Immunoglobulin variable domains

Abstract

VH domain, in which: (i) the amino acid residue at position 112 is one of K or Q; and/or (ii) the amino acid residue at position 89 is T; and/or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q; and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V; and in which said VH domain contains a C-terminal extension (X)n, in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an (preferably naturally occurring) amino acid residue that is independently chosen, and preferably independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

Claims

1. A nucleic acid encoding a polypeptide, wherein the polypeptide comprises a heavy chain immunoglobulin single variable domain (ISVD) in which the amino acid residue at Kabat position 11 is V and the amino acid residue at Kabat position 89 is L, and wherein the heavy chain ISVD specifically binds to: (A) IL-23 and comprises: (i) a CDR1 sequence that is the sequence of SEQ ID NO: 173; a CDR2 sequence that is the sequence of SEQ ID NO: 174; and a CDR3 sequence that is the sequence of SEQ ID NO: 175; or (ii) a CDR1 sequence that is the sequence of SEQ ID NO: 191; a CDR2 sequence that is the sequence of SEQ ID NO: 192; and a CDR3 sequence that is the sequence of SEQ ID NO: 193; or (B) TNF and comprises: a CDR1 sequence that is the sequence of SEQ ID NO: 335; a CDR2 sequence that is the sequence of SEQ ID NO: 336; and a CDR3 sequence that is the sequence of SEQ ID NO: 337.

2. The nucleic acid of claim 1, wherein the heavy chain ISVD comprises: an amino acid residue at Kabat position 14 that is selected from the group consisting of A and P; an amino acid residue at Kabat position 41 that is selected from the group consisting of A and P; an amino acid residue at Kabat position 108 that is selected from the group consisting of Q and L; and an amino acid residue at Kabat position 110 that is selected from the group consisting of T and Q.

3. The nucleic acid of claim 1, wherein the heavy chain ISVD specifically binds to IL-23 and comprises: a CDR1 sequence that is the sequence of SEQ ID NO: 173; a CDR2 sequence that is the sequence of SEQ ID NO: 174; and a CDR3 sequence that is the sequence of SEQ ID NO: 175.

4. The nucleic acid of claim 3, wherein the heavy chain ISVD comprises: an amino acid residue at Kabat position 14 that is selected from the group consisting of A and P; an amino acid residue at Kabat position 41 that is selected from the group consisting of A and P; an amino acid residue at Kabat position 108 that is selected from the group consisting of Q and L; and an amino acid residue at Kabat position 110 that is selected from the group consisting of T and Q.

5. The nucleic acid of claim 1, wherein the heavy chain ISVD specifically binds to IL-23 and comprises: a CDR1 sequence that is the sequence of SEQ ID NO: 191; a CDR2 sequence that is the sequence of SEQ ID NO: 192; and a CDR3 sequence that is the sequence of SEQ ID NO: 193.

6. The nucleic acid of claim 5, wherein the heavy chain ISVD comprises: an amino acid residue at Kabat position 14 that is selected from the group consisting of A and P; an amino acid residue at Kabat position 41 that is selected from the group consisting of A and P; an amino acid residue at Kabat position 108 that is selected from the group consisting of Q and L; and an amino acid residue at Kabat position 110 that is selected from the group consisting of T and Q.

7. The nucleic acid of claim 1, wherein the heavy chain ISVD specifically binds to TNF and comprises: a CDR1 sequence that is the sequence of SEQ ID NO: 335; a CDR2 sequence that is the sequence of SEQ ID NO: 336; and a CDR3 sequence that is the sequence of SEQ ID NO: 337.

8. The nucleic acid of claim 7, wherein the heavy chain ISVD comprises: an amino acid residue at Kabat position 14 that is selected from the group consisting of A and P; an amino acid residue at Kabat position 41 that is selected from the group consisting of A and P; an amino acid residue at Kabat position 108 that is selected from the group consisting of Q and L; and an amino acid residue at Kabat position 110 that is selected from the group consisting of T and Q.

9. The nucleic acid of claim 1, wherein the heavy chain ISVD is a VHH domain, a humanized VHH domain, or a camelized VH domain.

10. The nucleic acid of claim 1, wherein the polypeptide comprises: a first heavy chain ISVD, wherein the first heavy chain ISVD corresponds to the heavy chain ISVD that specifically binds to IL-23 or TNF; and a second heavy chain ISVD.

11. The nucleic acid of claim 10, wherein the first and the second ISVDs are linked directly or via a suitable linker.

12. The nucleic acid of claim 10, wherein the second heavy chain ISVD specifically binds to serum albumin.

13. The nucleic acid of claim 12, wherein the second heavy chain ISVD has at least 90% sequence identity with Alb-8 (SEQ ID NO:46) or Alb-23 (SEQ ID NO:61).

14. The nucleic acid of claim 12, wherein the second heavy chain ISVD comprises a CDR1, CDR2, and CDR3 in which: CDR1 comprises the amino acid sequence SFGMS (SEQ ID NO:41); CDR2 comprises the amino acid sequence SISGSGSDTLYADSVKG (SEQ ID NO:42); and CDR3 comprises the amino acid sequence GGSLSR (SEQ ID NO:43).

Description

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

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

(3) FIG. 2 lists the sequences referred to herein;

(4) FIG. 3 is a plot showing data points obtained in Example 4 when 96 serum samples were tested for binding a representative Nanobody with an S112K mutation (Reference A+S 112K+C-terminal alanine, indicated as (2) in FIG. 3), compared to a reference Nanobody without an S112K mutation (Reference A, SEQ ID NO: 44, indicated as (1) in FIG. 3);

(5) FIG. 4 is a plot showing data points obtained in Example 4 when 129 serum samples were tested for binding a representative Nanobody with an V89T mutation (Reference A+L11V+V89T+C-terminal alanine, indicated as (2) in FIG. 4), compared to a reference Nanobody without an V89T mutation (Reference A, SEQ ID NO: 44, indicated as (1) in FIG. 4);

(6) FIG. 5 is a plot showing data points obtained in Example 5 when 100 serum samples were tested for binding representative Nanobodies with V89L, T110K and/or T110Q mutations (Reference A+L11V+V89L+C-terminal Ala, indicated as (2) in FIG. 5; Reference A+L11V+V89L+T110K+C-terminal Ala, indicated as (3) in FIG. 5; Reference A+L11V+V89L+T110Q+C-terminal Ala, indicated as (4) in FIG. 5) and Reference A+L11V+T87A+V89L+C-terminal Ala, indicated as (5) in FIG. 5), compared to a reference Nanobody without any of these mutations (Reference A, SEQ ID NO: 44, indicated as (1) in FIG. 5).

(7) FIG. 6 is a plot showing data points obtained in Example 6 when 98 serum samples obtained from healthy volunteers were tested for binding representative trivalent Nanobody constructs. Each dot represents a data point collected by testing of the indicated construct against one of the 98 serum samples. Legenda: (1)=Reference X (Nanobody A-35GS-Nanobody A-35GS-Nanobody B); (2)=Reference X+C-terminal Ala; (3)=Reference X+L11V+V89L+C-terminal Ala; (4)=Reference X+L11V+T87A+V89L+C-terminal Ala; (5)=Reference X+L11V+V89L+T110K+C-terminal Ala; (6)=Reference X+L11V+V89L+T110Q+C-terminal Ala.

