OA17072A - CX3CR1-binding polypeptides. - Google Patents
CX3CR1-binding polypeptides. Download PDFInfo
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- OA17072A OA17072A OA1201400309 OA17072A OA 17072 A OA17072 A OA 17072A OA 1201400309 OA1201400309 OA 1201400309 OA 17072 A OA17072 A OA 17072A
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Abstract
The present invention relates to CX3CR1binding polypeptides, in particular polypeptides comprising specific immunoglobulin domains. The invention also relates to nucleic acids encoding such polypeptides; to methods for preparing such polypeptides; to host cells expressing or capable of expressing such polypeptides; to compositions comprising such polypeptides; and to uses of such polypeptides or such compositions, in particular for prophylactic, therapeutic and diagnostic purposes.
Description
The présent invention relates to CX3CR1-binding polypeptides, In particular 5 polypeptides comprising spécifie immunoglobulin domains. The invention also relates to nucleic acids encoding such polypeptides; to methods for preparing such polypeptides; to host celis expressing or capable of expressing such polypeptides; to compositions comprising such polypeptides; and to uses of such polypeptides or such compositions, in particular for prophylactic, therapeutic and 10 diagnostic purposes.
BACKGROUND OF THE INVENTION
CX3CR1 is a G-protein coupled intégral membrane protein, which is a chemokine receptor. It is predominantly expressed on cell types such as monocytes, is dendritic cells and T cells that hâve been associated with the initiation and progression of atherosclerotic plaques. It is upregulated on monocytes by oxidized liplds and médiates migration of these cells into and survival within plaques. Its unique ligand fractalkine (FKN) is expressed on the surface of vascular endothélial and smooth muscle cells in lésions where it modulâtes leukocyte adhesion. Fractalkine is also released into the circulation by proteolytic cleavage where it fonctions as a chemotactic agent.
In humans, a CX3CR1 variant (V249I/T280M) with decreased activity has been shown to be associated with a lower risk of cardiovascular disease (coronary heart disease, cerebrovascular disease or peripheral vascular diseaseXMcDermott, 2001 ; Cire Res 89:401 ), coronary artery disease (angiographie evidence of stenosis) (McDermott, 2003; J. Clin. Invest. 111:1241), and carotid artery occlusive disease (Ghilardi, 2004; Stroke 35:1276). CX3CR1 co-localized with fractalkine which showed enhanced immunostaining by polyclonal antibodies within atherosclerotic plaques (Wong, 2002 Cardiovasc.
Path. 11:332). No fractalkine staining was observed in non-plaque arterial régions.
-1 17072
CX3CR1-BINDING POLYPEPTIDES
FIELD OF THE INVENTION
The présent invention relates to CX3CR1-binding polypeptides, in particular polypeptides comprising spécifie immunoglobulin domains. The Invention also relates to nuclelc acids encoding such polypeptides; to methods for preparing such polypeptides; to host cells expressing or capable of expressing such polypeptides; to compositions comprising such polypeptides; and to uses of such polypeptides or such compositions, In particular for prophylactic, therapeutic and ro diagnostic purposes.
BACKGROUND OF THE INVENTION
CX3CR1 Is a G-protein coupled intégral membrane protein, which is a chemokine receptor. It is predominantly expressed on cell types such as monocytes, dendritic celis and T cells that hâve been associated with the Initiation and progression of atherosclerotic plaques. It Is upregulated on monocytes by oxidized lipids and médiates migration of these cells Into and survival within plaques. Its unique ligand fractalkine (FKN) is expressed on the surface of vascular endothélial and smooth muscle cells in lésions where it modulâtes leukocyte adhesion. Fractalkine is also released Into the circulation by proteolytic cleavage where It fonctions as a chemotactic agent.
In humans, a CX3CR1 variant (V249I/T280M) with decreased activity has been shown to be associated with a lower risk of cardiovascular disease (coronary heart disease, cerebrovascular disease or peripheral vascular disease)(McDermott, 2001 ; Cire Res 89:401 ), coronary artery disease (angiographie evidence of stenosis) (McDermott, 2003; J. Clin. Invest. 111:1241), and carotid artery occlusive disease (Ghilardi, 2004; Stroke 35:1276). CX3CR1 co-localized with fractalkine which showed enhanced immunostaining by polyclonal antibodies within atherosclerotic plaques (Wong, 2002 Cardiovasc.
Path. 11:332). No fractalkine stalning was observed In non-plaque arterial régions.
-1 ri
Several independent mouse genetic studies hâve shown a bénéficiai effect of CX3CR1 deficiency on atherosclerosls. A réduction in lésion area in the aortic arch and thoraclc aorta as well as a decrease in monocyte/macrophage accumulation in plaques was seen in two Independently derived strains of CXSCRI^' apoE7’ mice fed a high fat diet (Combadière, 2003; Circulation, 107:1009, Lesnik, 2003; J. Clin. Invest. 111:333).
This shows that CX3CR1 Is invoived in cardiovascular diseases and the modulation of Its activity could provide promislng thérapies. There Is therefore a need for antagonist molécules against CX3CR1 with bénéficiai pharmacological properties, which can be used as therapeutic agents to treat diseases, in particular cardiovascular diseases in humans.
Accordingly, one aim of the présent invention Is to provide anti-CX3CR1 antagonist molécules. In particular anti>CX3CR1 antagonist molécules, which hâve high binding affinity to CX3CR1.
A further aim of the présent invention is to provide anti-CX3CR1 antagonist molécules, which hâve high specificity for CX3CR1.
A further aim of the présent invention Is to provide anti-CX3CR1 antagonists, which hâve potent activity.
A further aim of the présent invention Is to provide antl-CX3CR1 antagonists, which hâve a favorable bloavaiiability and half-life.
A further aim of the présent invention is to provide anti-CX3CR1 antagonists, which hâve favorable biophysicai properties.
Further aims of the présent invention include combinations of any of the aims set forth above.
SUMMARY OF THE INVENTION
The Invention provides polypeptides which bind to human CX3CR1 and are capable of blocking the binding of human fractalkine to human CX3CR1. ln one aspect, the polypeptide Is an immunoglobulin comprising an antigen-binding domain comprising three complementarity determining régions CDR1, CDR2 and CDR3, wherein said immunoglobulin binds to human CX3CR1 and is capable of blocking the binding of human fractalkine to human CX3CR1. ln a further aspect,
-217072 the polypeptide comprises one or more anti-CX3CR1 immunoglobulin single variable domain, wherein said polypeptide is capable of blocking the binding of human fractalkine to human CX3CR1.
In one aspect, a polypeptide of the présent invention is characterized by one or more of the following properties:
• Bind with high affinity to human CX3CR1 ;
• Block the binding of soluble fractalkine to human CX3CR1 ;
• Inhibit fractalkine induced chemotaxls;
• Inhibit fractalkine Induced human CX3CR1 receptor Intemalization;
· Cross-react with cyno CX3CR1 within 10-foid of E/ICso for human CX3CR1 for binding and functional inhibition.
In a further aspect, a polypeptide of the présent Invention comprises an antlCX3CR1 immunoglobulin single variable domain and further comprises a half-life extending moiety, for example an aibumin binding moiety, a polyethylene glycol is molécule or a Fc domain. In a further aspect, a polypeptide of the présent invention comprises two or more antl-CX3CR1 Immunoglobulin single variable domains. In one aspect, the two anti-CX3CR1 Immunoglobulin single variable domains are covalently linked by a linker peptide. In one aspect, the two antiCX3CR1 Immunoglobulin single variable domains in a polypeptide of the présent 20 invention hâve the same amino add sequence. In another aspect, the two antl*
CX3CR1 Immunoglobulin single variable domains in a polypeptide of the présent invention hâve different amino acid sequences. In one aspect, a polypeptide of the présent Invention comprises two anti-CX3CR1 Immunoglobulin single variable domains and further comprises a haif-life extending moiety, for example an aibumin binding moiety, a polyethylene glycol molécule or a Fc domain.
In one aspect, a polypeptide of the présent Invention comprises a first antiCX3CR1 immunoglobulin single variable domain covalently linked to an aibumin binding moiety by a first linker peptide, wherein said aibumin binding moiety Is further covalently linked to a second anti-CX3CR1 Immunoglobulin single variable 30 domain by a second linker peptide.
In one aspect, a polypeptide of the présent Invention comprises an antl-CX3CR1 immunoglobulin single variable domain covalently linked to a Fc domain by a
-317072 linker peptide. In one aspect, such polypeptide comprising an anti-CX3CR1 immunoglobulin single variable domain covalently linked to a Fc domain by a linker peptide Is provided as a dimer, for example through disulfide bridges. The polypeptides of the présent invention are used for the prévention, treatment, aliénation and/or diagnosis of CX3CR1 -associated diseases, disorders or conditions, in particular cardiovascular diseases, such as atherosclerosis.
In a further aspect, the présent invention provides:
Embodiment 1 : An Immunoglobulin comprising an antigen-binding domain comprising three complementarity determining réglons CDR1, CDR2 and CDR3, wherein said immunoglobulin binds to human CX3CR1 and is capable of blocking the binding of human fractalkine to human CX3CR1.
rs Embodiment 2: A polypeptide comprising one or more anti-CX3CR1 immunoglobulin single variable domain, wherein said polypeptide is capable of blocking the binding of human fractalkine to human CX3CR1.
Embodiment 3: A polypeptide according to embodiment 2, wherein said anti20 CX3CR1 immunoglobulin single variable domain consists essentially of four framework régions (FR1, FR2, FR3 and FR4) and three complementary determining régions (CDR1, CDR2 and CDR3).
Embodiment 4: A polypeptide according to embodiment 3, wherein said anti25 CX3CR1 Immunoglobulin single variable domain has the structure FR1 - CDR1 FR2 - CDR2 - FR3 - CDR3 - FR4.
Embodiment 5: A polypeptide according to any one of embodiments 2 to 4, wherein said anti-CX3CR1 immunoglobulin single variable domain is an antibody 30 domain.
Embodiment 6: A polypeptide according to embodiment 5, wherein said antiCX3CR1 Immunoglobulin single variable domain is a VH, VL, VHH, camelized
-417072
VH, or VHH that Is optimlzed for stablllty, potency, manufacturabllity and/or slmilarity to human framework réglons.
Embodiment 7; A polypeptide according to any one of embodiments 1 to 6, wherein said polypeptide has an affinlty to human CX3CR1 at:
a) an EC50 of less than or equal to 10nM, less than or equal to 5nM, less than or equal to 2.5nM or less than or equal to 1 nM, as determined by cell blnding FACS; or
b) an IC50 of less than or equal to 10nM, less than or equal to 5nM, less than or equal to 2.5nM or less than or equal to 1 nM, as determined by compétition FACS.
Embodiment 8: A polypeptide according to any one of embodiments 1 to 7, wherein said polypeptide blocks the blnding of human fractalklne to human CX3CR1 at an IC50 of less than or equal to 300nM, or less than or equal to 10OnM, or less than or equal to 20nM, or less than or equal to 10nM, or less than or equal to 5nM, or less than or equal to 2.5nM or less than or equal to 1nM.
Embodiment 9: A polypeptide according to any one of embodiments 1 to 8, wherein said polypeptide Inhlblts fractalklne induced chemotaxls medlated by human CX3CR1 at an IC50 of less than or equal to 500 nM, or of less than or equal to 100 nM, or of less than or equal to 75 nM, or of less than or equal to 50 nM, or less than or equal to 10 nM or less than or equal to 5nM.
Embodiment 10: A polypeptide according to any one of embodiments 1 to 9, wherein said polypeptide inhibits fractalklne intemalization mediated by human CX3CR1 at an IC50 of less than or equal to 10 nM, or less than or equal to 5nM or or less than or equal to 1 nM.
Embodiment 11: A polypeptide according to any one of embodiments 3 to 10, wherein said CDR3 has the amino acid sequence of Asp-Xaa1-Arg-Arg-Gly-TrpXaa2-Xaa3-Xaa4-Xaa5 (SEQ ID NO: 197), wherein:
- Xaa1 Is Pro, Ala or Gly;
- Xaa2 Is Asp or Asn;
-517072
- Xaa3 1s Thr or Ser;
- Xaa4 1s Arg, Lys, Ala or Gly; and
- Xaa5 IsTyrorPhe.
Embodiment 12: A polypeptide according to any one of embodiments 3 to 11, wherein:
a)
- Xaa1 Is Pro, Ala or Gly;
- Xaa2 Is Asp or Asn;
w - Xaa3 Is Thr;
- Xaa4 Is Arg or Lys; and
- Xaa5 Is Tyr, and/or
b) wherein said CDR3 is selected from any of SEQ ID No’s: 186-190.
1S
Embodiment 13: A polypeptide according to any one of embodiments 3 to 12, wherein said CDR3 has the amino acid sequence of Asp-Pro-Arg-Arg-Gly-TrpAsp-Thr-Arg-Tyr (SEQ ID NO: 186).
Embodiment 14: A polypeptide according to any one of embodiments 3 to 10, wherein:
i) said CDR1:
a) has the amino acid sequence of SEQ ID NO: 141 ;
b) has an amlno acid sequence that has at least 80% amlno acid Identity with the amino acid sequence of SEQ ID NO: 141 ;
c) has an amino acid sequence that has 2, or 1 amino acld(s) différence with the amino acid sequence of SEQ ID NO: 141, wherein
- at position 2 the S has been changed into T, or G;
- at position 6 the S has been changed into R;
so - at position 7 the N has been changed into T; and/or
- at position 9 the M has been changed into K; or
d) has an amino acid sequence selected from any one of SEQ ID NO’s: 141-145 and 213;
-617072 ii) said CDR2:
a) has the amino acid sequence of SEQ ID NO: 164;
b) has an amino acid sequence that has at least 70% amino acid identity with the amino acid sequence of SEQ ID NO: 164;
c) has an amino acid sequence that has 4, 3,2, or 1 amino acid(s) différence with the amino acid sequence of SEQ ID NO: 164, wherein
- at position 1 the G has been changed into A, L, V or S;
- at position 3 the N has been changed into D, S, Q, G or T;
- at position 4 the S has been changed Into T, K, G or P;
- at position 5 the V has been changed Into A;
- at position 6 the G has been changed Into D;
- at position 7 the I has been changed into T, or V;
- at position 8 the T has been changed into A; and/or
- at position 9 the K has been changed Into R; or
d) has an amino acid sequence selected from any one of SEQ ID NO's: 162-175 and 214-221; and üi) said CDR3:
a) has the amino acid sequence of SEQ ID NO: 186;
b) has an amino acid sequence that has at least 70% amino acid Identity the amino acid sequence of SEQ ID NO: 186;
c) has an amino acid sequence that has 3, 2, or 1 amino acid(s) différence with the amino acid sequences of SEQ ID NO: 186, wherein
- at position 2 the P has been changed into A, or G;
- at position 7 the D has been changed into N; and/or
- at position 9 the R has been changed into K; or
d) has an amino acid sequence selected from any one of SEQ ID NO's: 186-190.
Embodiment 15: A polypeptide according to any one of embodiments 3 to 10, wherein
i) said CDR1 has the amino acid sequence of SEQ ID NO: 146;
ii) said CDR2 has an amino acid sequence that a) has at least 90% amino acid Identity with the amino acid sequence of SEQ ID NO:
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176 or b) has the amino acid sequence of SEQ ID NO: 176 or 177; and lii) said CDR3 has the amino acid sequence of SEQ ID NO: 191.
s Embodiment 16: A polypeptide according to any one of embodiments 3 to 10, wherein
I) said CDR1:
a) has the amino acid sequence of SEQ ID NO: 147; or
b) has an amino add sequence that has 6, 5, 4,3, 2, or 1 amino add(s) différence with the amino acid sequence of SEQ ID NO: 147, wherein
- at position 1 the G has been changed into K, R, or A;
- at position 2 the T has been changed into I, P, S or L;
- at position 3 the I has been changed into V, or T;
- at position 4 the F has been changed Into L;
- at position 5 the S has been changed Into R, or D;
- at position 6 the N has been changed into S, T, or D; and/or
- at position 7 the N has been changed Into T, or Y; or
c) has an amino acid sequence selected from any one of SEQ ID NO’s: 147-161;
II) said CDR2:
a) has the amino acid sequence of SEQ ID NO: 179; or
b) has an amino acid sequences that has 4, 3, 2, or 1 amino acid(s) différence with the amino acid sequence of SEQ ID NO: 179, wherein
- at position 3 the S has been changed into T, or G;
- at position 4 the N has been changed into S, or I;
- at position 5 the S has been changed into T;
- at position 6 the G has been changed into Y; and/or
- at position 8 the T has been changed into A; or
c) has an amino acid sequence selected from any one of SEQ ID NO’s: 178-185; and iii) said CDR3:
a) has the amino acid sequence of SEQ ID NO: 192; or
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b) has an amino acid sequence that has at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 192; or
c) has an amino acid sequence that has 2, or 1 amino acid(s) différence with the amino acid sequence of SEQ ID NO: 192, wherein
- at position 2 the A has been changed into G;
- at position 8 the T has been changed into S;
- at position 9 the A has been changed into G; and/or
- at position 10 the Y has been changed into F; or
d) has an amino acid sequence selected from any one of SEQ ID NO’s: 192-196.
Embodiment 17: A polypeptide according to embodiment 3, wherein the amino acid sequences of said CDR1, CDR2 and CDR3 are set forth in:
- SEQ ID No: 141,162 and 186, respectively; or
- SEQ ID No: 141,163 and 187, respectively; or
- SEQ ID No: 141,164 and 186, respectively; or
- SEQ ID No: 141,166 and 186, respectively; or
- SEQ ID No: 141,167 and 186, respectively; or
- SEQ ID No: 141,167 and 189, respectively; or
- SEQ ID No: 141,168 and 186, respectively; or
- SEQ ID No: 141,168 and 187, respectively; or
- SEQ ID No: 141,169 and 190, respectively; or
- SEQ ID No: 141,170 and 186, respectively; or
- SEQ ID No: 141,171 and 186, respectively; or
- SEQ ID No: 141,174 and 186, respectively; or
- SEQ ID No: 141,175 and 187, respectively; or
- SEQ ID No: 142,165 and 188, respectively; or
- SEQ ID No: 142,173 and 188, respectively; or
- SEQ ID No: 143,164 and 186, respectively; or
- SEQ ID No: 144,172 and 187, respectively; or
- SEQ ID No: 145,172 and 187, respectively; or
- SEQ ID No: 141,214 and 186, respectively; or
- SEQ ID No: 141,215 and 186, respectively; or
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- SEQ ID No: 141,216 and 186, respectiveiy; or
- SEQ ID No: 141,217 and 186, respectiveiy; or » SEQ ID No: 141,218 and 186, respectiveiy; or
- SEQ ID No: 141,219 and 186, respectiveiy; or
- SEQ ID No: 141,220 and 186, respectiveiy; or
- SEQ ID No: 213,221 and 186, respectiveiy; or
- SEQ ID No: 213,214 and 186, respectiveiy.
Embodiment 18: A polypeptide according to embodiment 3, wherein the amino acid sequences of said CDR1, CDR2 and CDR3 are set forth in:
- SEQ ID No: 146,176 and 191, respectiveiy; or
- SEQ ID No: 146,177 and 191, respectiveiy.
Embodiment 19: A polypeptide according to embodiment 3, wherein the amino acid sequences of said CDR1, CDR2 and CDR3 are set forth in:
- SEQ ID No: 147,178 and 192, respectiveiy; or
- SEQ ID No: 147,179 and 192, respectiveiy; or
- SEQ ID No: 147,179 and 194, respectiveiy; or
- SEQ ID No: 148,179 and 193, respectiveiy; or
- SEQ ID No: 149,179 and 192, respectiveiy; or
- SEQ ID No: 149,180 and 192, respectiveiy; or
- SEQ ID No: 149,181 and 192, respectiveiy; or
- SEQ ID No: 149,183 and 192, respectiveiy; or
- SEQ ID No: 149,185 and 192, respectiveiy; or
- SEQ ID No: 150,179 and 194, respectiveiy; or
- SEQ ID No: 150,182 and 194, respectiveiy; or
- SEQ ID No: 151,179 and 193, respectiveiy; or
- SEQ ID No: 151,182 and 194, respectiveiy; or
- SEQ ID No: 151,184 and 196, respectiveiy; or
- SEQ ID No: 152,179 and 195, respectiveiy; or
- SEQ ID No: 153,179 and 194, respectiveiy; or
- SEQ ID No: 154,182 and 194, respectiveiy; or
- SEQ ID No: 155,179 and 195, respectiveiy; or
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- SEQ ID No: 156,181 and 192, respectively; or
- SEQ ID No: 157,179 and 194, respectively: or
- SEQ ID No: 158,179 and 192, respectively; or
- SEQ ID No: 159,178 and 192, respectively; or
- SEQ ID No: 160,179 and 194, respectively; or
- SEQ ID No: 161,179 and 194, respectively.
Embodiment 20: A polypeptide according to embodiment 3, wherein the amino acid sequencesof said CDR1, CDR2 and CDR3 are setforth in: SEQ ID NO's: w 141,164 and 186, respectively, or SEQ ID NO’s: 141,162 and 186, respectively.
Embodiment 21: A polypeptide according to embodiment 3, wherein the amino acid sequencesof said CDR1, CDR2 and CDR3 are setforth in: SEQ ID NO’s: 213, 214 and 186 respectively, SEQ ID NO’s: 213, 221 and 186 respectively, or is SEQ ID NO’s: 141,162 and 186 respectively.
Embodiment 22: A polypeptide according to any one of embodiments 2 to 10, wherein said anti-CX3CR1 Immunoglobulin single variable domain is a VHH domain comprising the sequence set forth In:
a) the amino acid sequence of SEQ ID NO: 3;
b) amino acid sequences that hâve at least 90% amino acid identity with the amino acid sequences of SEQ ID NO: 3;
c) amino acid sequences that hâve 11,10, 9,8, 7, 6, 5, 4, 3, 2, or 1 amino acid différence with the amino acid sequences of SEQ ID NO: 3; or
d) an amino acid sequence of any one of SEQ ID NO: 1 -48,121 -140 or 222224.
Embodiment 23: A polypeptide according to any one of embodiments 2 to 10, wherein said anti-CX3CR1 immunoglobulin single variable domain Is a VHH
3o domain comprising the sequence set forth in:
a) the amino acid sequence of SEQ ID NO: 49;
b) an amino acid sequence that has at least 95% amino acid identity with the amino acid sequences of SEQ ID NO: 49;
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c) an amino acid sequence that has 5, 4, 3, 2, or 1 amino acid différence with the amino acid sequences of SEQ ID NO: 49; or
d) an amino acid sequence of any one of SEQ ID NO: 49-52.
Embodiment 24: A polypeptide according to any one of embodiments 2 to 10, wherein said anti-CX3CR1 Immunogiobuiin single variable domain is a VHH domain comprising the sequence set forth ln:
a) the amino acid sequence of SEQ ID NO: 67;
b) an amino acid sequence that has at least 90% amino acid identity with the amino acid sequences of SEQ ID NO: 67;
c) an amino acid sequence that has 12,11,10,9, 8, 7,6, 5, 4, 3,2, or 1 amino acid différence with the amino acid sequences of SEQ ID NO: 67; or
d) an amino acid sequence of any one of SEQ ID NO: 53-120.
Embodiment 25: A polypeptide according to embodiment 2, wherein said antiCX3CR1 immunogiobuiin single variable domain comprises the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3.
Embodiment 26: A polypeptide according to embodiment 2, wherein said antiCX3CR1 immunogiobuiin single variable domain comprises the sequence set forth ln any one of SEQ ID NO: 121-140 or SEQ ID NO: 222-224.
Embodiment 27: A polypeptide according to any of one of the embodiments above, which is humanized and/or optimlzed for stability, potency, manufacturability and/or similarity to human framework réglons.
Embodiment 28: A polypeptide according to embodiment 27, which Is humanized and/or sequence optimized in one or more of the following positions (according to Kabat numbering): 1,11,14,16, 74, 83,108.
Embodiment 29: A polypeptide according to embodiment 28, comprising one or more of the following mutations: E1D, S11L, A14P, E16G, A74S, K83R, Q108L.
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Embodiment 30: A polypeptide according to any one of embodiments 3-29, in which:
I) FR1 is selected from SEQ ID NO’s: 198-204;
II) FR2 is selected from SEQ ID NO’s: 205-208;
lii) FR3 Is selected form SEQ ID NO’s: 209-210; and/or
Iv) FR4 Is selected from SEQ ID NO’s: 211-212.
Embodiment 31: A polypeptide according to any one of embodiments 3-30, which is humanized and/or sequence optimlzed In one or more of the following positions (according to Kabat numbering): 52, 53.
Embodiment 32: A polypeptide according to embodiment 31, comprising one or more of the following mutations: N52S, S53T.
Embodiment 33: A polypeptide according to any one of embodiments 3-32, In which CDR2 Is selected from SEQ ID NO’s: 214-221.
Embodiment 34: A polypeptide according to any one of embodiments 2-33, wherein said antl-CX3CR1 immunoglobulin single variable domain comprises the sequence set forth In any of SEQ ID NO’s: 138-140 or 222-224.
Embodiment 35: A polypetide according to any one of embodiments 22 to 26, wherein said VHH domain consists of any one of said amino acid sequences.
Embodiment 36: A polypeptide according to any one of embodiments 2 to 35, wherein said immunoglobulin single variable domain cross-blocks the binding of at least one of the Immunoglobulin single variable domains of SEQ ID NO’s: 1120,121-140 and 222-224 to CX3CR1.
Embodiment 37: A polypeptide according to any one of embodiments 2 to 35, wherein said Immunoglobulin single variable domain Is cross-blocked from binding to CX3CR1 by at least one of the amino acid sequences of SEQ ID NO’s: 1-120,121-140 and 222-224.
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Embodiment 38: A polypeptide according to any one of embodiments 2 to 37, wherein the polypeptide further comprises a half-life extending moiety.
Embodiment 39: A polypeptide according to embodiment 38, wherein said half5 life extending moiety is covalently linked to said polypeptide and 1s selected from the group consisting of an albumln binding moiety, such as an anti-albumln Immunoglobulin domain, a transferrin binding moiety, such as an anti-transferrin Immunoglobulin domain, a polyethylene glycol molecuie, a recombinant polyethylene glycol molecuie, human sérum albumln, a fragment of human sérum io albumln, an albumln binding peptide or a Fc domain.
Embodiment 40: A polypeptide according to embodiment 38 or 39, whereln said half-life extending moiety conslsts of an anti-albumin immunoglobulin single variable domain.
Embodiment 41: A polypeptide according to embodiment 40, wherein the Immunoglobulin single variable domain is selected from a VHH domain, a humanized VHH domain, a camelized VH domain, a domain antibody, a single domain antibody and/or dAbs.
Embodiment 42: A polypeptide according to embodiment 41, wherein the antialbumin immunoglobulin single variable domain is selected from SEQ ID NO’s: 230-232.
Embodiment 43; A polypeptide according to any one of embodiment 2 to 39, wherein said polypeptide is linked to an Fc portion (such as a human Fc, for example as set forth in SEQ ID NO: 252), optionally via a suitable linker or hinge région.
a» Embodiment 44: A polypeptide according to any one of embodiments 2 to 39, wherein said polypeptide is further linked to one or more constant domains (for example, 2 or 3 constant domains that can be used as part of/to form an Fc portion), to an Fc portion and/or to one or more antibody parts, fragments or domains that confer one or more effector fonctions to the polypeptide of the
-1417072 invention and/or may confer the ability to bind to one or more Fc receptors, optionally via a suitable linker or hinge région.
Embodiment 45: A polypeptide according to any one of embodiments 2 to 37, wherein said polypeptide further comprises a second Immunoglobulin single variable domain, preferably a second anti-CX3CR1 immunoglobulin single variable domain.
Embodiment 46: A polypeptide according to embodiment 45, wherein said first and said second immunoglobulin single variable domains are covalently linked by a linker peptide.
Embodiment 47: A polypeptide according to embodiment 45 or 46, wherein said second Immunoglobulin single variable domains essentially consist of four framework régions (FR1 to FR4) and three complementary determining régions (CDR1 to CDR3).
Embodiment 48: A polypeptide according to any one of embodiments 45 to 47, wherein said first and said second Immunoglobulin single variable domains are antibody domains.
Embodiment 49: A polypeptide according to any one of embodiments 45 to 48, wherein said first and second immunoglobulin single variable domains are a VH, VL, VHH, camelized VH, or VHH that is optimized for stability, potency, manufacturability and/or similarity to human framework régions.
Embodiment 50: A polypeptide according to any one of embodiments 45 to 49, wherein said CDR1 to CDR3 of said second immunoglobulin single variable domain are set forth in any one of embodiments 11 to 21.
Embodiment 51 : A polypeptide according to any one of embodiments 45 to 50, wherein said first and said second immunoglobulin single variable domains comprise the same CDR3.
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Embodiment 52; A polypeptide according to embodiment 51, wherein said CDR3 is set forth In any one of embodiment 11 to 13.
Embodiment 53: A polypeptide according to any one of embodiments 45 to 53, wherein said first and said second Immunoglobulin single variable domains comprise the same CDR1, CDR2 and CDR3.
Embodiment 54: A polypeptide according to embodiment 53, wherein said CDR1 to CDR3 are set forth in any one of embodiments 11 to 21.
Embodiment 55: A polypeptide according to any one of embodiments 45 to 54, wherein said first and said second immunoglobulin single variable domains comprise the same VHH domain.
Embodiment 56: A The polypeptide according to any one of embodiments 45 to 55, wherein said VHH domain is set forth in any one of embodiments 22 to 37.
Embodiment 57: A polypeptide comprising a first immunoglobulin single variable domain comprising the CDR1, CDR2 and CDR3 set forth SEQ ID NO's: 141, 164 and 186 or SEQ ID NO’s: 141,162 and 186 and a second immunoglobulin single variable domain as set forth in any one of embodiments 2 to 37.
Such a polypeptide may in particular be a polypeptide according to any of embodiments 45 to 56.
Embodiment 58: A polypeptide according to embodiment 57, wherein said first immunoglobulin single variable domain comprises the CDR1, CDR2 and CDR3 set forth in SEQ ID NO’s: 213, 214 and 186, SEQ ID NO’s: 213, 221 and 186 or SEQ ID NO’s: 141,162 and 186.
Embodiment 59: A polypeptide according to embodiment 57 or 58, wherein said second immunoglobulin single variable domain comprises the CDR1, CDR2 and CDR3 set forth SEQ iD NO’s: 141,164 and 186 or SEQ ID NO’s: 141,162 and 186.
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Embodiment 60: A polypeptide according to embodiment 57 Or 58, wherein said second îmmunoglobulin single variable domain comprises the CDR1, CDR2 and CDR3 set forth ln: SEQ ID NO’s: 213, 214 and 186, SEQ ID NO’s: 213,221 and 186 or SEQ ID NO’s: 141,162 and 186.
Embodiment 61 : A polypeptide comprising a first Immunoglobulin single variable domain, wherein said first Immunoglobulin singie variable domain Is a VHH domain comprising the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 and a second immunoglobulin single variable domain according to any one of embodiments 2 to 37.
Such a polypeptide may in particular be a polypeptide according to any of embodiments 45 to 60.
Embodiment 62: A polypeptide according to embodiment 61, wherein said first Immunoglobulin single variable domain Is a VHH domain comprising the sequence set forth in any one of SEQ ID NO: 121-140 or 222-224.
Embodiment 63: A polypeptide according to embodiment 61 or 62, wherein said second Immunoglobulin single variable domain is a VHH domain comprising the sequence set forth In SEQ ID NO: 1 or SEQ iD NO: 3.
Embodiment 64: A polypeptide according to embodiment 63, wherein said second immunoglobulin single variable domain Is a VHH domain comprising the sequence set forth in any one of SEQ ID NO: 121-140 or 222-224.
Embodiment 65: A polypeptide according to any one of embodiments 45 to 64, wherein the polypeptide further comprises a half-life extending moiety.
Embodiment 66: A polypeptide according to embodiment 65, wherein said halflife extending moiety is covalently linked to said polypeptide and is selected from the group consisting of an albumin binding moiety, such as an anti-albumin immunoglobulin domain, a transferrin binding moiety, such as an anti-transferrin immunoglobulin domain, a polyethylene glycol molécule, a recombinant
-1717072 polyethylene glycol molécule, human sérum albumin, a fragment of human sérum albumin, an albumin binding peptide or a Fc domain.
Embodiment 67: A polypeptide according to embodiment 66, wherein said halflife extending moiety consists of an anti-albumin immunoglobulin single variable domain.
Embodiment 68: A polypeptide according to embodiment 67, wherein the immunoglobulin single variable domain is selected from a VHH domain, a humanized VHH domain, a camelized VH domain, a domain antibody, a single domain antibody and/or dAbs.
Embodiment 69: A polypeptide according to embodiment 68, wherein the antialbumin immunoglobulin single variable domain is selected from SEQ ID NO's: 230-232.
Embodiment 70: A polypeptide according to any one of embodiments 45 to 64, wherein said polypeptide is linked to an Fc portion (such as a human Fc, for example as set forth In SEQ ID NO: 252), optionally via a suitable linker or hinge région.
Embodiment 71 : A polypeptide according to any one of embodiments 45 to 66, wherein said polypeptide is further linked to one or more constant domains (for example, 2 or 3 constant domains that can be used as part of/to form an Fc portion), to an Fc portion and/or to one or more antibody parts, fragments or domains that confer one or more effector fonctions to the polypeptide of the invention and/or may confer the ability to bind to one or more Fc receptors, optionally via a suitable linker or hinge région.
Embodiment 72: A polypeptide comprising the amino acid sequence of any one ofSEQIDNO: 225-227.
Embodiment 73: A polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 249 or 277-281.
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Embodiment 74: A polypeptide comprising the amino acid sequence of any one ofSEQ ID NO: 257-262.
Embodiment 75: A polypeptide comprising the amino acid sequence of any one s ofSEQ ID NO: 253 or254.
Embodiment 76: A polypeptide comprising the amino acid sequence of any one ofSEQIDNO: 263 or 266.
io Embodiment 77: A polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 267-276 and 282.
Embodiment 78: A nucleic acid molécule comprising a région encoding a polypeptide according to any one of embodiments 1 to 77.
Embodiment 79: An expression vector comprising a nucleic acid molécule according to embodiment 78.
Embodiment 80: A host cell carrying an expression vector comprising a nucleic so acid molécule, said nucleic acid molécule comprising a région encoding a polypeptide according to any one of embodiments 1 to 77, wherein said host cell Is capable of expressing a polypeptide according to any one of embodiments 1 to 77, and wherein said host cell Is a prokaryotic or a eukaryotic cell.
Embodiment 81 : A pharmaceutical composition comprising (i) as the active ingrédient, one or more polypeptides according to any one of embodiments 1 to 77, and (ii) a pharmaceutically açceptable carrier, and optionally (iii) a diluent, excipient, adjuvant and/or stabilizer.
Embodiment 82: A method of manufacturing a polypeptide according to any one of embodiments 1 to 77, comprising the steps of
- culturing a host cell under conditions that allow expression of a polypeptide according to any one of embodiments 1 to 77, wherein said host cell carrying an expression vector comprising a nucleic 35 acid molécule, said nucleic acid molécule comprising a région encoding
- 1917072 a polypeptide according to any one of embodiments 1 to 77, and wherein said host cell is a prokaryotic or a eukaryotic cell;
- recovering said polypeptide; and
- purifying said polypeptide.
Embodiment 83: A method of using a polypeptide according to any one of embodiments 1 to 77 for the treatment, prévention or alleviation of a disease, disorder or condition, in particular in a human being.
Embodiment 84: The method of embodiment 83, wherein said disease, disorder or condition Is a CX3CR1 -associated disease, disorder or condition.
