SERUM ALBUMIN BINDERS

Abstract

The present invention relates to amino acid sequences that can bind to serum albumin. In particular, the present invention relates to immunoglobulin single variable domains, and in particular heavy-chain immunoglobulin single variable domains, that can bind to serum albumin. The invention also relates to proteins, polypeptides and other constructs, compounds, molecules or chemical entities that comprise at least one of the immunoglobulin single variable domains binding to serum albumin that are described herein.

Claims

1.-18. (canceled)

19. A method for the prevention and/or treatment of a disease or disorder comprising administering a recombinant protein to the circulation of a subject in an amount between 1 gram and 0.01 microgram per kg body weight per day, wherein the recombinant protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 18 or SEQ ID NO: 19.

20. An immunoglobulin single variable domain (ISVD) capable of binding to human serum albumin that comprises: a CDR1 according to Abm that is the amino acid sequence GGTFSTYVMG (SEQ ID NO: 12) or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 12; and a CDR2 according to Abm that is the amino acid sequence AISQNSIHTY (SEQ ID NO: 13) or an amino acid sequence that has 3, 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 13; and a CDR3 according to Abm that is the amino acid sequence SRFTSWYTADYEYDY (SEQ ID NO: 14) or an amino acid sequence that has 3, 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 14, wherein the immunoglobulin single variable domain capable of binding to human serum albumin binds to the same epitope on human serum albumin as SEQ ID NO:18.

21. The ISVD according to claim 20, that is a VHH, a humanized VHH or a camelized VH, optionally wherein the camelized VH is a camelized human VH.

22. The ISVD according to claim 20, that has: a degree of sequence identity with the sequence of SEQ ID NO: 18 and/or 19 of at least 85%, at least 90%, or at least 95%; and/or that has: no more than 7, no more than 5, no more than 3, no more than 2, or no more than 1 amino acid differences with the sequence of SEQ ID NO: 18 and/or 19; wherein the CDRs are not taken into account for determining the degree of sequence identity or the amino acid differences.

23. The ISVD according to claim 20, that contains, compared to the sequence of SEQ ID NO:18, one or more mutations that reduce the binding by pre-existing antibodies, such that after the mutation one of the following amino acid residues according to Kabat numbering is present: 11L, 11K, 11V, 14A, 14P, 41A, 41L, 41P, 41S, 41T, 42E, 42G, 87A, 87T, 89A, 89L, 89T, 108L, 110K, 110Q, 112K and/or 112Q; or any suitable combination of such substitutions, optionally wherein the ISVD comprises: 11V in combination with 89L or 89T; 11V in combination with 110K or 110Q; or 11V in combination with 89L and 110K or 110Q.

24. The ISVD according to claim 20, that is a VHH and that contains, compared to the sequence of SEQ ID NO:18, one or more humanizing substitutions that is Q108L or A14P or a suitable combination thereof.

25. A polypeptide that comprises at least one ISVD according to claim 20.

26. The polypeptide according to claim 25, that further comprises at least one therapeutic moiety or entity.

27. The polypeptide according to claim 25, that is such that: when it has an ISVD at its C-terminal end, then said C-terminal ISVD has a C-terminal extension (X)n, in which n is 1 to 10; and each X is an amino acid residue that is independently chosen from naturally occurring amino acid residues; and/or when it has an ISVD at its C-terminal end, then said C-terminal ISVD contains one or more mutations that reduce the binding of pre-existing antibodies; and/or when it has an ISVD at its N-terminal end, then said N-terminal ISVD contains a D at position 1; and/or each ISVD present in said polypeptide contains one or more mutations that reduce the binding of pre-existing antibodies.

28. A pharmaceutical composition comprising a polypeptide according to claim 25.

29. A nucleic acid that encodes an ISVD according to claim 20.

30. An expression vector comprising the nucleic acid of claim 29.

31. A host or host cell that comprises the expression vector of claim 30.

32. A method for preparing a polypeptide comprising cultivating or maintaining a host cell according to claim 31 under conditions such that said host cell produces or expresses the polypeptide, and optionally further comprising isolating the polypeptide.

Description

[0197] The invention will now be further described by means of the following non-limiting preferred aspects, examples and figures, in which:

[0198] 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);

[0199] FIG. 2 lists the amino acid sequences referred to herein;

[0200] FIGS. 3A and 3B show an alignment of the sequences of SEQ ID NOs:18 and 19 (invention) with the prior art sequences of SEQ ID NOs: 1 and 2;

[0201] FIG. 4 shows an alignment of SEQ ID NOs: 18 to 44;

[0202] FIG. 5 gives an alignment of serum albumin from different species of mammal. Mouse: SEQ ID NO: 54; Rat: SEQ ID NO: 56; Dog: SEQ ID NO: 59; Cat: SEQ ID NO: 60; Human: SEQ ID NO: 54; Cow: SEQ ID NO: 57; Sheep: SEQ ID NO: 61; Pig: SEQ ID NO: 58; Horse: SEQ ID NO: 62; Rabbit: SEQ ID NO: 63.

