimental Part.

[0402] The results are shown in FIG. 8. In FIG. 9, details are given for 4 representative SLE samples. FIG. 10 lists the results for each of the samples that forms one of the data points in FIG. 8.

[0403] Similar to Examples 1 and 2, it can be seen that for most of the 96 samples tested, introducing the mutations according to the invention for the great majority of samples leads to a reduction in pre-existing antibody binding, with the degree of reduction generally being dependent on the level to which the pre-existing antibodies in each sample were capable of binding to Reference C or Reference D.

[0404] The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference, in particular for the teaching that is referenced hereinabove.