Embodiment 85: The method of embodiment 83, wherein said disease, disorder or condition is atherosclerosis.
Embodiment 86: An injectable pharmaceutical composition comprising the polypeptide according to any one of embodiments 1 to 77, said composition being suitable for Intravenous or subcutaneous injection in a human being.
Embodiment 87: A method for preventing and/or treating a disease or disorder that Is associated with CX3CR1, wherein said method comprises administering to a subject in need thereof a pharmaceutically active amount of at least one polypeptide according to any one of embodiments 1 to 77.
Embodiment 88: A method of embodiment 85, further comprising administering an additional therapeutic agent selected from the group consisting of a statin, an antipiatelet, an anticoagulant, an antidiabetic and an antihypertensive.
Embodiment 89: A method for inhibiting the binding of CX3CR1 to fractalkine In a mammalian cell, comprising administering to the cell a polypeptide according to any one of embodiments 1 to 77, whereby signaling mediated by the fractalkine Is Inhibited.
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Embodiment 90: A method for detecting and/or quantifying CX3CR1 Ievels in a biological sample by contacting the sample with a polypeptide according to any one of embodiments 1 to 77 and detecting binding of the polypeptide with CX3CR1.
Embodiment 91 : A method for diagnosing an CX3CR1-associated disorder or for determining If a subject has an increased risk of developing an CX3CR1associated disorder, wherein the method comprises contacting a biological sample from a subject with a polypeptide according to any one of embodiments 1 io to 77 and detecting binding of the polypeptide to CX3CR1 to détermine the expression or concentration of CX3CR1.
Embodiment 92. A polypeptide according to any one of embodiments 1 to 77 for use in the treatment, prévention or alleviation of a disease, disorder or condition, is In a human being.
Embodiment 93. The polypeptide for use according to embodiment 92, wherein the disease, disorder or condition is a CX3CR1-associated disease, disorder or condition.
Embodiment 94. The polypeptide for use according to embodiment 92, wherein the disease, disorder or condition is selected from cardio- and cerebrovascular atherosclerotic disorders, peripheral artery disease, restenosis, diabetic nephropathy, glomerulomephritis, human crescentic glomerulonephritis, IgA 25 nephropathy, membranous nephropathy, lupus nephritis, vasculitis including
Henoch-Schonlein purpura and Wegener*s granulomatosis, rheumatoid arthritis, osteoarthritis, allograft rejection, systemic sclerosis, neurodegenerative disorders and demyelinating disease, muitiple sclerosis (MS), A!zheimer*s disease, puimonary diseases such as COPD, asthma,neuropathie pain, inflammatory pain, 30 or cancer.
Embodiment 95. The polypeptide for use according to embodiment 92, wherein the disease, disorder or condition ls atheroscierosis.
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Embodiment 96. Use of a polypeptide according to any of embodiments 1 to 77 for the manufacture of a médicament for the treatment, prévention or alleviation of a disease, disorder or condition, in a human being.
Embodiment 97. The method according to embodiment 87, wherein the disease or disorder is selected from cardio- and cerebrovascular atherosclerotic disorders, peripheral artery disease, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schonleln purpura and Wegeneris granulomatosis, rheumatoid arthritis, osteoarthritis, allograft rejection, systemic sclerosis, neurodegenerative disorders and demyelinating disease, multiple sclerosis (MS), Alzheimeris disease, pulmonary diseases such as COPD, asthma,neuropathie pain, inflammatory pain, or cancer.
Embodiment 98. The method according to embodiment 87, wherein the disease, disorder or condition is atherosclerosis.
Embodiment 99. A diagnostic kit or diagnostic method comprising a polypeptide according to any one of embodiments 1 to 77, or the use thereof.
Embodiment 100. A diagnostic kit or diagnostic method according to embodiment 99, for the diagnosis of at least one of cardio- and cerebrovascular atherosclerotic disorders, peripheral artery disease, restenosis, diabetic nephropathy, glomerulomephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schonlein purpura and Wegener*s granulomatosis, rheumatoid arthritis, osteoarthritis, allograft rejection, systemic sclerosis, neurodegenerative disorders and demyelinating disease, multiple sclerosis (MS), Alzheimer’s disease, pulmonary diseases such as COPD, asthma,neuropathie pain, inflammatory pain, or cancer.
DETAILED DESCRIPTION OF THE INVENTION
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Définitions
The above and other aspects and embodiments of the invention will become s clear from the further description herein, ln which:
a) Unless indicated or defined otherwise, ail terms used hâve their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al, Molecular Cloning: A
Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, Genes IV, Oxford University Press, New York, (1990), and Roltt et al., Immunology (2^ Ed.), Gower Medical Publishlng, London, New York (1989), as well as to the general background art clted herein; Furthermore, unless indicated otherwise, ail methods, steps, techniques and manipulations that is are not specifïcally described in detail can be performed and hâve been performed ln a manner known perse, as will be clearto the skilled person. Reference Is for example again made to the standard handbooks, to the générai background art referred to above and to the further référencés cited thereln;
b) Unless Indicated otherwise, the terms immunoglobulin” and * immunoglobu lin sequence* - whether used herein to refer to a heavy chaln antibody or to a conventional 4-chain antibody- are used as general terms to include both the fullslze antibody, the individual chains thereof, as well as ail parts, domains or fragments thereof (including but not limited to antigen-blnding domains or fragments such as VHH domains or VH/VL domains, respectively). ln addition, the term sequence as used herein (for example ln terms like immunoglobulin sequence, antibody sequence, (single) variable domain sequence, VHH sequence or protein sequence), shouid generally be understood to include both the relevant amino acid sequence as well as nucleic acid sequences or nucléotide sequences encoding the same, unless the context requlres a more limited interprétation;
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c) The term c/oma/n (of a polypeptide or protein) as used herein refers to a folded protein structure which has the ability to retain its tertiary structure independently of the rest of the protein. Generally, domains are responsible for discrète functional properties of proteins, and in many cases may be added, removed or transferred to other protelns without loss of fonction of the remalnder of the protein and/orofthe domain.
d) The term immunoglobulin domain* as used herein refers to a globular région of an antibody chain (such as e.g. a chain of a conventional 4-chaln antibody or of a heavy chain antibody), or to a polypeptide that essentially conslsts of such a globular région. Immunoglobulin domains are characterized in that they retain the Immunoglobulin fold characteristic of antibody moiecules, which consists of a 2-layer sandwich of about 7 antiparallel beta-strands arranged In two beta-sheets, optionally stabilized by a conserved disulphide bond.
e) The term immunoglobulin variable domain* as used herein means an Immunoglobulin domain essentially consisting of four Framework régions which are referred to In the art and herelnbelow as framework région Γ or FR1; as framework région 2 orFR2; as framework région 3 or FR3; and as framework région 4 or FR4, respectively; which framework réglons are Interrupted by three complementarity determining régions or CDRs, which are referred to in the art and herelnbelow as complementarity determining région 1orCDR1; as complementarity determining région 2 orCDR2; and as complementarity determining région 3 or CDR3, respectively. Thus, the general structure or sequence of an Immunoglobulin variable domain can be indicated as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. It Is the immunoglobulin variable domain(s) that confer specificity to an antibody for the antigen by carrying the antigen-binding site.
f) The terms immunoglobulin single variable domain* and “single variable domain as used herein mean an Immunoglobulin variable domain which is capable of specifically binding to an epitope of the antigen without patring with an additional variable immunoglobulin domain. One example of immunogiobulin
-2417072 single variable domains in the meaning of the présent invention are domain antibodies, such as the immunogiobulin singie variable domains VH and VL (VH domains and VL domains). Another example of immunoglobulin single variable domains are VHH domains (or simpiy VHHs) from camelids, as defined hereinafter.
In view of the above définition, the antigen-bindîng domain of a conventiona! 4chain antibody (such as an IgG, IgM, IgA, IgD or IgE molécule; known in the art) or of a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (ali known in the art) derived from such conventiona! 4-chaln antibody, would normally not be regarded as an Immunoglobulin single variable domain, as, In these cases, binding to the respective epitope of an antigen wouid normally not occur by one (single) Immunoglobulin domain but by a pair of (associating) immunoglobulin domains such as light and heavy chain variable domains, I.e. by a VH-VL pair of Immunoglobulin domains, which jointiy blnd to an epitope of the respective antigen.
f1 ) VHH domains, also known as VHHs, VhH domains, VHH antibody fragments, and VHH antibodies, hâve originally been described as the antigen binding immunoglobulin (variable) domain of heavy chain antibodies (i.e. of antibodies devold of light chains; Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: Naturally occurring antibodies devold of light chains; Nature 363, 446-448 (1993)). The term VHH domain has been chosen in order to dlstingulsh these variable domains from the heavy chain variable domains that are présent in conventional 4-chain antibodies (which are referred to herein as Vh domains or VH domains) and from the light chain variable domains that are présent In conventional 4-chaln antibodies (which are referred to herein as Vl domains or VL domains). VHH domains can specifically bind to an epitope without an additional antigen binding domain (as opposed to VH or VL domains In a conventional 4-chain antibody, in which case the epitope is recognized by a VL
-2517072 domain together with a VH domain). VHH domains are smali, robust and efficient antigen récognition units formed by a single immunogiobuiin domain.
ln the context of the présent invention, the terms VHH domain, VHH, VhH domain, VHH antibody fragment, VHH antibody, as well as Nanobody® and Nanobody® domain (Nanobody being a trademark of the company Ablynx N.V.; Ghent; Belgium) are used interchangeably and are représentatives of immunogiobuiin single variable domains (having the structure: FR1-CDR1-FR2CDR2-FR3-CDR3-FR4 and specifically binding to an epitope without requiring the presence of a second immunogiobuiin variable domain), and which are distinguished from VH domains by the so-called hallmark residues, as defined in e.g. W02009/109635, Fig. 1.
The amino acid residues of a VHH domain are numbered according to the general numbering for Vh domains given by Kabat et al. (Sequence of proteine of
Immunological interest, US Public Heaith Services, NIH Bethesda, MD, Publication No. 91), as applied to VHH domains from Camelids, as shown e.g. in Figure 2 of Riechmann and Muyidermans, J. immunol. Methods 231,25-38 (1999). According to this numbering,
- FR1 comprises the amino acid residues at positions 1-30,
- CDR1 comprises the amino acid residues at positions 31-35,
- FR2 comprises the amino acids at positions 36-49,
- CDR2 comprises the amino acid residues at positions 50-65,
- FR3 comprises the amino acid residues at positions 66-94,
-25 - CDR3 comprises the amino acid residues at positions 95-102, and
- FR4 comprises the amino acid residues at positions 103-113.
However, it should be noted that - as is well known in the art for Vh domains and for VHH domains - the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that Is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to
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Kabat may or may not correspond to the actual numbering of the amino acid residues In the actual sequence.
Alternative methods for numbering the amino acid residues of Vh domains, which methods can also be applied In an analogous manner to VHH domains, are known in the art. However, in the présent description, clalms and figures, the numbering according to Kabat and applied to VHH domains as described above wili be followed, unless indicated otherwise.
The total number of amino acid residues In a VHH domain wil! usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.
Further structural characteristics and functional properties of VHH domains and polypeptides containing the same can be summarized as follows:
VHH domains (which hâve been designed by nature to functionally blnd to an antigen without the presence of, and without any Interaction with, a light chain variable domain) can function as a single, relatively small, functional antigenblnding structural unit, domain or polypeptide. This distinguishes the VHH domains from the VH and VL domains of conventional 4-chain antibodies, which by themselves are generally not suited for practical application as single antigenbinding proteins or immunoglobulin single variable domains, but need to be combined in some form or another to provide a functional antigen-binding unit (as in for example conventional antibody fragments such as Fab fragments; in scFv*s, which consist of a VH domain covalently linked to a VL domain).
Because of these unique properties, the use of VHH domains - either alone or as part of a larger polypeptide - offers a number of significant advantages over the use of conventional VH and VL domains, scFVs or conventional antibody fragments (such as Fab- or F(ab')2-fragments):
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- only a single domain Is required to blnd an antigen with high affinity and with high seiectivity, so that there Is no need to hâve two separate domains présent, nor to assure that these two domains are présent in the right spacial conformation and configuration (i.e. through the use of especially designed linkers, as with scFv*s);
- VHH domains can be expressed from a singie gene and requlre no posttranslationai folding or modifications;
- VHH domains can easily be engineered into muitivaient and multispecific formats (as further discussed herein);
- VHH domains are highly soiuble and do not hâve a tendency to aggregate (as with the mouse-derived antigen-binding domains described by Ward étal., Nature 341:544-546(1989));
- VHH domains are highly stable to heat, pH, proteases and other denaturing agents or conditions and, thus, may be prepared, stored or transported without a the use of réfrigération equlpments, conveylng a cost, time and environmental savings;
- VHH domains are easy and reiatively cheap to préparé, even on a scale required for production. For example, VHH domains and polypeptides containing the same can be produced using microbial fermentation (e.g. as further described below) and do not require the use of mammaiian expression Systems, as with for example conventional antlbody fragments:
- VHH domains are reiatively small (approximately 15 kDa, or 10 times smaller than a conventional IgG) compared to conventional 4-chain antibodies and antigen-binding fragments thereof, and therefore — show hlgh(er) pénétration into tissues and
- can be administered in higher doses than such conventional 4-chaln antibodies and antigen-binding fragments thereof;
- VHH domains can show so-cailed cavity-binding properties (inter alia due to their extended CDR3 loop, compared to conventional VH domains) and can therefore also access targets and epitopes not accessible to conventional 4-chain antibodies and antigen-binding fragments thereof.
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Methods of obtaining VH H domains binding to a spécifie antigen or epitope hâve been described eariier, e.g. in W02006/040153 and W02006/122786. As also described therein in detail, VHH domains derived from camelids can be humanized by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence by one or more of the amino acid residues that occur at the corresponding position(s) In a VH domain from a conventional 4-chain antibody from a human being. A humanized VHH domain can contain one or more fully human framework région sequences, and, In an even more spécifie embodiment, can contain human framework région sequences derived from DP-29, DP-47, DP-51, or parts thereof, optionally combined with JH sequences, such as JH5.
f2) Domain antibodies?, also known as Dabs, Domain Antibodies, and dAbs (the terms Domain Antibodies and dAbs being used as trademarks by the GlaxoSmithKIine group of companles) hâve been described in e.g. Ward, E.S., et al.: Binding activities of a répertoire of single immunoglobulin variable domains secreted from Escherlchla coli; Nature 341: 544-546 (1989); Holt, L.J. et al.: Domain antibodies: proteins for therapy; TRENDS in Biotechnology 21(11 ): 484-490 (2003); and W02003/002609.
Domain antibodies essentially correspond to the VH or VL domains of noncameiid mammalians, in particular human 4-chain antibodies. In order to bind an epitope as a single antigen binding domain, i.e. without being paired with a VL or VH domain, respectively, spécifie sélection for such antigen binding properties Is required, e.g. by using libraries of human single VH or VL domain sequences. Domain antibodies hâve, like VHHs, a molecular weight of approximately 13 to approximately 16 kDa and, if derived from fully human sequences, do not requlre humanlzation for e.g. therapeutical use In humans. As In the case of VHH domains, they are well expressed also In prokaryotic expression Systems, providing a significant réduction in overall manufacturing cost.
Domain antibodies, as well as VHH domains, can be subjected to affînity maturation by introducing one or more alterations in the amino acid sequence of one or more CDRs, which alterations resuit in an improved affînity of the resulting
-2917072 immunoglobulin single variable domain for its respective antigen, as compared to the respective parent molécule. Affinity-matured immunoglobulin single variable domain molécules of the invention may be prepared by methods known in the art, for example, as described by Marks étal.,1992, Biotechnology 10:779-783, or Barbas, étal., 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813.; Shler et al., 1995, Gene 169:147-155; Yelton étal., 1995, Immunol. 155:1994-2004; Jackson étal., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J. Mol. Biol. 226(3): 889 896; KS Johnson and RE Hawkins, Affinity maturation of antibodies using phage display, Oxford University Press 1996.
f3) Furthermore, it wili also be clearto the skilled person that it Is possible to graft one or more of the CDR’s mentioned above onto other scaffolds, including but not limited to human scaffolds or non-lmmunoglobulin scaffolds. Suitable scaffolds and techniques for such CDR grafting are known in the art.
g) The terms epItope” and antigenle déterminant*, which can be used interchangeably, refer to the part of a macromolecule, such as a polypeptide, that is recognized by antigen-binding molécules, such as conventional antibodies or the polypeptides of the invention, and more particulariy by the antigen-binding site of said molécules. Epitopes define the minimum binding site for an immunoglobulin, and thus represent the target of specificity of an Immunoglobulin.
The part of an antigen-binding molécule (such as a conventional antibody or a polypeptide of the invention) that recognlzes the epitope is called a paratope.
h) The term biparatopic’' (antigen-)binding molécule or blparatopic polypeptide as used herein shall mean a polypeptide comprising a first immunoglobulin single variable domain and a second immunoglobulin single variable domain as herein defined, wherein these two variable domains are capable of binding to two different epitopes of one antigen, which epitopes are not normally bound at the same time by one monospecific immunoglobulin, such as e.g. a conventional antibody or one immunoglobulin single variable domain. Blparatopic polypeptides
-3017072 can be composed of variable domains which hâve different epitope specificities, and do not contain mutually complementary variable domain pairs which bind to the same epitope. The two variable domains do therefore not compete with each other for binding to the target.
I) A polypeptide (such as an Immunoglobulin, an antibody, an Immunoglobulin single variable domain, a polypeptide of the invention, or generally an antigen binding molecuie or a fragment thereof) that can bind to or specifically bind to”, that has affinity foi” and/or that has specificity fof a certain epitope, antigen or protein (or for at least one part, fragment or epitope thereof) Is said to be againsf or directed againsf said epitope, antigen or protein or is a binding” molecuie with respect to such epitope, antigen or protein, or is said to be anti”epitope, “anti’-antigen or antf-proteln (e.g antl-CX3CR1).
k) Generally, the term specificity refers to the number of different types of antigens or epitopes to which a particular antigen-binding molecuie or antigenbinding protein (such as an immunoglobulin, an antibody, an Immunoglobulin single variable domain, or a polypeptide ofthe Invention) can bind. The specificity of an antigen-binding protein can be determined based on its affinity and/or avldity. The affinity, represented by the equilîbrium constant for the dissociation of an antigen with an antigen-binding protein (KD), Is a measure for the binding strength between an epitope and an antigen-binding site on the antigen-binding protein: the lesser the value of the KD, the stronger the binding strength between an epitope and the antigen-binding molecuie (altematively, the affinity can aiso be expressed as the affinity constant (KA), which is 1/KD). As will be clear to the skilled person (for example on the basis of the further disclosure herein), affinity can be determined in a manner known per se, depending on the spécifie antigen of Interest. Avidity Is the measure of the strength of binding between an antigenbinding molecuie (such as an Immunoglobulin, an antibody, an Immunoglobulin single variable domain, or a polypeptide of the Invention) and the pertinent antigen. Avidity is related to both the affinity between an epitope and its antigen binding site on the antigen-binding molecuie and the number of pertinent binding sites présent on the antigen-binding molecuie.
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I) Amino acid residues will be Indicated according to the standard three-letter or one-letter amino acid code, as generally known and agreed upon In the art. When comparing two amino acid sequences, the term amino acid différence refers to insertions, délétions or substitutions of the Indicated number of amino acid residues at a position of the référencé sequence, compared to a second sequence. In case of substitution(s), such substitution(s) will preferably be conservative amino acid substitution(s), which means that an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence on the fonction, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example from WO 98/49185, wherein conservative amino acid substitutions preferably are substitutions in which one amino acid within the following groups (I) - (v) Is substituted by another amino acid residue within the same group: (i) small aliphatic, nonpolar or sllghtly polar residues: Ala, Ser, Thr, Pro and Gly; (ii) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gin; (iii) polar, positively charged residues: His, Arg and Lys; (iv) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (v) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative amino acid substitutions are as follows:
Ala into Gly or into Ser;
Arg Info Lys;
Asn Into Gin or Into His;
Asp Into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His Into Asn or into Gin; lie Into Leu or into Val; Leu into lie or into Val; Lys into Arg, into Gin or Into Glu; Met Into Leu, Into Tyr or into lie;
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Phe Into Met, into Leu or into Tyr;
Ser Into Thr,
Thr Into Ser,
Trp into Tyr
Tyr into Trp or into Phe;
Val Into Ile or Into Leu.
m) A nucieic acid or polypeptide molécule is considered to be (in) essentially isolated (form)” - for example, when compared to its native biological source and/or the reaction medium or cultivation medium from which It has been obtained - when it has been separated from at least one other component with which It is usually associated in said source or medium, such as another nucieic acid, another proteln/polypeptide, another biological component or macromolecule or at least one contaminant, Impurity or minor component. In is particular, a nucieic acid or polypeptide molécule is considered essentially
Isolated when it has been purified at least 2-fold, in particular at least 10- fold, more In particular at ieast 10O-fold, and up to 1000-fold or more. A nucieic acid or polypeptide molécule that Is In essentially Isolated form Is preferably essentially homogeneous, as determined using a suitable technique, such as a suitable
2o chromatographical technique, such as polyacrylamlde-gelelectrophoresls;
n) ''Sequence Identity between e.g. two immunoglobulin single variable domain sequences Indicates the percentage of amino acids that are Identlcal between these two sequences. it may be calculated or determined as described in paragraph f) on pages 49 and 50 of W008/020079. Sequence similarity indicates the percentage of amino acids that either are identlcal or that represent conservative amino acid substitutions.
Target specificity
The polypeptides ofthe invention hâve specificityforhuman CX3CR1. Thus, the polypeptides ofthe Invention preferably bind to human CX3CR1 (SEQ ID NO:
-3317072
255). ln one aspect, the polypeptides of the présent invention also blnd to cynomoigus CX3CR1 (SEQ ID NO: 256).
Polypeptides ofthe Invention
The invention provides novel pharmaceutically active agents for the prévention, treatment, aliénation and/or diagnosis of CX3CR1 associated diseases, disorders or conditions, such as cardiovascular diseases. In particular, the invention provides polypeptides which bind to human CX3CR1 and are capable of blocking the binding of human fractalkine to human CX3CR1. In one aspect, the polypeptide Is an immunoglobulin comprising an antigen-binding domain comprising three complementarity determining régions CDR1, CDR2 and CDR3, wherein said immunoglobulin bindsto human CX3CR1 and is capable of blocking the binding of human fractalkine to human CX3CR1. ln a further aspect, the polypeptide comprises one or more anti-CX3CR1 immunoglobulin single variable domain, wherein said polypeptide is capable of blocking the binding of human fractalkine to human CX3CR1.
In one aspect, a polypeptide of the présent invention is characterized by one or more of the foliowing properties:
• Bind with high affinity to human CX3CR1, for example at an EC50 of less than or equal to 10nM, less than or equal to 5nM, less than or equal to 2.5nM or less than or equal to 1nM, as determined by ceil binding FACS;
• Block the binding of human fractalkine to human CX3CR1, for example at an IC50 of less than or equai to 300nM, or less than or equal to 100nM, or less than or equal to 20nM, or less than or equal to 10nM, or less than or equal to 5nM, or less than or equal to 2.5nM or less than or equal to 1 nM;
• Inhibit fractalkine induced chemotaxis mediated by human CX3CR1, for example at an IC50 of less than or equal to 500 nM, or less than or equal to 100 nM, or of less than or equal to 75 nM, or less than or equal to 50 nM, or less than or equai to 10 nM or less than or equal to 5nM; the obtained efficacy of inhibition is more than or equal to 15%, or more than or equal to 50%, or more than or equal to 80%, or more than or equal to 95%;
-3417072 • Inhibit fractalkine Induced intemalization mediated by human CX3CR1, for example at an IC50 of less than or equai to 10 nM or less than or equal to 5nM;
• Cross-react with cynomolgus CX3CR1, for example within 10-fold of E/iCso for human CX3CR1 for binding and functionai inhibition.
In a further aspect, a polypeptide of the présent invention further comprises a half-life extending moiety, for example an albumin binding moiety, a polyethylene glycol molécule or a Fc domain. In a further aspect, a polypeptide ofthe présent 10 invention comprises two or more anti-CX3CR1 Immunoglobulin single variable domains. In one aspect, the two antî-CX3CR1 Immunoglobulin single variable domains are covalently linked by a linker peptide. In one aspect, the two antiCX3CR1 immunoglobulin single variable domains In a polypeptide ofthe présent invention hâve the same amino acid sequence. In another aspect, the two anti15 CX3CR1 Immunoglobulin single variable domains in a polypeptide of the présent invention hâve different amino acid sequences. In one aspect, a polypeptide of the présent Invention comprises two anti-CX3CR1 immunoglobulin single variable domains and further comprises a half-life extending moiety, for example an albumin binding moiety, a polyethylene glycol molécule or a Fc domain.
In one aspect, a polypeptide of the présent invention comprises a first antiCX3CR1 immunoglobulin single variable domain covalently linked to an albumin binding moiety by a first linker peptide, wherein said albumin binding moiety is further covalently linked to a second antl-CX3CR1 Immunoglobulin single variable domain by a second linker peptide.
In one aspect, a polypeptide of the présent invention comprises an anti-CX3CR1 immunoglobulin single variable domain covalently linked to a Fc domain by a linker peptide. In one aspect, such polypeptide comprising an anti-CX3CR1 immunoglobulin single variable domain covalently linked to a Fc domain by a linker peptide is provided as a dimer, for example through disulfide bridges.
Polypeptides according to the présent invention are obtalned as described hereinbelow. In summary, single variable domains of the présent Invention were
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Identified from a library expressing single variable domains (VHH) derived from a llama immunized with DNA encoding human CX3CR1. The phage library was panned on hCX3CR1 viral lipoparticles and binding phage were screened for their ability to compete for receptor binding with Alexa-fluor labeled fractalkine 5 (AF-FKN). Représentative single variable domains of the présent Invention are described herein ln further details.
ln one aspect, an Immunoglobulin single variable domain of the présent Invention consists essentially of fourframework régions (FR1, FR2, FR3 and FR4) and three complementary determlnlng réglons (CDR1, CDR2 and CDR3). In particular, the immunoglobulin single variable domain has the structure FR1 * CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. ln one aspect, the immunoglobulin single variable domain Is an antibody domain.
is ln one aspect, the CDR3 of a polypeptide of the présent invention, in particular a immunoglobulin single domain of the présent invention has the amino acid sequence of Asp-Xaa1-Arg-Arg-Gly-Trp-Xaa2-Xaa3-Xaa4-Xaa5 as set forth in SEQ ID NO: 197, wherein:
- Xaa1 Is Pro, Ala or Gly;
- Xaa2 Is Asp or Asn;
- Xaa3 Is Thr or Ser;
- Xaa4 Is Arg, Lys, Ala or Gly; and
- Xaa5 IsTyr or Phe.
ln one aspect, the CDR3 of a polypeptide of the présent invention, ln particular a Immunoglobulin single domain of the présent invention, has the amino acid sequence of Asp-Xaa1-Arg-Arg-Gly-Trp-Xaa2-Xaa3-Xaa4-Xaa5 as set forth in SEQ ID NO: 197, wherein:
- Xaa1 is Pro, Ala or Gly;
· Xaa2 is Asp or Asn;
- Xaa3 Is Thr;
- Xaa4 is Arg or Lys; and
- Xaa5 IsTyr.
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In one aspect, the CDR3 of a polypeptide ofthe présent invention, in particular an immunoglobuling single domain of the présent invention, has the amino acid sequence of Asp-Pro-Arg-Arg-Gly-Trp-Asp-Thr-Arg-Tyr as set forth in SEQ ID NO: 186.
In a further aspect, a polypeptide of the présent invention, in particular an immunoglobuling single domain of the présent Invention, has the following CDR1, CDR2 and CDR3:
- CDR1:
a) has the amino acid sequence of GSIFSSNAMA (SEQ ID NO: 141); or
b) has an amino acid sequence that has at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 141; or
c) has an amino acid sequence that has 2, or 1 amino acid(s) différence with the amino acid sequence of SEQ ID NO: 141, wherein
- at position 2 the S has been changed Into T, or G;
- at position 6 the S has been changed into R;
- at position 7 the N has been changed into T; and/or
- at position 9 the M has been changed Into K; or
d) has an amino acid sequence selected from any one of SEQ ID NO’s: 141-145 and 213;
- CDR2:
a) has the amino acid sequence of GINSVGITK (SEQ ID NO: 164); or
b) has an amino acid sequence that has at least 70% amino acid Identity with the amino acid sequence of SEQ ID NO: 164; or
c) has an amino acid sequence that has 4, 3, 2, or 1 amino acid(s) différence with the amino acid sequence of SEQ ID NO: 164, wherein
- at position 1 the G has been changed Into A, L, V or S;
- at position 3 the N has been changed into D, S, Q, G or T;
- at position 4 the S has been changed Into T, K, G or P;
- at position 5 the V has been changed into A;
- at position 6 the G has been changed into D;
- at position 7 the I has been changed into T, or V;
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- at position 8 the T has been changed into A; and/or
- at position 9 the K has been changed Into R; or
d) has an amino acid sequence selected from any one of SEQ ID NO's: 162-175 and 214-221; and
- CDR3:
a) has the amino acid sequence of DPRRGWDTRY (SEQ ID NO: 186); or
b) has an amino acid sequence that has at least 70% amino acid Identity the amino acid sequence of SEQ ID NO: 186; or
c) has an amino acid sequence that has 3, 2, or 1 amino acid(s) différence to with the amino acid sequences of SEQ ID NO: 186, wherein
- at position 2 the P has been changed Into A, or G;
- at position 7 the D has been changed Into N; and/or
- at position 9 the R has been changed Into K; or
d) has an amino acid sequence selected from any one of SEQ iD NO's:
is 186-190.
In a further aspect, a polypeptide of the présent Invention, In particular a Immunoglobuling single domain of the présent invention, has the following CDR1, CDR2 and CDR3, wherein:
- said CDR1 has the amino acid sequence of GRTFSSYAMG (SEQ ID NO:
146);
- said CDR2 has an amino acid sequence that a) has at least 90% amino acid Identity with the amino acid sequence of GISGSASRKY (SEQ ID NO: 176) or b) has the amino acid sequence ofSEQ ID NO: 176 or 177; and
- said CDR3 has the amino acid sequence of SNSYPKVQFDY (SEQ ID NO:
191). ’
In a further aspect, a polypeptide of the présent Invention, in particular an immunoglobuling single domain of the présent invention, has the following CDR1, 30 CDR2 and CDR3:
- saidCDRI: .
a) has the amino acid sequence of GTIFSNNAMG (SEQ ID NO: 147); or
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b) has an amlno acid sequence that has 6, 5, 4,3, 2, or 1 amino acid(s) différence with the amino acid sequence of SEQ ID NO: 147, wherein
- at position 1 the G has been changed into K, R, or A;
- at position 2 the T has been changed Into I, P, S or L;
- at position 3 the I has been changed into V, or T;
- at position 4 the F has been changed Into L;
- at position 5 the S has been changed into R, or D;
- at position 6 the N has been changed into S, T, or D; and/or
- at position 7 the N has been changed Into T, or Y; or w c) has an amino acid sequence selected from any one of SEQ ID NO's:
147-161:
- said CDR2:
a) has the amino acid sequence of SISNSGSTN (SEQ ID NO: 179); or
b) has an amino acid sequences that has 4, 3, 2, or 1 amlno acld(s) is différence with the amino acid sequence of SEQ ID NO: 179, wherein
- at position 3 the S has been changed into T, or G;
- at position 4 the N has been changed Into S, or I;
- at position 5 the S has been changed Into T;
- at position 6 the G has been changed Into Y; and/or
- at position 8 the T has been changed into A; or
c) has an amino acid sequence selected from any one of SEQ ID NO’s: 178-185; and
- said CDR3:
a) has the amino acid sequence of DARRGWNTAY (SEQ ID NO: 192); or
b) has an amino acid sequence that has at least 80% amlno acid Identity with the amlno acid sequence of SEQ ID NO: 192; or
c) has an amino acid sequence that has 2, or 1 amino acid(s) différence with the amlno acid sequence of SEQ ID NO: 192, wherein
- at position 2 the A has been changed Into G;
- at position 8 the T has been changed into S;
- at position 9 the A has been changed into G; and/or
- at position 10 the Y has been changed into F; or
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d) has an amino acid sequence selected from any one of SEQ ID NO’s: 192-196.
ln a further aspect, a polypeptide of the présent invention, in particular an immunoglobuling single domain of the présent invention, has the foilowing CDR1, CDR2 and CDR3:
- SEQ ID No: 141,162 and 186, respectively; or
- SEQ ID No: 141,163 and 187, respectively; or
- SEQ ID No: 141,164 and 186, respectively; or
- SEQ ID No: 141,166 and 186, respectively; or
- SEQ ID No: 141,167 and 186, respectively; or
- SEQ ID No: 141,167 and 189, respectively; or
- SEQ ID No: 141,168 and 186, respectively; or
- SEQ ID No: 141,168 and 187, respectively; or
- SEQ ID No: 141,169 and 190, respectively; or
- SEQ ID No: 141,170 and 186, respectively; or
- SEQ ID No: 141,171 and 186, respectively; or
- SEQ ID No: 141,174 and 186, respectively; or
- SEQ ID No: 141,175 and 187, respectively; or
- SEQ ID No: 142,165 and 188, respectively; or
- SEQ ID No: 142,173 and 188, respectively; or
- SEQ ID No: 143,164 and 186, respectively; or
- SEQ ID No: 144,172 and 187, respectively; or
- SEQ ID No: 145,172 and 187, respectively; or
- SEQ ID No: 141,214 and 186, respectively; or
- SEQ ID No: 141,215 and 186, respectively; or
- SEQ ID No: 141,216 and 186, respectively; or
- SEQ ID No: 141,217 and 186, respectively; or
- SEQ ID No: 141,218 and 186, respectively; or
- SEQ ID No: 141,219 and 186, respectively; or
- SEQ ID No: 141, 220 and 186, respectively; or
- SEQ ID No: 213, 221 and 186, respectively; or
- SEQ ID No: 213, 214 and 186, respectively.
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In a further aspect, a polypeptide of the présent Invention, in particular an Immunoglobuling single domain of the présent invention, has the following CDR1, CDR2 and CDR3:
- SEQ ID No: 146,176 and 191, respectively; or
- SEQ ID No: 146,177 and 191, respectively.
In a further aspect, a polypeptide of the présent Invention, In particular an immunoglobuling single domain of the présent Invention, has the following CDR1, CDR2 and CDR3:
- SEQ ID No: 147,178 and 192, respectively; or
- SEQ ID No: 147,179 and 192, respectively; or
- SEQ ID No: 147,179 and 194, respectively; or
- SEQ ID No: 148,179 and 193, respectively; or
- SEQ ID No: 149,179 and 192, respectively; or
- SEQ ID No: 149,180 and 192, respectively; or
- SEQ ID No: 149,181 and 192, respectively; or
- SEQ ID No: 149,183 and 192, respectively; or
- SEQ ID No: 149,185 and 192, respectively; or
- SEQ ID No: 150,179 and 194, respectively; or
- SEQ ID No: 150,182 and 194, respectively; or
- SEQ ID No: 151,179 and 193, respectively; or
- SEQ ID No: 151,182 and 194, respectively; or
- SEQ ID No: 151,184 and 196, respectively; or
- SEQ ID No: 152,179 and 195, respectively; or
- SEQ ID No: 153,179 and 194, respectively; or
- SEQ ID No: 154,182 and 194, respectively; or
- SEQ ID No: 155,179 and 195, respectively; or
- SEQ ID No: 156,181 and 192, respectively; or
- SEQ ID No: 157,179 and 194, respectively; or
- SEQ ID No: 158,179 and 192, respectively; or
- SEQ ID No: 159,178 and 192, respectively; or
- SEQ ID No: 160,179 and 194, respectively; or
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- SEQ ID No: 161,179 and 194, respectively.