[0203] FIG. 6 is a graph showing binding of SEQ ID NO:1 (reference)-cMycHis6 to coated HSA in presence of the serum albumin binder of SEQ ID NO:19 (invention). 1.5 nM SEQ ID NO:1-cMycHis6 and concentration series of His6Flag3-SEQ ID NO:1 (reference), Flag3His6-SEQ ID NO:19 (invention) or cAblys3-Flag3His6 (reference) were incubated on coated HSA. Bound SEQ ID NO:1-cMycHis6 was detected with goat anti-cMyc and HRP-labelled rabbit anti-goat antibodies.

[0204] FIG. 7 is a graph showing binding of HSA to FcRn in presence of the serum albumin binder of SEQ ID NO:19 (invention). Human FcRn-human 32 microglobulin heterodimer was immobilized on CM5 chip. Binding of 1 μM HSA in absence or presence of 2 μM Nanobody in 50 mM NaPO4+150 mM NaCl+0.05% Tween-20 pH 6.0 was monitored on a Biacore T100 instrument.

[0205] 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.

EXPERIMENTAL PART

Example 1 Affinity for Serum Albumin

[0206] The affinity of the serum albumin binder of SEQ ID NO:19 (invention) for human (Sigma-Aldrich A3782), cynomolgus monkey (generated in-house), mouse (Albumin Bioscience 2601), rat (Sigma-Aldrich A4538), rabbit (Sigma-Aldrich A0764), guinea pig (Gentaur GPSA62), pig (Sigma-Aldrich A4414), sheep (Sigma-Aldrich A3264), dog (Abcam 119814) and bovine (Sigma-Aldrich A3059) serum albumin (SA) was measured via Surface Plasmon Resonance (SPR) on a ProteOn XPR36 (BioRad) instrument. Serum albumin was immobilized via amine coupling on GLC ProteOn chip using ProteOn Amine Coupling Kit (BioRad). Different concentrations (300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM and 1.23 nM) of the serum albumin binder of SEQ ID NO:19 (invention) were injected in HBS-P+ pH 7.4 buffer (GE Healthcare) at 45 μL/min for 120 s, followed by dissociation for 900 s. There was no or very low binding observed for the serum albumin binder of SEQ ID NO:19 (invention) on rabbit, sheep and bovine SA. The affinities of the serum albumin binder of SEQ ID NO:19 (invention) for human, cynomolgus monkey, rat, mouse and guinea pig SA were higher compared to the respective affinities of the albumin binder of SEQ ID NO: 1. The results are given in Table 1 below and show that the serum albumin binder of SEQ ID NO: 19 (invention) is cross-reactive with dog and pig SA.

TABLE-US-00003 TABLE 1 Kinetic parameters for binding of SEQ ID NO: 19 (invention) on SA from different species. SEQ ID NO: 19 (invention) SEQ ID NO: 1 (reference) ka Kd KD ka Kd KD SA (s−1M−1) (s−1) (M) (s−1M−1) (s−1) (M) human 6.1E+05 2.7E−04 4.4E−10 4.9E+05 1.6E−03 3.3E−09 cyno 5.2E+05 2.3E−04 4.5E−10 4.6E+05 1.4E−03 3.1E−09 rat 1.2E+06 8.0E−05 6.7E−11 3.9E+05 2.6E−01 6.7E−07 mouse 9.1E+05 2.5E−04 2.7E−10 6.6E+05 3.0E−02 3.9E−08 guinea 1.0E+06 3.1E−04 3.0E−10 9.4E+05 1.9E−02 2.0E−08 pig dog 7.2E+05 8.4E−04 1.2E−09 no binding pig 1.0E+05 4.6E−03 4.5E−08 no binding The serum albumin binder of SEQ ID NO: 19 (invention) was injected at different concentrations on immobilized SA on a ProteOn instrument. Binding and dissociation kinetics were analysed at pH 7.4. Kinetic parameters for SEQ ID NO: 1 are listed as a reference and were determined in separate experiments.

[0207] The long half-life of albumin in blood is mainly driven by two characteristics: (i) the large size (65 kDa) of albumin limits its glomerular filtration and (ii) albumin binds to FcRn at low pH (pH 6), which protects albumin from degradation in the lysosomes after passive endocytosis in endothelial and epithelial cells, by recycling from early endosome back to the extracellular environment. For albumin-binding Nanobodies to result in long serum half-life through albumin binding and subsequent recycling, these should stay bound to albumin in the pH range from 5.0 to 7.4. The dissociation rate of the serum albumin binder of SEQ ID NO:19 (invention) from HSA at pH 5, pH 6 and pH 7.4 was measured on a ProteOn instrument as described above, including SEQ ID NO:1 (reference) as a reference. the serum albumin binder of SEQ ID NO:19 (invention) and SEQ ID NO:1 (reference) were injected at 500 nM and 300 nM respectively in HBS-P+ pH 7.4 buffer. Dissociation buffers were 50 mM NaOAc/HOAc+150 mM NaCl+0.05% Tween-20 pH 5.0, 50 mM NaOAc/HOAc+150 mM NaCl+0.05% Tween-20 pH 6.0 and HBS-P+pH 7.4 respectively. Dissociation was analysed for 2700 s. Dissociation rates for the serum albumin binder of SEQ ID NO:19 (invention) do not differ significantly across the pH range from 5.0 to 7.4. The results are shown in Table 2.