(8) FIG. 7 is a plot showing data points obtained in Example 6 when 30 serum samples obtained from healthy volunteers (samples selected for high titers of pre-existing antibodies or presence of pre-existing antibodies with high degree of binding even in the presence of a C-terminal alanine extension) were tested for binding representative trivalent Nanobody constructs. Each dot represents a data point collected by testing of the indicated construct against one of the 30 serum samples. Legenda: (1)=Reference X+C-terminal Ala; (2)=Reference X+L11V+V89L+C-terminal Ala; (4)=Reference X+L11V+T87A+V89L+C-terminal Ala; (4)=Reference X+L11V+V89L+T110K+C-terminal Ala.

(9) FIG. 8 is a plot showing data points obtained in Example 6 when 98 serum samples obtained from healthy volunteers were tested for binding representative bivalent Nanobody constructs. Each dot represents a data point collected by testing of the indicated construct against one of the 30 serum samples. Legenda: (1)=Reference Y (Nanobody A-35GS-Nanobody B); (2)=Reference Y+C-terminal Ala; (3)=Reference Y+L11V+V89L+C-terminal Ala; (4)=Reference Y+L11V+T87A+V89L+C-terminal Ala; (5)=Reference Y+L11V+V89L+T110K+C-terminal Ala; (6)=Reference Y+L11V+V89L+T110Q+C-terminal Ala;

(10) FIGS. 9A and 9B show preferred but non-limiting examples of monovalent Nanobodies of the invention (FIG. 9A) and trivalent bispecific half-life extended compounds of the invention (FIG. 9B) against the ion channel Kv1.3; and FIG. 9C lists some preferred CDRs (classification according to Kabat and Abm, respectively) for ISVDs against Kv1.3.

(11) FIG. 10 is a plot showing data points obtained in Example 7 when 47 serum samples obtained from diabetic human subjects were tested for binding representative trivalent bispecific half-life extended compounds of the invention against Kv1.3. Each dot represents a data point collected by testing of the indicated construct against one of the 47 serum samples. The SEQ ID NO's refer to the relevant sequences listed in FIG. 9;

(12) FIG. 11 is a plot showing data points obtained in Example 7 when 90 serum samples obtained from healthy volunteers were tested for binding representative trivalent bispecific half-life extended compounds of the invention against Kv1.3. Each dot represents a data point collected by testing of the indicated construct against one of the 47 serum samples. The SEQ ID NO's refer to the relevant sequences listed in FIG. 9.

(13) FIGS. 12A and 12B list the CDR's and amino sequences of some preferred, but non-limiting examples of Nanobodies of the invention against IL-23 that are based on the indicated reference sequences. See also Example 8;

(14) FIG. 13 lists the CDR's and amino sequences of some preferred, but non-limiting examples of Nanobodies of the invention against OX40-L that are based on the indicated reference sequence. See also Example 9;

(15) FIG. 14 lists the CDR's and amino sequences of some preferred, but non-limiting examples of Nanobodies of the invention against IgE that are based on the indicated reference sequence. See also Example 10;

(16) FIGS. 15A and 15B list the CDR's and amino sequences of some preferred, but non-limiting examples of Nanobodies of the invention against CXCR-4 that are based on the indicated reference sequences. See also Example 11;

(17) FIGS. 16A and 16B list the CDR's and amino sequences of some preferred, but non-limiting examples of Nanobodies of the invention against HER-3 that are based on the indicated reference sequences. See also Example 12;

(18) FIGS. 17A and 17B list the CDR's and amino sequences of some preferred, but non-limiting examples of Nanobodies of the invention against TNF that are based on the indicated reference sequences. See also Examples 13 and 14;

(19) FIGS. 18A and 18B list the CDR's and amino sequences of some preferred, but non-limiting examples of Nanobodies of the invention against c-Met that are based on the indicated reference sequences. See also Example 15;

(20) FIG. 19 lists the CDR's and amino sequences of some preferred, but non-limiting examples of Nanobodies of the invention against RANK-L that are based on the indicated reference sequence. See also Example 16;

(21) FIGS. 20A to 20C list the CDR's and amino sequences of some preferred, but non-limiting examples of Nanobodies of the invention against CXCR-7 that are based on the indicated reference sequences. See also Example 17;

(22) FIGS. 21A and 21B list the CDR's and amino sequences of some preferred, but non-limiting examples of Nanobodies of the invention against A-beta that are based on the indicated reference sequences. See also Example 18;

(23) FIG. 22 gives the amino acid sequences of some preferred but non-limiting examples of compounds of the invention against IL-23;

(24) FIG. 23 gives the amino acid sequences of some preferred but non-limiting examples of compounds of the invention against OX40-L;

(25) FIG. 24 gives the amino acid sequences of some preferred but non-limiting examples of compounds of the invention against IgE;

(26) FIG. 25 gives the amino acid sequences of some preferred but non-limiting examples of compounds of the invention against CXCR-4;

(27) FIG. 26 gives the amino acid sequences of some preferred but non-limiting examples of compounds of the invention against HER-3;

(28) FIG. 27 gives the amino acid sequences of some preferred but non-limiting examples of compounds of the invention against TNF;

(29) FIGS. 28A and 28B give the amino acid sequences of some preferred but non-limiting examples of compounds of the invention against c-Met;

(30) FIG. 29 gives the amino acid sequences of some preferred but non-limiting examples of compounds of the invention against RANK-L;

(31) FIG. 30 gives the amino acid sequences of some preferred but non-limiting examples of compounds of the invention against A-beta;

(32) FIG. 31A is a plot showing data points obtained in Example 19 when 92 serum samples obtained from healthy volunteers were tested for binding representative trivalent bispecific half-life extended compounds of the invention against A-beta. Each dot represents a data point collected by testing of the indicated construct against one of the 92 serum samples. The reference numbers are as listed in Table CC-1. Similarly, FIG. 31B is a plot showing data points obtained in Example 19 when 92 serum samples obtained from healthy volunteers were tested for binding the (monovalent) C-terminal Nanobodies that are present in the constructs tested in FIG. 31A. Each dot represents a data point collected by testing of the indicated construct against one of the 92 serum samples. The reference numbers are as listed in Table CC-2.