In a further aspect, a polypeptide of the présent Invention, In particular an Immunoglobuiing single domain of the présent invention, has the CDR1, CDR2 s and CDR3 set forth In:
- SEQ ID NO's: 141,164 and 186; or
- SEQ ID NO's: 141,162 and 186.
In a further aspect, a polypeptide of the présent invention, in particular an to immunoglobuiing single domain of the présent Invention, has the CDR1, CDR2 and CDR3 set forth in:
- SEQ ID NO's: 213, 214 and 186; or
- SEQ ID NO’s: 213, 221 and 186; or
- SEQ ID NO’s: 141,162 and 186.
Représentative polypeptides of the présent Invention having the CDRs described above are shown in Tables 1,2, 3 (représentative polypeptides of families 101, 9 and 13, respectively) and 4 (représentative polypeptides of optimlzed variants of family 101.
Table 1: Family 101
| Nanobody | SEQ | CDR1* | SEQ CDR1 | CDR2* | SEQ CDR2 | CDR3* | SEQ CDR3 |
| CX3CR1BII PMP66B02 | 1 | GSIFSSNA MA | 141 | AINSVGVT K | 162 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP54A12 | 2 | GSIFSSNA MA | 141 | VINSVGIT K | 163 | DARRGW DTRY | 187 |
| CX3CR1BII PMP54A3 | 3 | GSIFSSNA MA | 141 | GINSVGIT K | 164 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP54A4 | 4 | GSIFSSNA MA | 141 | GINSVGIT K | 164 | DPRRGW DTRY | 186 |
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| CX3CR1BII PMP54A5 | 5 | GSIFSSNA MA | 141 | GINSVGIT K | 164 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP54A7 | 6 | GTIFSSNA MA | 142 | GINSVDIT K | 165 | DPRRGW NTRY | 188 |
| CX3CR1BI! PMP54B1 | 7 | GSIFSSNA MA | 141 | GINSVGIT K | 164 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP54B2 | 8 | GTIFSSNA MA | 142 | GINSVDIT K | 165 | DPRRGW NTRY | 188 |
| CX3CR1BII PMP54B3 | 9 | GSIFSSNA MA | 141 | AINSVGIT K | 166 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP54B5 | 10 | GSIFSSNA MA | 141 | GINSVGIT K | 164 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP54D5 | 11 | GSIFSSNA MA | 141 | LINSVGIT K | 167 | DGRRGW DTRY | 189 |
| CX3CR1BII PMP54D8 | 12 | GSIFSSNA MA | 141 | GINSVGIT K | 164 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP54F6 | 13 | GSIFSSNA MA | 141 | AINSVGIT K | 166 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP54G3 | 14 | GSIFSSNA MA | 141 | LINSVGIT K | 167 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP54H1 | 15 | GTIFSSNA MA | 142 | GINSVDIT K | 165 | DPRRGW NTRY | 188 |
| CX3CR1BII PMP54H4 | 16 | GSIFSSNA MA | 141 | VINSVGIT K | 163 | DARRGW DTRY | 187 |
| CX3CR1BII PMP61F10 | 17 | GTIFSSNA MA | 142 | GINSVDIT K | 165 | DPRRGW NTRY | 188 |
| CX3CR1BII PMP61D1 | 18 | GSIFSSNA MA | 141 | LINSVGIT K | 167 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP61D5 | 19 | GSIFSSNA MA | 141 | LINSVGIT K | 167 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP61E2 | 20 | GSIFSSNA MA | 141 | GINSVGIT K | 164 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP61F11 | 21 | GSIFSSNA MA | 141 | AINSVGIT K | 166 | DPRRGW DTRY | 186 |
| CX3CR1BII | 22 | GSIFSSNA | 141 | LINSVGIT | 167 | DPRRGW | 186 |
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| PMP61G2 | MA | K | DTRY | ||||
| CX3CR1BII PMP61G3 | 23 | GSIFSSNA MA | 141 | AINSVGIT K | 166 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP61G4 | 24 | GSIFSSNA MA | 141 | AINSVGIT K | 166 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP61F4 | 25 | GSIFSSNA MA | 141 | VINTVGIT K | 168 | DARRGW DTRY | 187 |
| CX3CR1BII PMP61A11 | 26 | GSIFSSNA MA | 141 | VINSVGIT K | 163 | DARRGW DTRY | 187 |
| CX3CR1BII PMP61B2 | 27 | GSIFSSNA MA | 141 | VINTVGIT K | 168 | DARRGW DTRY | 187 |
| CX3CR1BII PMP61C9 | 28 | GSIFSSNA MA | 141 | LIDSAGIT K | 169 | DARRGW NTKY | 190 |
| CX3CR1BII PMP65H02 | 29 | GSIFSSNA MA | 141 | AINSVGIT K | 166 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP65E11 | 30 | GSIFSSNA MA | 141 | GINSVGIA K | 170 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP65E10 | 31 | GSIFSSNA KA | 143 | GINSVGIT K | 164 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP65E05 | 32 | GSIFSSNA MA | 141 | GINSVGIT K | 164 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP65B11 | 33 | GSIFSSNA MA | 141 | VINKVGIT K | 171 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP65B07 | 34 | GSIFSSNA MA | 141 | AINSVGIT K | 166 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP65B09 | 35 | GSIFSRNA MA | 144 | SINSVGIT K | 172 | DARRGW DTRY | 187 |
| CX3CR1BII PMP65H01 | 36 | GGIFSRN AMA | 145 | SINSVGIT K | 172 | DARRGW DTRY « | 187 |
| CX3CR1BII PMP65G07 | 37 | GTIFSSNA MA | 142 | GINSVDIT R | 173 | DPRRGW NTRY | 188 |
| CX3CR1BII PMP66H08 | 38 | GSIFSSNA MA | 141 | LINSVGIT K | 167 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP66H04 | 39 | GSIFSSNA MA | 141 | AINSVGIT K | 166 | DPRRGW DTRY | 186 |
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| CX3CR1BII PMP66F02 | 40 | GSIFSSNA MA | 141 | LINSVGIT K | 167 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP66E11 | 41 | GSIFSSNA MA | 141 | AINSVGTT K | 174 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP66D10 | 42 | GSIFSSNA MA | 141 | LINSVGIT K | 167 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP66D08 | 43 | GSIFSSNA MA | 141 | GINSVGIT K | 164 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP66A04 | 44 | GSIFSSNA MA | 141 | LINSVGIT K | 167 | DPRRGW DTRY | 186 |
| CX3CR1BII PMP66D04 | 45 | GTIFSSNA MA | 142 | GINSVDIT K | 165 | DPRRGW NTRY | 188 |
| CX3CR1BII PMP66D02 | 46 | GSIFSSNA MA | 141 | VINSVGIT K | 163 | DARRGW DTRY | 187 |
| CX3CR1BII PMP66D06 | 47 | GSIFSSNA MA | 141 | SIDSVGIT K | 175 | DARRGW DTRY | 187 |
| CX3CR1BII PMP66G01 | 48 | GSIFSSNA MA | 141 | LINSVGIT K | 167 | DGRRGW DTRY | 189 |
*CDR sequences were determined according to Antibody Engineering, vol 2 by Konetermann & Dübel (Eds.), Springer Veriag Heidelberg Berlin, 2010. The sequence numbers In the table (SEQ) refer to the sequences in the sequence 5 listing of the Instant application.
Table 2: Family 9
| Nanobody | SEQ | CDR1* | SEQ CDR1 | CDR2* | SEQ CDR2 | CDR3* | SEQ CDR3 |
| CX3CR1BII PMP11H11 | 49 | GRTFSSY AMG | 146 | GISGSAS RKY | 176 | SNSYPKV QFDY | 191 |
| CX3CR1BII PMP12B6 | 50 | GRTFSSY AMG | 146 | GISGSAS RKY | 176 | SNSYPKV QFDY | 191 |
| CX3CR1BII PMP12G9 | 51 | GRTFSSY AMG | 146 | GISGSGS RKY | 177 | SNSYPKV QFDY | 191 |
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| CX3CR1BII | 52 | GRTFSSY | 146 | GISGSGS | 177 | SNSYPKV | 191 |
| PMP15G11 | AMG | RKY | QFDY |
*CDR sequences were determined according to Antibody Engineering, vol 2 by Konetermann & Dûbel (Eds.), Springer Veriag Heidelberg Berlin, 2010. The sequence numbers In the table (SEQ) refer to the sequences In the sequence 5 listing of the instant application.
Table 3: Famlly 13
| Nanobody | SEQ | CDR1* | SEQ CDR1 | CDR2* | SEQ CDR2 | CDR3* | SEQ CDR3 |
| CX3CR1BII PMP18E6 | 53 | GTIFSNNA MG | 147 | SISSSGST N | 178 | DARRGW NTAY | 192 |
| CX3CR1BII PMP12C2 | 54 | GTIFSNTA MG | 148 | SISNSGST N | 179 | DARRGW NSGY | 193 |
| CX3CR1BII PMP18A10 | 55 | GIIFSNNA MG | 149 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP18A2 | 56 | GIIFSNNA MG | 149 | SIGSTYST N | 180 | DARRGW NTAY | 192 |
| CX3CR1BII PMP18A8 | 57 | RTIFRSNA MG | 150 | SISNSGST N | 179 | DARRGW NTGY | 194 |
| CX3CR1BII PMP18A9 | 58 | GIIFSNNA MG | 149 | SISSTYST N | 181 | DARRGW NTAY | 192 |
| CX3CR1BII PMP18B7 | 59 | GTIFRSNA MG | 151 | SISNSGST N | 179 | DARRGW NSGY | 193 |
| CX3CR1BII PMP18B9 | 60 | GTIFSNNA MG | 147 | SISSSGST N | 178 | DARRGW NTAY | 192 |
| CX3CR1BII PMP18C6 | 61 | GTIFSNNA MG | 147 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP18C9 | 62 | GIIFSNNA MG | 149 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP18D1 | 63 | GIIFSNNA MG | 149 | SISNSGST N | 179 | DARRGW NTAY | 192 |
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| CX3CR1BII PMP18D10 | 64 | GTIFSNNA MG | 147 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP18D12 | 65 | GTIFSNNA MG | 147 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP18F1 | 66 | GTIFSNNA MG | 147 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP18F5 | 67 | GTIFSNNA MG | 147 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP18F6 | 68 | GTIFSNNA MG | 147 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP18F9 | 69 | GTIFRTNA MG | 152 | SISNSGST N | 179 | DGRRGW NTGY | 195 |
| CX3CR1BII PMP18G5 | 70 | RTIFRSNA MG | 150 | SISNSGST N | 179 | DARRGW NTGY | 194 |
| CX3CR1BII PMP18H1 | 71 | GTIFSNNA MG | 147 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP18H10 | 72 | KTIFRSNA MG | 153 | SISNSGST N | 179 | DARRGW NTGY | 194 |
| CX3CR1BII PMP18H7 | 73 | GIIFSNNA MG | 149 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP18H9 | 74 | GTIFSNNA MG | 147 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP20B3 | 75 | GIIFSNNA MG | 149 | SIGSTYST N | 180 | DARRGW NTAY | 192 |
| CX3CR1BII PMP20C12 | 76 | GTIFRSNA MG | 151 | SISNSGST N | 179 | DARRGW NSGY | 193 |
| CX3CR1BII PMP20C3 | 77 | GIIFSNNA MG | 149 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP20C6 | 78 | GTIFSNNA MG | 147 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP20D8 | 79 | GTTFRSN AMG | 154 | SITNSGST N | 182 | DARRGW NTGY | 194 |
| CX3CR1BII PMP20E11 | 80 . | RTIFRSNA MG | 150 | SITNSGST N | 182 | DARRGW NTGY | 194 |
| CX3CR1BII | 81. | GTIFSNNA | 147 | SISNSGST | 179 | DARRGW | 194 |
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| PMP20E5 | MG | N | NTGY | ||||
| CX3CR1BII PMP20F3 | 82 | GTIFSNNA MG | 147 | SISSSGST N | 178 | DARRGW NTAY | 192 |
| CX3CR1BII PMP20F4 | 83 | ATIFRSNA MG | 155 | SISNSGST N | 179 | DGRRGW NTGY | 195 |
| CX3CR1BII PMP20F5 | 84 | ATIFRSNA MG | 155 | SISNSGST N | 179 | DGRRGW NTGY | 195 |
| CX3CR1BII PMP21B6 | 85 | GTIFSNNA MG | 147 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP24A12 | 86 | GIIFSNNA MG | 149 | SISNSGSA N | 183 | DARRGW NTAY | 192 |
| CX3CR1BII PMP24A6 | 87 | GTIFSNNA MG | 147 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP24B9 | 88 | GTIFRSNA MG | 151 | SISISGST N | 184 | DARRGW NTGF | 196 |
| CX3CR1BII PMP24D3 | 89 | GIIFSNNA MG | 149 | SISSTYST N | 181 | DARRGW NTAY | 192 |
| CX3CR1BII PMP24F7 | 90 | GLIFSNNA MG | 156 | SISSTYST N | 181 | DARRGW NTAY | 192 |
| CX3CR1BII PMP28B4 | 91 | ATIFRSNA MG | 155 | SISNSGST N | 179 | DGRRGW NTGY | 195 |
| CX3CR1BII PMP28F1 | 92 | GIIFSNNA MG | 149 | SIGSTYST N | 180 | DARRGW NTAY | 192 |
| CX3CR1BII PMP28F6 | 93 | GIIFSNNA MG | 149 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP28F9 | 94 | GTIFSNNA MG | 147 | SISNSGST N | 179 | DARRGW NTGY | 194 |
| CX3CR1BII PMP29A5 | 95 | GTIFSNNA MG | 147 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP29D5 | 96 | GTIFRSNA MG | 151 | SISNSGST N | 179 | DARRGW NSGY | 193 |
| CX3CR1BII PMP29E3 | 97 | KTIFRSNA MG | 153 | SISNSGST N | 179 | DARRGW NTGY | 194 |
| CX3CR1BI1 PMP29E7 | 98 | KTIFRSNA MG | 153 | SISNSGST N | 179 | DARRGW NTGY | 194 |
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| CX3CR1BII PMP29G10 | 99 | GTIFRSNA MG | 151 | SITNSGST N | 182 | DARRGW NTGY | 194 |
| CX3CR1BII PMP29G7 | 100 | GIIFSNNA MG | 149 | SITNTGST N | 185 | DARRGW NTAY | 192 |
| CX3CR1BII PMP29H1 | 101 | GTIFSNNA MG | 147 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP37A8 | 102 | RTIFRSNA MG | 150 | SISNSGST N | 179 | DARRGW NTGY | 194 |
| CX3CR1BII PMP37B9 | 103 | GTIFSNNA MG | 147 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP37C12 | 104 | GSIFRSNA MG | 157 | SISNSGST N | 179 | DARRGW NTGY | 194 |
| CX3CR1BII PMP37C7 | 105 | RTIFSNNA MG | 158 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP37D9 | 106 | GTVFSNN AMG | 159 | SISSSGST N | 178 | DARRGW NTAY | 192 |
| CX3CR1BII PMP37E12 | 107 | KPIFRSNA MG | 160 | SISNSGST N | 179 | DARRGW NTGY | 194 |
| CX3CR1BII PMP41B10 | 108 | GTIFSNNA MG | 147 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP41B11 | 109 | GTIFSNNA MG | 147 | SISNSGST N | 179 | DARRGW NTAY | 192 |
| CX3CR1BII PMP41B8 | 110 | GIIFSNNA MG | 149 | SIGSTYST N | 180 | DARRGW NTAY | 192 |
| CX3CR1BII PMP41C10 | 111 | RTIFRSNA MG | 150 | SISNSGST N | 179 | DARRGW NTGY | 194 |
| CX3CR1BII PMP41F9 | 112 | GIIFSNNA MG | 149 | SIGSTYST N | 180 | DARRGW NTAY | 192 |
| CX3CR1BII PMP41H10 | 113 | GLTLDDY AMG | 161 | SISNSGST N | 179 | DARRGW NTGY | 194 |
| CX3CR1BII PMP46B5 | 114 | RTIFRSNA MG | 150 | SISNSGST N | 179 | DARRGW NTGY | 194 |
| CX3CR1BII PMP46D3 | 115 | GTIFSNNA MG | 147 | SISNSGST N | 179 | DARRGW NTGY | 194 |
| CX3CR1BII | 116 | GIIFSNNA | 149 | SISSTYST | 181 | DARRGW | 192 |
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| PMP46H5 | MG | N | NTAY | ||||
| CX3CR1BII PMP48B8 | 117 | KTIFRSNA MG | 153 | SISNSGST N | 179 | DARRGW NTGY | 194 |
| CX3CR1BII PMP48D11 | 118 | RTIFRSNA MG | 150 | SISNSGST N | 179 | DARRGW NTGY | 194 |
| CX3CR1BII PMP48G8 | 119 | RTIFRSNA MG | 150 | SISNSGST N | 179 | DARRGW NTGY | 194 |
| CX3CR1BII PMP48H9 | 120 | GTIFSNNA MG | 147 | SISNSGST N | 179 | DARRGW NTAY | 192 |
*CDR sequences were determined according to Antibody Engineering, vol 2 by Konetermann & Dübel (Eds.), Springer Verlag Heidelberg Berlin, 2010. The sequence numbers In the table (SEQ) refer to the sequences in the sequence 5 listing of the instant application.
Table 4: Optimlzed variants
| Nanobody | SEQ | CDR1 | SEQ CDR1 | CDR2 | SEQ CDR2 | CDR3 | SEQ CDR3 |
| CX3CR1BII PMP66B02 | 1 | GSIFSSNA MA | 141 | AINSVGVT K | 162 | DPRRGW DTRY | 186 |
| CX3CR1BII 043 | 121 | GSIFSSNA MA | 141 | AINSVGVT K | 162 | DPRRGW DTRY | 186 |
| CX3CR1BII 045 | 122 | GSIFSSNA MA | 141 | AINSVGVT K | 162 | DPRRGW DTRY | 186 |
| CX3CR1BII 047 | 123 | GSIFSSNA MA | 141 | AINSVGVT K | 162 | DPRRGW DTRY | 186 |
| CX3CR1BII 048 | 124 | GSIFSSNA MA | 141 | AINSVGVT K | 162 | DPRRGW DTRY | 186 |
| CX3CR1BII 049 | 125 | GSIFSSNA MA | 141 | AINSVGVT K | 162 | DPRRGW DTRY | 186 |
| CX3CR1BII | 126 | GSIFSSNA | 141 | AINSVGVT | 162 | DPRRGW | 186 |
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| 050 | MA | K | DTRY | ||||
| CX3CR1BII 061 | 127 | GSIFSSNA MA | 141 | AINSVGVT K | 162 | DPRRGW DTRY | 186 |
| CX3CR1BII 056 | 128 | GSIFSSNA MA | 141 | AINSVGVT K | 162 | DPRRGW DTRY | 186 |
| CX3CR1BII 057 | 129 | GSIFSSNA MA | 141 | AINSVGVT K | 162 | DPRRGW DTRY | 186 |
| CX3CR1BII 060 | 130 | GSIFSSNA MA | 141 | AINSVGVT K | 162 | DPRRGW DTRY | 186 |
| CX3CR1BII 065 | 131 | GSIFSSNA MA | 141 | AISSVGVT K | 214 | DPRRGW DTRY | 186 |
| CX3CR1BII 067 | 132 | GSIFSSNA MA | 141 | AIQSVGVT K | 215 | DPRRGW DTRY | 186 |
| CX3CR1B1I 068 | 133 | GSIFSSNA MA | 141 | AIGSVGVT K | 216 | DPRRGW DTRY | 186 |
| CX3CR1BII 074 | 134 | GSIFSSNA MA | 141 | AITSVGVT K | 217 | DPRRGW DTRY | 186 |
| CX3CR1BII 118 | 135 | GSIFSSNA MA | 141 | AINTVGVT K | 218 | DPRRGW DTRY | 186 |
| CX3CR1BII 129 | 136 | GSIFSSNA MA | 141 | AINGVGV TK | 219 | DPRRGW DTRY | 186 |
| CX3CR1BII 158 | 137 | GSIFSSNA MA | 141 | AINPVGVT K | 220 | DPRRGW DTRY | 186 |
| CX3CR1BII 306 | 138 | GSIFSSTA MA | 213 | AISSVGVT K | 214 | DPRRGW DTRY | 186 |
| CX3CR1BII 307 | 139 | GSIFSSTA MA | 213 | AISTVGVT K | 221 | DPRRGW DTRY | 186 |
| CX3CR1BII 308 | 140 | GSIFSSNA MA | 141 | AINSVGVT K | 162 | DPRRGW DTRY | 186 |
-51 17072 *CDR sequences were determined according to Antibody Engineering, vol 2 by Konetermann & Dübel (Eds.), Springer Veriag Heidelberg Berlin, 2010. The sequence numbers in the table (SEQ) refer to the sequences in the sequence listing of the Instant application.
ln a further aspect, the présent invention provides polypeptides having one or more VHH domains.
ln one aspect, a VHH domain of the présent Invention comprises or essentially consiste of the sequence set forth in:
a) the amino acid sequence of SEQ ID NO: 3; or
b) amino acid sequences that hâve at least 90% amino acid identity with the amino acid sequences of SEQ ID NO: 3; or
c) amino acid sequences that hâve 11,10, 9, 8, 7,6, 5,4, 3, 2, or 1 amino acid différence with the amino acid sequences of SEQ ID NO: 3 or
d) an amino acid sequence of any one of SEQ ID NO: 1-48, or SEQ ID NO: 121-140, or SEQ ID NO: 222-224.
ln a further aspect, a VHH domain of the présent invention comprises or essentially conslsts of the sequence set forth ln:
a) the amino acid sequence of SEQ ID NO: 49; or
b) an amino acid sequence that has at least 95% amino acid identity with the amino acid sequences of SEQ ID NO: 49; or
c) an amino acid sequence that has 5, 4, 3,2, or 1 amino acid différence with the amino acid sequences of SEQ ID NO: 49; or
d) an amino acid sequence of any one of SEQ ID NO: 49-52.
In a further aspect, a VHH domain of the présent invention comprises or essentially conslsts of the sequence set forth ln:
a) the amino acid sequence of SEQ ID NO: 67; or
b) an amino acid sequence that has at least 90% amino acid Identity with the amino acid sequences of SEQ ID NO: 67; or
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c) an amîno acid sequence that has 12,11,10, 9,8, 7,6, 5, 4, 3,2, or 1 amino acid différence with the amino acid sequences of SEQ ID NO: 67; or
d) an amino acid sequence of any one of SEQ ID NO: 53-120.
In a further aspect, a VHH domain ofthe présent invention comprises or essentially consists of the amino acid sequence set forth In any one of SEQ ID NO: 121-140, or SEQ ID NO: 222-224.
w In a further aspect, a VHH domain of the présent Invention comprises or essentially consists of the amino acid sequence set forth in any one of SEQ ID NO: 138-140.
In a further aspect, a VHH domain of the présent Invention comprises or is essentially consists of the amino acid sequence set forth In any one of SEQ ID NO: 222-224.
Représentative VHH domains of the présent invention are shown in Table 5 and représentative optlmlzed VHH domains of the présent invention are shown in
Table 6 below;
Table 5: VHH domains
SEQ ID NO: 1-48 are VHH domains of family 101. SEQ ID NO: 49-52 are VHH domains of family 9. SEQ ID NO: 53-120 are VHH domains of family 13.
| CX3CR1BII PMP66B02 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS | SEQ ID NO: | 1 |
| CX3CR1BII PMP54A12 | EVQLVESGGGSVQAGESLRLSCAASGS1FSSNAM AWYRQAPGKQRDLVAVINSVGITKYADSVKGRFTI SGDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGW | SEQ ID NO: | 2 |
-5317072
| DTRYWGQGTQVTVSS | |||
| CX3CR1BII PMP54A3 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS | SEQID NO: | 3 |
| CX3CR1BII PMP54A4 | EVQLVESGRGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS | SEQ ID NO: | 4 |
| CX3CR1BII PMP54A5 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQID NO: | 5 |
| CX3CR1BII PMP54A7 | EVQLVESGGGSVQAGESLRLSCAASGT1FSSNAM AWYRQAPGKQRDLVAGINSVDITKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW NTRYWGQGTQVTVSS | SEQID NO: | 6 |
| CX3CR1BII PMP54B1 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTI SRDNAKNTAYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS | SEQID NO: | 7 |
| CX3CR1BI1 PMP54B2 | EVQLVESGGGSVQAGESLRLSCAASGTIFSSNAM AWYRQAPGKQRDLVAGINSVDITKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW NTRYWGQGTLVTVSS | SEQ ID NO: | 8 |
| CX3CR1BII PMP54B3 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAAINSVGITKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS | SEQ ID NO: | 9 |
| CX3CR1BII PMP54B5 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTI SRDNAKNTAYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQID NO: | 10 |
| CX3CR1BI1 PMP54D5 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPPGKQRDLVALINSVGITKYADSVKGRFT ISSDNAKNTVYLEMNSLKPEDTAVYYCTSDGRRG | SEQID NO: | 11 |
-5417072
| WDTRYWGQGTQVTVSS | |||
| CX3CR1BII PMP54D8 | EVQLVESGGGSVQAGGSLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQID NO: | 12 |
| CX3CR1BII PMP54F6 | KVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAAINSVGITKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQID NO: | 13 |
| CX3CR1BII PMP54G3 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVALINSVGITKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQID NO: | 14 |
| CX3CR1BII PMP54H1 | EVQLVESGGGSVQAGESLRLSCAASGTIFSSNAM AWYRQAPGKQRDLVAGINSVDITKYADSVKGRFTV SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW NTRYWGQGTQVTVSS | SEQ ID NO: | 15 |
| CX3CR1BII PMP54H4 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAVINSVGITKYADSVKGRFTI SGDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGW DTRYWGQGTLVTVSS | SEQ ID NO: | 16 |
| CX3CR1BII PMP61F10 | KVQLVESGGGSVQAGESLRLSCAASGTIFSSNAM AWYRQAPGKQRDLVAGINSVDITKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW NTRYWGQGTQVTVSS | SEQ ID NO: * ..«W | 17 |
| CX3CR1BII PMP61D1 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAFGKQRDLVALINSVGITKYADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWD TRYWGQGTQVTVSS | SEQ ID NO: | 18 |
| CX3CR1BII PMP61D5 | KVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAFGKQRDLVALINSVGITKYADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWD TRYWGQGTQVTVSS | SEQ ID NO: | 19 |
| CX3CR1BII PMP61E2 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDMAVYYCTSDPRRG | SEQ ID NO: | 20 |
-5517072
| WDTRYWGQGTQVTVSS | |||
| CX3CR1BII PMP61F11 | KVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQPPGKQRDLVAAINSVGITKYADSVKGRFTI FRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS | SEQ ID NO: | 21 |
| CX3CR1B1I PMP61G2 | EVQLVKSGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVALINSVGITKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS | SEQ ID NO: | 22 |
| CX3CR1B1I PMP61G3 | KVQLVESGGGSMQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAAINSVGITKYADSVKGRFTI SRDNAKNTVYLQMMSLKPEDTAVYYCTSDPRRG WDTRYWGQGTQVTVSS | SEQID NO: | 23 |
| CX3CR1BII PMP61G4 | KVQLVESGGGSVQAGGSLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAAINSVGITKYADSVKGRFTI SRDNAKNTVYLQMMSLKPEDTAVYYCTSDPRRG WDTRYWGQGTQVTVSS | SEQID NO: | 24 |
| CX3CR1B1I PMP61F4 | EVQLVESGGGSVQAGASLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAVINTVGITKYADSVKGRFTl SRDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGW DTRYWGQGTLVTVSS | SEQ ID NO: | 25 |
| CX3CR1B1I PMP61A11 | EVQLVESRGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAVINSVGITKYADSVKGRFTI SGDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGW DTRYWGQGTQVTVSS | SEQ ID NO: | 26 |
| CX3CR1BII PMP61B2 | EVQLVESRGGSVQAGASLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAVINTVGITKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGW DTRYWGQGTQVTVSS | SEQ ID NO: | 27 |
| CX3CR1BII PMP61C9 | EVQLVKSGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQALGKQRDLVALIDSAGITKYADSVKGRFTIS RDNAKNTVYLQMNRLKPEDTAVYYCASDARRGW NTKYWGQGTLVTVSS | SEQID NO: | 28 |
| CX3CR1BII PMP65H02 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAAINSVGITKYADSVKGRFTI SRDNAKNTVHLQMNSLKPEDTAVYYCTSDPRRGW | SEQID NO: | 29 |
-5617072
| DTRYWGQGTLVTVSS | |||
| CX3CR1BII PMP65E11 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAGINSVGIAKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQ ID NO: | 30 |
| CX3CR1B1I PMP65E10 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAKA WYRQAPGKQRDLVAGINSVGITKYADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWD TRYWGQGTLVTVSS | SEQID NO: | 31 |
| CX3CR1BII PMP65E05 | KVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQID NO: | 32 |
| CX3CR1B1I PMP65B11 | EVQLVKSGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAVINKVGITKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS | SEQID NO: | 33 |
| CX3CR1BII PMP65B07 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAAINSVGITKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQID NO: | 34 |
| CX3CR1BII PMP65B09 | EVQLVESGGGSVQAGESLRLSCAASGSIFSRNAM AWYRQAPGKQRDLVASINSVGITKYGDSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGW DTRYWGQGTLVTVSS | SEQID NO: | 35 |
| CX3CR1BÜ PMP65H01 | EVQLVESGGGSVQAGESLRLSCAASGGIFSRNAM AWYRQAPGKQRDLVASINSVGITKYGDSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGW DTRYWGQGTQVTVSS | SEQID NO: | 36 |
| CX3CR1BH PMP65G07 | EVQLVESGGGSVQAGESLRLSCAASGTIFSSNAM AWYRQAPGKQRDLVAGINSVDITRYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW NTRYWGQGTQVTVSS | SEQID NO: | 37 |
| « — CX3CR1BI! PMP66H08 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVALINSVGITKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW | SEQID NO: | 38 |
-5717072
| DTRYWGQGTQVTVSS | |||
| CX3CR1BII PMP66H04 | EVQLVESGGGSVQAGGSLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAAINSVGITKYADSVKGRFTI SRDNAKNTVYLQMMSLKPEDTAVYYCTSDPRRG WDTRYWGQGTLVTVSS | SEQ ID NO: | 39 |
| CX3CR1BII PMP66F02 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVALINSVGITKYAGSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS | SEQ ID NO: | 40 |
| CX3CR1BII PMP66E11 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAAINSVGTTKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS | SEQID NO: | 41 |
| CX3CR1BII PMP66D10 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQALGKQRDLVALINSVGITKYADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWD TRYWGQGTQVTVSS | SEQID NO: | 42 |
| CX3CR1BII PMP66D08 | EVQLMESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS | SEQID NO: | 43 |
| CX3CR1BII PMP66A04 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQALGKQRDLVALINSVGITKYADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWD TRYWGQGTLVTVSS | SEQ ID NO: | 44 |
| CX3CR1BII PMP66D04 | KVQLVESGGGSVQAGESLRLSCAASGTIFSSNAM AWYRQAPGKQRDLVAGINSVDITKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW NTRYWGQGTLVTVSS | SEQID NO: | 45 |
| CX3CR1BII PMP66D02 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAVINSVGITKYADSVKGRFTT SGDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGW DTRYWGQGTQVTVSS | SEQID NO: | 46 |
| CX3CR1BII PMP66D06 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVASIDSVGITKYRDSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGW | SEQID NO: | 47 |
-5817072
| DTRYWGQGTQVTVSS | |||
| CX3CR1BII PMP66G01 | EMQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVALINSVG1TKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDGRRG WDTRYWGQGTQVTVSS | SEQ ID NO: | 48 |
| CX3CR1BII PMP11H11 | EVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAM GWFRQAPGKERAFVAGISGSASRKYYADSVKGRF TVSRDNARNTVYLQMNSLKPEDTAVYYCAASNSY PKVQFDYYGQGTQVTVSS | SEQ1D NO: | 49 |
| CX3CR1BII PMP12B6 | EVQLVQSGGGLVQAGGSLRLSCVASGRTFSSYAM GWFRQAPGRERAFVAGISGSASRKYYADSVKGRF TVSRDNARNTVYLQMNSLKPEDTAVYYCAASNSY PKVQFDYYGQGTQVTVSS | SEQID NO: | 50 |
| CX3CR1BII PMP12G9 | EVQLVESGGGLVQPGGSLRLSCVASGRTFSSYAM GWFRQAPGKEREFVAGISGSGSRKYYADSVKGRF TISRDNARNTVYLQMNSLKPEDRAVYYCAASNSYP KVQFDYYGQGTQVTVSS | SEQ ID NO: | 51 |
| CX3CR1BII PMP15G11 | EVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAM GWFRQAPGKEREFVAG1SGSGSRKYYADSVKGRF TISRDNARNTVYLQMNSLKPEDRAVYYCAASNSYP KVQFDYYGQGTQVTVSS | SEQID NO: | 52 |
| CX3CR1BII PMP18E6 | KVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM GWYRQAPGKKRDLVASISSSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTLDARRG WNTAYWGQGAQVTVSS | SEQID NO: | 53 |
| CX3CR1BI1 PMP12C2 | EVQLVESGGGLVQPGGSLRLSCATSGTIFSNTAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNSGYWGQGTQVTVSS | SEQ ID NO: | 54 |
| CX3CR1BI1 PMP18A10 | EVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMG WYRQAPGKKRDLVASISNSGSTNYADSAKGRFTV SRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS | SEQ ID NO: | 55 |
| CX3CR1BII PMP18A2 | EVQLVESGGGWQPGGSLRLSCVTSG1IFSNNAMG WYRQGPGKKRDLVAS1GSTYSTNYADSVKGRFTV SRDNDKNTGYLQMNSLKPEDTGVYYCT1DARRGW | SEQ ID NO: | 56 |
-5917072
| NTAYWGQGTPVTVSS | |||
| CX3CR1BII PMP18A8 | EVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRG WNTGYWGQGTQVTVSS | SEQID NO: | 57 |
| CX3CR1BII PMP18A9 | EVQLVESGGGWQPGGSLRLSCVTSGIIFSNNAMG WYRQGPGKKRDLVASISSTYSTNYADSVKG RFTV SRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGW NTAYWGQGTPVTVSS | SEQ ID NO: | 58 |
| CX3CR1BII ΡΜΡ18Β7 | EVQLVESGGGLVQPGGSLRLSCATSGTIFRSNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNSGYWGQGTQVTVSS | SEQ ID NO: | 59 |
| CX3CR1BII PMP18B9 | EVQLVESRGGLVQPGGSLRLSCATSGTIFSNNAM GWYRQAPGKKRDLVASISSSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTLDARRG WNTAYWGQGTQVTVSS | SEQID NO: | 60 |
| CX3CR1BII PMP18C6 | EVQLMESGGGLVQPGGSLRLSCATSGTIFSNNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS | SEQID NO: | 61 |
| CX3CR1BII PMP18C9 | EVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMG WYRQAPGKKRDLVASISNSGSTNYADSVKGRFTV SRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS | SEQID NO: | 62 |
| CX3CR1BII PMP18D1 | EVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMG WYRQAPGKKRDLVASISNSGSTNYADSVKGRFTV SRDNDKSTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS | SEQID NO: | 63 |
| CX3CR1BII PMP18D10 | EVQLVESGGG LVQPGGSLG LSCATSGTIFSNNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS | SEQID NO: | 64 |
| CX3CR1BII PMP18D12 | EVQLVESGGGLVQPGGSLRLSCTTSGTIFSNNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNNLKPEDTGVYYCTLDARRG | SEQID NO: | 65 |
-6017072
| WNTAYWGQGTQVTVSS | |||
| CX3CR1BII PMP18F1 | KVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS | SEQID NO: | 66 |
| CX3CR1BII PMP18F5 | EVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS | SEQ ID NO: | 67 |
| CX3CR1BII PMP18F6 | EVQLVDSGGGLVQPGGSLRLSCATSGTIFSNNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS | SEQID NO: | 68 |
| CX3CR1BII PMP18F9 | EVQLVESGGGLVQPGGSLRLSCATSGTIFRTNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTAYLQMNSLKPEDTGVYYCTIDGRRG WNTGYWGQGTQVTVSS | SEQID NO: | 69 |
| CX3CR1BII PMP18G5 | EVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS | SEQID NO: | 70 |
| CX3CR1BII PMP18H1 | EVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM GWYRQALGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS | SEQ ID NO: | 71 |
| CX3CR1BII PMP18H10 | EVQLVESGGGLVQPGGSLRLSCATSKTIFRSNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS | SEQID NO: | 72 |
| CX3CR1BII PMP18H7 | EVQLVESRGGLVQPGGSLRLSCATSGIIFSNNAMG WYRQAPGKKRDLVASISNSGSTNYADSVKGRFTV SRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS | SEQID NO: | 73 |
| CX3CR1BII PMP18H9 | EVQLVKSGGGLVQPGGSLRLSCTTSGTIFSNNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNNLKPEDTGVYYCTLDARRG | SEQ ID NO: | 74 |
-61 17072
| WNTAYWGQGTQVTVSS | |||
| CX3CR1BII PMP20B3 | EVQLVESGGGLVQAGGSLRLSCVTSGIIFSNNAMG WYRQGPGKKRDLVASIGSTYSTNYADSVKGRFTV SRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGW NTAYWGQGTPVTVSS | SEQID NO: | 75 |
| CX3CR1BII PMP20C12 | EVQLVESGGGLVQPGGSLRLSCATSGTIFRSNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNSGYWGQGTRVTVSS | SEQID NO: | 76 |
| CX3CR1BII PMP20C3 | KVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMG WYRQAPGKKRDLVASISNSGSTNYADSVKGRFTV SRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS | SEQ ID NO: | 77 |
| CX3CR1BII PMP20C6 | EVQLVESGGGLVQAGGSLRLSCATSGTIFSNNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS | SEQ ID NO: | 78 |
| CX3CR1BII PMP20D8 | EVQLVESGGGLVQPGRSLRLSCATSGTTFRSNAM GWYRQGPGKKRDLVASITNSGSTNYADSVKGRFT VSRDNDKNTGYLQMSSLKPEDTGVYYCTLDARRG WNTGYWGQGTQVTVSS | SEQ ID NO: | 79 |
| CX3CR1BII PMP20E11 | EVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAM GWYRQGPGKKRDLVASITNSGSTNYADSVKGRFT VSRDNDRNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS | SEQ ID NO: | 80 |
| CX3CR1BII PMP20E5 | KVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM GWYRQVPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS | SEQ ID NO: | 81 |
| CX3CR1BII PMP20F3 | EVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM GWYRQAPGKKRDLVASISSSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTLDARRG WNTAYWGQGTQVTVSS | SEQ ID NO: | 82 |