TABLE-US-00004 TABLE 2 Dissociation rate of the serum albumin binder of SEQ ID NO: 19 (invention) from HSA at different pH. kd (s−1) pH SEQ ID NO: 19 (invention) SEQ ID NO: 1 (reference) pH 7.4 2.0E−04 1.3E−03 pH 6.0 3.2E−04 9.2E−04 pH 5.0 5.1E−04 1.1E−03 The serum albumin binders of SEQ ID NO: 19 (invention) and SEQ ID NO: 1 (reference) were injected on immobilized HSA. Dissociation was monitored at pH 5.0, 6.0 and 7.4 on a ProteOn instrument.

Example 2 Epitope

[0208] Epitope binning was analysed in a competition ELISA. Human serum albumin was coated at 125 ng/ml in PBS at 4° C. overnight. After blocking with PBS+1% casein, 1.5 nM SEQ ID NO:1 (reference)-cMycHis6 and a concentration series of competitors (His6Flag3-the serum albumin binder of SEQ ID NO:19 (invention), His6Flag3-SEQ ID NO:1 (reference) as positive control or hen egg lysozyme binding single domain antibody cAblys3-Flag3His6 as negative control) were added. Bound SEQ ID NO:1 (reference)-cMycHis6 was detected with goat anti-cMyc (Abcam ab19234) and HRP-labelled rabbit anti-goat (Genway 18-511-244226) antibodies. The serum albumin binder of SEQ ID NO:19 (invention) and SEQ ID NO:1 (reference) do not bind identical epitopes on HSA (FIG. 6).

Example 3 Interference with Interaction Between SA and FcRn

[0209] For the serum albumin binder of SEQ ID NO:19 (invention) to result in long half-life via albumin binding and subsequent recycling, it should not interfere with the binding of albumin to FcRn. This was analysed in SPR on a Biacore T100 (GE Healthcare) instrument. Human FcRn-human 32 microglobulin heterodimer (Sino Biological CT009-H08H) was immobilized on CM5 chip via standard amine coupling (Biacore amine coupling kit). A mixture of 1 μM HSA and 2 μM Nanobody (His6Flag3-the serum albumin binder of SEQ ID NO:19 (invention), His6Flag3-SEQ ID NO:1 (reference) or cAblys3-Flag3His6) in 50 mM NaPO4+150 mM NaCl+0.05% Tween-20 pH 6.0 was injected at 10 μl/min for 120 s, followed by dissociation for 600 s. Binding curves were qualitatively compared with binding curve of 1 μM HSA in absence of Nanobody. the serum albumin binder of SEQ ID NO:19 (invention) did not interfere with the binding of HSA to FcRn (FIG. 7).

Example 4 PK Profile in Dog

[0210] Pharmacokinetics of a trivalent Nanobody construct (SEQ ID NO:53) after single i.v. dose are studied in dog. The trivalent construct was produced in Pichia pastoris and comprises two Nanobodies against respiratorial syncytial virus (RSV) with the albumin binding Nanobody of SEQ ID NO:21 at the C-terminal end (in which the three Nanobodies are linked to each other using (Gly4Ser)7 linkers).

[0211] The pharmacokinetic profile of the construct is evaluated in a pharmacokinetic study (non-crossover, single dose) in twelve fasted healthy male Beagle dogs (healthy as determined by a pre-study health examination). The construct is administered intravenously.

[0212] The animals are fasted (with uninterrupted access to water) overnight prior to dosing, and prior to collection of blood samples at pre-dose, 24, 168, and 336 hours after dosing (total fasting time no longer than 24 hours). Food is returned 4 hours post-dose, or immediately after blood collection at 24, 168, and 336 hours. Body weight is also recorded and tracked during the study.

[0213] 1 ml of whole blood (or 2-3 ml pre-dose and at 24, 168 and 336 hours) for plasma is collected from a peripheral vessel pre-dose and at 5, 15, 30 min and 1, 4, 8, 12, 24, 72, 168, 240, 336, 504, and 672 hours post initial dose. Collected samples for serum processing are kept at room temperature and are allowed to clot. Blood is be centrifuged at 10,000 rpm for 2 minutes using a refrigerated centrifuge (set to maintain 4° C.). Samples are frozen within 30 minutes from collection until they are submitted for analysis (including measurement of concentration of the relevant Nanobody construct).