EXPERIMENTAL PART

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

(34) In the Examples below, the binding of pre-existing antibodies that are present in the samples used (i.e. from healthy volunteers, rheumatoid arthritis (RA) patients and SLE patients) to the Nanobodies tested was determined using ProteOn as follows: Binding of pre-existing antibodies on Nanobodies captured on human serum albumin (HSA) was evaluated using the ProteOn XPR36 (Bio-Rad Laboratories, Inc.). PBS/Tween (phosphate buffered saline, pH7.4, 0.005% Tween20) was used as running buffer and the experiments were performed at 25° C. The ligand lanes of a ProteOn GLC Sensor Chip were activated with EDC/NHS (flow rate 30 μl/min) and HSA was injected at 10 μg/ml in ProteOn Acetate buffer pH4.5 (flow rate 100 μl/min) to render immobilization levels of approximately 3200 RU. After immobilization, surfaces were deactivated with ethanolamine HCl (flow rate 300/min). Nanobodies were injected for 2 minutes at 45 μl/min over the HSA surface to render a Nanobody capture level of approximately 200 RU. The samples containing pre-existing antibodies were centrifuged for 2 minutes at 14,000 rpm and supernatant was diluted 1:10 in PBS-Tween20 (0.005%) before being injected for 2 minutes at 45 μl/min followed by a subsequent 400 seconds dissociation step. After each cycle (i.e. before a new Nanobody capture and blood sample injection step) the HSA surfaces were regenerated with a 2 minute injection of HCl (100 mM) at 45 μl/min. Sensorgram processing and data analysis was performed with ProteOn Manager 3.1.0 (Bio-Rad Laboratories, Inc.). Sensorgrams showing pre-existing antibody binding were obtained after double referencing by subtracting 1) Nanobody-HSA dissociation and 2) non-specific binding to reference ligand lane. Binding levels of pre-existing antibodies were determined by setting report points at 125 seconds (5 seconds after end of association). Percentage reduction in pre-existing antibody binding was calculated relative to the binding levels at 125 seconds of a reference Nanobody.

Example 1: S112K Mutation Inhibits Binding of Pre-Existing Antibodies

(35) The influence of a substitution at position 112 on the binding of pre-existing antibodies in human samples to Nanobodies was determined and compared to the influence of a C-terminal alanine extension as described in WO 12/175741.

(36) Two reference compounds (Reference A without a C-terminal alanine extension and Reference B with a C-terminal alanine extension) and variants of these reference compounds with different mutations at position 112 were tested against sera obtained from six different RA patients and eight sera obtained from different healthy human subjects. Binding of pre-existing antibodies in the samples to the Nanobodies tested was measured on ProteOn according to the general protocol outlined above. The results are shown in Table A below.

(37) As can be seen, of the mutations at position 112 that were tested, the S112K mutation gave a reduction of binding of the pre-existing antibodies that were present in the tested sera that was comparable to that of the C-terminal alanine extension (even without the C-terminal alanine extension being present in the S112K variant). Similar results were obtained with three human plasma samples (data not shown).

(38) TABLE-US-00006 TABLE A comparison of mutations at position 112 to a C-terminal alanine extension on binding of pre-existing antibodies present in sera from RA patients and human volunteers RA sera healthy human sera Average binding on Reference A (RU) Reference A 75 96 44 11 117 141 242 81 121 179 93 92 91 92 Inhibition compared to average binding on Reference A (%) Reference A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Reference B 70 90 100 88 84 97 53 78 83 93 93 81 86 86 Reference A (S112E) 51 67 63 65 66 84 31 82 72 59 73 100 74 70 Reference A (S112F) 75 51 85 100 79 39 56 86 69 60 78 93 76 79 Reference A (S112K) 88 88 94 100 94 100 86 93 87 87 95 100 84 91 Reference A (S112L) 69 45 63 54 50 81 37 74 59 42 75 97 68 83

Example 2: Influence of S112K Mutation on Binding of Pre-Existing Antibodies that are Present in Human SLE Samples

(39) The same Nanobody variants as used in Example 1 were tested for binding by pre-existing antibodies from 7 serum samples obtained from patients who were confirmed positive for systemic lupus erythematosus (SLE). For comparison, plasma samples from two healthy human volunteers were included.

(40) Binding of pre-existing antibodies in the samples to the Nanobodies tested was measured on ProteOn according to the general protocol outlined above. The results are shown in Table B below.

(41) As can be seen from a comparison of the binding data for Reference A and Reference B and nanobodies of the invention, the samples obtained from some of the SLE patients appear to contain certain pre-existing antibodies that can still bind to Nanobodies even in the presence of a C-terminal alanine residue (the C-terminal alanine residue did essentially prevent/remove (partially or essentially fully) all binding of the pre-existing antibodies that were present in the plasma samples from healthy volunteers).

(42) It can further be seen that the binding of these pre-existing antibodies from SLE samples could be greatly reduced by mutations at positions 11 and 112 (and in case of position 112, in particular S112K).

(43) TABLE-US-00007 TABLE B comparison of mutations at positions 11 and 112 to a C-terminal alanine extension on binding of pre-existing antibodies present in sera from SLE patients Plasma samples obtained from healthy Serum samples obtained from SLE patients volunteers Average binding on Reference A (RU) Reference A 45 61 38 40 43 20 69 128 171 Inhibition compared to binding on Reference A (%) Reference B 20 16 13 45 53 86 101 95 90 Reference A (L11E) 63 88 117 61 87 88 92 68 21 Reference A (L11K) 87 97 107 54 106 79 102 100 61 Reference A (L11V) 68 84 49 56 95 91 21 23 6 Reference A (L11Y) 27 71 111 37 84 74 72 13 3 Reference A (S112E) 13 56 91 77 74 91 94 84 22 Reference A (S112F) −6 18 26 −13 62 69 117 74 43 Reference A (S112K) 71 77 105 80 116 86 120 87 62 Reference A (S112L) −36 36 48 −24 123 19 84 91 3

Example 3: Influence of Combined Framework Mutations and C-Terminal Extensions on Binding of Pre-Existing Antibodies that are Present in Human SLE Samples

(44) Four different Nanobodies (with specific framework mutations and with or without C-terminal alanine extension) were tested for binding of pre-existing antibodies from 5 serum samples obtained from patients who were confirmed positive for systemic lupus erythematosus (SLE). For comparison, one plasma sample from a healthy human volunteer was included.

(45) Binding of pre-existing antibodies in the samples to the Nanobodies tested was measured on ProteOn according to the general protocol outlined above. The results are shown in Tables C and D below.

(46) As can be seen from a comparison of the binding data for Reference A and Reference B, the samples obtained from SLE patients appear to contain a certain pre-existing antibodies that can still bind to Nanobodies even in the presence of a C-terminal alanine residue (the C-terminal alanine residue did essentially prevent/remove all binding of the pre-existing antibodies that were present in the plasma samples from the healthy volunteer).

(47) It can further be seen that the binding of these pre-existing antibodies from SLE samples could be greatly reduced by mutations at positions 11 and 112 (and in case of position 112, in particular S112K).

Example 4: Influence of a V89T Mutation on Binding of Pre-Existing Antibodies in Samples from SLE Patients

(48) As described herein, samples obtained from certain SLE patients appear to contain pre-existing antibodies/factors that can bind to the exposed C-terminal end of a VH domain, even when a C-terminal extension is present. It was investigated whether a V89T mutation could reduce or prevent/remove such binding, with or without the presence of a C-terminal extension. The results are also shown in Tables C and E below.