| CX3CR1BII PMP20F4 | EVQLVESGGGLVQPGGSLRLSCATSATIFRSNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTAYLQMNSLKPEDTGVYYCTIDGRRG | SEQ ID NO: | 83 |
-6217072
| WNTGYWGQGTQVTVSS | |||
| CX3CR1BII PMP20F5 | EVQLVESGGGLVQPGGSLRLSCATSATIFRSNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRST VSRDNDKNTAYLQMNSLKPEDTGVYYCTIDGRRG WNTGYWGQGTQVTVSS | SEQ ID NO: | 84 |
| CX3CR1BII PMP21B6 | EVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDMGVYYCTVDARR GWNTAYWGQGTQVTVSS | SEQ ID NO: | 85 |
| CX3CR1BII PMP24A12 | EVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMG WYRQAPGKKRDLVASISNSGSANYADSVKGRFTV SRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS | SEQ ID NO: | 86 |
| CX3CR1BII PMP24A6 | EVQLVESGGGLVQPGGSLRLSCTTSGTIFSNNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSGDNDKNTGYLQMNNLKPEDTGVYYCTLDARRG WNTAYWGQGTQVTVSS | SEQID NO: | 87 |
| CX3CR1BII PMP24B9 | EVQLVESGGGLVQPGGSLRLSCATSGTIFRSNAM GWYRQAPGKKRDLVASISISGSTNYADSVKGRFTV SRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGFWGQGTQVTVSS | SEQID NO: | 88 |
| CX3CR1BII PMP24D3 | EVQLVESGGGLVQPGGSLRLSCVTSGIIFSNNAMG WYRQGPGKKRDLVASISSTYSTNYADSVKGRFTV SRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGW NTAYWGQGTPVTVSS | SEQ ID NO: | 89 |
| CX3CR1BII PMP24F7 | EVQLMESGGGMVQVGGSLRLSCTASGLIFSNNAM GWYRQGPGKKRDLVASISSTYSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRG WNTAYWGQGTPVTVSS | SEQID NO: | 90 |
| CX3CR1BII PMP28B4 | EVQLVESGGGLVQPGGSLRLSCAISATIFRSNAMG WYRQAPGKKRDLVASISNSGSTNYADSVKGRFTV SRDNDKNTAYLQMNSLKPEDTGVYYCTIDGRRGW NTGYWGQGTQVTVSS | SEQID NO: | 91 |
| CX3CR1BII PMP28F1 | EMQLVESGGGWQPGGSLRLSCVTSGIIFSNNAM GWYRQGPGKKRDLVASIGSTYSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRG | SEQID NO: | 92 |
-6317072
| WNTAYWGQGTPVTVSS | |||
| CX3CR1BII PMP28F6 | EVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMG WYRQAPGKKRDLVASISNSGSTNHADSVKGRFTV SRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS | SEQ ID NO: | 93 |
| CX3CR1BII PMP28F9 | EVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM GWYRQVPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS | SEQ ID NO: | 94 |
| CX3CR1BII PMP29A5 | EVQLVESRGGLVQPGGSLRLSCATSGTIFSNNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS | SEQID NO: | 95 |
| CX3CR1BII PMP29D5 | KVQLVESGGGLVQPGGSLRLSCATSGTIFRSNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNSGYWGQGTQVTVSS | SEQID NO: | 96 |
| CX3CR1BII PMP29E3 | EVQLVESEGGLVQPGGSLRLPCATSKTIFRSNAMG WYRQAPGKKRDLVASISNSGSTNYADSVKGRFTV SRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS | SEQID NO: | 97 |
| CX3CR1BII PMP29E7 | EVQLVESGGGLVQPGGSLRLSCATSKTIFRSNAM GWYRQAPGKKRGLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS | SEQID NO: | 98 |
| CX3CR1BII PMP29G10 | EVQLMESGGGLVQPGGSLRLSCATSGTIFRSNAM GWYRQGPGKKRDLVASITNSGSTNYADSVKGRFT VSRDNDKNTGYLQMSSLKPEDTGVYYCTLDARRG WNTGYWGQGTQVTVSS | SEQID NO: | 99 |
| CX3CR1BII PMP29G7 | EVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMG WYRQGPGKKRDLVASITNTGSTNYADSVKGRFTV SRDNDRNTVYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS | SEQ ID NO: | 100 |
| CX3CR1BII PMP29H1 | EVQLVESGGGLVQAGGSLRLSCTTSGTIFSNNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNNLKPEDTGVYYCTLDARRG | SEQ ID NO: | 101 |
-6417072
| WNTAYWGQGTQVTVSS | |||
| CX3CR1BII PMP37A8 | EVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAM GWYRQAPGKKRDLVASISNSGSTNYADSAKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS | SEQID NO: | 102 |
| CX3CR1BII PMP37B9 | EVQLVESGGLVQPGGSLRLSCATSGTIFSNNAMG WYRQAPGKKRDLVASISNSGSTNYADSVKGRFTV SRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS | SEQID NO: | 103 |
| CX3CR1BII PMP37C12 | EVQLVESGGGLVQAGGSLRLSCVASGSIFRSNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRG WNTGYWGQGTQVTVSS | SEQ ID NO: | 104 |
| CX3CR1BII PMP37C7 | EVQLVESGGGLVQPGGSLRLSCATSRTIFSNNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS | SEQID NO: | 105 |
| CX3CR1BII PMP37D9 | EVQLVESGGGLVQPGGSLRLSCATSGTVFSNNAM GWYRQAPGKKRDLVASISSSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTLDARRG WNTAYWGQGTQVTVSS | SEQID NO: | 106 |
| CX3CR1BII PMP37E12 | EVQLVESGGGLVQPGGSLRLSCATSKPIFRSNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS | SEQID NO: | 107 |
| CX3CR1BII PMP41B10 | EVQLVESEGGLVQPGGSLRLSCTTSGTIFSNNAMG WYRQAPGKKRDLVASISNSGSTNYADSVKGRFTV SRDNDKNTGYLQMNNLKPEDTGVYYCTLDARRG WNTAYWGQGTQVTVSS | SEQ ID NO: | 108 |
| CX3CR1BII PMP41B11 | EVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSPKPEDTGVYYCTVDARR GWNTAYWGQGTQVTVSS | SEQID NO: | 109 |
| CX3CR1BII PMP41B8 | EVQLVESEGGWQPGGSLRLSCVTSGIIFSNNAMG WYRQGPGKKRDLVASIGSTYSTNYADSVKGRFTV SRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGW | SEQID NO: | 110 |
-6517072
| NTAYWGQGTPVTVSS | |||
| CX3CR1BII PMP41C10 | EMQLVESGGGLVQPGGSLRLSCATSRTIFRSNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKSTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS | SEQID NO: | 111 |
| CX3CR1BII PMP41F9 | EVQLVESGGGWQPGESLRLSCVTSGIIFSNNAMG WYRQGPGKKRDLVASIGSTYSTNYADSVKGRFTV SRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGW NTAYWGQGTPVTVSS | SEQID NO: | 112 |
| CX3CR1BII PMP41H10 | KVQLVESGGGLVQPGDSLRLSCAASGLTLDDYAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRG WNTGYWGQGTQVTVSS | SEQID NO: | 113 |
| CX3CR1BII PMP46B5 | KVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRG WNTGYWGQGTQVTVSS | SEQID NO: | 114 |
| CX3CR1BII PMP46D3 | EVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM GWYRQVPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLRMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS | SEQID NO: | 115 |
| CX3CR1BII PMP46H5 | EVQLVESGGGLVQAGGSLRLSCVTSGIIFSNNAMG WYRQGPGKKRDLVASISSTYSTNYADSVKGRFTV SRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGW NTAYWGQGTPVTVSS | SEQID NO: | 116 |
| CX3CR1BII PMP48B8 | EVQLVESGGGLVQPGGSLRLSCATSKTIFRSNAM GWYRQAPGKKRDLVASISNSGSTNYTDSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS | SEQID NO: | 117 |
| CX3CR1BII PMP48D11 | KVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS | SEQID NO: | 118 |
| CX3CR1BII PMP48G8 | EVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAM GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFA VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG | SEQID NO: | 119 |
-6617072
| WNTGYWGQGTQVTVSS | |||
| CX3CR1BII PMP48H9 | EVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM GWYRQAPGKKRDLVASISNSGSTNYADFVKGRFT VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS | SEQ ID NO: | 120 |
Table 6: Optlmlzed VHH domains
| CX3CR1BII 043 | EVQLVESGGGSVQPGESLRLSCAASGSIFSSNAM AWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTI SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQ ID NO: | 121 |
| CX3CR1BII 045 | DVQLVESGGGSVQPGESLRLSCAASGSIFSSNAM AWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTI SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQ ID NO: | 122 |
| CX3CR1BII 047 | EVQLVESGGGLVQPGESLRLSCAASGSIFSSNAMA WYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTIS RDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQ ID NO: | 123 |
| CX3CR1BII 048 | EVQLVESGGGSVQPGGSLRLSCAASGSIFSSNAM AWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTI SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQ ID NO: | 124 |
| CX3CR1BII 049 | EVQLVESGGGSVQPGESLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAAINSVGVTKYADSVKGRFTI SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQ ID NO: | 125 |
| CX3CR1BII 050 | EVQLVESGGGSVQPGESLRLSCAASGSIFSSNAM AWYRQAPGKRRELVAAINSVGVTKYADSVKGRFTI SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQID NO: | 126 |
| CX3CR1BII 061 | EVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAM AWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTI SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW | SEQ ID NO: | 127 |
-6717072
| DTRYWGQGTLVTVSS | |||
| CX3CR1BII 056 | EVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAM AWYRQAPGKQRDLVAAINSVGVTKYADSVKGRFTI SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQ ID NO: | 128 |
| CX3CR1BII 057 | EVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAM AWYRQAPGKRRELVAAINSVGVTKYADSVKGRFTI SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQ ID NO: | 129 |
| CX3CR1BII 060 | EVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAM AWYRQAPGKQRELVAAINSVGVTKYADSVKGRFTI SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQ ID NO: | 130 |
| CX3CR1BII 065 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKRRDLVAAISSVGVTKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQID NO: | 131 |
| CX3CR1BII 067 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKRRDLVAAIQSVGVTKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQID NO: | 132 |
| CX3CR1BII 066 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKRRDLVAAIGSVGVTKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQID NO: | 133 |
| CX3CR1BII 074 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKRRDLVAAITSVGVTKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQID NO: | 134 |
| CX3CR1BII 118 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKRRDLVAAINTVGVTKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQ ID NO: | 135 |
| CX3CR1BII 129 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKRRDLVAAINGVGVTKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW | SEQID NO: | 136 |
-6817072
| DTRYWGQGTLVTVSS | |||
| CX3CR1BII 158 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM AWYRQAPGKRRDLVAAINPVGVTKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQ ID NO: | 137 |
| CX3CR1BII 306 | DVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAM AWYRQAPGKRRDLVAAISSVGVTKYADSVKGRFTI SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQID NO: | 138 |
| CX3CR1BII 307 | DVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAM AWYRQAPGKRRDLVAAISTVGVTKYADSVKGRFTI SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQID NO: | 139 |
| CX3CR1BII 308 | DVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAM AWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTI SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQID NO: | 140 |
| CX3CR1BII 00306 (D1E) | EVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAMA WYRQAPGKRRDLVAAISSVGVTKYADSVKGRFTIS RDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQID NO: | 222 |
| CX3CR1BII 00307 (D1E) | EVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAMA WYRQAPGKRRDLVAAISTVGVTKYADSVKGRFTIS RDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQID NO: | 223 |
| CX3CR1BII 00308 (D1E) | EVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAM AWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTI SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS | SEQ ID NO: | 224 |
In a further aspect, a polypeptide according to the présent Invention, In particular an Immunoglobulin single variable domain of the présent invention, is humanized 5 and/or optimized for stability, potency, manufacturabllity and/or similarity to human framework régions. For example, the polypeptide is humanized and/or
-6917072 sequence optimized in one or more of the following positions (according to Kabat numbering): 1,11,14,16, 74,83,108. In one aspect, the polypeptide comprises one or more of the following mutations: E1D, S11L, A14P, E16G, A74S, K83R, Q108L.
In one aspect, one or more framework régions of a polypeptide according to the présent invention, in particular an immunoglobulin single variable domain of the présent invention, are humanized and/or sequence optimized. In one aspect, a polypeptide according to the présent invention, in particular an immunoglobulin w single variable domain of the présent invention, comprises framework régions (FR) for exampie as set forth below:
I) FR1 is selected from any one of SEQ ID NO's: 198-204;
li) FR2 is selected from any one of SEQ ID NO’s: 205-208; lii) FR3 is selected form any one of SEQ ID NO's: 209-210; and/or 15 Iv) FR4 is selected from any one of SEQ ID NO’s: 211-212.
Human immunoglobulin framework région sequences (FR) that can also be used as framework région sequences for the immunoglobulin single variable domains as described above are known in the art. Also known in the art are methods for 20 humanizing framework régions of immunoglobulin single variable domains derived from species other than humans.
In a further aspect, one or more CDR régions of a polypeptide according to the présent invention, in particular an Immunoglobulin single variable domain of the 25 présent invention, is humanized and/or sequence optimized. In one aspect, a polypeptide according to the présent invention, in particular an immunoglobulin single variable domain of the présent invention, is humanized and/or sequence optimized in one or more of the following positions (according to Kabat numbering): 52, 53.
In a further aspect, a polypeptide according to the présent invention, in particular an Immunoglobulin single variable domain of the présent Invention, comprises one or more of the following mutations: N52S, S53T.
-7017072 ln a further aspect, a polypeptide according to the présent invention, in particular an Immunogiobuiin single variable domain of the présent Invention, comprises a CDR2 selected from any one of SEQ ID NO's: 214-221.
Représentative humanized and/or optlmized sequences of the présent Invention are shown in Table 4 and 6 hereinabove and ln Table 7 herein below.
io Table 7: Sequence optlmized variants
Table 7a shows the FR1-CDR1-FR2-CRD2 of the sequence optimized variants, table 7b shows FR3-CDR3-FR4-CDR4 of said variants. The sequence numbers in the tables (SEQ) refer to the sequences in the sequence listing of the instant application.
Table 7a: Sequence optlmized variants (FR1-CDR1-FR2-CDR2)
| Nano body | SEQ | FR1 | SEQ FR1 | CDR1 | SEQ CDR 1 | FR2 | SEQ FR2 | CDR2 | SEQ CDR 2 |
| CX3CR1 BIIPMP6 6B02 | 1 | EVQLVES GGGSVQ AGESLRL SCAAS | 198 | GSIFS SNAM A | 141 | WYRQ APGKR RDLVA | 205 | AINSV GVTK | 162 |
| CX3CR1 BII043 | 121 | EVQLVES GGGSVQ PGESLRL SCAAS | 199 | GSIFS SNAM A | 141 | WYRQ APGKR RDLVA | 205 | AINSV GVTK | 162 |
| CX3CR1 B1I045 | 122 | DVQLVES GGGSVQ PGESLRL | 200 | GSIFS SNAM A | 141 | WYRQ APGKR RDLVA | 205 | AINSV GVTK | 162 |
-71 17072
| SCAAS | |||||||||
| CX3CR1 BII047 | 123 | EVQLVES GGGLVQ PGESLRL SCAAS | 201 | GSIFS SNAM A | 141 | WYRQ APGKR RDLVA | 205 | AINSV GVTK | 162 |
| CX3CR1 BII048 | 124 | EVQLVES GGGSVQ PGGSLRL SCAAS | 202 | GSIFS SNAM A | 141 | WYRQ APGKR RDLVA | 205 | AINSV GVTK | 162 |
| CX3CR1 BII049 | 125 | EVQLVES GGGSVQ PGESLRL SCAAS | 199 | GSIFS SNAM A | 141 | WYRQ APGK QRDLV A | 206 | AINSV GVTK | 162 |
| CX3CR1 BII050 | 126 | EVQLVES GGGSVQ PGESLRL SCAAS | 199 | GSIFS SNAM A | 141 | WYRQ APGKR RELVA | 207 | AINSV GVTK | 162 |
| CX3CR1 BII061 | 127 | EVQLVES GGGLVQ PGGSLRL SCAAS | 203 | GSIFS SNAM A | 141 | WYRQ APGKR RDLVA | 205 | AINSV GVTK | 162 |
| CX3CR1 BII056 | 128 | EVQLVES GGGLVQ PGGSLRL SCAAS | 203 | GSIFS SNAM A | 141 | WYRQ APGK QRDLV A | 206 | AINSV GVTK | 162 |
| CX3CR1 BII057 | 129 | EVQLVES GGGLVQ PGGSLRL SCAAS | 203 | GSIFS SNAM A | 141 | WYRQ APGKR RELVA | 207 | AINSV GVTK | 162 |
| CX3CR1 BII060 | 130 | EVQLVES GGGLVQ PGGSLRL SCAAS | 203 | GSIFS SNAM A | 141 | WYRQ APGK QRELV A | 208 | AINSV GVTK | 162 |
| CX3CR1 BII065 | 131 | EVQLVES GGGSVQ AGESLRL | 198 | GSIFS SNAM A | 141 | WYRQ APGKR RDLVA | 205 | AISSV GVTK | 214 |
-7217072
| SCAAS | |||||||||
| CX3CR1 B1I067 | 132 | EVQLVES GGGSVQ AGESLRL SCAAS | 198 | GSIFS SNAM A | 141 | WYRQ APGKR RDLVA | 205 | AIQSV GVTK | 215 |
| CX3CR1 BII068 | 133 | EVQLVES GGGSVQ AGESLRL SCAAS | 198 | GSIFS SNAM A | 141 | WYRQ APGKR RDLVA | 205 | AIGSV GVTK | 216 |
| CX3CR1 BII074 | 134 | EVQLVES GGGSVQ AGESLRL SCAAS | 198 | GSIFS SNAM A | 141 | WYRQ APGKR RDLVA | 205 | AITSV GVTK | 217 |
| CX3CR1 BII118 | 135 | EVQLVES GGGSVQ AGESLRL SCAAS | 198 | GSIFS SNAM A | 141 | WYRQ APGKR RDLVA | 205 | AINTV GVTK | 218 |
| CX3CR1 BII129 | 136 | EVQLVES GGGSVQ AGESLRL SCAAS | 198 | GSIFS SNAM A | 141 | WYRQ APGKR RDLVA | 205 | AING VGVT K | 219 |
| CX3CR1 BII158 | 137 | EVQLVES GGGSVQ AGESLRL SCAAS | 198 | GSIFS SNAM A | 141 | WYRQ APGKR RDLVA | 205 | AINPV GVTK | 220 |
| CX3CR1 BII306 | 138 | DVQLVES GGGLVQ PGGSLRL SCAAS | 204 | GSIFS STAMA | 213 | WYRQ APGKR RDLVA | 205 | AISSV GVTK | 214 |
| CX3CR1 BII307 | 139 | DVQLVES GGGLVQ PGGSLRL SCAAS | 204 | GSIFS STAMA | 213 | WYRQ APGKR RDLVA | 205 | AISTV GVTK | 221 |
| CX3CR1 BII308 | 140 | DVQLVES GGGLVQ PGGSLRL | 204 | GSIFS SNAM A | 141 | WYRQ APGKR RDLVA | 205 | AINSV GVTK | 162 |
-7317072
| SCAAS | 1 |
Table 7b: Sequence optlmlzed variants (FR3-CDR3-FR4)
| Nano body | SEQ | FR3 | SEQ FR3 | CDR3 | SEQ CDR3 | FR4 | SEQ FR4 |
| CX3CR1 BIIPMP6 6B02 | 1 | YADSVKGRFTI SRDNAKNTVYL QMNSLKPEDTA VYYCTS | 209 | DPRRGW DTRY | 186 | WGQGTQ VTVSS | 211 |
| CX3CR1 BII043 | 121 | YADSVKGRFTI SRDNSKNTVYL QMNSLRPEDT AVYYCTS | 210 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
| CX3CR1 BII045 | 122 | YADSVKGRFTI SRDNSKNTVYL QMNSLRPEDT AVYYCTS | 210 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
| CX3CR1 BII047 | 123 | YADSVKGRFTI SRDNSKNTVYL QMNSLRPEDT AVYYCTS | 210 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
| CX3CR1 BII048 | 124 | YADSVKGRFTI SRDNSKNTVYL QMNSLRPEDT AVYYCTS | 210 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
| CX3CR1 BII049 | 125 | YADSVKGRFTI SRDNSKNTVYL QMNSLRPEDT AVYYCTS | 210 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
| CX3CR1 BII050 | 126 | YADSVKGRFTI SRDNSKNTVYL QMNSLRPEDT | 210 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
-7417072
| AVYYCTS | |||||||
| CX3CR1 BII061 | 127 | YADSVKGRFTI SRDNSKNTVYL QMNSLRPEDT AVYYCTS | 210 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
| CX3CR1 BII056 | 128 | YADSVKGRFTI SRDNSKNTVYL QMNSLRPEDT AVYYCTS | 210 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
| CX3CR1 BII057 | 129 | YADSVKGRFTI SRDNSKNTVYL QMNSLRPEDT AVYYCTS | 210 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
| CX3CR1 BII060 | 130 | YADSVKGRFTI SRDNSKNTVYL QMNSLRPEDT AVYYCTS | 210 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
| CX3CR1 BII065 | 131 | YADSVKGRFTI SRDNAKNTVYL QMNSLKPEDTA VYYCTS | 209 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
| CX3CR1 BII067 | 132 | YADSVKGRFTI SRDNAKNTVYL QMNSLKPEDTA VYYCTS | 209 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
| CX3CR1 BII068 | 133 | YADSVKGRFTI SRDNAKNTVYL QMNSLKPEDTA VYYCTS | 209 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
| CX3CR1 BII074 | 134 | YADSVKGRFTI SRDNAKNTVYL QMNSLKPEDTA VYYCTS | 209 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
| CX3CR1 B1I118 | 135 | YADSVKGRFTI SRDNAKNTVYL QMNSLKPEDTA | 209 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
-7517072
| VYYCTS | |||||||
| CX3CR1 BII129 | 136 | YADSVKGRFTI SRDNAKNTVYL QMNSLKPEDTA VYYCTS | 209 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
| CX3CR1 BII158 | 137 | YADSVKGRFTI SRDNAKNTVYL QMNSLKPEDTA VYYCTS | 209 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
| CX3CR1 BII306 | 138 | YADSVKGRFTI SRDNSKNTVYL QMNSLRPEDT AVYYCTS | 210 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
| CX3CR1 BII307 | 139 | YADSVKGRFTI SRDNSKNTVYL QMNSLRPEDT AVYYCTS | 210 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
| CX3CR1 BII308 | 140 | YADSVKGRFTI SRDNSKNTVYL QMNSLRPEDT AVYYCTS | 210 | DPRRGW DTRY | 186 | WGQGTL VTVSS | 212 |
In one aspect of the présent Invention, a polypeptide of the Invention can additionaily contain modifications such as glycosyl residues, modified amino acid 5 side chains, and the like.
It will be ciear to the skllled person that for pharmaceutical uses in humans, the polypeptides of the Invention are preferably directed against human CX3CR1, whereas for veterinary purposes, the polypeptides of the invention are preferably 10 directed against CX3CR1 from the species to be treated.
It will also be ciear to the skiiled person that when used as a therapeutic agent in humans, the immunoglobulin single variable domains comprised In the
-7617072 polypeptides according to the invention are preferably humanized immunoglobulin single variable domains.
According to the invention, an immunoglobulin single variable domain can be a domain antibody, I.e. VL or VH antibody, and/or VHH domains as described above, and/or any other sort of Immunoglobulin single variable domain, for example camelized VH, provided that these immunoglobulin single variable domains are anti-CX3CR1 Immunoglobulin single variable domains.
ln one aspect of the Invention, the immunoglobulin single variable domain essentially consists of either a domain antibody sequence or a VHH domain sequence as described above. In particular, the immunoglobulin single variable domain essentially consists of a VHH domain sequences.
In a further aspect, a polypeptide of the présent invention comprises two or more anti-CX3CR1 immunoglobulin single variable domains. In a further aspect, a polypeptide of the présent invention comprises two anti-CX3CR1 immunoglobulin single variable domains, for example anti-CX3CR1 VHHs. In one aspect, the two anti-CX3CR1 immunoglobulin single variable domains In a polypeptide of the présent invention hâve the same amino add sequence. In another aspect, the two anti-CX3CR1 Immunoglobulin single variable domains in a polypeptide of the présent Invention hâve different amino acid sequences.
According to another embodiment of the invention, the at least two immunoglobulin single variable domains présent in a polypeptide of the invention can be llnked to each other directly (I.e. without use of a linker) or via a linker. The linker is preferably a linker peptide and will, according to the Invention, be selected so as to allow blnding of the at least two immunoglobulin single variable domains to CX3CR1, either within one and the same CX3CR1 molécule, or within two different molécules.
Suitable linkers will inter a//a dépend on the epitopes and, specifically, the distance between the epitopes on CX3CR1 to which the immunoglobulin single
-7717072 variable domains bind, and will be clear to the skilled person based on the disclosure herein, optionally after some limited degree of routine expérimentation.
Also, when the two or more anti-CX3CR1 Immunoglobulin single variable domains are domain antibodies or VHH domains, they may also be linked to each other via a third domain antibody or VHH domain (in which the two or more Immunoglobulin single variable domains may be linked directly to the third domain antibody or VHH domain or via suitable linkers). Such a third domain antibody or VHH domain may for example be a domain antibody or VHH domain 10 that provides for an increased half-lîfe, as further described herein. For example, the latter domain antibody or VHH domain may be a domain antibody or VHH domain that is capable of binding to a (human) sérum proteln such as (human) sérum albumin or (human) transferrin, as further described herein.
Altematively, the two or more anti-CX3CR1 immunoglobulin single variable domains may be linked in sériés (either directly or via a suitable linker) and the third (single) domain antibody or VHH domain (which may provide for increased half-life, as decribed above) may be connected directly or via a linker to one of these two or more aforementioned immunoglobulin sequences.
Suitable linkers are described herein ln connection with spécifie polypeptides of the invention and may - for example and without limitation - comprise an amino acid sequence, which amino acid sequence preferably has a length of 5 or more amino acids, 7 or more amino acids, 9 or more amino acids, 11 or more amino 25 acids, 15 or more amino acids or at least 17 amino acids, such as about 20 to 40 amino acids. However, the upper limlt Is not critlcal but is chosen for reasons of convenlence regarding e.g. biopharmaceutlcal production of such polypeptides.
The linker sequence may be a naturally occurring sequence or a non-naturally occurring sequence. If used for therapeutical purposes, the linker is preferably non-lmmunogenic In the subject to which the polypeptide of the invention is adminlstered.
-7817072
One useful group of linker sequences are linkers derived from the hinge région of heavy chain antibodies as described In WO 96/34103 and WO 94/04678.
Other exampies are poly-alanine linker sequences such as Ala-Ala-Ala.
Further preferred examples of linker sequences are Gly/Ser linkers of different length such as (glyxsery)z linkers, inciuding (gly4ser)3, (gly4ser)4, (gly4ser), (gly3ser), gly3, and (gly3ser2)3.
If the polypeptide of the Invention Is modified by the attachment of a polymer, for example a polyethylene glycol (PEG) moiety, the linker sequence preferably includes an amino acid residue, such as a cysteine or a lysine, allowing such modification, e.g. PEGylation, In the linker région.
Examples of linkers are:
GGGGS (5 GS linker, SEQ ID NO: 233)
SGGSGGS (7GS linker, SEQ ID NO: 234)
GGGGCGGGS (8GS linker, SEQ ID NO: 235)
GGGGSGGGS (9GS linker, SEQ iD NO: 236)
GGGGSGGGGS (10GS linker, SEQ ID NO: 237) GGGGSGGGGSGGGGS (15GS linker, SEQ ID NO: 238) GGGGSGGGGSGGGGGGGS (18GS linker, SEQ ID NO: 239) GGGGSGGGGSGGGGSGGGGS (20GS linker, SEQ ID NO: 240) GGGGSGGGGSGGGGSGGGGSGGGGS (25GS linker, SEQ ID NO: 241) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (30GS linker, SEQ ID NO: 242)
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (35GS linker, SEQ ID NO: 243) ‘ EPKSCDKTHTCPPCP (G 1 hinge linker, SEQ ID NO: 244)
GGGGSGGGSEPKSCDKTHTCPPCP (9GS-G1 hinge linker, SEQ ID NO: 245) EPKTPKPQPAAA (Llama upper long hinge région, SEQ ID NO: 246)
-7917072
ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCD
TPPPCPRCP (G3 hinge, SEQ ID NO: 247)
AAA (Ala iinker, SEQ ID NO: 248)
Furthermore, the Iinker may also be a poly(ethy!ene glycol) molety, as shown In e.g. W004/081026.
Non-limiting examples of polypeptides comprising or consisting of two or more antl-CX3CR1 Immunoglobulin single variable domains are given In Table 8a.
o
Table 8a: Bivalent antl-CX3CR1 polypeptides
| CX3CR1 BII007 | EVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAMG WFRQAPGKERAFVAGISGSASRKYYADSVKGRFTV SRDNARNTVYLQMNSLKPEDTAVYYCAASNSYPKV QFDYYGQGTQVTVSSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSKVQLVESGGGLVQPGG SLRLSCATSGTIFSNNAMGWYRQAPGKKRDLVASIS SSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKP EDTGVYYCTLDARRGWNTAYWGQGAQVTVSS | SEQ ID NO: | 267 |
| CX3CR1 BII009 | KVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMG WYRQAPGKKRDLVASISSSGSTNYADSVKGRFTVS RDNDKNTGYLQMNSLKPEDTGVYYCTLDARRGWNT AYWGQGAQVTVSSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGS LRLSCVASGRTFSSYAMGWFRQAPGKERAFVAGIS GSASRKYYADSVKGRFTVSRDNARNTVYLQMNSLK PEDTAVYYCAASNSYPKVQFDYYGQGTQVTVSS | SEQ ID NO: | 268 |
| CX3CR1 BII012 | EVQLVESGGGSVQAGGSLRLSCAASGSIFSSNAMA WYRQAPGKQRDLVAGINSVGITKYADSVKGRFTISR DNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTR YWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSKVQLVESGGGLVQPGGSLRL SCATSGTIFSNNAMGWYRQAPGKKRDLVASISSSGS | SEQ ID NO: | 269 |
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| TNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTG VYYCTLDARRGWNTAYWGQGAQVTVSS | |||
| CX3CR1 BII016 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMA WYRQAPGKQRDLVAVINSVGITKYADSVKGRFTISG DNAKNTVYLQMNSLKPEDTAVYYCTSDARRGWDTR YWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSEVQLVESGGGSVQAGESL RLSCAASGSIFSSNAMAWYRQAPGKQRDLVAVINSV GITKYADSVKGRFTISGDNAKNTVYLQMNSLKPEDT AVYYCTSDARRGWDTRYWGQGTQVTVSS | SEQ ID NO: | 270 |
| CX3CR1 BII017 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMA WYRQAPPGKQRDLVALINSVGITKYADSVKGRFTISS DNAKNTVYLEMNSLKPEDTAVYYCTSDGRRGWDTR YWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSEVQLVESGGGSVQAGESL RLSCAASGSIFSSNAMAWYRQAPPGKQRDLVAL1NS VG1TKYADSVKGRFTISSDNAKNTVYLEMNSLKPEDT AVYYCTSDGRRGWDTRYWGQGTQVTVSS | SEQ ID NO: | 271 |
| CX3CR1 BII018 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMA WYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTISR DNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTR YWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSEVQLVESGGGSVQAGESL RLSCAASGSIFSSNAMAWYRQAPGKRRDLVAAINSV GVTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCTSDPRRGWDTRYWGQGTQVTVSS | SEQ ID NO: | 272 |
| CX3CR1 BI1019 | EMQLVESGGGSVQAGESLRLSCAASGSIFSSNAMA WYRQAPGKQRDLVALINSVGITKYADSVKGRFTISR DNAKNTVYLQMNSLKPEDTAVYYCTSDGRRGWDTR YWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSEMQLVESGGGSVQAGESL RLSCAASGSIFSSNAMAWYRQAPGKQRDLVALINSV GITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCTSDGRRGWDTRYWGQGTQVTVSS | SEQ ID NO: | 273 |
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| CX3CR1 BII020 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMA WYRQAPGKQRDLVAGINSVGITKYADSVKGRFTISR DNAKNTAYLQMNSLKPEDTAVYYCTSDPRRGWDTR YWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSEVQLVESGGGSVQAGESLRL SCAASGSIFSSNAMAWYRQAPGKQRDLVAGINSVGl TKYADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAV YYCTSDPRRGWDTRYWGQGTLVTVSS | SEQ ID NO: | 274 |
| CX3CR1 BII026 | EVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAMG WFRQAPGKERAFVAGISGSASRKYYADSVKGRFTV SRDNARNTVYLQMNSLKPEDTAVYYCAASNSYPKV QFDYYGQGTQVTVSSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGE SLRLSCAASGSIFSSNAMAWYRQAPGKRRDLVAA1N SVGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKPE DTAVYYCTSDPRRGWDTRYWGQGTQVTVSS | SEQ ID NO: | 275 |
| CX3CR1 BII027 | EVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAMG WFRQAPGKERAFVAG1SGSASRKYYADSVKGRFTV SRDNARNTVYLQMNSLKPEDTAVYYCAASNSYPKV QFDYYGQGTQVTVSSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGE SLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAG IN SVG1TKYADSVKGRFTISRDNAKNTAYLQMNSLKPE DTAVYYCTSDPRRGWDTRYWGQGTLVTVSS | SEQ ID NO: | 276 |
| CX3CR1 BII006 | EVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAMG WFRQAPGKERAFVAG1SGSASRKYYADSVKGRFTV SRDNARNTVYLQMNSLKPEDTAVYYCAASNSYPKV QFDYYGQGTLVTVSSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGG SLRLSCVASGRTFSSYAMGWFRQAPGKERAFVAGI SGSASRKYYADSVKGRFTVSRDNARNTVYLQMNSL KPEDTAVYYCAASNSYPKVQFDYYGQGTLVTVSS | SEQ ID NO: | 282 |
-8217072 ln another embodiment, the at least two immunoglobulin single variable domains of the polypeptide of the Invention are linked to each other via another moiety (optionally via one or two linkers), such as another polypeptide which, ln a preferred but non-limltlng embodiment, may be a further Immunoglobulin single s variable domain as already described above. Such moiety may either be essentially Inactive or may hâve a biologicai effect such as Improving the desired properties of the polypeptide or may confer one or more additional desired properties to the polypeptide. For example, and without limitation, the moiety may improve the half-life of the protein or polypeptide, and/or may reduce its
Immunogenlcity or Improve any other desired property.
ln one aspect, a polypeptide of the invention includes, especially when used as a therapeutlc agent, a moiety which extends the half-life of the polypeptide of the Invention in sérum or other body fluids of a patient. The term half-life means the 15 time taken for the sérum concentration of the (modified) polypeptide to reduce by
50%, in vivo, for example due to dégradation of the polypeptide and/or clearance and/or séquestration by natural mechanisms.
According to a further embodiment of the invention, the two immunoglobulin 20 single variable domains may be fused to a sérum albumln molécule, such as described e.g. ln WO01/79271 and WO03/59934.
Altematively, such half-life extending moiety can be covalently linked or fused to said polypeptide and may be, without limitation, an Fc portion, an albumln moiety, 25 a fragment of an albumln moiety, an albumln blndlng moiety, such as an antlalbumln Immunoglobulin single variable domain, a transferrin blnding moiety, such as an anti-transferrin immunoglobulin single variable domain, a polyoxyalkylene molécule, such as a polyethylene glycol molécule, an albumln blnding peptide, or hydroxyethyl starch (HES) dérivatives.
ln another aspect, the polypeptide of the invention comprises a moiety which binds to an antigen found ln blood, such as sérum albumin, sérum immunoglobulins, thyroxine-binding protein, fibrinogen or transferrin, thereby
-8317072 conferring an Increased half-life in vivo to the resulting polypeptide of the invention. According to one embodiment, such moiety is an albumin-binding immunoglobulin and, in particular, an albumin-binding Immunoglobulin single variable domain such as an albumin-binding VHH domain.
In another embodiment, the polypeptide of the invention comprises a moiety which binds to sérum albumin, wherein such moiety Is an albumin binding peptide, as described e.g. in international patent publications W02008/068280 and W02009/127691.
If Intended for use in humans, such albumin-binding immunoglobulin single variable domain (also called anti-albumin Immunoglobulin single variable domain) will preferably bind to human sérum albumin and will preferably be a humanized albumin-binding VHH domain.
Immunoglobulin single variable domains binding to human sérum albumin are known In the art and are described in further detail in e.g. W02006/122786. A specifically useful albumin binding VHH domain consists of or contains the amino acid sequence as set forth ln any one of SEQ ID NO: 230-232:
Table 8b
| ALB-1 | AVQLVESGGGLVQPGNSLRLSCAASGFTFRSFG MSWVRQAPGKEPEWVSSISGSGSDTLYADSVK GRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIG GSLSRSSQGTQVTVSS | SEQ ID NO: | 230 |
| ALB-11 (humanized ALB-1) | EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFG MSWVRQAPGKGLEWVSSISGSGSDTLYADSVK GRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIG GSLSRSSQGTLVTVSS | SEQ ID NO: | 231 |
| ALB-2 | AVQLVESGGGLVQGGGSLRLACAASERIFDLNL MGWYRQGPGNERELVATCITVGDSTNYADSVK GRFTISMDYTKQTVYLHMNSLRPEDTGLYYCKIR | SEQ ID NO: | 232 |
-8417072
| RTWHSELWGQGTQVTVSS |
According to one embodiment, a polypeptide of the invention may be linked to one or more antibody parts, fragments or domains that confer one or more effector fonctions to the polypeptide of the Invention and/or may confer the abiiity to bind to one or more Fc receptors. For example, for this purpose, and without being limited thereto, the antibody parts may be or may comprise CH2 and/or CH3 domains of an antibody, such as from a heavy chain antibody (as described hereabove) and more preferably from a conventional human 4-chain antibody;
specifically, the polypeptide of the Invention may be linked to an Fc région, for example from human IgG, from human IgE or from another human Ig. For example, WO 94/04678 describes heavy chain antlbodies comprising a Camelid VHH domain or a humanized dérivative thereof, In which the Camelidae CH2 and/or CH3 domain hâve been replaced by human CH2 and/or CH3 domains, so is as to provide an Immunoglobulin that constats of 2 heavy chains each comprising a - optionally humanized - VHH domain and human CH2 and CH3 domains (but no CH1 domain), which immunoglobulin has the effector fonction provided by the CH2 and CH3 domains, can fonction without the presence of any light chains, and has an increased half-life as compared to the corresponding VHH domains 20 without such modification.
In one aspect, a polypeptide of the présent Invention comprises two antl-CX3CR1 VHHs and a VHH capable of binding to sérum albumin. In one aspect, the VHHs are fused using linker peptides. Représentative examples of such polypeptides of 25 the présent invention are shown herelnbelow.
In one aspect, a polypeptide of the présent Invention comprises a first antlCX3CR1 VHH fused to a first linker peptide, which ls Itself fused to a VHH capable of binding to sérum albumin, which ls Itself fused to a second linker peptide, which ls itself fused to a second anti-CX3CR1 VHH. In one aspect, the first or the second linker peptide ta a 9GS linker, in one aspect, the first and the
-8517072 second linker peptide Is a 9GS linker. ln one aspect, the VHH capable of binding to sérum albumin is capable of binding to human sérum albumin. In one aspect, the VHH capable of binding to sérum albumin has the amino acid sequence set forth ln SEQ ID NO: 231. In one aspect, the first and the second anti-CX3CR1
VHH hâve the same amino acid sequence. ln one aspect, the first or the second antl-CX3CR1 VHH has the CDR1, CDR2 and CDR3 set forth ln:
- SEQ ID NO’s: 213, 214 and 186; or
- SEQ ID NO’s: 213, 221 and 186; or
- SEQ ID NO’s: 141,162 and 186.
ln one aspect, the first and the second anti-CX3CR1 VHH hâve the CDR1, CDR2 and CDR3 set forth ln:
- SEQ ID NO’s: 213, 214 and 186; or
- SEQ ID NO’s; 213, 221 and 186; or
- SEQ ID NO’s: 141,162 and 186.
ln one aspect, the first or the second anti-CX3CR1 VHH has the amino acid sequence set forth in any one of SEQ ID NO: 138 to 140 or SEQ ID NO: 222 to 224. In one aspect, the first and the second anti-CX3CR1 VHH hâve the same amino acid sequence, wherein said amino acid sequence is the sequence set forth In any one of SEQ ID NO: 138 to 140 or SEQ ID NO: 222 to 224.
Non-limiting examples of polypeptides of the présent Invention are the polypeptides of any one of SEQ ID NO: 225 to 227, 249 or 277 to 281.
Table 9
| CX3CR1BII 00312 | DVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA MAWYRQAPGKRRDLVAAISSVGVTKYADSVKGR FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPR RGWDTRYWGQGTLVTVSSGGGGSGGGSEVQL VESGGGLVQPGNSLRLSCAASGFTFSSFGMSW VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR | SEQ ID NO: | 225 |
-8617072
| SSQGTLVTVSSGGGGSGGGSEVQLVESGGGLV QPGGSLRLSCAASGSIFSSTAMAWYRQAPGKRR DLVAAISSVGVTKYADSVKGRFTISRDNSKNTVYL QMNSLRPEDTAVYYCTSDPRRGWDTRYWGQG TLVTVSS | |||
| CX3CR1BII 00313 | DVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA MAWYRQAPGKRRDLVAAISTVGVTKYADSVKGR FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPR RGWDTRYWGQGTLVTVSSGGGGSGGGSEVQL VESGGGLVQPGNSLRLSCAASGFTFSSFGMSW VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR SSQGTLVTVSSGGGGSGGGSEVQLVESGGGLV QPGGSLRLSCAASGSIFSSTAMAWYRQAPGKRR DLVAAISTVGVTKYADSVKGRFTISRDNSKNTVYL QMNSLRPEDTAVYYCTSDPRRGWDTRYWGQG TLVTVSS | SEQID NO: | 226 |
| CX3CR1BII 00314 | DVQLVESGGGLVQPGGSLRLSCAASGSIFSSNA MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPR RGWDTRYWGQGTLVTVSSGGGGSGGGSEVQL VESGGGLVQPGNSLRLSCAASGFTFSSFGMSW VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR SSQGTLVTVSSGGGGSGGGSEVQLVESGGGLV QPGGSLRLSCAASGSIFSSNAMAWYRQAPGKR RDLVAAINSVGVTKYADSVKGRFTISRDNSKNTV YLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQ GTLVTVSS | SEQID NO: | 227 |
| CX3CR1BII 032 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPR RGWDTRYWGQGTQVTVSSGGGGSGGGSEVQL VESGGGSVQAGESLRLSCAASGSIFSSNAMAWY RQAPGKRRDLVAAINSVGVTKYADSVKGRFTISR DNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSSGGGGSGGGSEVQLVES GGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQ APGKGLEWVSSISGSGSDTLYADSVKGRFTISRD NAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQ | SEQID NO: | 277 |
-8717072
| GTLVTVSS | |||
| CX3CR1BI1 034 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPR RGWDTRYWGQGTQVTVSSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSEVQLVES GGGSVQAGESLRLSCAASGSIFSSNAMAWYRQ APGKRRDLVAAINSVGVTKYADSVKGRFTISRDN AKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTR YWGQGTQVTVSSGGGGSGGGSEVQLVESGGG LVQPGNSLRLSCAASGFTFSSFGMSWVRQAPG KGLEWVSSISGSGSDTLYADSVKGRFTISRDNAK TTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTL VTVSS | SEQ ID NO: | 278 |
| CX3CR1BII 036 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPR RGWDTRYWGQGTLVTVSSGGGGSGGGSEVQL VESGGGLVQPGNSLRLSCAASGFTFSSFGMSW VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR SSQGTLVTVSSGGGGSGGGSEVQLVESGGGSV QAGESLRLSCAASGSIFSSNAMAWYRQAPGKRR DLVAAINSVGVTKYADSVKGRFTISRDNAKNTVY LQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQG TLVTVSS | SEQ ID NO: | 249 |
| CX3CR1BII 040 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPR RGWDTRYWGQGTQVTVSSGGGGSGGGSEVQL VESGGGSVQAGESLRLSCAASGSIFSSNAMAWY RQAPGKRRDLVAAINSVGVTKYADSVKGRFTISR DNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGGL VQPGNSLRLSCAASGFTFSSFGMSWVRQAPGK GLEWVSSISGSGSDTLYADSVKGRFTISRDNAKT TLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLV TVSS | SEQ ID NO: | 279 |
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| CX3CR1BII 041 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPR RGWDTRYWGQGTQVTVSSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSEVQLVES GGGSVQAGESLRLSCAASGSIFSSNAMAWYRQ APGKRRDLVAAINSVGVTKYADSVKGRFTISRDN AKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTR YWGQGTQVTVSSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSEVQLVESGGGLVQP GNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLY LQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVS S | SEQID NO: | 280 |
| CX3CR1BII 042 | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPR RGWDTRYWGQGTQVTVSSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSEVQLVES GGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQ APGKGLEWVSSISGSGSDTLYADSVKGRFTISRD NAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQ GTLVTVSSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSEVQLVESGGGSVQAGESLRL SCAASGSIFSSNAMAWYRQAPGKRRDLVAAINS VGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKP EDTAVYYCTSDPRRGWDTRYWGQGTQVTVSS | SEQ ID NO: | 281 |
In another aspect, a polypeptide of the présent Invention comprises an antiCX3CR1 VHH and a Fc domain. In one aspect, a polypeptide of the présent invention comprises an anti-CX3CR1 VHH fused to a linker peptide, which Is itself fused to a Fc domain. In one aspect, the linker peptide is a 15GS linker. In one aspect, the Fc domain has the amino acid sequence set forth in SEQ ID NO: 250 or 252. In one aspect, the VHH has the CDR1, CDR2 and CDR3 set forth In:
- SEQ ID NO's: 213, 214 and 186; or io - SEQ ID NO’s: 213, 221 and 186; or * , SEQ ID NO’s: 141,162 and 186. - -8917072
In one aspect, the VHH has the amino acid sequence set forth in any one of SEQ ID NO: 138 to 140 or SEQ ID NO: 222 to 224. In one aspect the polypeptide ls in the form of a dimer, for example wherein the dimer is formed by one or more disuifide bridge.
Non-limiting examples of polypeptides ofthe présent invention are the polypeptides of SEQ ID NO: 251, 253 or 254.
io Table 10
| Mouse Fc domain | PPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVT CVWAVSEDDPDVQISWFVNNVEVHTAQTQTHR EDYNSTLRWSALPIQHQDWMSGKEFKCKVNNK DLPAPiERTISKPKGSVRAPQVYVLPPPEEEMTK KQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKN TEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCS WHEGLHNHHTTKSFSRTPGK | SEQ ID NO: | 250 |
| 66B02-mFc | EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPR RGWDTRYWGQGTLVTVSSGGGGSGGGGSGG GGSPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLS PIVTCVWAVSEDDPDVQISWFVNNVEVHTAQTQ THREDYNSTLRWSALPIQHQDWMSGKEFKCKV NNKDLPAPIERTiSKPKGSVRAPQVYVLPPPEEE MTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELN YKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSY SCSWHEGLHNHHTTKSFSRTPGK | SEQID NO: | 251 |
| Human Fc Domain | CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEV TCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK | SEQID NO: | 252 |
| 306D-hFc | DVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA | SEQID | 253 |
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| MAWYRQAPGKRRDLVAAISSVGVTKYADSVKGR FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPR RGWDTRYWGQGTLVTVSSGGGGSGGGGSGG GGSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK | NO: | ||
| 307D-hFc | DVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA MAWYRQAPGKRRDLVAAISTVGVTKYADSVKGR FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPR RGWDTRYWGQGTLVTVSSGGGGSGGGGSGG GGSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK | SEQID NO: | 254 |
A polypeptide of the Invention may be modified to improve Its properties. In one aspect, a polypeptide of the présent Invention may be modified to Increase Its s stability upon storage. In one aspect, a polypeptide of the présent invention may be modified to facilitate Its expression in a particular host system. For example, the first codon of a polypeptide ofthe présent Invention may be modified. In one aspect, a polypeptide of the présent invention beglns with a glutamlc acid (glu) as Its firstamino acid. In another aspect, a polypeptide ofthe présent Invention 10 beglns with an aspartlc acid (asp) as Its first amino acid, for example to reduce pyroglutamate formation at the N-termlnus during storage and hence increase product stability. In another aspect, a polypeptide of the présent invention beglns with an alanine (ala) or a valine (val) as Its first amino acid, for example to facilitate the expression ofthe polypeptide in a prokaryotic expression system,
-91 17072 such as Escherichia coii. Such modification of a polypeptide according to the présent invention are made using techniques known in the art.
Représentative examples of polypeptides according to the présent invention with a modified first codon are set forth In any one of SEQ ID ND: 257-262 and 2635 266 and are shown in Tables 11 and 12 below;
Table 11
| CX3CR1BII 00312 (D1A) | AVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA MAWYRQAPGKRRDLVAAISSVGVTKYADSVKGR FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPR RGWDTRYWGQGTLVTVSSGGGGSGGGSEVQL VESGGGLVQPGNSLRLSCAASGFTFSSFGMSW VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR SSQGTLVTVSSGGGGSGGGSEVQLVESGGGLV QPGGSLRLSCAASGSIFSSTAMAWYRQAPGKRR DLVAAISSVGVTKYADSVKGRFTISRDNSKNTVYL QMNSLRPEDTAVYYCTSDPRRGWDTRYWGQG TLVTVSS | SEQ ID NO: | 257 |
| CX3CR1BII 00313 (D1A) | AVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA MAWYRQAPGKRRDLVAAISTVGVTKYADSVKGR FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPR RGWDTRYWGQGTLVTVSSGGGGSGGGSEVQL VESGGGLVQPGNSLRLSCAASGFTFSSFGMSW VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR SSQGTLVTVSSGGGGSGGGSEVQLVESGGGLV QPGGSLRLSCAASGSIFSSTAMAWYRQAPGKRR DLVAAISTVGVTKYADSVKGRFTISRDNSKNTVYL QMNSLRPEDTAVYYCTSDPRRGWDTRYWGQG TLVTVSS | SEQ ID NO: | 258 |
| CX3CR1BII 00314 (D1A) | AVQLVESGGGLVQPGGSLRLSCAASGSIFSSNA MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPR RGWDTRYWGQGTLVTVSSGGGGSGGGSEVQL VESGGGLVQPGNSLRLSCAASGFTFSSFGMSW VRQAPGKGLEVWSSISGSGSDTLYADSVKGRFTI | SEQ ID NO: | 259 |
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| SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR SSQGTLVTVSSGGGGSGGGSEVQLVESGGGLV QPGGSLRLSCAASGSIFSSNAMAWYRQAPGKR RDLVAAINSVGVTKYADSVKGRFTISRDNSKNTV YLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQ GTLVTVSS | |||
| CX3CR1BII 00312 (D1V) | VQLVESGGGLVQPGGSLRLSCAASGSIFSSTAM AWYRQAPGKRRDLVAAISSVGVTKYADSVKGRF TISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRR GWDTRYWGQGTLVTVSSGGGGSGGGSEVQLV ESGGGLVQPGNSLRLSCAASGFTFSSFGMSWV RQAPGKGLEWVSSISGSGSDTLYADSVKGRFTIS RDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQ PGGSLRLSCAASGSIFSSTAMAWYRQAPGKRRD LVAAISSVGVTKYADSVKGRFTISRDNSKNTVYL QMNSLRPEDTAVYYCTSDPRRGWDTRYWGQG TLVTVSS | SEQ ID NO: | 260 |
| CX3CR1BII 00313 (D1V) | VQLVESGGGLVQPGGSLRLSCAASGSIFSSTAM AWYRQAPGKRRDLVAAISTVGVTKYADSVKGRF TISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRR GWDTRYWGQGTLVTVSSGGGGSGGGSEVQLV ESGGGLVQPGNSLRLSCAASGFTFSSFGMSWV RQAPGKGLEWVSSISGSGSDTLYADSVKGRFTIS RDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQ PGGSLRLSCAASGSIFSSTAMAWYRQAPGKRRD LVAAISTVGVTKYADSVKGRFTISRDNSKNTVYLQ MNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTL VTVSS | SEQID NO: | 261 |
| CX3CR1BII 00314 (D1V) | VQLVESGGGLVQPGGSLRLSCAASGSIFSSNAM AWYRQAPGKRRDLVAAINSVGVTKYADSVKGRF TISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRR GWDTRYWGQGTLVTVSSGGGGSGGGSEVQLV ESGGGLVQPGNSLRLSCAASGFTFSSFGMSWV RQAPGKGLEWVSSISGSGSDTLYADSVKGRFTIS RDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQ PGGSLRLSCAASGSIFSSNAMAWYRQAPGKRRD LVAAINSVGVTKYADSVKGRFTISRDNSKNTVYL | SEQ ID NO: | 262 |
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| QMNSLRPEDTAVYYCTSDPRRGWDTRYWGQG TLVTVSS |
Table 12
| 306D-hFc (D1A) | AVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA MAWYRQAPGKRRDLVAAISSVGVTKYADSVKGR FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPR RGWDTRYWGQGTLVTVSSGGGGSGGGGSGG GGSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK | SEQID NO: | 263 |
| 307D-hFc (D1A) | AVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA MAWYRQAPGKRRDLVAAISTVGVTKYADSVKGR FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPR RGWDTRYWGQGTLVTVSSGGGGSGGGGSGG GGSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK | SEQ ID NO: | 264 |
| 306D-hFc (D1V) | VQLVESGGGLVQPGGSLRLSCAASGSIFSSTAM AWYRQAPGKRRDLVAAISSVGVTKYADSVKGRF TISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRR GWDTRYWGQGTLVTVSSGGGGSGGGGSGGG GSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV | SEQ ID NO: | 265 |
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| SNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK | |||
| 307D-hFc (D1V) | VQLVESGGGLVQPGGSLRLSCAASGSIFSSTAM AWYRQAPGKRRDLVAAISTVGVTKYADSVKGRF TISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRR GWDTRYWGQGTLVTVSSGGGGSGGGGSGGG GSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK | SEQID NO: | 266 |
ln one further aspect, a polypeptide of the présent invention is characterized by one or more of the following properties:
· Bind with high affinity to human CX3CR1 :
• Inhlblt blnding of soluble fractalkine to human CX3CR1 ;
• Inhibit fractalkine Induced chemotaxis;
• Inhibit fractalkine induced human CX3CR1 receptor intemalization;
• Cross-react with cyno CX3CR1 within 10-fold of E/IC50 for human CX3CR1 for binding and functionai Inhibition.
Accordingly, ln one aspect, a polypeptide of the présent invention has an affinity to human CX3CR1 at an IC50 less than or equal to 10nM, or less than or equal to 5nM, or less than or equal to 2.5nM or less than or equal to 1 nM, as determined 15 by compétition FACS.
ln a further aspect, a polypeptide of the présent invention has an affinity to human CX3CR1 at an EC50 of less than or equal to 10nM, or less than or equal to 5nM, or less than or equal to 2.5nM or less than or equal to 1 nM, as determined by cell 20 binding FACS.
-9517072 ln a further aspect, a polypeptide of the présent invention blocks the binding of human CX3CR1 to human fractalkine at or above 50%, or at or above 60%, or at or above 70%, or at or above 80%, or at or above 90%, or at or above 95% as determined by compétition FACS with human fractalkine.
In a further aspect, a polypeptide of the présent invention blocks the binding of human fractalkine to human CX3CR1 at an IC50 of less than or equal to 300nM, or less than or equal to 100nM, or less than or equal to 20nM, or less than or equal to 10nM, less than or equal to 5nM, less than or equal to 2.5nM or less than or equal to 1nM as determined by compétition FACS with human fractalkine.
in a further aspect, a polypeptide of the présent invention inhibits fractalkine induced chemotaxis mediated by human CX3CR1 at or above 10%, or at or above 30%, or at or above 40%, or at or above 50%, or at or above 60%, or at or above 70%, or at or above 80%, or at or above 90%.
In a further aspect, a polypeptide of the présent invention inhibits fractalkine induced chemotaxis mediated by human CX3CR1 at an IC50 of less than or equal to 500 nM, or of iess than or equal to 100 nM, or iess than or equal to 75 nM, or less than or equal to 50 nM, or less than or equal to 10 nM or less than or equal to 5nM.
• In a further aspect, a polypeptide of the présent invention inhibits fractalkine induced human CX3CR1 receptor intemalization at an IC50 of less than or equal to 10 nM, or iess than or equal to 5nM or or less than or equal to 1 nM.
According to still another embodiment, a haif-life extending modification of a polypeptide of the invention (such modification also reducing immunogenicity of the polypeptide) comprises attachment of a suitable pharmacologically acceptable poiymer, such as straight or branched chain poly(ethyiene glycol) (PEG) or dérivatives thereof (such as methoxypoly(ethylene glycol) or mPEG). Generally, any suitable form of PEGylation can be used, such as the PEGylation used in the art for antibodies and antibody fragments (including but not limited to domain antibodies and scFv*s); reference is made, for example, to: Chapman,
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Nat. Biotechnol., 54, 531-545 (2002); Veronese and Harris, Adv. Drug Deliv. Rev. 54,453-456 (2003); Harris and Chess, Nat. Rev. Drug. Discov. 2 (2003); WO 04/060965; and US6,875,841.
s Various reagents for PEGylation of polypeptides are also commercially available, for example from Nektar Therapeutics, USA, or NOF Corporation, Japan, such as the Sunbright® EA Sériés, SH Sériés, MA Sériés, CA Sériés, and ME Sériés, such as Sunbright® ME-100MA, Sunbright® ME-200MA, and Sunbright® ME400MA.
Preferably, site-directed PEGylation is used, in particular via a cysteine-residue (see for example Yang et al., Protein Engineering 16, 761-770 (2003)). For example, for this purpose, PEG may be attached to a cysteine residue that naturally occurs in a polypeptide of the invention, a polypeptide of the invention 15 may be modified so as to suitably Introduce one or more cysteine residues for attachment of PEG, or an amino acid sequence comprising one or more cysteine residues for attachment of PEG may be fused to the N- and/or C-terminus and/or PEG may be attached to a linker région that bridges two or more functional domains of a polypeptide of the Invention, ail using techniques of protein 20 engineering known per se to the skilled person.
Preferably, for the polypeptides of the invention, a PEG is used with a moiecular weight of more than 5 kDa, such as more than 10 kDa and less than 200 kDa, such as less than 100 kDa; for example in the range of 20 kDa to 80 kDa.
With regard to PEGylation, it should be noted that generally, the invention also encompasses any polypeptide of the invention that has been PEGylated at one or more amino acid positions, preferably in such a way that said PEGylation either (1) Increases the half-life in vivo; (2) reduces immunogenicity; (3) provides one or 30 more further bénéficiai properties known per se for PEGylation; (4) does not essentially affect the affinity of the polypeptide for CX3CR1 (e.g. does not reduce said affinity by more than 50 %, and more preferably not by more than 10%, as determined by a suitable assay, such as those described in the Examples below);
-9717072 and/or (4) does not affect any of the other desired properties of the polypeptides of the invention. Suitable PEG-groups and methods for attaching them, either specifically or non-specifically, will be clear to the skilled person.
According to a specifically preferred embodiment of the invention, a PEGylated polypeptide of the invention includes one PEG moiety of linear PEG having a molecular weight of 40 kDa or 60 kDa, wherein the PEG moiety is attached to the polypeptide In a linker région and, specifially, at a Cys residue, for example at position 5 of a GS8-linker peptide as shown in SEQ ID NO:235.
Preferred examples of PEGylated polypeptides of the invention are PEGylated preferably with one of the PEG reagents as mentioned above, such as Sunbright® ME-400MA as shown in the following chemical formula:
o.
?
CHjO—(C H2CH2O)n—CH2CH2CH2NHC(CH2)2>N which has an average molecular weight of 40 kDa.
Therapeutic uses
In one aspect, the présent invention provides a polypeptide of the présent 20 invention or a pharmaceutical composition comprising said polypeptide for use as a médicament.
In one aspect, the présent invention provides the use of a polypeptide of the présent invention or a pharmaceutical composition comprising said polypeptide 25 for the treatment or prophylaxie of cardio- and cerebrovascular atherosclerotic disorders, peripheral artery disease, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schonlein purpura and Wegeneris granulomatosis, rheumatoid arthritis, osteoarthritis, allograft rejection, systemic scierosis, neurodegenerative disorders and
-9817072 demyelinating disease, multiple sclerosis (MS), Alzheimer*s disease, pulmonary diseases such as COPD, asthma, neuropathie pain, inflammatory pain, or cancer.
In another aspect, the présent Invention provides the use of a polypeptide of the présent Invention or a pharmaceutical composition comprising said polypeptide for the treatment or prophylaxis of atherosclerosis.
In another aspect, the présent invention provides the use of a polypeptide of the présent invention or a pharmaceutical composition comprising said polypeptide for the treatment or prophylaxis of atherosclerosis by preventing and/or reducing the formation of new atheroscierotic lésions or plaques and/or by preventing or slowing progression of existing lésions and plaques.
In another aspect, the présent invention provides the use of a polypeptide of the présent invention or a pharmaceutical composition comprising said polypeptide for the treatment or prophylaxis of atherosclerosis by changing the composition of the plaques to reduce the risk of plaque rupture and atherothrombotic events.
In one aspect, the présent Invention also provides a method of treating, or reducing the risk of, cardlo- and cerebrovascular atheroscierotic disorders, peripheral artery disease, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schonlein purpura and Wegeneris granulomatosis, rheumatoid arthritis, osteoarthritis, allograft rejection, systemic sclerosis, neurodegenerative disorders and demyelinating disease, multiple sclerosis (MS), Alzheimeris disease, pulmonary diseases such as COPD, asthma, neuropathie pain, inflammatory pain, or cancer, In a person suffering from or at risk of, said disease or condition, wherein the method comprises administering to the person a therapeutically effective amount of a polypeptide according to the présent invention or a pharmaceutical composition comprising said polypeptide.
-9917072 ln one aspect, the présent Invention also provides a method of treating, or reduclng the risk of atherosclerosis in a person suffering from or at risk of, said disease or condition, wherein the method comprises admlnlsterlng to the person a therapeutically effective amount of polypeptide of the présent invention or a pharmaceutical composition comprising said polypeptide.
In one aspect, the présent Invention also provides a method of treating, or reduclng the risk of atherosclerosis by preventlng and/or reduclng the formation of new atherosclerotic lésions or plaques and/or by preventing or slowing progression of exlsting lésions and plaques in a person suffering from or at risk of, said disease or condition, wherein the method comprises administering to the person a therapeutically effective amount of polypeptide of the présent invention or a pharmaceutical composition comprising said polypeptide.
is In one aspect, the présent Invention also provides a method of treating, or reducing the risk of atherosclerosis by changing the composition of the plaques so as to reduce the risk of plaque rupture and atherothrombotic events ln a person suffering from or at risk of, said disease or condition, wherein the method comprises administering to the person a therapeutically effective amount of a polypeptide of the présent invention or a pharmaceutical composition comprising said polypeptide.
In one aspect; a polypeptide of the présent invention is indicated for use in the treatment or prophyIaxis of a disease or disorder that is associated with CX3CR1.
In one aspect, a polypeptide of the présent invention is indicated for use In the treatment or prophylaxie of diseases or conditions in which modulation of activity at the CX3CR1 receptor Is désirable. In one aspect, the présent invention also provides a method of treating, or reducing the risk of, diseases or conditions in 30 which antagonism of the CX3CR1 receptor is bénéficiai which comprises administering to a person suffering from or at risk of, said disease or condition, a polypeptide of the présent Invention.
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Prophylaxis Is expected to be particulariy relevant to the treatment of persons who hâve suffered a previous épisode of, or are otherwise considered to be at increased risk of, the disease or condition ln question. Persons at risk of developlng a particular disease or condition generally include those having a family hlstory of the disease or condition, or those who hâve been Identified by genetlc testing or screening to be particulariy susceptible to developlng the disease or condition.
ln the context of the présent Invention, the term prévention, treatment and/or aliénation not only comprises preventing and/or treating and/or alleviating the disease, but also generally comprises preventing the onset of the disease, slowing or reversing the progress of disease, preventing or slowing the onset of one or more symptoms assoclated with the disease, reducing and/or alleviating one or more symptoms assoclated with the disease, reducing the severity and/or the duration of the disease and/or of any symptoms assoclated therewith and/or preventing a further increase In the severity of the disease and/or of any symptoms associated therewith, preventing, reducing or reversing any physiologicai damage caused by the disease, and generally any pharmacological action that Is bénéficiai to the patient being treated.
The subject to be treated will be a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will ln particular be a person suffering from, or at risk from, the diseases, disorders or conditions mentloned herein.
It will also be clear to the skilled person that the above methods of treatment of a disease Include the préparation of a médicament for the treatment of said disease. Furthermore, It Is clear that the polypeptides of the Invention can be used as an active Ingrédient in a médicament or pharmaceutical composition Intended for the treatment of the above diseases. Thus, the invention also relates to the use of a polypeptide of the invention ln the préparation of a pharmaceutical composition for the prévention, treatment and/or aliénation of any of the diseases, disorders or conditions mentioned hereinabove. The Invention further
-101 17072 relates to a polypeptide of the Invention for therapeutic or pnophylactic use and, specifically, for the prévention, treatment and/or aliénation of any of the diseases, disorders or conditions mentioned hereinabove. The invention further relates to a pharmaceutical composition for the prévention, treatment and/or alleviation of the diseases, disorders or conditions mentioned hereinabove, wherein such composition comprises at least one polypeptide of the invention.
The polypeptides of the invention and/or the compositions comprising the same can be administered to a patient ln need thereof ln any suitable manner, depending on the spécifie pharmaceutical formulation or composition to be used. Thus, the polypeptides of the invention and/or the compositions comprising the same can for example be administered intravenously, subcutaneously, Intramusculariy, Intraperitoneally, transdermally, orally, sublingually (e.g. ln the form of a sublingual tablet, spray or drop placed under the tongue and adsorbed through the mucus membranes Into the capillary network under the tongue), (intra-)nasally (e.g. ln the form of a nasal spray and/or as an aérosol), topically, by means of a suppository, by inhalation, Intravitreally (esp. for the treatment of dry AMD or glaucoma), or any other suitable manner ln an effective amount or dose.
The polypeptides of the invention and/or the compositions comprising the same are administered according to a regimen of treatment that Is suitable for preventing, treating and/or alleviating the disease, disorder or condition to be prevented, treated or alleviated. The clinician will generally be able to détermine a suitable treatment regimen, depending on factors such as the disease, disorder or condition to be prevented, treated or alleviated, the severity of the disease, the severity of the symptoms thereof, the spécifie polypeptide of the invention to be used, the spécifie route of administration and pharmaceutical formulation or composition to be used, the âge, gender, weight, diet, general condition of the patient, and similar factors well known to the clinician. Generally, the treatment regimen will comprise the administration of one or more polypeptides of the invention, or of one or more compositions comprising the same, ln therapeutlcally and/or prohylactically effective amounts or doses.
-10217072
Generally, for the prévention, treatment and/or allégation of the diseases, disorders and conditions mentioned herein and depending on the spécifie disease, disorder or condition to be treated, the potency of the spécifie 5 polypeptide of the invention to be used, the spécifie route of administration and the spécifie pharmaceuticai formulation or composition used, the polypeptides of the invention will generally be administered In an amount between 0.005 and 20.0 mg per kilogram of body weight and dose, preferably between 0.05 and 10.0 mg/kg/dose, and more preferably between 0.5 and 10 mg/kg/dose, either w continuously (e.g. by infusion) or as single doses (such as e.g. daily, weekly, or monthly doses; cf. below), but can significantly vary, especially, depending on the before-mentioned parameters.
For prophylactic applications, compositions containing the polypeptides of the invention may also be administered In similar or slightly lower dosages. The dosage can also be adjusted by the individual physician in the event of any complication.