(49) As can be seen, a V89T mutation could essentially prevent/remove binding of pre-existing antibodies that are present in samples obtained from SLE patients, to a similar degree as an S112K mutation. However, as can be seen from comparing the data given in Tables C and E for nanobodies with a V89T mutation without a C-terminal extension with similar nanobodies with an S112K mutation and without a C-terminal extension, having a mutation at position 112 in a nanobody without a C-terminal extension generally reduces binding of pre-existing antibodies in samples from a healthy volunteer to a larger degree than a V89T mutation (i.e. 100%, 85% and 64% of S112K nanobodies vs. 9%, 11% and 16% for V89T nanobodies, respectively). For this reason, the use of a mutation at position 112 (and in particular S112K or S112K) will often be preferred over the use of a mutation at position 89 (such as V89T).

(50) However, as can also be seen from the data in Tables C and E, adding a C-terminal alanine to a V89T nanobody completely prevented/removed the binding of pre-existing antibodies in a sample obtained from healthy volunteers, and for this reason a combination of a V89T mutation and a C-terminal extension as described herein will usually be preferred (i.e. over the use of a V89T without C-terminal extension) if the V89T nanobody or VH domain has, or is intended to have, an exposed C-terminal region in the protein or polypeptide in which it will be present (for example, because if forms the C-terminal end of the same).

(51) TABLE-US-00008 TABLE C influence of different mutations of binding by pre-existing antibodies in samples obtained from SLE patients and human volunteers Sample obtained from healthy Mutation(s) Samples obtained from SLE patients volunteer L11K L11V V89T S112K 114A SLE25 SLE37 SLE39 SLE41 NB13025-14 004-030-ABL-02 Average binding to Reference A Average binding for 38 66 30 41 45 175 Reference A Inhibition compared to average binding to Reference A captured on HSA (%) Reference A + V89T, x x 100 98 100 100 98 9 no C-terminal extension Reference A + V89T + x x x 97 98 100 98 100 100 C-terminal alanine Reference A + S112 K, X x 100 100 100 100 98 100 no C-terminal extension Reference A + S 112K + X x x 100 100 100 99 99 100 C-terminal alanine (*) (*) Note: this Nanobody was also used to generate the data shown in FIG. 3 and Table F (see below)

(52) TABLE-US-00009 TABLE D influence of different mutations of binding by pre-existing antibodies in samples obtained from SLE patients and human volunteers Sample obtained from healthy Mutation Samples obtained from SLE patients volunteer L11V V89L S112Q 114A SLE25 SLE37 SLE39 SLE41 NB13025-14 004-030-ABL-02 Average binding to Reference A Average binding for ND 71 51 ND 41 180 Reference A Inhibition compared to average binding to Reference A captured on HSA (%) Reference A + V89L + x x x ND 100 100 ND 100 97 S 112Q + C-terminal alanine Reference A + L11V + x x x ND 100 100 ND 100 99 S 112Q + C-terminal alanine Reference A + S 112Q + x x ND 92 85 ND 94 100 C-terminal alanine

(53) TABLE-US-00010 TABLE E influence of different mutations on binding by pre-existing antibodies in samples from SLE patients and human volunteers Sample from healthy Mutation(s) Samples obtained from SLE patients volunteer L11V V89L V89T S112K 114A SLE25 SLE37 SLE39 SLE41 NB13025-14 004-030-ABL-02 Average binding to Reference A Reference A 28 44 26 33 30 151 Inhibition compared to average binding to Reference A captured on HSA (%) Reference A + V89L, x 77 64 53 63 41 35 no C-terminal extension Reference A + V89L + x x 35 27 63 42 46 83 C-terminal alanine Reference A + V89T, x 68 12 84 100 71 11 no C-terminal extension Reference A + V89T + x x 46 35 71 100 97 99 C-terminal alanine Reference A + V89T + x x 100 97 100 100 100 16 L11V, no C-terminal extension Reference A + V89T + x x x 100 100 100 100 100 67 L11V + C-terminal alanine (*) Reference A + S112K + x x 100 100 100 100 100 85 V89L, no C-terminal extension Reference A + S112K + x x x 100 100 100 100 100 100 V89L C-terminal alanine Reference A + S112K + x x 100 100 100 100 100 64 L11V, no C-terminal extension Reference A + S112K + x x x 100 100 100 100 100 100 L11V + C-terminal alanine (*) Note: this Nanobody was also used to generate the data shown in FIG. 4 and Table G (see below)

(54) To confirm that the results/findings from the table above are broadly applicable, representative Nanobodies with S112K and/or V89T mutations were tested against a test panel of 96 (S112k) and 129 (V89T) human serum samples. Binding was determined on ProteOn using the protocol set out above.

(55) The results are summarized FIG. 3 and Table F (representative Nanobody with an S112K mutation) and FIG. 4 and Table G (representative Nanobody with a V89T mutation).

(56) In FIG. 3, a Nanobody with an S112K mutation (Reference A+S112K+C-terminal alanine—see Table C above) was compared to a reference Nanobody (Reference A; SEQ ID NO:44). The Nanobody with the S112K mutation and Reference A were both tested against each of the serum samples, and the binding level at 125 seconds (RU) was determined. The data was then plotted in FIG. 3, with each point presenting the binding measured in one sample for either Reference A (indicated as (1) in FIG. 3) or for the S112K mutant (indicated as (2) in FIG. 3). The dotted line indicates a measured binding level of 20 RU.

(57) The same data is also represented numerically in Table F, which mentions—for Reference A and the S112K mutant, respectively—the total number of samples tested that gave a level of binding at 125 seconds of more than 20 RU, less than 20 RU (i.e. between 0 and 20 RU) and less than 10 RU.

(58) As can be seen from the data plotted in FIG. 3 and shown in Table F, for Reference A, more than half of the 96 samples tested gave a binding level of more than 20 RU (in some cases, as high as 150-200 RU), indicating that the pre-existing antibodies present in the sample were binding to Reference A. By comparison, for the S112K mutant, no sample gave a binding level of more than 20 RU (and most less than 10 RU), indicating that the S122K mutation was essentially capable of reducing/preventing binding of the pre-existing antibodies in all of the 96 samples tested.

(59) A similar plot and similar data is shown in FIG. 4 and Table G, respectively, for a representative Nanobody with a V89T mutation (Reference A+L11V+V89T+C-terminal alanine; see Table E above), tested against 129 serum samples and again compared to Reference A (indicated by (1) in FIG. 4; the V89T mutant is indicated by (2) in FIG. 4). Again, from the plot in FIG. 4 and the data in Table G, it can be seen that with a few exceptions (i.e. less than 10% of samples tested, which then each gave an absolute binding value after 125 seconds of about 100 RU or less), the V89T mutation was capable of reducing/preventing binding of the pre-existing antibodies in most of the 129 samples tested, whereas the reference without the V89T mutation was bound by pre-existing antibodies in most of the samples tested.