Depending on the spécifie polypeptide of the invention and its spécifie 20 pharmacokinetic and other properties, it may be administered daily, every second, third, fourth, fifth or sixth day, weekiy, monthly, and the like. An administration regimen could include long-term, weekly treatment. By long-term is meant at least two weeks and preferably months, or years of duration.
The efficacy of the polypeptides of the invention, and of compositions comprising the same, can be tested using any suitable In vitro assay, cell-based assay, in vivo assay and/or animal modei known per se, or any combination thereof, depending on the spécifie disease involved. Suitable assays and animal models will be clear to the skilled person, and for example include the assays and animal 30 models used in the Examples beiow.
For pharmaceuticai use, the polypeptides of the invention may be formuiated as a pharmaceuticai préparation comprising (i) at least one polypeptideofthe
-10317072 invention and (li) at least one pharmaceutically acceptable carrier, diluent, excipient, adjuvant, and/or stabilizer, and (iii) optionally one or more further pharmaceutically active polypeptides and/or compounds. By pharmaceutically acceptable is meant that the respective material does not show any blological or otherwise undesirabie effects when administered to an individual and does not Internet In a deleterious manner with any of the other components of the pharmaceutical composition (such as e.g. the pharmaceutically active ingrédient) In which it is contained. Spécifie examples can be found in standard handbooks, such as e.g. Remlngton's Pharmaceutical Sciences, 18111 Ed., Mack Publishing Company, USA (1990). For example, the polypeptides of the Invention may be formulated and administered in any manner known per se for conventional antibodies and antibody fragments and other pharmaceutically active proteins. Thus, according to a further embodiment, the invention relates to a pharmaceutical composition or préparation that contains at least one polypeptide of the invention and at least one pharmaceuticaliy acceptable carrier, diluent, excipient, adjuvant and/or stabilizer, and optionally one or more further pharmaceuticaliy active substances.
By means of non-iimiting examples, such a formulation may be in a form suitable for oral administration, for parentéral administration (such as by intravenous, intramuscular, subeutaneous, intrathecal, intracavemosal or intraperitoneal Injection or intravenous Infusion), for topical administration, for sublingual administration, for administration by inhalation, by a skin patch, by an implant, by a supposltory, for transdermal, nasal, intravitreai, rectal or vaginal administration, and the like. Such suitable administration forms - which may be solid, seml-solid or liquid, depending on the manner of administration - as well as methods and carriers for use in the préparation thereof, wili be clear to the skilled person.
Pharmaceutical préparations for parentéral administration, such as intravenous, intramuscular, subeutaneous Injection or intravenous infusion may for exampie be steriie solutions, suspensions, dispersions, émulsions, or powders which comprise the active ingrédient and which are suitable, optionally after a further dissolution or dilution step, for Infusion or injection. Suitable carriers or diiuents
-10417072 for such préparations for example include, without limitation, stérile water and pharmaceutically acceptable aqueous buffers and solutions such as physiological phosphate-buffered saline, Ringerts solutions, dextrose solution, and Hank's solution; water oils; glycerol; éthanol; glycols such as propylene glycol, as well as 5 minerai oils, animal oils and vegetable oils, for example peanut oil, soybean oil, as well as suitable mixtures thereof.
Solutions of the active compound or its salts may also contain a preservative to prevent the growth of microorganisms, such as antibacterial and antifungal το agents, for example, parabens, chlorobutanol, phénol, sorbic acid, thlmerosal (thlomersal), and the like. In many cases, It will be préférable to Include Isotonie agents, for example, sugars, buffers or sodium chloride. The proper fluidity can be maintalned, for example, by the formation of liposomes, by the maintenance of the required particle size In the case of dispersions or by the use of surfactants. is Other agents delaying absorption, for example, aluminum monostearate and gelatin, may also be added.
In ail cases, the ultimate dosage form must be stérile, fluid and stable under the conditions of manufacture and storage. Stérile Injectable solutions are prepared 2o by incorporating the active compound in the required amount In the appropriate solventwith various ofthe otherIngrédients enumerated above, as required, followed by filter sterilization. In the case of stérile powders for the préparation of stérile injectable solutions, the preferred methods of préparation are vacuum drylng and the freeze drying techniques, which yleld a powder of the active
Ingrédient plus any additional desired Ingrédient présent In the previously sterilefiltered solutions.
Usually, aqueous solutions or suspensions will be preferred. Generally, suitable formulations for therapeutic proteins such as the polypeptides of the Invention are 30 buffered protein solutions, such as solutions including the protein in a suitable concentration (such as from 0.001 to 400 mg/ml, preferably from 0.005 to 200 mg/ml, more preferably 0.01 to 200 mg/ml, more preferably 1.0 -100 mg/ml, such
-10517072 as 1.0 mg/ml (l.v. administration) or 100 mg/ml (s.c. administration) and an aqueous buffer such as:
- phosphate buffered saline, pH 7.4,
- other phosphate buffers, pH 6.2 to 8.2,
- histidine buffers, pH 5.5 to 7.0,
- succlnate buffers, pH 3.2 to 6.6, and
- citrate buffers, pH 2.1 to 6.2, and, optionally, salts (e.g. NaCI) and/or sugars or polyalcohols (such as trehalose, mannîtol, or glycerol) for providing isotonicity of the solution.
Preferred buffered protein solutions are solutions Including about 0.05 mg/ml of the polypeptide of the invention dissolved ln 25 mM phosphate buffer, pH 6.5, adjusted to Isotonicity by adding 220 mM trehalose. ln addition, other agents such as a detergent, e.g. 0.02 % Tween-20 orTween-80, may be included in such solutions. Formulations for subcutaneous application may include significantly higher concentrations of the polypeptide of the invention, such as up to 100 mg/ml or even above 100 mg/mi. However, It will be clear to the person skilled ln the art that the Ingrédients and the amounts thereof as given above do only represent one, preferred option. Alternatives and variations thereof will be immediately apparent to the skilled person, or can easily be conceived starting from the above disclosure.
The polypeptides of the invention may also be administered using suitable depot, slow-release or sustained-release formulations, e.g. suitable for injection, using controiled-release devices for implantation under the skin, and/or using a doslng pump or other devices known per se for the administration of pharmaceutically active substances or principles. ln addition, the polypeptides of the invention may be formulated in the form of a gel, cream, spray, drop, patch or film which, If placed on the skin, passes through the skin.
Also, compared to conventional antibodies or antibody fragments, one major advantage of the use of the polypeptides of the invention is that they can also be easily administered via routes other than parentéral administration and can be
-10617072 easily formulated for such administration. For example, as described in the International application W02004/041867, such polypeptides may be formulated for oral, Intranasal, Intrapulmonary and transdermal administration.
According to another embodiment of the invention there Is provided a pharmaceutical combination comprising at ieast one polypeptide of the Invention as disclosed herein and at least one other therapeutic agent selected from the group consisting of statins, antiplateiets, anticoagulants, antidiabetics and antihypertensives.
Such pharmaceutical combination may optionally additionally comprise a diluent, excipient, adjuvant and/or stabilizer.
When two or more substances or principles are to be used as part of a combined treatment regimen, they can be administered via the same route of administration or via different routes of administration, at essentialiy the same time or at different times (e.g. essentialiy slmultaneously, consecutively, or according to an altematlng régime). When the substances or principles are to be administered slmultaneously via the same route of administration, they may be administered as different pharmaceutical formulations or compositions or part of a combined pharmaceutical formulation or composition. Also, when two or more active substances or principles are to be used as part of a combined treatment regimen, each ofthe substances or principles may be administered In the same amount and according to the same regimen as used when the compound or principle Is used on its own, and such combined use may or may not lead to a synergistic effect. However, when the combined use of the two or more active substances or principles leads to a synergistic effect, it may also be possible to reduce the amount of one, more or ail of the substances or principles to be administered, whiie still achieving the desired therapeutic action. This may for exampie be useful for avoiding, limiting or reducing any unwanted slde-effects that are associated with the use of one or more of the substances or principles when they
-10717072 are used in their usual amounts, while still obtaining the desired pharmaceutical or therapeutic effect.
Yet, a further embodiment of the invention is a method for treating the diseases and disorders as set out above, comprising administering to an individual, simultaneously, separately or sequentially, an effective amount of at least one polypeptide of the Invention and at least one agent selected from the group consisting of a statin, an antiplatelet, an anticoagulant, an antidiabetic and an antihypertensive.
According to a further aspect of the invention, the polypeptide of the invention ls prepared to be administered in combination with other drugs used for the treatment of the diseases and disorders set out above, such other drugs being selected from the group consisting of a statin, an antiplatelet, an anticoagulant, an antidiabetic and an antihypertensive.
According to still another aspect of the Invention, drugs used for the treatment of the diseases and disorders set out above, such drugs being selected from the group consisting of a statin, an antiplatelet, an anticoagulant, an antidiabetic and an antihypertensive are prepared to be administered In combination with the polypeptide of the invention.
According to a further aspect of the invention, the polypeptide of the invention is used in combination with a device useful for the administration of the polypeptide, such as a syringe, Injector pen, or other device.
According to still another embodiment of the invention, there is provided a method of diagnosing a disease, disorder or condition mediated by CX3CR1 dysfunction comprising the steps of:
a) obtaining a sample from a subject, and
b) contacting, in vitro, the sample with a polypeptide of the invention as defined above, and
c) detecting the binding of said polypeptide to said sample, and
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d) comparing the binding detected in step (c) with a standard, wherein a différence in binding relative to said sample Is diagnostic of a disease, disorder or condition characterised by CX3CR1 dysfonction.
According to another embodiment of the invention, there Is provîded a method of diagnoslng a disease, disorder or condition mediated by CX3CR1 dysfonction comprising the steps of:
a) obtaining a sample from a subject, and
b) contacting the sample with a polypeptide of the invention as defined above;
c) determining the amount of CX3CR1 In the sample; and
d) comparing the amount determined in step (c) with a standard, wherein a différence in amount relative to said sample is diagnostic of a disease, disorder or condition characterised by CX3CR1 dysfonction.
The above diagnostic methods can also be used for monitoring the effectiveness of a therapeutic treatment of a subject.
According to another embodiment of the invention, there Is provîded a kit for diagnosing a disease, disorder or condition mediated by CX3CR1 dysfonction, for use in a method as defined above, such kit comprising at least one polypeptide of the invention and, optionally, one or more media, détection means and/or In vitro or in vivo imaging agents, and, further optionally, instructions of use. Suitable in vivo imaging agents include 99mTc, 111 Indium, 123lodine, and, for magnetic résonance Imaging, paramagnetic compounds.
The invention further provides a kit comprising at ieast one polypeptide of the Invention and, additionaily, one or more other components selected from the group conslsting of other drugs used for the treatment of the diseases and disorders as described above, and devices as described above.
The invention further provides methods of manufacturing a polypeptide of the invention, such methods generally comprising the steps of:
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- culturing host cells comprising a nucleic acid capable of encoding a polypeptide of the invention (hereinafter: nucleic acid of the invention) under conditions that allow expression of the polypeptide of the invention; and,
- recovering or Isolating the polypeptide expressed by the host cells from the culture; and
- optionally further purifying and/or modifying and/or formulating the polypeptide of the invention.
A nucleic acid of the Invention can be genomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage that has been specifically adapted for expression in the Intended host cell or host organism). According to one embodiment of the Invention, the nucleic acid of the invention is In essentially isolated form, as defined hereabove.
is The nucleic acid of the invention may also be In the form of, be présent In and/or be part of a vector, such as for example a plasmid, cosmid or YAC, which agaln may be In essentially isolated form. The vector may especially be an expression vector, i.e. a vector that can provide for expression of the polypeptide in vitro and/or in vivo (e.g. in a suitabie host cell, host organism and/or expression
System). Such expression vector generally comprises at least one nucleic acid of the invention that is operably linked to one or more suitable regulatory element(s), such as promoterfs), enhancer(s), terminator(s), and the like. Spécifie examples of such regulatory éléments and other éléments, such as Intégration factor(s), sélection marker(s), signal or leader sequence(s), reporter gene(s), and the like, useful or necessary for expressing polypeptides of the Invention, are disclosed e.g. on pp. 131 to 133ofW02006/040153.
The nucleic acids of the invention can be prepared or obtained in a manner known per se (e.g. by automated DNA synthesis and/or recombinant DNA
3o technology), based on the information on the amino acid sequences for the polypeptides of the invention given herein, and/or can be Isolated from a suitable naturel source.
-11017072
According to another embodiment, the Invention relates to a host or host cell that expresses or Is capable of expressing a polypeptide of the'invention; and/or that contains a nucleic acid encoding a polypeptide of the invention. According to a particularly preferred embodiment, said host cells are bacterial cells, yeast cells, fungal cells or mammalian cells.
Suitable bacterial cells Include cells from gram-negative bacterial strains such as strains of Escherichia coli, Proteus, and Pseudomonas, and gram-positive bacterial strains such as strains of Bacillus, Streptomyces, Staphylococcus, and Lactococcus. Suitable fungal cell include cells from species of Trichoderma, Neurospora, and Aspergillus. Suitable yeast cells include cells from species of Saccharomyces (for example Saccharomyces cerevlslae), Schlzosaccharomyces (for example Schlzosaccharomyces pombe), Pichia (for example Pichla pastoris and Pichia methanolica), and Hansenula.
Suitable mammalian cells include for example CHO celis, BHK cells, HeLa cells, COS cells, NSO cells, HEK cells, and the like. However, amphibian ceils, insect cells, plant cells, and any other cells used in the art for the expression of heterologous proteins can be used as well.
For production on Industrial scale, preferred heterologous hosts for the (industrial) production of immunoglobulin single variable domain polypeptides and protein therapeutics containing them include strains of E. coll, Pichia pastoris, and S. cerevislae that are suitable for large scale expression, production and fermentation, and in particular for large scale (bio-)pharmaceutical expression, production and fermentation.
The choice of the spécifie expression System would dépend ln part on the requirement for certain post-translational modifications, more specifically giycosyiation. The production of a polypeptide of the Invention for which glycosylation is desired or required would necessltate the use of mammalian expression hosts that hâve the ability to glycosylate the expressed protein. ln this respect, it will be clear to the skilled person that the glycosylation pattern obtained
-111 17072 (Le. the kind, number and position of residues attached) will dépend on the cell or cell line that Is used for the expression.
Polypeptides of the Invention produced In a cell as set out above can be produced either intracellullarly (e.g. In the cytosol, In the periplasma or in inclusion bodies) and then Isolated from the host cells and optionally further purified; or they can be produced extracellularly (secreted into the medium In which the host cells are cultured) and then isolated from the culture medium and optionally further purified.
Further methods and reagents used for the recombinant production of polypeptides, such as suitable expression vectors, transformation or transfection methods, sélection markers, methods of Induction of protein expression, culture conditions, and the like, are known in the art. Similarly, protein isolation and is purification techniques useful in a method of manufacture of a polypeptide of the invention are well known to the skilled person.
Production of the poiypeptldes of the invention through fermentation in convenient recombinant host organisme such as E. coli and yeast is cost20 effective, as compared to conventional antibodies which also require expensive mammalian cell culture facllitles. Furthermore, achievabie levels of expression are high and yields of the polypeptides of the Invention are in the range of 1 to 10 g/l (E. coli) and up to 10 g/l (yeast) and more.
-11217072
EXAMPLES
Génération CHO, Baf/3, Caki and HEK293 cell Unes overexpressing human CX3CR1 or cynomolgus CX3CR1
CHO and Baf/3 cells overexpressing human or cynomolgus CX3CR1 were s generated using techniques known in the art. Cells expressing human CCR2 or
CCR5 were also generated using techniques known in the art.
The cDNA was cloned Into pCDNA3.1(+)-neo for human CX3CR1 whereas pcDNA-DEST40-neo was used for mouse CX3CR1.
The amino acid sequences of humanCX3CR1 and cynomolgus CX3CR1 are depicted In SEQ ID NO: 255 and 256, respectively.
To establish Camel Kidney (Cakl) cells overexpressing human CX3CR1 or mouse CX3CR1, parental Cakl cells were electroporated with pCDNA3.1(+)-neohCX3CR1 or pcDNA-DEST40-neo-mCX3CR1, respectively. For ail conditions, is transfectants were selected by adding 1 mg/mL geneticin (Invitrogen, Carlsbad,
CA, USA).
Human Embyonic Kidney (HEK293) cells overexpressing human CX3CR1 or cynomolgus CX3CR1 were generated by lipid-mediated transfection with Fugene (Roche) of pCDNA3.1(+)-neo-hCX3CR1 orcyCX3CR1 plasmids, respectively, In 20 the HEK293 parental cell line. These cells were used as translent transfectants and as such not put under sélection. In brlef, 2*10E6 cells were seeded per T75 and incubated ovemight before transfection. After removal of the culture medium, cells were transfected with the respective plasmids (9 pg) and Fugene (27 pl) according to manufacturées instructions. 48 hours post transfection, cells were 25 harvested and frozen for further usage.
Example 1: Immunizatlon with CX3CR1 induces a humoral Immune response in llama
1.1. Immunlzations
-11317072
After approval of the Ethlcal Commlttee (Unlversity Antwerp, Belgium, UA2008A1, 2008/096,2007/068), 9 Hamas (designated No. 368, 369, 370, 381, 382, 384, 312, 313 and 314) were immunized.
Six Hamas (312, 313, 314, 381, 382 and 384) were immunized with s 4 Intramuscular Injections (2mg/dose at weekly or biweekly Intervals) of pVAX1 huCX3CR1 plasmld vector (Invitrogen, Carlsbad, CA, USA ). Three Hamas (381, 382 and 384) subsequently received 4 subcutaneous Injections of human CX3CR1 overexpresslng Cakl cells which were established as described above. Cells were re-suspended in D-PBS and kept on Ice prior to Injection.
io Three additional Hamas (designated No. 368, 369 and 370) were Immunized according to standard protocols with 4 subcutaneous Injections of human CX3CR1 overexpresslng Cakl cells which were established as described above. Cells were re-suspended ln D-PBS and kept on Ice prior to injection. Subsequently, these Hamas were administered two Injections with recombinant is CX3CR1 NT/EC3 fragment coupled to BSA (Table 13). Peptides were ordered at NeoMPS (Polypeptidegroup, Strasbourg, France) and coupled to BSA according to standard protocols.
Table 13 Sequence of peptide fragments used for Immunizatlon boost
| Fragment | sequence | SEQ ID NO: |
| CX3CR1-NT | Ac-Met-Asp-GIn-Phe-Pro-Glu-Ser-Val-Thr-Glu-Asn- Phe-Glu-Tyr-Asp-Asp-Leu-Ala-Glu-Ala-Cys-NH2 | 228 |
| CX3CR1- EC3 | Ac-Lys-Leu-Tyr-Asp-Phe-Phe-Pro-Ser-Cys-Asp- Met-Arg-Lys-Asp-Leu-Arg-Leu-NH2 | 229 |
The first Injection was formulated ln Complété Freund's Adjuvant (Difco, Detroit, Ml, USA), while the subséquent Injection was formulated ln Incomplète Freund’s Adjuvant (Difco, Detroit, Ml, USA).
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1.2. Evaluation of induced immune responses In llama
To evaluate the Induction of Immune responses in the animais against human CX3CR1 by ELISA or FACS, sera were collected from Hamas 312,313 and 314 at day 0 (pre-immune), and different time points ln the Immunization schedule (time s of peripheral blood lymphocyte [PBL] collection).
In short, Neutravidîn (2pg/ml) was immobilized ovemight at 4*C in a 96-well Maxisorb piate (Nunc, Wiesbaden, Germany). Wells were biocked with a casein solution (1%) ln PBS. Subsequently blotinylated recombinant NT fragment (Polypeptide, Strasbourg, France) or biotlnylated EC3 fragments of CX3CR1 io (Polypeptide, Strasbourg, France) were captured at 2 pg/ml. After addition of sérum dilutions, specifically bound immunoglobulins were detected using a horseradish peroxldase (HRP)-conjugated goat antl-ilama immunoglobulin (Bethyl Laboratories Inc., Montgomery, TX, USA) and a subséquent enzymatic reaction ln the presence of the substrate TMB One (3,3’,5,5’is tetramentylbenzidine) (Promega, Mannheim, Germany), showing that a significant antibody-dependent immune response against CX3CR1 was induced after the peptide immunlzations.
Additionaily, sérum titers of cell immunized animais were confïrmed by FACS analysis on actively growing human CX3CR1 overexpressing CHO cells. The 20 CX3CR1 sérum titer responses for Hamas 368, 369 and 370 were determined with sérum sampled after 4 ceil immunizatlons (day 49), 4 cell immunizations and 1 peptide boost (day 77) and 4 cell immunlzations and 2 peptide boosts (day 81). Cells were harvested and washed before incubation with the sérum dilutions. Détection was performed with goat anti-llama IgG (Bethyl, Montgomery, TX,
USA) followed by donkey anti-goat coupled with PE (Jackson Laboratories, Suffolk, UK) and read out by analysis on FACSArray (BD Biosciences). A summary of the obtained sérum responses as determined by either ELISA or FACS Is shown ln Table 14 and Table 15.
-11517072
Table 14 Sérum titer analysis for the cell/peptide immunized animais
| ELISA | FACS | ||||
| Llama | Immunogen | Recombinant NT | Recombinant EC3 | After cell immunlzatlon | After peptide boosts |
| 368 | CakihuCX3CR1 + NT/EC3 peptide | + | +/- | - | - |
| 369 | CaklhuCX3CR1 + NT/EC3 peptide | + | +/- | + | + |
| 370 | CakihuCX3CR1 + NT/EC3 peptide | ++ | ++ | - | + |
Table 15 Sérum titer analysis for the DNA/ceil Immunized animais
| EL | SA | FACS | |||
| Llama | Immunogen | Recombinant NT | Recombinant EC3 | After DNA Immunlzatlon | After Cell boosts |
| 381 | DNA + CaklhuCX3CR1 | ++ | + | ++ | ++ |
| 382 | DNA + CakihuCX3CR1 | + | - | - | - |
| 384 | DNA + CakihuCX3CR1 | ++ | - | - | - |
For the DNA only Immunized Hamas (312, 313 and 314) no sérum titer was determined.
Example 2: Cloning of the heavy-chain only antibody fragment répertoires and préparation of phage
-11617072
Following the final Immunogen Injection of each subset, Immune tissues as the source of B-cells that produce the heavy-chain antibodies were collected from the immunized Hamas. For llama 312,313 and 314, two 150-ml blood samples, collected 4 and 8 days after the last antigen injection were collected per animal, s For Hamas 368, 369 and 370 four 150 ml blood samples were collected, 5 and 7 days after the last cell immunization and additionally 4 and 8 days after the last peptide immunization. Next to those, two lymph node biopsies were taken, 12 days after the last cell immunization and 12 days after the last peptide Immunization. For Hamas 381,382 and 384 five 150 ml blood samples were w collected, 8 days after the last DNA Immunization and additionally 4 days after the first cell boost, 8 and 11 days after the second cell boost and 8 days after the last cell Immunization. Next to those, one lymph node biopsy was taken, 8 days after the second cell Immunization.
From the blood samples, peripheral blood lymphocytes (PBLs) were prepared is using Ficoll-Hypaque according to the manufacturées instructions (Amersham
Biosciences, Plscataway, NJ, USA). From the PBLs and the lymph node biopsy (LN), total RNA was extracted, which was used as starting material for RT-PCR to amplify the VHH encoding DNA segments.
For each immunized llama, libraries were constructed by pooling the total RNA 20 Isolated from samples originatïng from a certain subset of the immunization schedule l.e. after one type of immunization antigen, and for some Hamas samples from the different animais were pooled into one library (Table 16).
Table 16 Pooling of the different sample for library construction
| Library Na me | Llama | Sample |
| 368-PBL1+2+LN-V-100209 | 368 | PBL1 and 2, LN |
| 369+370-PBL1+2+LN-V-100209 | 369, 370 | PBL1 and 2, LN |
| 368-PBL3+4-V-280909 | 368 | PBL 3 and 4 |
| 369-PBL3+4-V-070409 | 369 | PBL 3 and 4 |
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| 370-PBL3+4-V-070409 | 370 | PBL 3 and 4 |
| 381-PBL1-V-180310 | 381 | PBL 1 |
| 382-PBL1-V-180310 | 382 | PBL1 |
| 384-PBL1-V-180310 | 384 | PBL1 |
| 381-PBL1+2+3+4+5+LN-V-280909 | 381 | PBL 1,2, 3, 4, 5 and LN |
| 382-PBL1+2+3+4+5+LN-V-280909 | 382 | PBL 1,2, 3, 4, 5 and LN |
| 384-PBL1 +2+3+4+5+Ln-V-280909 | 384 | PBL 1,2, 3, 4, 5 and LN |
| 312+313+314-PBL1+2-V-220210 | 312, 313 and 314 | PBL 1 and 2 |
| 312-PBL1+2-V-180310 | 312 | PBL 1 and 2 |
| 313-PBL1+2-V-180310 | 313 | PBL 1 and 2 |
| 314-PBL1+2-V-180310 | 314 | PBL 1 and 2 |
In short, the PCR-amplified VHH répertoire was cloned via spécifie restriction sites into a vector designed to facilitate phage display of the VHH library. The vector was derived from pUC119 and contains the LacZ promoter, a M13 phage s glll protêtn coding sequence, a résistance gene for ampicillin or carbeniciliin, a multiple cloning site and a hybrld glil-pelB leader sequence (pAX050). In frame with the VHH coding sequence, the vector encodes a C-terminal c-myc tag and a His6 tag. Phage were prepared according to standard protocols and stored after fiiter sterilization at 4°C or at -80°C in 20 % glycerol for further use.
to
Example 3: Sélection of CX3CR1 spécifie VHHs via phage display
VHH répertoires obtained from ali Hamas and cloned as phage library were used In different sélection strategies, applying a multiplicity of sélection conditions. Variables Include I) the présentation form of the CX3CR1 protein (on different cell 15 backgrounds or on liposomes/VLPs), li) the antigen présentation method (In solution when using cells or coated onto plates when using VLPs), Iii) the antigen
-11817072 concentration iv) the orthologue used (human or cynomolgus) v) the number of sélection rounds and vi) different elution methods (non-specific via trypsln or spécifie via the ligand Fractalkine). Ail solid coated phase sélections were done in Maxisorp 96-well plates (Nunc, Wiesbaden, Germany).
Sélections were performed as follows: CX3CR1 antigen préparations for solid and solution phase sélection formats were presented as described above at multiple concentrations. After 2h Incubation with the phage libraries followed by extensive washing, bound phages were eluted with trypsln (1 mg/mL) for 15 minutes. When trypsln was used for phage elution, the protease activity was Immediately neutraiized by applying 0.8 mM protease Inhibitor ABSF. As control, sélections without antigen were performed in parallei.
Phage outputs were used to Infect E. coli which were then In tum used to préparé phage for the next sélection round (phage rescue) After the second round sélection the phage outputs were used to infect E. co//which were then plated on agar plates (LB+carb+glucose2%) for analysis of individual VHH clones. In order to screen a sélection output for spécifie binders, single colonies were plcked from the agar plates and grown In 1 mL 96-deep-well plates. LacZ-controlled VHH expression was Induced by adding IPTG (1mM final) in the absence of glucose. Periplasmic extracts (in a volume of ~ 80 uL) were prepared according to standard protocols.
Example 4: Screening of periplasmic extracts In CX3CR1-Fraktalklne compétition FACS assay
Periplasmic extracts were screened in a human CX3CR1/human Fractalkine FACS compétition assay to assess the blocking capacity of the expressed VHHs. Human CX3CR1 was presented on CHO cells overexpressing CX3CR1. Both a setup using cells harvested from an actively growing culture and a setup using frozen cells was used. As a détection reagent labeled fractalkine was used (R&D Systems, Minneapolis, MN, USA) labeled with alexa647 (A647-Fractalkine) at a degree of labellng of 1. To setup the assay, first a titration sériés of the labeled
-11917072 fractalklne was performed on the CHO-huCX3CR1 cells in order to détermine the EC50 value for binding. Initially screening was performed at a higher concentration of fractalkine (3 nM) to increase the assay robustness. To increase the sensitïvity of the screening to a maximum, the EC30 concentration (1 nM) was chosen for subséquent screening. In brief 50 pi of periplasmlc extract was added to 6 nM labeled fractalklne (50μΙ) and 200 000 CHO-huCX3CR1 cells. After one hour Incubation at 4 C, cells were washed three times before read out was performed on a FACS Array (Becton Dickinson). First a gâte was set on the Intact cells as determined from the scatter profile. Next, dead cells were gated out by their fluorescence profile from the PI staln (Sigma, St Louis, US). The fluorescence profile from the alexa647 label was determined for each sample and used for calculation of blocking capacity. As contrats, conditions were taken along where there was no VHH présent in the péri extract or a known irrelevant VHH and samples were included where excess cold fractalklne was Included. For each sample the percentage block was determined using the control samples to détermine the assay window.
From this screening, VHHs were selected and sequence analysis revealed 120 unique VHHs belonging to 3 different B-cell lineages. The total number of variants found for each B-cell lineage isdepicted in Table 17.
Table 17 Sélection parameters used for the identification of the humanCX3CR1 spécifie VHH B-cell lineages
| B-cell lineage | Représentative VHH ID | # variants | librarles |
| 9 | CX3CR1BII11H11 | 4 | 368-PBL1+2+LN-V-100209 |
| 13 | CX3CR1BII18E06 | 68 | 368-PBL1+2+LN-V-100209 368-PBL3+4-V-280909 |
| 101 | CX3CR1BII66B02 | 48 | 312+313+314-PBL1+2-V-220210 314-PBL1+2-V-180310 |
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An overview of the sélection procedure and performance during initial screening is given for all VHHs in Table 18.
Table 18 Sélection conditions and primary screening resuit for the huCX3CR1 spécifie VHH
| VHH ID | Family | Libre ry | Sélections | % block | |||
| first round | second | round | |||||
| CX3CR1BII PMP11H11 | 9 | 368PBL1+2+LN -V-100209 | BA/F3 hCX 3CR1 | total (trypsin) | CHOK1 hCX3C R1 | total (trypsin) | 99.0 |
| CX3CR1BII PMP18E6 | 13 | 368PBL3+4-V280909 | BA/F3 hCX 3CR1 | total (trypsin) | CHOK1 hCX3C R1 | total (trypsin) | 53.1 |
| CX3CR1BII PMP54A12 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 yM) | VLPshCX3CR1 (10U) | hFrac (2 yM) | 93.8 |
| CX3CR1BII PMP54A3 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 yM) | VLPshCX3CR1 (10U) | hFrac (2 yM) | 90.8 |
| CX3CR1BII PMP54A4 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 yM) | VLPshCX3CR1 (10U) | hFrac (2 yM) | 86.6 |
| CX3CR1BII PMP54A5 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 yM) | VLPshCX3CR1 (10U) | hFrac (2 yM) | 92.5 |
| CX3CR1BII PMP54A7 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | total (trypsin) | VLPshCX3CR1 (10U) | total (trypsin) | 68.9 |
| CX3CR1BII PMP54B1 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 yM) | VLPshCX3CR1 (10U) | hFrac (2 yM) | 92.1 |
| CX3CR1BII PMP54B2 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 yM) | VLPshCX3CR1 (10U) | hFrac (2 yM) | 65.3 |
| CX3CR1BII PMP54B3 | 101 | 312+313+3 14-PBL1+2- | VLPshCX3CR1 | hFrac (2 KM) | VLPshCX3CR1 | hFrac (2 hM) | 90.1 |
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| V-220210 | (10U) | (10U) | |||||
| CX3CR1BII PMP54B5 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 yM) | VLPshCX3CR1 (10U) | hFrac (2 yM) | 92.6 |
| CX3CR1BII PMP54D5 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 yM) | VLPshCX3CR1 (10U) | hFrac (2 yM) | 87.8 |
| CX3CR1BII PMP54D8 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | total (trypsln) | VLPshCX3CR1 (10U) | total (trypsln) | 64.1 |
| CX3CR1BII PMP54F6 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | VLPshCX3CR1 (10U) | hFrac (2 PM) | 96.6 |
| CX3CR1BII PMP54G3 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 <1OU) | hFrac (2 yM) | VLPshCX3CR1 (10U) | hFrac (2 yM) | 74.7 |
| CX3CR1BII PMP54H1 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 yM) | VLPshCX3CR1 (10U) | hFrac (2 yM) | 74.6 |
| CX3CR1BII PMP54H4 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 yM) | VLPshCX3CR1 (10U) | hFrac (2 yM) | 96.0 |
| CX3CR1BII PMP61F10 | 101 | 714. PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 yM) | VLPshCX3CR1 (10U) | hFrac (2 yM) | 73.5 |
| CX3CR1BII PMP61D1 | 101 | 714. PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 yM) | VLPshCX3CR1 (10U) | hFrac (2 yM) | 68.4 |
| CX3CR1BII PMP61D5 | 101 | 714PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 yM) | VLPshCX3CR1 (10U) | hFrac (2 yM) | 94.9 |
| CX3CR1BII PMP61E2 | 101 | 314PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 yM) | VLPshCX3CR1 (10U) | hFrac (2 yM) | 70.3 |
| CX3CR1BII PMP61F11 | 101 | 314PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 yM) | VLPshCX3CR1 (10U) | hFrac (2 yM) | 96.5 |
| CX3CR1BII PMP61G2 | 101 | 314PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 yM) | VLPshCX3CR1 (10U) | hFrac (2 PM) | 82.0 |
| CX3CR1BII | 101 | 314- | VLPs- | hFrac (2 | VLPs- | hFrac (2 | 92.1 |
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| PMP61G3 | PBL1+2-V180310 | hCX3CR1 (10U) | μΜ) | hCX3CR1 (10U) | μΜ) | ||
| CX3CR1BII PMP61G4 | 101 | 314PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 MM) | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | 94.5 |
| CX3CR1BII PMP61F4 | 101 | 314PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 MM) | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | 94.4 |
| CX3CR1BII PMP61A11 | 101 | 314PBL1+2-V180310 | VLPshCX3CRl (10U) | hFrac (2 μΜ) | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | 78.0 |
| CX3CR1BII PMP61B2 | 101 | 314PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | 94.5 |
| CX3CR1BII PMP61C9 | 101 | 314PBL1+2-V180310 | VLPshCX3CR1 (10U) | total (trypsln) | VLPshCX3CR1 (10U) | total (trypsln) | 69.4 |
| CX3CR1BII PMP65H02 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | #N/A |
| CX3CR1BII PMP65E11 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | #N/A |
| CX3CR1BII PMP65E10 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | #N/A |
| CX3CR1BII PMP65E05 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | #N/A |
| CX3CR1BII PMP65B11 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | #N/A |
| CX3CR1BII PMP65B07 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | #N/A |
| CX3CR1BII PMP65B09 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | #N/A |
| CX3CR1BII PMP65H01 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 μΜ) | VLPshCX3CR1 (wyi | hFrac (2 μΜ) | #N/A |
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| CX3CR1BII PMP65G07 | 101 | 312+313+3 14-PBL1+2V-220210 | VLPshCX3CR1 (10U) | hFrac (2 pM) | VLPshCX3CR1 (10U) | hFrac (2 pM) | #N/A |
| CX3CR1BII PMP66H08 | 101 | T14PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 pM) | VLPshCX3CR1 (10U) | hFrac (2 pM) | #N/A |
| CX3CR1BII PMP66H04 | 101 | T14PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 pM) | VLPshCX3CR1 (10U) | hFrac (2 pM) | #N/A |
| CX3CR1BII PMP66F02 | 101 | ^14PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 pM) | VLPshCX3CR1 (10U) | hFrac (2 PM) | #N/A |
| CX3CR1BII PMP66E11 | 101 | 314PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 pM) | VLPshCX3CR1 (1011) | hFrac (2 PM) | #N/A |
| CX3CR1BII PMP66D10 | 101 | 314PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 pM) | VLPshCX3CR1 (10U) | hFrac (2 pM) | #N/A |
| CX3CR1BII PMP66D08 | 101 | 314PBL1+2-V180310 | VLPshCX3CR1 (10U> | hFrac (2 pM) | VLPshCX3CR1 (10U) | hFrac (2 pM) | #N/A |
| CX3CR1BII PMP66B02 | 101 | 314PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 pM) | VLPshCX3CR1 (10U) | hFrac (2 pM) | #N/A |
| CX3CR1BII PMP66A04 | 101 | 314PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 pM) | VLPshCX3CR1 (10U) | hFrac (2 pM) | #N/A |
| CX3CR1BII PMP66D04 | 101 | T14PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 pM) | VLPshCX3CR1 (1011) | hFrac (2 pM) | #N/A |
| CX3CR1BII PMP66D02 | 101 | 314PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 pM) | VLPshCX3CR1 (10U) | hFrac (2 PM) | #N/A |
| CX3CR1BII PMP66D06 | 101 | 314PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 pM) | VLPshCX3CR1 (10U) | hFrac (2 PM) | #N/A |
| CX3CR1BII PMP66G01 | 101 | T14PBL1+2-V180310 | VLPshCX3CR1 (10U) | hFrac (2 pM) | VLPshCX3CR1 (10U) | hFrac (2 pM) | #N/A |
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The amino acid sequences of ali obtained unique VHHs are shown in the Sequence Listing and above (CDRs and framework régions were indicated).
Example 5: Characterization of purified VHHs
Inhibitory anti-CX3CR1 VHHs selected from the screening described in Example 4 were further purified and characterized. Selected VHHs were expressed in E. coliTGI as c-myc, His6-tagged proteins. Expression was induced by addition of 1 mM IPTG and allowed to continue for 4 hours at 37'C. After spinning the cell cultures, periplasmic extracts were prepared by freeze-thawing the pellets. These extracts were used as starting material and VHHs were purified via IMAC and size exclusion chromatography (SEC) resulting in 95% purity as assessed via SDS-PAGE.
Inhibition by anti-CX3CR1 VHHs ofhuman Fractalkine binding to human CX3CR1 expressed on the BA/F3 cells
The blocking capacitytowards the ligand fractalkine ofthe VHHs was evaluated in a human CX3CR1 compétition FACS as outlined in Example 4. Either CHOhuCX3CR1 cells, BA/F3-huCX3CR1 cells or transientiy transfected HEK293T cells were used. The amount of labeled ligand used in the different compétition setups was aiso varied. The IC5o values for VHHs blocking the interaction of human fractalkine to human CX3CR1 are depicted in Table 19.
Table 19 Potency and efficacy ofthe VHH In a ligand compétition FACS
| VHHID | Family | Cell line | IC50 | % block | Repeats |
| 11H11 | 9 | CHO-huCX3CR1 | 1.7 E-8 | 100 | 4 |
| 18E06 | 13 | CHO-huCX3CR1 | 1.8 E-9 | 33 | 4 |
| 54A12 | 101 | CHO-huCX3CR1 | 2.1 E-9 | 104 | 2 |
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| 54D08 | 101 | CHO-huCX3CR1 | 1.5 E-8 | 101 | 2 |
| 54A07 | 101 | CHO-huCX3CR1 | 1.1 E-8 | 78 | 2 |
| 54D05 | 101 | CHO-huCX3CR1 | 2.7 E-9 | 102 | 2 |
| 54B03 | 101 | CHO-huCX3CR1 | 2.5 E-8 | 108 | 1 |
| 54G03 | 101 | CHO-huCX3CR1 | 5.6 E-8 | 107 | 1 |
| 11H11 | 9 | BA/F3-huCX3CR1 | 8.1 E-9 | 100 | 3 |
| 18E06 | 13 | BA/F3-huCX3CR1 | 2.8 E-9 | 71 | 3 |
| 54A12 | 101 | BA/F3-huCX3CR1 | 4.0 E-9 | 100 | 4 |
| 54D08 | 101 | BA/F3-huCX3CR1 | 3.8 E-8 | 99 | 1 |
| 54A07 | 101 | BA/F3-huCX3CR1 | 1.5 E-8 | 81 | 4 |
| 54D05 | 101 | BA/F3-huCX3CR1 | 5.5 E-9 | 99 | 1 |
| 54B03 | 101 | BA/F3-huCX3CR1 | 3.3 E-8 | 99 | 1 |
| 54G03 | 101 | BA/F3-huCX3CR1 | 9.8 E-8 | 98 | 1 |
| 54A12 | 101 | HEK293-huCX3CR1 | 6.8 E-9 | 96 | 5 |
| 54D08 | 101 | HEK293-huCX3CR1 | 8.4 E-8 | 95 | 2 |
| 54A07 | 101 | HEK293-huCX3CR1 | 2.3 E-8 | 52 | 2 |
| 54D05 | 101 | HEK293-huCX3CR1 | 5.3 E-9 | 94 | 5 |
| 54B03 | 101 | HEK293-huCX3CR1 | 6.7 E-8 | 92 | 2 |
| 54G03 | 101 | HEK293-huCX3CR1 | 2.7E-7 | 89 | 2 |
| 61E02 | 101 | HEK293-huCX3CR1 | 8.2 E-8 | 98 | 2 |
| 61B04 | 101 | HEK293-huCX3CR1 | 5.7 E-8 | 97 | 2 |
| 61B02 | 101 | HEK293-huCX3CR1 | 1.0 E-8 | 94 | 2 |
| 54H01 | 101 | HEK293-huCX3CR1 | 1.0 E-8 | 64 | 2 |
| 54A04 | 101 | HEK293-huCX3CRl | 4.9 E-8 | 100 | 2 |
| 61F11 | 101 | HEK293-huCX3CR1 | 4.6 E-8 | 96 | 2 |
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| 61G03 | 101 | HEK293-huCX3CR1 | 6.0 E-8 | 96 | 2 |
| 61G04 | 101 | HEK293-huCX3CR1 | 4.2 E-8 | 96 | 2 |
| 66B02 | 101 | HEK293-huCX3CR1 | 2.5 E-9 | 102 | 2 |
| 66G01 | 101 | HEK293-huCX3CR1 | 1.4 E-8 | 99 | 2 |
Inhibition by anti-CX3CR1 VHHs of human Fractalkine Induced chemotaxis of BA/F3 cells overexpresslng human CX3CR1
To evaluate Inhibition of Fractalkine Induced chemotaxis, a chemotaxis assay was setup using the ChemoTx disposable chamber with 5 pm poreslze (Neuroprobe, Gaithersburg, US). Cells were harvested from an actively growing culture and washed before use In assay medium, RPMI (Gibco, Carlsbad, US) suppiemented with 0.1% BSA. The bottom chamber was filled with 320 pM human Fractalkine In a total volume of 300 μΙ. Upon application of the membrane, 0.13E6 cells were deposlted on top of the membrane In a total volume of 70 pl. Chemotaxis was allowed for 3 hours at 37’C in a humidifïed chamber with CO2. After this incubation perlod, the membrane was removed and cells in the bottom chamber were resuspended. The amount of ATP présent in the wells was determined using the CelITiter-GIo kit (Promega, Madison Wl, US). Read out was performed on an Envlslon (Perkin Elmer, Massachusetts, US) with the standard settings for luminescence read out. Titration sériés were performed in triplIcate and each plate contained control samples in triplicate as weli. As control, a sample without VHH was inciuded as well as asample where nohuman Fractalkine was added to the bottom chamber. A summary of the results Is shown In Table 20. ·
Table 20 Potency and efficacy of the VHH'ln blocklng the fractalkine
Induced chemotaxis :
| VHH ID | Fam | IC50 | % block · - | flepeatS^ |
< S
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| 11H11 | 9 | 2E-7 | 89 | 5 |
| 18E06 | 13 | NA | 17 | 4 |
| 54A12 | 101 | 8E-8 | 84 | 6 |
| 54D08 | 101 | NA | 33 | 2 |
| 54A07 | 101 | 5E-8 | 45 | 4 |
| 54D05 | 101 | 7E-8 | 81 | 4 |
| 54B03 | 101 | 1 E-7 | 73 | 1 |
| 54G03 | 101 | NA | 40 | 1 |
| 66B02 | 101 | 2E-8 | 87 | 2 |
| 66G01 | 101 | 4 E-7 | 54 | 2 |
Evaluation ofthe cross reactivity ofthe anti-CX3CR1 VHHs against cynomolgus CX3CR1
Initiaily, a FACS based binding setup was used to evaluate the cynomolgus cross reactivity. For this, the VHHs were incubated with the respective cells for 30 minutes at 4’C followed by three wash steps and subsequently incubated with the détection reagents. As détection, a mouse anti-cmyc antibody (Serotec, MCA2200) followed by a goat anti-mouse antibody coupled to PE (Jackson 115116-071 ) was used, each incubation for 30 minutes at 4eC, followed by three io wash steps. Results of the assay are shown ln Table 21.
Table 21 EC50 value for binding of the respective VHH on human CX3CR1 or on cynomolgus CX3CR1
| VHH ID | Famiiy | ECSO(M) Human | ECSO (M) Cynomolgus | ratio | Repeats |
| 11H11 | 9 | 8.0E-10 | NA | NA | 2 |
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| 18E06 | 13 | 1.5 E-9 | 1.4 E-8 | 9 | 2 |
| 54A12 | 101 | 3.5 E-8 | 1.1 E-7 | 3.3 | 1 |
| 54A07 | 101 | 5.4 E-7 | 3.0 E-8 | 0.1 | 1 |
| 54D05 | 101 | 8.4E-10 | 5.0 E-8 | 59.6 | 1 |
For later Identified VHHs, a human Fractalklne compétition FACS was set up using human or cynomolgus CX3CR1 expressed ln HEK293T cells. Both the human and the cynomolgus receptor was transiently transfected in HEK293T s cells and transférions were matched by the binding of the Iabeled ligand, human fractalkine. The compétition was evaluated using the EC30 concentration of fractalkine and as such obtained IC50 values are a good estimate ofthe Kl value, a measure for affinity (Table 22). The experiment was performed as described in Example 4. The ratio ofthe IC50 values on cynomolgus monkey and human io CX3CR1 was used to evaluate potential différences in affinity for CX3CR1 ln both species.
Table 22 Efflcacy and potency of VHHs ln ligand compétition FACS towards human and cynomolgus CX3CR1
| VHH ID | Famlly | Human | Cynomolgus | ratio | Repeats | ||
| IC50 (M) | % block | IC50 (M) | % block | ||||
| 54A12 | 101 | 6.8 E-9 | 96 | 6.7 E-8 | 94 | 9.85 | 5 |
| 54D08 | 101 | 8.4 E-8 | 95 | 7.2 E-8 | 91 | 0.85 | 2 |
| 54A07 | 101 | 2.3 E-8 | 52 | 2.8 E-7 | 69 | 12.3 | 2 |
| 54D05 | 101 | 5.3 E-9 | 94 | 6.4 E-8 | 91 | 12.25 | 5 |
| 54B03 | 101 | 6.7 E-8 | 92 | 4.7 E-7 | 95 | 7.05 | 2 |
| 54G03 | 101 | 2.7E-7 | 89 | 4.7 E-7 | 89 | 1.74 | 2 |
| 61E02 | 101 | 8.2 E-8 | 98 | 4.6 E-8 | 96 | 0.55 | 2 |
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| 61B04 | 101 | 5.7 E-8 | 97 | 8.7 E-8 | 93 | 1.53 | 2 |
| 61B02 | 101 | 1.0 E-8 | 94 | 4.5 E-8 | 92 | 4.37 | 2 |
| 54H01 | 101 | 1.0 E-8 | 64 | 6.8 E-8 | 92 | 6.48 | 2 |
| 54A04 | 101 | 4.9 E-8 | 100 | 3.1 E-8 | 91 | 0.64 | 2 |
| 61F11 | 101 | 4.6 E-8 | 96 | 1.6 E-7 | 95 | 3.58 | 2 |
| 61G03 | 101 | 6.0 E-8 | 96 | 3.0 E-7 | 89 | 5.02 | 2 |
| 61G04 | 101 | 4.2 E-8 | 96 | 2.0 E-7 | 100 | 4.8 | 2 |
| 66B02 | 101 | 2.5 E-9 | 102 | 1.9 E-8 | 97 | 7.5 | 2 |
| 66G01 | 101 | 1.4 E-8 | 99 | 1.2 E-7 | 100 | 8.29 | 2 |
Binding of the anti-human CX3CR1 VHHs to human CCR2, human CCR5 or mouse CX3CR1
Specifîcity for the huCX3CR1 receptor was evaluated by performing a FACS binding experiment on CH0-K1 parental cells or CHO cells expressing huCCR2, huCCRS or msCX3CR1. The VHHs were Incubated with the respective cell Unes for 30 minutes at 4°C followed by three wash steps and subsequently Incubated with the détection reagents. As détection, a mouse antl-cmyc antibody (Serotec, MCA2200) followed by a goat antl-mouse antibody coupled to PE (Jackson 115w 116-071 ) was used, each Incubation for 30 minutes at 4°C, followed by three wash steps. For each cell line a quality control with receptor-speclfic antibody was included. ln addition, the highest concentration of each VHH was also Incubated with CHO cells expressing huCX3CR1 as a positive control. No binding to msCX3CR1, huCCR2 or huCCRS could be observed.
is Détermination ofthe epitope bin
A compétitive binding experiment was setup In order to détermine whether the VHHs bind overiapplng epitopes on CX3CR1. For this, the VHH 66B02 labeled with alexa647 was used ln a compétition FACS on the BA/F3 cells expressing huCX3CR1. Représentative VHHs from the three functional familles were used
-13017072 as competitors for the binding of the labeled 66B02. The obtained IC50 values are shown ln Table 23.
Table 23 Compétition FACS based epitope blnning
| VHH ID | famlly | IC50 (M) | % block |
| 11H11 | 9 | 4.9E-09 | 100 |
| 18E06 | 13 | 2.3E-09 | 100 |
| 66B02 | 101 | 1.5E-09 | 100 |
As a complété inhibition of 66B02 binding could be obtained by ail représentative VHHs from the different ligand blocking families, it can be concluded that ail functional families bind in close enough proximity of each other such that they compete with binding of 66B02.
Example 6: Formattlng of VHHs to blvalency
Construction of bivalents
In order to Increase potency and/or efficacy from a sélection of the obtained VHHs, bivalent molécules were constructed by genetic engineering. Two VHHs is were genetically linked together with a 35GS linker in between the two building blocks and subsequently expressed ln E.coli as described above for the monovalent VHHs. Different bivalent constructs were made as listed ln Table 24.
Table 24 Représentative bivalent formats
| Construct ID | VHH Identity | Famlly | Linker | VHH Identity | Famlly |
| CX3CR1BII007 | CX3CR1BII11H11 | 9 | 35GS | CX3CR1BII18E6 | 13 |
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| CX3CR1BII009 | CX3CR1BII18E6 | 13 | 35GS | CX3CR1BII11H11 | 9 |
| CX3CR1BII012 | CX3CR1BII54D08 | 101 | 35GS | CX3CR1BII18E06 | 101 |
| CX3CR1BII016 | CX3CR1BII54A12 | 101 | 35GS | CX3CR1BII54A12 | 101 |
| CX3CR1BII017 | CX3CR1BII54D5 | 101 | 35GS | CX3CR1BII54D5 | 101 |
| CX3CR1BII018 | CX3CR1BII66B02 | 101 | 35GS | CX3CR1BII66B02 | 101 |
| CX3CR1BII019 | CX3CR1BII66G01 | 101 | 35GS | CX3CR1BII66G01 | 101 |
| CX3CR1BII020 | CX3CR1BII54B5 | 101 | 35GS | CX3CR1BII54B5 | 101 |
| CX3CR1BII026 | CX3CR1BII11H11 | 9 | 35GS | CX3CR1BII66B02 | 101 |
| CX3CR1BII027 | CX3CR1BII11H11 | 9 | 35GS | CX3CR1BII54B5 | 101 |
Inhibition by antl-CX3CR1 VHHs of human Fractalkine blnding to human
CX3CR1 expressed on the BA/F3 cells
The Inhibition of ligand binding to human CX3CR1 was Investigated for the s different formats as described in Example 4. For this characterization the BA/F3huCX3CRl cell line was used showing stable expression of the human CX3CR1 receptor. The alexa647 labeled ligand fractalkine was used at its EC30 concentration and thereby obtained IC50 values are reflective of the Kl values. An overview of the obtained data Is shown In Table 25.
Table 25 Potency of bivalent formats In ligand compétition
| Construct ID | Cell line | IC50 (M) | % block | Repeats |
| CX3CR1BII007 | CHO-huCX3CR1 | 3.8 E-10 | 100 | 2 |
| CX3CR1BII009 | CHO-huCX3CR1 | 7.0 E-10 | 91 | 2 |
| CX3CR1BII012 | CHO-huCX3CR1 | 8.0 E-10 | 93 | 1 |
| CX3CR1BII016 | HEK293-huCX3CR1 | 1.9 E-10 | 102 | 2 |
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| CX3CR1BII017 | HEK293-huCX3CR1 | 3.1 E-10 | 99 | 2 |
| CX3CR1BII018 | HEK293-huCX3CR1 | 3.0 E-10 | 102 | 2 |
| CX3CR1BII019 | HEK293-huCX3CR1 | 2.9 E-10 | 100 | 2 |
| CX3CR1BII020 | HEK293-huCX3CR1 | 2.2 E-10 | 102 | 2 |
| CX3CR1BII026 | BA/F3-huCX3CR1 | 7.0 E-10 | 100 | 3 |
| CX3CR1BII027 | BA/F3-huCX3CR1 | 6.7 E-10 | 100 | 3 |
Inhibition by anti-CX3CR1 VHHs of human Fractalkine induced chemotaxis of BA/F3 cells overexpresslng human CX3CR1
Similar to what was described for the monovalent anti-CX3CR1 VHHs, the s Inhibition of fractalkine induced chemotaxis on the BA/F3-huCX3CR1 cells was evaluated for the bivalent constructs. An identical assay setup was used as described above and the obtained results are summarized In Table 26.
Table 26 Inhibition of fractalkine Induced chemotaxis by bivalent VHH io constructs
| Construct ID | Cell line | IC50 (M) | % block | Repeats |
| CX3CR1BII007 | BA/F3-huCX3CR1 | 4E-9 | 101 | 5 |
| CX3CR1BII009 | BA/F3-huCX3CR1 | 2E-8 | 79 | 5 |
| CX3CR1BII012 | BA/F3-huCX3CR1 | 4E-9 | 78 | 1 |
| CX3CR1BII016 | BA/F3-huCX3CR1 | 2E-9 | 88 | 3 |
| CX3CR1BII017 | BA/F3-huCX3CR1 | 3E-9 | 89 | 3 |
| CX3CR1BII018 | BA/F3-huCX3CR1 | 6 E-10 | 98 | 6 |
| CX3CR1BII019 | BA/F3-huCX3CR1 | 2E-9 | 85 | 3 |
| CX3CR1BII020 | BA/F3-huCX3CR1 | 2E-9 | 85 | 3 |
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| CX3CR1B1I026 | BA/F3-huCX3CR1 | 3 E-10 | 98 | 1 |
| CX3CR1BII027 | BA/F3-huCX3CR1 | 9E-10 | 98 | 1 |
Evaluation ofthe cross reactivity ofthe antl-CX3CR1 VHHs against cynomolgus CX3CR1
Also for the bivalent constructs the cross reactivity towards cynomolgus CX3CR1 was evaluated and compared with the human reactivity. As described earlier, either a binding setup (Table 27) or a ligand compétition setup (Table 28) were applied using transient transfected HEK293T cells. Batches of transient transfected cells were matched by their receptor expression level.
w Table 27 Binding of bivalent constructs to human or cynomolgus CX3CR1
| Construct ID | Cell Une | EC50 (M) Human | EC50 (M) Cynomolgus | ratio | Repeats |
| CX3CR1BII007 | HEK293T | 3.1 E-10 | 4.8 E-8 | 154 | 2 |
| CX3CR1BII009 | HEK293T | 2.0 E-9 | 6.8 E-9 | 3.3 | 2 |
| CX3CR1BII012 | HEK293T | 5.6 E-11 | 6.3 E-11 | 1.1 | 1 |
Table 28 Ligand compétition of bivalent constructs on human or ts cynomolgus CX3CR1
| Construct ID | Cell Une | Human | Cynomolgus | ratio | Repeats | ||
| IC50 (M) | % block | IC50 (M) | % block | ||||
| CX3CR1BII016 | HEK293T | 1.9 E-10 | 102 | 6.9 E-10 | 98 | 3.67 | 2 |
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| CX3CR1BII017 | HEK293T | 3.1 E-10 | 99 | 1.6 E-9 | 95 | 5.34 | 2 |
| CX3CR1BII018 | HEK293T | 3.0 E-11 | 102 | 1.0 E-10 | 97 | 3.33 | 2 |
| CX3CR1BII019 | HEK293T | 2.9 E-10 | 100 | 8.2 E-10 | 96 | 2.86 | 2 |
| CX3CR1BII020 | HEK293T | 2.2 E-10 | 102 | 3.2 E-10 | 97 | 1.47 | 2 |
Example 7: Exploration of linker length and half life extension
Evaluation ofthe linker length and positioning ofthe alb11 VHH
As the linker length used In a bivalent format can impact drastically on the obtained potency, different linker lengths were evaluated.
In addition, Alb11, a Nanobody binding to human sérum albumin was induded to Increase the in vivo half-life of the formatted molécules (WO 06/122787). Different formats were made including variations on the linker lengths used, but also the positioning of the different composlng VHHs. A summary of the explored formats ls shown In Table 29.
Table 29 Exploration of half llfe extension and linker length
| Construct ID | VHH Identlty | Linker | VHH Identlty | Linker | VHH Identlty |
| CX3CR1B II032 | CX3CR1BII66B02 | 9GS | CX3CR1BII66B0 2 | 9GS | Alb11 |
| CX3CR1B II034 | CX3CR1BII66B02 | 35GS | CX3CR1BII66B0 2 | 9GS | Alb11 |
| CX3CR1B II036 | CX3CR1BII66B02 | 9GS | Alb11 | 9GS | CX3CR1BII66B0 2 |
| CX3CR1B Il 040 | CX3CR1BII66B02 | 9GS | CX3CR1BII66B0 2 | 35GS | Alb11 |
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| CX3CR1B 11041 | CX3CR1BII66B02 | 35GS | CX3CR1BII66B0 2 | 35GS | Aîb11 |
| CX3CR1B Il 042 | CX3CR1BII66B02 | 35GS | Alb11 | 35GS | CX3CR1BII66B0 2 |
Coding sequences for the formatted VHH were cloned Into an ln-house constructed plasmid allowing expression In Pichia pastoris and sécrétion Into the cultivation medium. The expression vector was derived from pPICZa (Invitrogen) 5 and contained the A0X1 promotor for tightly regulated, methanol Induced expression, a restance gene for Zeocin™, a multicloning site and the a-factor sécrétion signal. Upon transformation expression cultures were grown and VHH expression was Induced by addition of methanol and allowed to continue for 48 hours at 30°C.
io The potency of these different formats was evaluated using the ligand compétition assay as described above. Seeing that the ligand concentration used Is below the EC50 value, the obtained IC50 values are équivalent to the Kl values. The obtained Ki for the different formats is summarized in Table 30.
is Table 30 Potency of half life extended formats in ligand compétition
| Construct ID | Cell line | IC50 (M) | % block | Repeats |
| CX3CR1BII032 | BA/F3-huCX3CR1 | 5.8E-10 | 99 | 2 |
| CX3CR1BII034 | BA/F3-huCX3CR1 | 5.2E-10 | 99 | 2 |
| CX3CR1BII036 | BA/F3-huCX3CR1 | 5.6E-10 | 99 | 2 |
| CX3CR1BII040 | BA/F3-huCX3CR1 | 5.9E-10 | 104 | 1 |
| CX3CR1BII041 | BA/F3-huCX3CR1 | 6.4E-10 | 102 | 1 |
| CX3CR1BII042 | BA/F3-huCX3CR1 | 8.9E-10 | 100 | 1 |
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Impact of human sérum albumln on the potency
The binding of human sérum albumin (HSA) to the alb11 VHH could impact on the potency of the format and therefore ligand compétition was repeated in presence of HSA. Briefly, to allow the binding of HSA to the alb11 VHH, the s constructs under évaluation and fractalkine were pre-incubated with HSA for 30 minutes before addition to the cells. Also the cells were resuspended in FACS buffer supplemented with HSA. The final concentration HSA used was a 50 fold excess above the highest VHH concentration used. Subsequentiy, compétition was allowed for 2 hours and further processing was as described in Example 4.
| Construct ID | Cell line | IC50 (M) | % block | Repeats |
| CX3CR1BII032 | BA/F3-huCX3CR1 | 1.4 E-9 | 100 | 2 |
| CX3CR1BII034 | BA/F3-huCX3CR1 | 1.3 E-9 | 100 | 2 |
| CX3CR1BII036 | BA/F3-huCX3CR1 | 1.4 E-9 | 100 | 2 |
The potential Interférence of HSA was aiso evaluated in an adapted chemotaxis setup, including HSA in the different compartments of the assay. The concentration HSA used was again a 50 fold excess over the highest 15 concentration of construct used and constructs were loaded with HSA for 30 minutes before start of the assay. The assay buffer was aiso supplemented with HSA such that HSA is présent during the entire span of the experiment. As described above, the disposable ChemoTx chamber with 5 pm poresize (Neuroprobe, Gaithersburg, MD, USA) was used. Cells were harvested from an 20 actively growing culture and washed before use in assay medium, RPMI (Gibco, Carisbad, US) supplemented with 0.1% BSA and 62.5μΜ HSA (Sigma, A8763).
The bottom chamber was filled with 320 pM human Fractalkine in a total volume of 300 μΙ. Upon application of the membrane, 0.13E6 celis were deposited on top of the membrane in a total volume of 70 μΙ. Chemotaxis was allowed for 3 hours 25 at 37 C in a humidified chamber with CO2. After this incubation period, the membrane was removed and celis in the bottom chamber were resuspended.
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The amount of ATP présent In well was determined using the CelITiter-GIo kit (Promega, Madlson Wl, USA). Read out was performed on an Envision (Perkin Elmer, Waltham, MA, USA) with the standard factory settings for luminescence read out. Titration sériés were performed in triplicate and each plate contained 5 control samples In triplicate as well. As control, a sample without VHH was included as well as a sample where no human Fractalkine was added to the bottom chamber. The obtained IC50 values are listed In Table 31.
Table 31 Fractalkine Induced chemotaxis In the presence of HSA
| Construct ID | Cell line | IC50 (M) | % block | Repeats |
| CX3CR1BII032 | BA/F3-huCX3CR1 | 6E-10 | 98 | 2 |
| CX3CR1BII034 | BA/F3-huCX3CR1 | 9E-10 | 100 | 2 |
| CX3CR1BII036 | BA/F3-huCX3CR1 | 6E-10 | 102 | 2 |
Inhibition of Fractalkine internalization by the formatted bivalent half-life extended polypeptides
Additional functlonai assays were performed to demonstrate the antagonist activity of the bivalent half-life extended polypeptides. The polypeptides were is evaluated for their ability to inhibit the Internalization of A647-Fractalklne In CHO huCX3CR1 cells. Briefly, 1E4 cells/weii were plated In black clear bottom, 96 well plates (BD, Franklin Lakes, NJ, USA) and grown ovemight. The cells were washed once and then equilibrated in assay buffer (HBSS with calcium and magnésium (Glbco) supplemented with 10 mM HEPES and 0.1% BSA). The 20 formatted polypeptide constructs were added and the plates were incubated for minutes at 37 C. A647-Fractalklne was then added at a final concentration of nM and the cells were incubated for 60 minutes at 37 C. The media was removed and the cells were fixed for 10 minutes with 3.7% formaldéhyde solution (Polysciences, Warrington, PA, USA). The cells were rinsed once with PBS and the nuclei were labeled with Hoechst dye (Life Technologies, Grand Island, NY,
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USA). To quantitate the intemalized labeled Fractalkine, the cells were imaged using the BD Pathway bloimaging system. Image segmentation was performed by Identifying the labeled cell nucléus and drawing a 3 pixel ring around that mask. Mean A647 intensity was measured in the cytoplasmic ring. The formatted 5 polypeptides potently inhibited Fractalkine Intemalization as summarized In Table 32:
Table 32 Inhibition of A647*Fractalklne Intemalization
| Construct ID | Cell line | IC50 (M) | Repeats |
| CX3CR1BII032 | CHO-huCX3CR1 | 4.0E-10 | 5 |
| CX3CR1BII034 | CHO-huCX3CR1 | 7.4E-10 | 3 |
| CX3CR1BII036 | CHO-huCX3CR1 | 4.9E-10 | 8 |
| CX3CR1BII040 | CHO-huCX3CR1 | 1.0 E-9 | 3 |
| CX3CR1BII041 | CHO-huCX3CR1 | 1.1 E-9 | 2 |
| CX3CR1BII042 | CHO-huCX3CR1 | 8.5E-10 | 2 |
An antl-CX3CR1 formatted bivalent half-life extended polypeptide Is devold of agonist activity
In order to confirm that a bivalent anti-CX3CR1 half-life extended polypeptide did not hâve agonist activity, CX3CR1BII036 was evaluated for Induction of calcium influx In the CHO huCX3CR1 cells. Fractalkine mediated Increases in cytosolic calcium levels in these cells In a CX3CR1 dépendent manner and CX3CR1BII036 Inhibited this response.
The CHO huCX3CR1 cells were plated at 5E4 cells/well in black clear bottom, 96 well plates (BD) and grown ovemight. The cells were incubated with Calcium-4 dye/2 mM probenicid (Molecular Devices, Sunnyvale, CA, USA) In HBSS supplemented with 20 mM HEPES for 60 minutes at 37 °C. For demonstrating polypeptide antagonism, CX3CR1BII036 was preincubated with the cells for 15
-13917072 minutes prior to the addition of Fractalklne at Its EC80 value. Calcium mobilization was monitored on a FLIPR Tetra system (Molecular Devices) as per the manufacturées Instructions. For determlnlng agonism, there was no preincubation with the polypeptide and Instead, CX3CR1BII036 was used in place of Fractalkine stimulation. While CX3CR1BII036 inhlbited Fractalkine mediated calcium influx with an IC50 of 1.3 nM, no increase In cytosolic calcium Ievels were observed when the polypeptide alone was added at concentrations up to 1 μΜ.
Example 8: Exploration of half life extension formats using mouse Fc
To investigate alternative half-life extension modalities, the 66B02 VHH domain was produced as a fusion protein with a mouse lgG2 Fc domain (66B02-mFc). An aspartic acid to alanine mutation (D265A) was Incorporated In the CH2 domain to abrogate potential Fc-mediated effector fonction In this construct (Baudino, J. Immunol., 181, 6664-6669 (2008)). 66B02-mFc was expressed in HEK293T cells or NSO cells and purified by Protein A affinity chromatography followed by ion exchange chromatography. This molécule was tested for activity utilizing the assay formats described In Example 7. The results are summarized In Table 33:
Table 33 66B02-mFc Activity
| Assay | Cell line | IC50 (M) | Repeats |
| Ligand compétition | BA/F3-huCX3CR1 | 5.7 E-10 | 2 |
| Chemotaxls | BA/F3-huCX3CR1 | 8.9 E-10 | 3 |
| Ligand intemalization | CHO-huCX3CR1 | 5.2 E-10 | 5 |
| Calcium influx | CHO-huCX3CR1 | 9.8 E-10 | 5 |
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While 66B02-mFc potently inhibited Fractalkine medlated CX3CR1 activation, it did not display agonlst activity. No increase in cytosolic calcium levels was observed with treatment with up to 1 μΜ of this molécule.
Example 9: Inhibition of plaque progression in a mouse atherosclerosis model bivalent half-Hfe extended polypeptides
Génération of human CX3CR1 knock-in Apo E mice
Given the lack of cross reactivity of the identified VHHs for mouse CX3CR1 (Example 5), a human CX3CR1 knock-in mouse line (hu CX3CR1 Kl) was generated at TaconicArtemis (Koein, Germany) to enable testing of these molécules in mouse disease models. A strategy was employed that allowed the expression of the human chemokine receptor under the control of the corresponding mouse promoter while disrupting the expression of the endogenous mouse protein. Briefly, a targeting vectorwas constructed where the mouse CX3CR1 coding région in exon 2 was replaced with the complété human CX3CR1 open reading frame and flanked by sélection markers and loxP sites. The targeting vectorwas Introduced into mouse ES cells and clones that had successfully undergone homologous recombination were used to generate chimeric mice. These mice were bred to highly efficient Flp-deleter mice to achieve removal of the sélection marker and germline transmission. The resulting hu CX3CR1 Kl mice in a C57BL/6 background were then crossed to Apo E * mice (The Jackson Laboratory, Bar Harbor, Maine, USA) to generate hu CX3CR1 Kl Apo E7 mice. The Apo E+ mouse model provides a robust method to elicit extensive atherosclerotic plaque formation that is grossly similar to the human disease with respect to the site-specific localization of plaque formation, histological composition, and the known rlsk factors (cholestérol, inflammation, hypertension, etc).
Evalutation ofthe anti-CX3CR1 bivalent half-life extended polypeptides in the mouse Apo E*' atherosclerosis model
Female hu ÇX3CR1KI ApoE * mice were fed a hlgh fat/high cholestérol diet containing 1.5% cholestérol for 16 weeks beginning at four weeks of âge. After 10
-141 17072 weeks, the animais were administered by l.p. Injection vehicle (20 mM NaCitrate pH 6.0,115 mM NaCI), 10 mg/kg 66B02-mFc once or twice per week or 30 mg/kg CX3CR1BII036 twice per week for 6 weeks. The animais were anesthetized by gas anaesthesia and perfused with 0.9% saline. The descending aorta to the ileac bifurcation was carefully removed and fixed in formalin. It was then opened longitudinally, and stained with Sudan IV for 15 minutes, followed by 70% methanol for 2 minutes. The vessels were washed under running water and covered with PBS. The tissues were photographed with a digital caméra using SPOT Advanced software (SPOT Imaglng Solutions, Sterling Heights, Mi, USA).
io The percentage of lipid staining was determined with image analysis software (Image-Pro Plus, MediaCybemetics, Rockville, MD, USA) and expressed as a percentage positive staining of the vessei. The results from this study are summarized in Table 34:
Table 34 Quantification of Plaque Size ln the Descending Aorta in female hu CX3CR1 Kl Apo E Mice
| Group | Dose | # Animais | % Plaque Area | % Réduction ln Plaque Area |
| Control (10 weeks) | N/A | 6 | 3.4 | N/A |
| Control (16 weeks) | Vehicle | 17 | 14.8 | N/A |
| 66B02-mFc | 10 mg/kg (Ixweek) | 17 | 13.0 | 16 |
| 66B02-mFc | 10 mg/kg (2x/week) | 17 | 10.3 | 39 (p<0.05) |
| CX3CR1BII036 | 30 mg/kg (2x/week) | 17 | 10.1 | 41 (p<0.05) |
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Both 66B02-mFc and CX3CR1BII036 significantly inhibited plaque progression when dosed twice weekly. This correlated with coverage as plasma levels of these molécules could be confirmed to be maintained throughout the study. For once weekly dosing of 66B02-mFc, détectable plasma levels were not maintained 5 and this correlated with the lack of significant efficacy observed after 6 weeks of treatment. Neither molécule significantly affected plasma cholestérol or triglycéride levels.