(60) TABLE-US-00011 TABLE F testing of representative Nanobody with S112K mutation against 96 serum samples. Binding Binding Binding level at 125 level at 125 level at 125 seconds <10 seconds <20 seconds >20 Nanobody tested RU RU RU Reference A 34 41 55 Reference A + S112K + 92 96 0 C-terminal alanine

(61) TABLE-US-00012 TABLE G testing of Nanobody mutants with V89L and/or T100K/Q mutations against 129 serum samples. Binding Binding Binding level at 125 level at 125 level at 125 seconds <10 seconds <20 seconds >20 Nanobody tested RU RU RU Reference A 27 40 89 Reference A + L11V + 110 119 10 V89T + C-terminal alanine

Example 5: Influence of a V89L Mutation Combined with a T110K or T110Q Mutation on Binding of Pre-Existing Antibodies in Samples from SLE Patients

(62) As described herein, samples obtained from certain SLE patients appear to contain pre-existing antibodies/factors that can bind to the exposed C-terminal end of a VH domain, even when a C-terminal extension is present. It was investigated whether a V89L and/or a T110Q or T110K mutation (or a combination thereof) could reduce or prevent/remove such binding, with or without the presence of a C-terminal extension. The results are also shown in Tables H and I, which present the data from two separate experiments (Table D above also shows data for an S112Q mutation of the invention combined with a V89L mutation). The Nanobodies used in Table I were also used in generating the data shown in FIG. 5 and Table J.

(63) TABLE-US-00013 TABLE H influence of different mutations on binding by pre-existing antibodies in samples from SLE patients and human volunteers Sample from healthy Mutation(s) Samples obtained from SLE patients volunteer L11V V89L T110K T110Q 114A SLE25 SLE37 SLE39 SLE41 NB13025-14 004-030-ABL-02 Average binding to Reference A Reference A 28 44 26 33 30 151 Inhibition compared to average binding to Reference A captured on HSA (%) Reference A + V89L, x 77 64 53 63 41 35 no C-terminal extension Reference A + V89L + x x 35 27 63 42 46 83 C-terminal alanine Reference A + L11V + x x 100 100 8 95 100 29 V89L, no C-terminal extension Reference A + L11V + x x x 94 70 100 78 100 89 V89L + C-terminal alanine (*) Reference A + V89L + x x 100 77 100 100 100 65 T110K, no C-terminal extension Reference A + V89L + x x x 96 70 100 100 100 100 T110K, C-terminal alanine Reference A + L11V + x x 100 0 100 100 100 43 T110K, no C-terminal extension Reference A + L11V + x x x 96 0 100 100 100 99 T110K + C-terminal alanine (*) This Nanobody was also used in generating the data shown in FIG. 5 and Table J

(64) TABLE-US-00014 TABLE I influence of different mutations on binding by pre-existing antibodies in samples from SLE patients and human volunteers. Mutation(s) Samples obtained from SLE patients L11V T87A V89L T110K T110Q 114A SLE25 SLE37 SLE39 SLE41 NB13025-14 Average binding to Reference A Reference A 50 72 55 56 58 Inhibition compared to average binding to Reference A captured on HSA (%) Reference A + L11V + x x x 100 100 98 100 99 V89L + C-terminal alanine Reference A + L11V + x x x x 100 81 98 99 98 V89L + T110K + C-terminal alanine Reference A + L11V + x x x x 100 100 87 98 81 V89L + T110Q + C-terminal alanine Reference A + L11V + x x x x 100 100 100 100 100 T87A + V89L + C-terminal alanine

(65) To confirm that the results/findings from the table above are broadly applicable, representative Nanobodies with V89L, T110K and/or T110Q mutations were again tested against a panel of 99 human serum samples. Binding data was again obtained and plotted as indicated in Example 4 for the results and data shown in FIGS. 3 and 4 and Tables F and G.

(66) The Nanobodies tested were (the numbers correspond to the numbering used in FIG. 5): (1) Reference A; (2) Reference A+L11V+V89L+C-terminal Ala; (3) Reference A+L11V+V89L+T110K+C-terminal Ala; (4) Reference A+L11V+V89L+T110Q+C-terminal Ala; (5) Reference A+L11V+T87A+V89L+C-terminal Ala. The results are shown in FIG. 5 and Table I.

(67) As can be seen, introducing the mutations tested again greatly reduced the number of samples in which the pre-existing antibodies were capable of binding the Nanobody tested. It can also be seen that, for Nanobody (2) in FIG. 5 (Reference A+L11V+V89L+C-terminal Ala), still some of the samples showed binding of pre-existing antibodies after 125 seconds at levels of more than 20 RU (but already much less than 100 RU). However, when the V89L mutation was combined with a T110K mutation (Nanobody (3)) or a T100Q mutation (Nanobody (4)), then essentially all of the 99 samples tested showed a binding level of less than 20 RU (and in fact less than 10 RU, see Table J).

(68) TABLE-US-00015 TABLE J testing of representative Nanobodies with V89L, T110K and/or T110Q mutations against 99 serum samples. Binding Binding Binding level at 125 level at 125 level at 125 seconds <10 seconds <20 seconds >20 Nanobody tested RU RU RU Reference A 52 64 35 Reference A + L11V + 94 95 4 V89L + C-terminal Ala Reference A + L11V + 99 99 0 V89L + T110K + C-terminal Ala Reference A + L11V + 99 99 0 V89L + T110Q + C-terminal Ala Reference A + L11V + 99 99 0 T87A + V89L + C-terminal Ala

Example 6: Testing of Multivalent Constructs for Binding of Pre-Existing Antibodies

(69) Multivalent constructs are made based on the following nanobodies:

(70) Nanobody A (Directed Against a Therapeutic Target):

(71) TABLE-US-00016 (SEQ ID NO: 92) EVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMGWFRQAPGKEREFVAAI TRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAA SAIGSGALRRFEYDYSGQGTLVTVSS
Nanobody B (Directed Against Serum Albumin):

(72) TABLE-US-00017 (SEQ ID NO: 93) EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSI SGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSL SRSSQGTLVTVSS
Nanobody C (Directed Against a Therapeutic Target):

(73) TABLE-US-00018 (SEQ ID NO: 94) EVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATI TGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVT SRDTWGQGTLVTVSS 

(74) The constructs made are listed in Table K below (with in each case, the introduced substitution(s) in the relevant nanobody (if any) mentioned between parentheses. “HIS6” denotes an N-terminal his-tag of 6 histidine residues, and “—Ala” denotes a C-terminal extension of 1 alanine residue).