Exampie 10: Sequence optimization of the parental VHH w In general, during VHH sequence optimization, parental wild type VHH sequences are mutated to yield VHH sequences that are more Identical to human VH3-JH germline consensus sequences. Spécifie amino acids in the framework régions that differ between the VHH and the human VH3-JH germline consensus are altered to the human counterpart In such a way that the protein structure, is activity and stability are kept intact. To investigate this, ail sequence optimization variants were compared with the parental VHH In three different assays: (I) détermination of the melting température (Tm) In a Thermal Shift Assay (TSA), (il) analysis of In vitro potency In fractalkine compétition FACS, and for some constructs (iii) analysis of in vitro potency in the fractalkine induced chemotaxis m assay.
Mutation of framework residues
For sequence optimization, the following mutations were investigated: E1D, S11L, A14P, E16G, R44Q, D46E, A74S, K83R and Q108L. The individual 25 mutants that were generated In the parental sequence of CX3CR1BII66B02 are depicted in Table 35:
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Table 35 Investlgated mutations during sequence optlmlzatlon of 66B02
| Clone number | Mutations Introduced |
| C100CX3CR1BI1043 | A14P,A74S,K83R,Q108L |
| C100CX3CR1BI1045 | E1D.A14P.A74S.K83R.Q108L |
| C100CX3CR1BI1047 | S11L,A14P,A74S,K83R,Q108L |
| C100CX3CR1BI1048 | A14P,E16G(A74S,K83R,Q108L |
| C100CX3CR1BI1049 | A14P,R44QlA74S,K83R,Q108L |
| C100CX3CR1BI1050 | A14P,D46E,A74S,K83R,Q108L |
| C100CX3CR1BI1061 | S11 L,A14P,E16G.A74S.K83R.Q108L |
| C100CX3CR1BI1056 | S11 L,A14P.E16G.R44Q.A74S.K83R.Q108L |
| C100CX3CR1BI1057 | S11L(A14P,E16G,D46E,A74S,K83R,Q108L |
| C100CX3CR1BI1060 | S11 L,A14P.E16G.R44Q.D46E, A74S.K83R.Q108L |
Ail constructs were cioned In an E. coli expression vector, and expressed in E coli as myc/His-tagged proteins ln a culture volume of 0.25L to 0.5L TB medium.
Expression was Induced by addition of 1mM IPTG and allowed to continue for 4 hours at 37°C and 250 rpm. Ceiis were pelleted, and periplasmic extracts were prepared by freeze-thawing and resuspension in dPBS. These extracts were used as starting material for Immobilized métal affinity chromatography (IMAC) using Hlstrap FF crude columns (GE healthcare). Nanobodies were eluted from w the column with 250mM imldazole and subsequently desalted towards dPBS. The purity and Integrity of Nanobodies was verified by reducing SDS-PAGE.
As summarized in Table 36, A14P, A74S, K83R and Q108L mutations had no clear effect on potency as determined from compétition FACS. Similariy, the additional mutations E1D, S11L and E16G dîd not affect potency. The is introduction of either R44Q or D46E on the other hand resulted in a signifîcant drop in potency that was even more pronounced If both mutations were Introduced.
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Table 36 Potency of sequence optlmlzatlon constructs determined by ligand compétition FACS
| Clone number | IC50 | % block | Tm at pH7 |
| CX3CR1BII66B02 | 2.6E-09 | 101.0 | 65.66 |
| C100CX3CR1BH043 | 2.2E-09 | 101 | 66.49 |
| C100CX3CR1BII045 | 2.2E-09 | 101.2 | 66.07 |
| C100CX3CR1BII047 | 2.3E-09 | 101.2 | 66.49 |
| C100CX3CR1BII048 | 1.9E-09 | 101.2 | 67.74 |
| C100CX3CR1BII049 | 1.8E-08 | 101.2 | 66.07 |
| C100CX3CR1BII050 | 1.7E-08 | 101.1 | 71.90 |
| C100CX3CR1BII061 | 1.4E-09 | 98.9 | 68.57 |
| C100CX3CR1BH056 | 1.6E-08 | 101.1 | 68.57 |
| C100CX3CR1BH057 | 1.4E-08 | 99.4 | 74,39 |
| C100CX3CR1BII060 | 1.9E-07 | 98.4 | 74.81 |
Also the melting température, prédictive for the stability of the VHH, was evaluated. Most indivldual mutations had limited to no effect except for the D46E mutation which raised the melting température by approximately 6°C. The introduction of the combined mutations also enhanced the thermal stability, cfr 057 and 060.
io Due to the major effects on the potency in ligand compétition FACS, the mutations R44Q and D46E were not included ln the final sequence.
Mutation of CDR residues
Based on the in siüco analysis of the parental sequence, a glycosylation site was predîcted at position 52. Therefore two libraries were constructed; one for position
-14517072 and one for position 53, which was designed to include ail possible amino acids at the respective position. The libraries were screened as periplasmlc extracts In a ligand compétition FACS. First, a dilution sériés was made of periplasmic material from the parental sequence and three dilutions were selected for further screening. A first dilution point (two fold) was chosen to give full block of the ligand Interaction whereas the other two dilution points (128 and 512 fold) should resuit In 70 % and 40% block respectively. Upon production of the periplasmic extracts from the llbrary, ail samples were split In two and one of them was subjected to a heat treatment. Both the non-treated and the heat to treated samples were subsequentiy analyzed In the ligand compétition FACS at the three dilution points. The Impact of the mutation could be estimated by comparing the obtained blockage with that from the parental sequence. The analysis of the heat treated samples provides a measure for a potential Impact on stability of the mutation.
is Based upon the Initiai screening results, seven mutations were selected for further characterization. The obtained potency in ligand compétition FACS Is shown In Table 37.
Table 37 Removal glycosylation site at position 52
| Construct | IC50(M) | % block | Tm at pH7 |
| C100CX3CR1BII66B02 | 2.5E-09 | 98.0 | 65.66 |
| CX3CR1BII66B02 (N52S, Q108L) | 1.7E-09 | 98.0 | 66.07 |
| CX3CR1BII66B02 (N52Q, Q108L) | 2.1E-09 | 97.9 | 59.83 |
| CX3CR1BII66B02 (N52G, Q108L) | 1.1E-09 | 98.0 | 59.83 |
| CX3CR1BII66B02 (N52T, Q108L) | 2.8E-09 | 98.0 | 66.07 |
| CX3CR1BII66B02 (S53T, Q108L) | 1.3E-09 | 98.1 | 66.07 |
| CX3CR1BII66B02 (S53G, Q108L) | 1.2E-09 | 98.3 | 64.83 |
| CX3CR1BII66B02 (S53P, Q108L) | 8.0E-10 | 98.2 | 66.91 |
-14617072
From this analysis, sequence alignment with the human référencé sequence and based upon an In silico T cell epitope récognition prédiction program, it was decided to include the mutations N52S and S53T in the sequence.
Because of stability reasons an additional library was made for position 32.The ligand compétition screening was set up In a similar fashion as described above. Again three dilutions of the periplasmlc extracts were screened and the obtained % block was compared with that obtained for the parental sequence. Upon analysis of the various mutants, the substitution of N32T was chosen and included in the final sequence optimized variant.
Example 11: Analysis of the optimized variants
In a final characterization round the constructs listed In Table 38 were characterized.
Table 38 Sequence optimized variants of the tead VHH 66B02
| Clone number | Mutation introduced | SEQ ID NO: |
| CX3CR1BII00306 | CX3CR1 Bl 16 6B02(E1 D,S11 L.A14P.E16G,N32T,N5 2S,A74S,K86R,Q113L) | 138 |
| CX3CR1BII00307 | CX3CR1 Bl I6 6B02(E1 D,S11 L,A14P.E16G,N32T,N5 2S,S53T,A74S,K86R,Q113L) | 139 |
| CX3CR1BII00308 | CX3CR1 Bl 166 B02( E1 D,S11 L,A14P.E16G,A74S,K8 6R.Q113L) | 140 |
| CX3CR1BII00312 | CX3CR1 Bl I66 B02( E1 D,S11 L,A14P.E16G,N32T,N5 2S,A74S,K86R,Q113L)-9GS-Alb11-9GS- CX3CR1 Bl I66 B02( S11 L,A14P,E16G,N32T,N52S,A 74S,K86R,Q113L) | 225 |
| CX3CR1BII00313 | CX3CR1 Bl 166B02(E1 D,S11 L,A14P,E16G.N32T.N5 2S,S53T,A74S,K86R,Q113L)-9GS-Alb11-9GS- | 226 |
-147I W
| CX3CR1BII66B02(S11L,A14PlE16G,N32T,N52S,S 53T,A74S,K86R,Q113L) | ||
| CX3CR1BII00314 | CX3CR1 BII66B02(E1 D.S11 L.A14P.E16G,A74S,K8 6R,Q113L)-9GS-Alb11-9GS- CX3CR1 BII66B02(S11 L,A14P.E16G,A74S,K86R,Q 113L) | 227 |
A compétition FACS experiment was performed as described above as well as a détermination of the melting température. The obtained values are represented in Table 39.
Table 39 Compétition FACS and Tm of the sequence optlmized variants
| Construct | IC50 (M) | % block | Tm at pH7 |
| C100CX3CR1BII66B02 | 2.5E-09 | 98.0 | 65.05 |
| CX3CR1BII00306 | 1.7E-09 | 97.0 | 68.54 |
| CX3CR1BII00307 | 1.9E-09 | 97.0 | 68.13 |
| CX3CR1BII00308 | 1.6E-09 | 97.0 | 68.13 |
| CX3CR1BII00312 | 4.6E-10 | 100.0 | 59.37 |
| CX3CR1BII00313 | 4.0E-10 | 100.0 | 58.88 |
| CX3CR1BII00314 | 6.5E-10 | 100.0 | 58.40 |
These constructs were aiso characterized In fractalkine Induced chemotaxis as described above (Table 40).
Table 40 Ligand induced chemotaxis with sequence optlmized variants
| Construct | IC50(M) | % block | n |
| C100CX3CR1BII66B02 | 3.6E-08 | 91 | 3 |
- 14817072
| CX3CR1BII00306 | 6.3E-08 | 95 | 2 |
| CX3CR1BII00307 | 6.3E-08 | 100 | 2 |
| CX3CR1BII00308 | 4.4E-08 | 89 | 2 |
| CX3CR1BII00312 | 2.7E-09 | 99 | 3 |
| CX3CR1BII00313 | 2.7E-09 | 99 | 3 |
| CX3CR1BII00314 | 3.6E-09 | 100 | 3 |
Selected constructs were evaluated for inhibition of A647-Fractalkine induced Intemalization in CHO huCX3CR1 cells. The results are summarized in Table 41:
Table 41 Ligand Induced Intemalization with sequence optimized variants
| Construct | IC50(M) | n |
| CX3CR1BII00312 | 5.5 E-10 | 1 |
| CX3CR1BII00313 | 3.3E-10 | 6 |
A sequence optimized anti-CX3CR1 half-life extended polypeptide Is devoid of agonist actlvity io In order to confirm that sequence optimized anti-CX3CR1 haif-life extended polypeptide does not hâve agonist activity, CX3CR1BII00313 was evaluated for Induction of calcium Influx in the CHO huCX3CR1 cells. While prelncubatlon with CX3CR1BII00313 inhiblted Fractalkine-mediated calcium influx with an IC50 of 1.3 nM, no increase In cytosolic calcium ievels were observed when the is polypeptide alone was added at concentrations up to 1 μΜ.
Example 12: Exploration of half life extension formats using human Fc
To Investigate additional half-iife extension modaiities, the CX3CR1BII00306and CX3CR1BII00307 sequence optimized VHH domains were produced as fusion
-14917072 proteins with a human IgG 1 Fc domain (306D-hFc and 307D-hFc). Two mutations were incorporated in the CH2 domain to abrogate potential Fcmediated effector fonction in this construct. 306D-hFc and 307D-hFc were expressed in HEK293T cells or NSO celis and purified by Protein A affinity chromatography followed by ion exchange chromatography. These molécules were tested for functîonal activity utilizing the assay formats described in Example 7. The results are summarized in Table 42:
Table 42 Activity of hFc Fusion Proteins
| Assay | Cell line | 306D-hFc IC50 | 306D-hFc Repeats | 307DhFc IC50 | 307DhFc Repeats |
| Ligand compétition | BA/F3-huCX3CR1 | 6.9 E-10 | 2 | 7.0 E-10 | 2 |
| Chemotaxis | BA/F3-huCX3CR1 | 2.9 E-9 | 2 | 3.0 E-9 | 3 |
| Ligand intemalization | CHO-huCX3CR1 | 4.8 E-10 | 3 | 3.7 E-10 | 3 |
| Calcium influx | CHO-huCX3CR1 | 1.3 E-9 | 3 | 3.2 E-9 | 3 |
While these molécules potently Inhibited Fractalkine mediated CX3CR1 activation, they did not display agonist activity. No increases in cytosolic calcium levels were observed with treatment with up to 1 μΜ of these Nanobodies alone.
Example 13: Inhibition of plaque progression in a mouse atherosclerosls 15 model by a sequence optlmlzed antl-CX3CR1 Nanobody
Female hu CX3CR1KI ApoE ψ mice were fed a high fat/high cholestérol diet containing 1.5% cholestérol for 16 weeks beginning at four weeks of age. After 10 weeks, the animais were administered by i.p. injection vehicle (20 mM NaCitrate pH 6.0,115 mM NaCI), 30 mg/kg CX3CR1BII00313 once ortwice per week or 30 mg/kg CX3CR1B11036 twice per week for 6 weeks. The animais were sacrificed
-15017072 and the percentage of plaque area In the descending aorta was quantitated as described above. The results from this study are summarized In Table 43:
Table 43 Quantification of Plaque Slze In the Descending Aorta In s female hu CX3CR1 Kl Apo E ψ Mlce
| Group | Dose | # Animais | % Plaque Area | % Réduction In Plaque Area |
| Control (10 weeks) | N/A | 6 | 2.1 | N/A |
| Control (16 weeks) | Vehicie | 18 | 12.0 | N/A |
| CX3CR1BII00313 | 30 mg/kg (Ixweek) | 17 | 10.7 | 13 |
| CX3CR1BII00313 | 30 mg/kg (2x/week) | 18 | 5.9 | 62(p<0.01) |
| CX3CR1BII036 | 30 mg/kg (2x/week) | 17 | 6.8 | 52(p<0.01) |
Both CX3CR1BII00313 and CX3CR1BII036 signlficantly Inhibited plaque progression when dosed twîce weekly. This correlated with coverage as plasma levels of these molécules could be confirmed to be maintained throughout the study. For once weekly doslng of CX3CR1BII00313, détectable plasma levels
7 - - . - ’ - to were not maintained and this correlated with the lack of slgnificaht efficacy observed after 6 weeks of treatment. Neither molécule sîgnificantly affected plasma cholestérol or triglycéride levels.
Example 14: Nanobody binding to primary human and cynomolgus monkey . -. CD14+ cells In whole blood ‘ « Compétition FACS with formatted sequence optimized anti-CX3CR1 Nanobody
To confirm binding of the formatted sequence optimized anti-CX3CRl Nanobody to human primary cells, CX3CR1B1100313 was demonstrated to compete for the
-151 17072 binding of A647 labeled CX3CR1BII018 (A647-018) to CD14+ cells In a compétition FACS assay in whole blood. Briefly, a mouse antl-human CD14 antibody conjugated with eFluor 450 (eBioscience, San Diego, CA, USA) was diluted 1:10 in EDTA treated whole blood from a healthy human donor. 40 μΙ/well 5 was added to 96 well polystyrène round bottom plate followed by 10 μΙ/well of CX3CR1B1100313 diluted In Stain Buffer with BSA (BD Pharmingen) at a final concentration ranging from 100 nM to 0.002 pM and the sampies were incubated for 20 minutes at room température. 10 μΙ/well of A647-018 in Stain Buffer was then added to yield a final concentration of 1 nM (the EC80 of A647-018 binding) io and the sampies were incubated for an additional 20 minutes at room température. 220 μΙ/well of 1-Step Fix/Lyse solution (eBioscience) was then added. After a 10 minute room température incubation the cells were pelleted, washed twlce In Stain buffer and resuspended in this buffer. The sampies were anaiyzed on a BD LSR II flow cytometer. The médian fluorescence Intensity for 15 AlexaFluor 647 was quantified for the gâte CD14 positive cell population.
CX3CR1BII00313 potently inhibited the binding of A647-018 to CD14 positive cells in human blood with an IC50 of 0.35 nM (n=8).
To confirm binding ofthe formatted sequence optimized anti-CX3CR1 Nanobody to cynomolgus monkey primary cells, CX3CR1BII00313 was demonstrated to compete for the binding of A647 labeled CX3CR1BII018 (A647-018) to CD14+ cells in a compétition FACS assay In cynomolgus monkey whole blood. The method used was analogous to that outlined above except the final concentration of A647-018 was 3 nM (the EC80 of A647-018 binding) and ACK lysïng buffer (Life Technologies) was used instead of the 1-Step Fix/Lyse solution. The ceils were resuspended In Stain buffer supplemented with 1% formaldéhyde prior to analysis. CX3CR1BII00313 potently Inhibited the binding of A647-018 to CD14 positive cells In cynomolgus monkey blood with an IC50 of 0.43 nM (n=4). Example 15: Pharmacoklnetlcs (PK) In cynomolgus monkeys
A pharmacokinetic study was conducted in naïve male cynomolgus monkeys (Macaca fascîcularis) 2 -5 years of âge with a body weight range between 2.4 3.5 kg. The monkeys were divided Into four treatment groups. Group 1 (n=3) received 0.2 mg/kg of CX3CR1BII00313 i.v.; Group 2 (n=3) received 2 mg/kg of
-15217072
CX3CR1BII00313 l.v.; Group 3 (n=3) received 2 mg/kg CX3CR1BII00313 s.c. and Group 4 (n=3) received 5 mg/kg CX3CR1BII00313 l.v. CX3CR1BI100313 was administered as a 2 mg/ml solution ln citrate buffer (20 mM sodium citrate/115 mM sodium chloride, pH 6.0). Blood samples were coliected over 6 s weeks from a peripheral veln Into sérum separator tubes for PK analysis.
Sérum samples were analyzed using a MSD (Meso Scale Discovery) format. Briefly, a biotinylated anti-Nanobody antibody was bound to a MSD standard streptavidin plate (Meso Scale Discovery, Rockville, MD, USA). The plates were washed with 0.05% Tween 20 ln phosphate buffered saline and blocked with 5% io w/v of SeraCare BSA (SeraCare Life Sciences, Milford, MA, USA) prior to
Incubation with sérum samples. CX3CR1BII00313 was detected utilizing a sulfolabeled anti-Nanobody Nanobody and the plates were analyzed on a Sector Imager 2400 (Meso Scale Discovery). Varying concentrations of
CX3CR1BII0313 from 5000 to 0.5 ng/ml in 5% monkey sérum were used as standards. Target engagement was assessed by monitoring levels of free CX3CR1 on CD14+ gated monocytes. This assay was analogous to the compétition FACS assay summarized ln Example 14 except no additional CX3CR1B1100313 was added. Sérum samples were also monitored for the presence of primate anti-human antibodies (PAHA) as they may Impact assessment of PK and free CX3CR1.
ForteBio RED96 was used for détection of PAHA. Briefly, biotinylated CX3CR1BII0313 was captured over streptavidin sensors. Pooled naïve monkey sérum was then used as a négative control to calculate cut-off value (defîned as two fold above the average binding signal of naïve sera). Ail sérum samples were 25 diluted 20 fold in buffer and the PAHA response was determined to be positive if the binding signal was greater than the cut-off value.
Data for time points following détection of PAHA were exduded from PK/PD analysis. The PK data are summarized ln Table 44 below.
-15317072
Table 44 Pharmacoklnetlc parameters of CX3CR1BII00313
| Dose (mg/kg) | CL (mL/day/kg) | Tm (day) | MRT (day) | Dose normallzed AUC(014d) (nMd) | F% |
| IV 0.2 | 113 | 1 | 1 | ** | |
| IV 2.0 | 9±1 | 9±2 | 8±2 | 56530 | |
| IV 5.0 | ** | ** | ** | 58604 | |
| SC 2.0 | 54 |
**insufficient data for characterization of terminal phase
Clearance and half-life at 2.0 mg/kg i.v. were 9.4 mL/d/kg and 9.6 days, respectiveiy. At 0.2 mg/kg i.v., clearance was substantially higher (113 mL/d/kg) consistent with saturable target-mediated disposition (TMD) phamnacokinetics. Dose-adjusted AUC(o-i4d) was comparable between the 2 and 5 mg/kg i.v. doses suggesting saturation of TMD at the 2 mg/kg dose. Exposure at 2 weeks following either I.v. or s.c. Nanobody administration was > 70 nM and bioavailability after s.c. administration was 54 %. Free receptortracked with exposure with greater w than 90 % target coverage maintained at exposures >10 nM.
Claims (26)
- Claims1. A polypeptide comprising an anti-CX3CR1 Immunoglobulin single variable domain, wherein said polypeptide is capable of blocking the binding of human fractalkine to human CX3CR1.
- 2. The polypeptide according to claim 1, wherein said anti-CX3CR1 Immunoglobulin single variable domain conslsts essentially of four framework régions (FR1, FR2, FR3 and FR4) and three complementary determining réglons (CDR1.CDR2 and CDR3).
- 3. The polypeptide according to claim 1 or 2, wherein said antl-CX3CR1 Immunoglobulin single variable domain Is a VH, VL, VHH, camelized VH, or VHH that Is optlmized for stabiiity, potency, manufacturability and/or slmilarity to human framework réglons.
- 4. The polypeptide according to any one of claims 1 to 3, wherein said polypeptide has an affinity to human CX3CR1 at:c) an EC50 of less than or equal to 10nM, less than or equal to 5nM, less than or equal to 2.5nM or less than or equal to 1nM, as determined by cell binding FACS; ord) an IC50 of less than or equai to 10nM, less than or equal to 5nM, less than or equal to 2.5nM or less than or equal to 1nM, as determined by compétition FACS.
- 5. The polypeptide according to any one of claims 1 to 4, wherein said polypeptide blocks the binding of human fractalkine to human CX3CR1 at an IC50 of less than or equal to 300nM, or less than or equal to 100nM, or less than or equal to 20nM, or less than or equal to 10nM, or less than or equal to 5nM, or less than or equal to 2.5nM or less than or equai to 1nM.
- 6. The polypeptide according to any one of claims 1 to 5, wherein said polypeptide comprises a CDR3 that has the amino acid sequence of Asp-Xaa1Arg-Arg-Gly-Trp-Xaa2-Xaa3-Xaa4-Xaa5 (SEQ ID NO: 197), wherein:-15517072- Xaa1 is Pro, Ala or Gly;- Xaa2 is Asp or Asn;- Xaa3 is Thr or Ser;- Xaa4 is Arg, Lys, Ala or Gly; and s - Xaa5 is Tyr or Phe.
- 7. The polypeptide according to claim 6, wherein said CDR3 has the amino acid sequence of Asp-Pro-Arg-Arg-Gly-Trp-Asp-Thr-Arg-Tyr(SEQ ID NO: 186).10
- 8. The polypeptide according to any of daims 1-6, wherein said polypeptide comprises a CDR1, CDR2 and CDR3 having the amino acid sequences set forth in:- SEQ ID No: 213,221 and 186, respectively; or- SEQ ID No: 141,162 and 186, respectively; or is - SEQ ID No: 141,163 and 187, respectively; or- SEQ ID No: 141,164 and 186, respectively; or- SEQ ID No: 141,166 and 186, respectively; or- SEQ ID No: 141,167 and 186, respectively; or- SEQ iD No: 141,167 and 189, respectively; or20 - SEQ ID No: 141,168 and 186, respectively; or- SEQ iD No: 141,168 and 187, respectively; or- SEQ iD No: 141,169 and 190, respectively; or- SEQ ID No: 141,170 and 186, respectively; or- SEQ ID No: 141,171 and 186, respectively; or25 - SEQ ID No: 141,174 and 186, respectively; or- SEQ ID No: 141,175 and 187, respectively; or- SEQ ID No: 142,165 and 188, respectively; orZ- SEQ ID No: 142,173 and 188, respectively; or- SEQ ID No: 143,164 and 186, respectively; or30 - SEQ ID No: 144,172 and 187, respectively; or- SEQ ID No: 145,172 and 187, respectively; or- SEQ ID No: 141, 214 and 186, respectively; or- SEQ ID No: 141, 215 and 186, respectively; or-15617072- SEQ ID No: 141,216 and 186, respectively; or- SEQ ID No: 141,217 and 186, respectively; or- SEQ ID No: 141,218 and 186, respectively; or- SEQ ID No: 141,219 and 186, respectively; or5 - SEQ ID No: 141,220 and 186, respectively; or- SEQ ID No: 213,214 and 186, respectively.
- 9. The polypeptide according to any of clalms 1 to 5, wherein said polypeptide comprises a CDR1, CDR2 and CDR3 having the amino acid sequences set forth
- 10 In:- SEQ ID No: 146,176 and 191, respectively; or- SEQ ID No: 146,177 and 191, respectively.10. The polypeptide according to claim any of clalms 1 to 5, wherein said is polypeptide comprises a CDR1, CDR2 and CDR3 having the amino acid sequences set forth in:- SEQ ID No: 147,178 and 192, respectively; or- SEQ ID No: 147,179 and 192, respectively; or- SEQ ID No: 147,179 and 194, respectively; or20 - SEQ ID No: 148,179 and 193, respectively; or- SEQ ID No: 149,179 and 192, respectively; or- SEQ ID No: 149,180 and 192, respectively; or- SEQ ID No: 149,181 and 192, respectively; or- SEQ ID No: 149,183 and 192, respectively; or25 - SEQ ID No: 149,185 and 192, respectively; or- SEQ ID No: 150,179 and 194, respectively; or- SEQ ID No: 150,182 and 194, respectively; or- SEQ ID No: 151,179 and 193, respectively; or- SEQ ID No: 151,182 and 194, respectively; or30 - SEQ ID No: 151,184 and 196, respectively; or- SEQ ID No: 152,179 and 195, respectively; or- SEQ ID No: 153,179 and 194, respectively; or- SEQ ID No: 154,182 and 194, respectively; or-15717072- SEQ ID No: 155,179 and 195, respectively; or- SEQ ID No: 156,181 and 192, respectively; or- SEQ ID No: 157,179 and 194, respectively; or- SEQ ID No: 158,179 and 192, respectively; or s - SEQ ID No: 159,178 and 192, respectively; or- SEQ ID No: 160,179 and 194, respectively; or- SEQ ID No: 161,179 and 194, respectively.
- 11. The polypeptide according to claim 1, wherein the polypeptide comprises a10 CDR1, CDR2 and CDR3 having amino acid sequences set forth In: SEQ ID NO’s: 141,164 and 186, respectively, SEQ ID NO’s: 141,162 and 186, respectively, SEQ ID NO’s: 213, 214 and 186 respectively, or SEQ ID NO’s: 213,221 and 186 respectively.is
- 12. The polypeptide according to any one of claims 1 to 5, wherein said antiCX3CR1 immunoglobulin single variable domain is a VHH domain comprising the sequence set forth In:a) the amino acid sequence of SEQ ID NO: 3;b) an amino acid sequence that has at least 90% amino acid identlty with20 the amino acid sequences of SEQ ID NO: 3; orc) an amino acid sequence of any one of SEQ ID NO: 1-48,121-140 or 222-224.
- 13: The polypeptide according to any one of claims 1 to 5, wherein said anti25 CX3CR1 immunoglobulin single variable domain ls a VHH domain comprising the sequence set forth in:a) the amino acid sequence of SEQ ID NO: 49;b) an amino acid sequence that has at least 95% amino acid Identlty with the amino acid sequences of SEQ ID NO: 49; or30 c) an amino acid sequence of any one of SEQ ID NO: 49-52.-15817072
- 14. The polypeptide according to any one of claims 1 to 5, wherein sald antiCX3CR1 immunoglobulin single variable domain is a VHH domain comprising the sequence set forth ln:a) the amino acid sequence of SEQ ID NO: 67;s b) an amino acid sequence that has at least 90% amino acid Identity with the amino acid sequences of SEQ ID NO: 67; orc) an amino acid sequence of any one of SEQ ID NO: 53-120.
- 15. The polypeptide according to claim 1, wherein sald antl-CX3CR1 io immunoglobulin single variable domain comprises the sequence set forth ln SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 121-140 or SEQ ID NO: 222-224.
- 16. The polypeptide according to any one of claims 1 to 15, wherein said Immunoglobulin single variable domain eithera) cross-blocks the binding of, orb) is cross blocked from binding by at least one of the immunoglobulin single variable domains of SEQ ID NO's: 1 20 120.121 -140 and 222-224 to CX3CR1.
- 17. The polypeptide according to any one of claims 1 to 16, wherein the polypeptide further comprises a haif-life extending moiety, which Is covalently linked to sald polypeptide and is selected from the group consisting of an albumin25 binding moiety, such as an anti-albumin Immunoglobulin domain, a transferrin binding moiety, such as an anti-transferrin Immunoglobulin domain, a polyethylene giycol molécule, a recombinant poiyethylene glycoi molécule, human sérum albumln, a fragment of human sérum albumin, an albumin binding peptide or a Fc domain and further wherein said half-life extending moiety30 consists of an antl-albumin immunoglobulin single variable domain.
- 18. The polypeptide according to claim 17, wherein the immunoglobulin singie variable domain Is selected from a VHH domain, a humanlzed VHH domain, a-15917072 camelized VH domain, a domain antibody, a single domain antibody and/or dAb’s and which comprises a sequence selected from any one of SEQ ID NO’s: 230-232.
- 19. The polypeptide according to any one of daims 1 to 17, wherein said polypeptide Is iinked to an Fc portion, optionally via a suitable linkeror hinge région.
- 20. The polypeptide according to any one of daims 1 to 16, wherein said polypeptide further comprises a second immunoglobulin single variable domain, comprising a second anti-CX3CR1 Immunoglobulin single variable domain.
- 21. The polypeptide according to claim 20, wherein said first and second immunoglobulin single variable domains are a VH, VL, VHH, camelized VH, or VHH.
- 22. The polypeptide according to daim 20 or 21, wherein said second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 amino add sequences set forth In any one of daims 6 to 11 and wherein said first and said second Immunoglobulin single variable domains comprise the same CDR1,CDR2and CDR3.
- 23. The polypeptide according to any of daims 20 to 22, wherein said first and second immunoglobulin single variable domains comprise a VHH domain having an amino acid sequence set forth In any one of daims 12-15, more particularly, wherein said first and said second immunoglobulin single variable domains comprise the same VHH domain.
- 24. The polypeptide according to daim 1, comprising a first Immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 having amino add sequences set forth In SEQ ID NO’s: 141,164 and 186, or SEQ ID NO's: 141, 162 and 186, or SEQ ID NO’s: 213, 214 and 186, or SEQ ID NO’s: 213, 221 and 186, and a second immunoglobulin single variable domain as set forth In any one of daims 1 to 16, more particularly, wherein said second Immunoglobulin single-16017072 variable domain comprises CDR1, CDR2 and CDR3 having amino acid sequences set forth SEQ ID NO’s: 141,164 and 186, or SEQ ID NO’s: 141,162 and 186, or SEQ ID NO’s: 213, 214 and 186, or SEQ ID NO’s: 213, 221 and 186.s 25. The polypeptide according to claim 1, comprising a first Immunoglobulin single variable domain, wherein said first immunoglobulin single variable domain is a VHH domain comprising the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, or any of SEQ ID NO: 121-140 or 222-224 and a second immunoglobulin single variable domain according to any one of claims 1-16, more particulariy, 10 wherein said second Immunoglobulin single variable domain Is a VHH domain comprising the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, or any one of SEQ ID NO: 121-140 or 222-224.26. The polypeptide according to any one of claims 20-25, wherein the is polypeptide further comprises a haif-iife extending moiety, which Is covalently linked to said polypeptide and is selected from the group consisting of an albumin binding moiety, such as an anti-aibumin Immunoglobulin domain, a transferrin binding moiety, such as an antl-transfemn immunoglobulin domain, a polyethylene glycol molecuie, a recombinant polyethyiene glycol molecuie, x human sérum albumin, a fragment of human sérum albumin, an albumin binding peptide or a Fc domain and further wherein said haif-iife extending moiety constats of an anti-albumln Immunoglobulin single variable domain, which is selected from a VHH domain, a humanized VHH domain, a camelized VH domain, a domain antibody, a single domain antibody and/or dAbs.27. The polypeptide according to claim 26, wherein the anti-aibumin Immunoglobulin single variable domain is selected from SEQ ID NO’s: 230-232.28. The polypeptide according to claim 26, wherein said polypeptide is linked to 30 an Fc portion, optionally via a suitable linker or hlnge région.29. The polypeptide according to any one of claims 1 to 5 comprising the amino acid sequence of any one of SEQ ID NO: 225-227 or 257-262.-161 1707230. The polypeptide according to any one of daims 1 to 5 comprising the amino acid sequence of any one of SEQ ID NO: 253,254, or 263 to 266.31. The polypeptide according to any one of daims 1 to 5 comprising the amino s add sequence of any one of SEQ ID NO: 249,277-281, and 267-276.32. A nucleic acid molécule encoding a polypeptide according to any one of daims 1 to 42.10 33. An expression vedor comprising a nucleic add molécule according to daim43.34. A host cell comprising a nucleic acid molécule encoding a polypeptide according to any one of daims 1 to 42, wherein said host cell Is capable of is expressing said polypeptide.35. A pharmaceutical composition comprising (I) a polypeptide according to any one of clalms 1 to 31, and (ii) a pharmaceutically acceptable carrier, and optionally (iil) a diluent, excipient, adjuvant and/or stabilizer, which20 pharmaceutical composition Is suitable for intravenous or subcutaneous injection in a human being.36. A method of manufaduring a polypeptide according to any one of clalms 1 to 31, comprising the steps of
- 25 - culturing a host cell under conditions that allow expression of a polypeptide according to any one of clalms 1 to 31, wherein said host cell Is carrying an expression vedor comprising a nucleic acid molécule, said nucleic acid molécule comprising a région encoding a polypeptide according to any one of daims 1 to 31, and
- 30 wherein said host cell is a prokaryotic or a eukaryotic cell;- recovering said polypeptide; and- purifying said polypeptide.-1621707237. A polypeptide according to any of claims 1 to 31 for use in the treatment, prévention or aliénation of a disease, disorder or condition, in a human being, wherein the disease, disorder or condition Is a CX3CR1-associated disease, disorder or condition and more specifically wherein the disease, disorder or condition is selected from cardio- and cerebrovascular atherosclerotlc disorders, peripheral artery disease, restenosis, dlabetlc nephropathy, glomerulomephritis, human crescentic glomerulonephritls, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schonlein purpura and Wegener*s granuiomatosis, rheumatoid arthritis, osteoarthritis, allograft rejection, systemic sclerosls, neurodegenerative disorders and demyelinating disease, multiple sclerosls (MS), Alzhelmer*s disease, puimonary diseases such as COPD, asthma,neuropathie pain, Inflammatory pain, or cancer, and even more specifically wherein the disease, disorder or condition Is atherosclerosis.38. A diagnostic kit or diagnostic method comprising a polypeptide according to any one of claims 1 to 31, or the use thereof for the dïagnosis of at least one of cardio- and cerebrovascular atherosclerotlc disorders, peripheral artery disease, restenosis, diabetic nephropathy, glomerulomephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schonlein purpura and Wegeneris granuiomatosis, rheumatoid arthritis, osteoarthritis, allograft rejection, systemic sclerosls, neurodegenerative disorders and demyelinating disease, multiple sclerosls (MS), Alzheimer*s disease, puimonary diseases such as COPD, asthma,neuropathie pain, Inflammatory pain, or cancer.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US61/603622 | 2012-02-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| OA17072A true OA17072A (en) | 2016-03-23 |
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