(75) TABLE-US-00019 TABLE K multivalent constructs tested HIS6-Nanobody A(E1D, L11V, S112K)-35GS-Nanobody B-Ala HIS6-Nanobody A(E1D, L11V, S112K)-35GS-Nanobody B(L11V, S112K)-Ala HIS6-Nanobody A(E1D)-35GS-Nanobody B HIS6-Nanobody A(E1D)-35GS-Nanobody B-Ala HIS6-Nanobody A(E1D, L11V, S112K)-35GS-Nanobody B(L11V, S112K) HIS6-Nanobody A(E1D, S112K)-35GS-Nanobody B(S112K)-Ala HIS6-Nanobody A(E1D, S112Q)-35GS-Nanobody B(S112Q)-Ala HIS6-Nanobody A(E1D, V89L, S112K)-35GS-Nanobody B(V89L, S112K)-Ala HIS6-Nanobody A(E1D, L11V, S112Q)-35GS-Nanobody B(L11V, S112Q)-Ala HIS6-Nanobody B(E1D)-35GS-Nanobody A HIS6-Nanobody B(E1D)-35GS-Nanobody A-Ala HIS6-Nanobody A(E1D)-35GS-Nanobody B(L11V, S112K)-Ala HIS6-Nanobody B(E1D, L11V, S112K)-35GS-Nanobody A-Ala HIS6-Nanobody B(E1D,)-35GS-Nanobody A(L11V, S112K)-Ala HIS6-Nanobody A(E1D, V89L, S112Q)-35GS-Nanobody B(V89L, S112Q)-Ala HIS6-Nanobody B(E1D, L11V, S112K)-35GS-Nanobody A(L11V, S112K)-Ala HIS6-Nanobody B(S112K)-Ala Nanobody B(L11V, S112K)-Ala Nanobody B(L11V, S112Q)-Ala Nanobody B(S112Q)-Ala HIS6-Nanobody A(E1D, S112K)-35GS-Nanobody A(S112K)-35GS-Nanobody B(S112K)- Ala HIS6-Nanobody A(E1D, S112Q)-35GS-Nanobody A(S112Q)-35GS-Nanobody B(S112Q)- Ala HIS6-Nanobody A(E1D, L11V, S112K)-35GS-Nanobody A(L11V, S112K)-35GS-Nanobody B(L11V, S112K)-Ala HIS6-Nanobody A(E1D, V89L, S112K)-35GS-Nanobody A(V89L, S112K)-35GS-Nanobody B(V89L, S112K)-Ala HIS6-Nanobody A(E1D)-35GS-Nanobody A-35GS-Nanobody B HIS6-Nanobody A(E1D)-35GS-Nanobody A -35GS-Nanobody B-Ala Nanobody C(E1D)-9GS-AlaLB11-9GS-Nanobody C HIS6-Nanobody A(E1D, L11V, S112K)-35GS-Nanobody A(L11V, S112K)-35GS-Nanobody B-Ala HIS6-Nanobody C(E1D, L11V, S112K)-9GS-Nanobody B(L11V, S112K)-9GS-Nanobody C(L11V, S112K)-Ala Nanobody C(E1D)-9GS-AlaLB11-9GS-Nanobody C-Ala HIS6-Nanobody A(E1D, L11V, S112Q)-35GS-Nanobody A(L11V, S112Q)-35GS-Nanobody B(L11V, S112Q)-Ala HIS6-Nanobody A(E1D, V89L, S112Q)-35GS-Nanobody A(V89L, S112Q)-35GS-Nanobody B(V89L, S112Q)-Ala HIS6-Nanobody C(E1D, S112K)-9GS-Nanobody B(S112K)-9GS-Nanobody C(S112K)-Ala HIS6-Nanobody C(E1D, S112Q)-9GS-Nanobody B(S112Q)-9GS-Nanobody C(S112Q)-Ala HIS6-Nanobody C(E1D, L11V, S112Q)-9GS-Nanobody B(L11V, S112Q)-9GS-Nanobody C(L11V, S112Q)-Ala HIS6-Nanobody C(E1D, V89L, S112K)-9GS-Nanobody B(V89L, S112K)-9GS-Nanobody C(V89L, S112K)-Ala HIS6-Nanobody C(E1D, V89L, S112Q)-9GS-Nanobody B(V89L, S112Q)-9GS-Nanobody C(V89L, S112Q)-Ala HIS6-Nanobody A(E1D, L11V, A14P, R39Q, K83R, V89L, T91Y, Q108L)-35GS-Nanobody B(L11V, R30S, E44G, P45L, K83R, V89L, Q108L)-Ala HIS6-Nanobody A(E1D, L11V, A14P, R39Q, K83R, T87A, V89L, T91Y, Q108L)-35GS- Nanobody B(L11V, R30S, E44G, P45L, K83R, T87A, V89L, Q108L)-Ala HIS6-Nanobody A(E1D, L11V, A14P, R39Q, K83R, V89L, T91Y, Q108L, T110Q)-35GS- Nanobody B(L11V, R30S, E44G, P45L, K83R, V89L, Q108L, T110Q)-Ala HIS6-Nanobody A(E1D, L11V, A14P, R39Q, K83R, V89L, T91Y, Q108L, T110K)-35GS- Nanobody B(L11V, R30S, E44G, P45L, K83R, V89L, Q108L, T110K)-Ala HIS6-Nanobody A(E1D, L11V, A14P, R39Q, K83R, V89L, T91Y, Q108L)-35GS-Nanobody A(E1D, L11V, A14P, R39Q, K83R, V89L, T91Y, Q108L)-35GS-Nanobody B(L11V, R30S, E44G, P45L, K83R, V89L, Q108L)-Ala HIS6-Nanobody A(E1D, L11V, A14P, R39Q, K83R, T87A, V89L, T91Y, Q108L)-35GS- Nanobody A(E1D, L11V, A14P, R39Q, K83R, T87A, V89L, T91Y, Q108L)-35GS-Nanobody B(L11V, R30S, E44G, P45L, K83R, T87A, V89L, Q108L)-Ala HIS6-Nanobody A(E1D, L11V, A14P, R39Q, K83R, V89L, T91Y, Q108L, T110Q)-35GS- Nanobody A(E1D, L11V, A14P, R39Q, K83R, V89L, T91Y, Q108L, T110Q)-35GS-Nanobody B(L11V, R30S, E44G, P45L, K83R, V89L, Q108L, T110Q)-Ala HIS6-Nanobody A(E1D, L11V, A14P, R39Q, K83R, V89L, T91Y, Q108L, T110K)-35GS- Nanobody A(E1D, L11V, A14P, R39Q, K83R, V89L, T91Y, Q108L, T110K)-35GS-Nanobody B(L11V, R30S, E44G, P45L, K83R, V89L, Q108L, T110K)-Ala

(76) Representative multivalent constructs were tested for binding by pre-existing antibodies that are present in a blood or serum sample obtained from patients with SLE and healthy volunteers. Both were determined using ProteOn, essentially as described above.

(77) The representative constructs tested are listed in Tables L (trivalent constructs) and M (bivalent constructs), and the results are shown in FIGS. 6-8 and Tables N-Q. The trivalent constructs tested were derived from the reference construct Nanobody A-35GS-Nanobody A-35GS-Nanobody B (“Reference X”) and the bivalent constructs were derived from the reference construct Nanobody A-35GS-Nanobody B (“Reference X”). All of the constructs (except for the reference constructs) had, were indicated, a C-terminal alanine residue as well as, in each of the “Nanobody A” and “Nanobody B” building blocks, the indicated mutations.

(78) TABLE-US-00020 TABLE L trivalent constructs tested. Trivalent constructs The constructs (except for the reference constructs) had, were indicated, a C-terminal alanine residue as well as, in each of the building blocks, Mutation(s) the indicated mutations. L11V T87A V89L T110K T110Q 114A Reference X (=Nanobody A- 35GS-Nanobody A-35GS-Nanobody B) Reference X + C-terminal Ala x Reference X + L11V + x x x V89L + C-terminal Ala Reference X + L11V + x x x x V89L + T110K + C-terminal Ala Reference X + L11V + x x x x T87A + V89L + C-terminal Ala Reference X + L11V + x x x x V89L + T110Q + C-terminal Ala

(79) TABLE-US-00021 TABLE M bivalent constructs tested. Bivalent constructs The constructs (except for the reference constructs) had, were indicated, a C-terminal alanine residue as well as, in each of the building blocks, Mutation(s) the indicated mutations. L11V T87A V89L T110K T110Q 114A Reference Y (=Nanobody A- 35GS-Nanobody B) Reference Y + C-terminal Ala x Reference Y + L11V + x x x V89L + C-terminal Ala Reference Y + L11V + x x x x V89L + T110K + C-terminal Ala Reference Y + L11V + x x x x T87A + V89L + C-terminal Ala Reference Y + L11V + x x x x V89L + T110Q + C-terminal Ala

(80) TABLE-US-00022 TABLE N results of testing trivalent constructs for binding by pre-existing antibodies present in 98 serum samples obtained from healthy human volunteers. Results are also represented in FIG. 6 as a plot in which each dot represents a data point collected by testing of the indicated construct against one of the 98 serum samples. Lane number in Binding Binding Binding Nanobody tested the plot level at 125 level at 125 level at 125 on 98 samples shown in seconds <10 seconds <20 seconds >20 (healthy subjects) FIG. 6 RU RU RU Reference X (1) 38 48 50 Reference X + C- (2) 64 75 23 terminal Ala Reference X + L11V + (3) 94 95 3 V89L + C-terminal Ala Reference X + L11V + (5) 95 96 2 V89L + T110K + C-terminal Ala Reference X + L11V + (6) 95 96 2 V89L + T110Q + C-terminal Ala Reference X + L11V + (4) 92 95 3 T87A + V89L + C-terminal Ala

(81) TABLE-US-00023 TABLE O results of testing trivalent constructs for binding by pre-existing antibodies present in 30 selected serum samples obtained from healthy human volunteers. The 30 samples used were pre-selected for either having a known high titer of pre-existing antibodies or because it was known that the pre-existing antibodies present in the sample still has a high degree of binding even if a C-terminal alanine residue is present. Results are also represented in FIG. 7 as a plot in which each dot represents a data point collected by testing of the indicated construct against one of the selected 30 serum samples. Lane number in Binding Binding Binding Nanobody tested the plot level at 125 level at 125 level at 125 on selected set shown in seconds <10 seconds <20 seconds >20 of 30 samples FIG. 7 RU RU RU Reference X + C- (1) 12 13 17 terminal Ala Reference X + L11V + (2) 18 22 8 V89L + C-terminal Ala Reference X + L11V + (3) 27 27 3 T87A + V89L + C-terminal Ala Reference X + L11V + (4) 26 26 4 V89L + T110K + C-terminal Ala

(82) TABLE-US-00024 TABLE P results of testing bivalent constructs for binding by pre-existing antibodies present in 98 serum samples obtained from healthy human volunteers. Results are also represented in FIG. 8 as a plot in which each dot represents a data point collected by testing of the indicated construct against one of the 98 serum samples. Lane number in Binding Binding Binding Nanobody tested the plot level at 125 level at 125 level at 125 on 98 samples shown in seconds <10 seconds <20 seconds >20 (healthy subjects) FIG. 8 RU RU RU Reference Y (1) 54 67 31 Reference Y + C- (2) 90 92 6 terminal Ala Reference Y + L11V + (3) 97 98 0 V89L + C-terminal Ala Reference Y + L11V + (4) 98 98 0 T87A + V89L + C-terminal Ala Reference Y + L11V + (5) 96 98 0 V89L + T110K + C-terminal Ala Reference Y + L11V + (6) 98 98 0 V89L + T110Q + C-terminal Ala
Three representative trivalent constructs were also tested against serum samples obtained from SLE patients. The results are shown in Table Q.

(83) TABLE-US-00025 TABLE Q testing of representative trivalent constructs against serum samples obtained from HLE patients. The constructs (had, were indicated, a C-terminal Sample alanine residue as well as, from healthy in each of the building blocks, Mutation(s) Samples obtained from SLE patients volunteer the indicated mutations. L11V T87A V89L T110K T110Q 114A SLE25 SLE37 SLE39 SLE41 NB13025-14 004-030-ABL-02 Average binding to [Reference X + C-terminal Ala] Reference X + C-terminal 142 194 126 108 102 27 alanine Inhibition compared to average binding to [Reference X + C-terminal Ala] captured on HSA (%) Reference X + L11V + x x x 100 95 100 88 100 0 V89L + C-terminal alanine Reference X + L11V + x x x x 100 76 100 100 100 100 V89L + T110K + C-terminal alanine Reference X + L11V + x x x x 100 100 100 100 97 100 87A + V89L + C-terminal alanine

Example 7: Nanobodies and Nanobody-Constructs Against the Ion Channel Kv1.3

(84) In one specific aspect, the VH domains of the invention (and in particular ISVDs of the invention and more in particular Nanobodies of the invention) and compounds of the invention may be directed against the ion channel Kv.1.3.

(85) The co-pending US provisional application U.S. Ser. No. 62/014,023 (title: “Kv1.3 binding immunoglobulins”; assignee: Ablynx N. V.; filing date: Jun. 18, 2014) as well as the subsequent filed US provisional application of the same title filed on Mar. 16, 2015 (assignee: Ablynx N. V.) inter alia describe immunoglobulin single variable domains (and in particular Nanobodies) that are directed against the potassium selective voltage-gated ion channel Kv1.3, as well as proteins, polypeptides and other Nanobody-based constructs that comprise at least one such Nanobody against Kv1.3.

(86) The mutations described in the present application (optionally suitably combined with a C-terminal extension as described in herein and/or in WO 12/175741) may also be suitably applied to the Nanobodies, proteins, polypeptides and other Nanobody-based constructs against Kv1.3 that are described in these two US provisional applications.

(87) Thus, in one aspect, the invention relates to a VH domain that is directed against Kv1.3 and that is as further described herein for the ISVD's of the invention (i.e. comprising the amino acid residues/mutations as described herein).

(88) Again, as with other aspects and embodiments of the invention described herein, when a specific ISVD (such as the ISVD against Kv1.3 described in this Example) or compound comprising the same is said to be “according to the invention” or “as further described herein”, the preferred aspects/embodiments and preferences that are generally described herein for the ISVD's or compounds of the invention also specifically apply to said specific ISVD or compound, respectively, unless explicitly indicated otherwise or unless the specific technical context requires otherwise.

(89) Thus, in a particular aspect, the present invention relates to a VH domain that is directed against Kv1.3, in which (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). In particular, in such VH domains against Kv1.3: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

(90) The VH domains against Kv1.3 referred to in this Example may in particular have CDR's that are as described on pages 5-10 of U.S. Ser. No. 62/014,023 (including any preferred aspects/embodiments of such CDRs) or sequence-optimized versions thereof, as described in the other US provisional application referred to above.

(91) In particular, the VH domains against Kv 1.3 referred to in this Example may in particular have a combination of CDR1, CDR2 and CDR3 that is chosen from one of the combination of CDR 1, CDR2 and CDR3 that are listed as preferred aspects in the list that bridges pages 9 and 10 of U.S. Ser. No. 62/014,023.

(92) In some preferred, but non-limiting aspect of the inventions: in a VH domain of the invention: (i) CDR1 (according to Kabat) is the sequence of SEQ ID NO:166 or an amino acid sequence that has one or two amino acid differences with the sequence of SEQ ID NO:166; (ii) CDR2 (according to Kabat) is the sequence of SEQ ID NO:167 or an amino acid sequence that has one or two amino acid differences with the sequence of SEQ ID NO:167; and (iii) CDR3 (according to Kabat) is the sequence of SEQ ID NO:168 or an amino acid sequence that has one or two amino acid differences with the sequence of SEQ ID NO:168; and even more preferably: (i) CDR1 (according to Kabat) is the sequence of SEQ ID NO:166; (ii) CDR2 (according to Kabat) is the sequence of SEQ ID NO:167; and (iii) CDR3 (according to Kabat) is the sequence of SEQ ID NO:168; and/or in a VH domain of the invention: (i) CDR1 (according to Abm) is the sequence of SEQ ID NO:169 or an amino acid sequence that has one or two amino acid differences with the sequence of SEQ ID NO:169; (ii) CDR2 (according to Abm) is the sequence of SEQ ID NO:170 or an amino acid sequence that has one or two amino acid differences with the sequence of SEQ ID NO:170; and (iii) CDR3 (according to Abm) is the sequence of SEQ ID NO:171 or an amino acid sequence that has one or two amino acid differences with the sequence of SEQ ID NO:171; and even more preferably: (i) CDR1 (according to Abm) is the sequence of SEQ ID NO:169; (ii) CDR2 (according to Abm) is the sequence of SEQ ID NO:170; and (iii) CDR3 (according to Abm) is the sequence of SEQ ID NO:178.

(93) The VH domains of the invention against Kv1.3 may further be as described herein and may again in particular be an ISVD (and more in particular a Nanobody) against Kv1.3 or a protein, polypeptide or other compound or construct that comprises as least one such ISVD. Such a protein, polypeptide or other compound or construct may also be as further described herein, and may for example have an increased half-life (i.e. as described herein, e.g. a half-life—expressed as t½ beta—in human subjects of in human subjects of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days), and for this purpose may for example comprise a serum-albumin binding Nanobody, which may also be a serum-albumin binding Nanobody of the invention (again, as described herein).

(94) Also, such an ISVD may suitably have a C-terminal extension (as further described herein and in WO 12/175741), in particular when said ISVD forms the C-terminal end of a protein, polypeptide or other compound or construct comprising the same (again, as further described herein).

(95) For example and without limitation, the VH domains of the invention against Kv1.3 may be one of the sequences listed in Table A-1 of U.S. Ser. No. 62/014,023 (SEQ ID NO's: 1 to 123 in U.S. Ser. No. 62/014,023) or one of the sequences of listed in Table A-1 of US provisional application entitled “Kv1.3 binding immunoglobulins” (assignee: Ablynx N. V.; filing date: Mar. 16, 2015) (SEQ ID NO's: 1 to 123, 495, 498 to 513 or 523 to 540 in said US provisional; and in particular the sequence of SEQ ID NO: 495), but suitably with the mutations/specific amino acid residues described herein for the ISVDs of the invention, and optionally suitably with a C-terminal extension.

(96) In one specific aspect, a Nanobody of the invention against Kv1.3 is a variant of the Nanobody of SEQ ID NO:137 (with at least 90% sequence identity with SEQ ID NO:137), in which: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

(97) Some specifically preferred, but non-limiting examples of Nanobodies of the invention against Kv1.3 and of compounds of the invention comprising the same are listed in FIG. 9A (monovalent Nanobodies: SEQ ID NO's: 138 to 155) and FIG. 9B (trivalent bispecific half-life extended constructs: SEQ ID NO's: 156 to 164). Compounds of the invention that comprise or essentially consist of at least one (such as one, two or three) anti-Kv1.3 Nanobody chosen from the anti-Kv1.3 Nanobodies of SEQ ID NO's: 138 to 155 form a further aspect of the invention. Also, each of the compounds of SEQ ID NO's: 156 to 164 forms a further aspect of the invention. In one specific aspect, such compounds contain two such Nanobodies of the invention against Kv1.3 and one Nanobody against human serum albumin (which is preferably also a Nanobody of the invention). Also, again, such a construct may contain suitable linkers and a C-terminal extension.

(98) The monovalent anti-Kv1.3 Nanobodies of SEQ ID NO's: 138 to 155 were generated by introducing the L11V and V89L mutations of the invention into the starting sequence of SEQ ID NO: 137 (reference). In addition, different combinations of humanizing (or other sequence-optimizing) mutations were introduced, such as E1D, A14P, G19R, M53A or M53Q or M53Y, T62S, A74S, K83R, S94G and/or T97E). The specific mutations introduced in each of the sequences of SEQ ID NO: 138 to 155 is given in FIG. 9A.

(99) Some of the monovalent anti-Kv1.3 Nanobodies from FIG. 9A were also formatted as trivalent bispecific constructs comprising two Nanobodies of the invention against Kv1.3 and one half-life extending Nanobody of the invention against human serum albumin (SEQ ID NO:109, also referred to as “ALB-82” in FIG. 9B). 35GS linkers were used, and all the constructs have a C-terminal extension (a single C-terminal alanine residue). The sequences of the resulting constructs are given in SEQ ID NO's 156 to 164. Of these, three constructs (SEQ ID NOs: 156, 157 and 160) were tested for binding by pre-existing antibodies in samples obtained from 47 diabetic human subjects and 90 healthy human subjects, using the general protocol described herein. The binding by pre-existing antibodies by samples from these two sets were compared to the reference construct of SEQ ID NO:165, which is a corresponding trivalent bispecific construct based on the reference anti-Kv1.3 building block of SEQ ID NO:137 and the serum albumin binder Alb-8 (SEQ ID NO:46), again combined with a C-terminal alanine extension. The results are shown in FIG. 10 (samples from 47 diabetic patients) and FIG. 11 (samples from 90 healthy volunteers). In each case, the constructs with the L11V and V89L mutations of the invention showed reduced binding by pre-existing antibodies compared to the reference construct.

Example 8: VH Domains (and in Particular Nanobodies) Against IL-23, and Compounds of the Invention Comprising the Same

(100) In one specific aspect, the VH domains of the invention (and in particular ISVDs of the invention and more in particular Nanobodies of the invention) and compounds of the invention may be directed against IL-23.

(101) Such a VH domain of the invention against IL-23 will generally comprise: (i) suitable framework sequences that suitably comprise the amino acid residues/mutations of the invention as described herein; as well as (ii) CDR sequences that allow the VH domain of the invention to specifically bind to IL-23. In addition, such a VH domain of the invention against IL-23 may also suitably have a C-terminal extension as described herein, in particular when said VH domain is monovalent or forms the C-terminal end of the compound of the invention in which said VH domain is present (again, as further described herein). Such VH domains of the invention against IL-23 may further be as further described herein, and may in particular be ISVD's.

(102) Again, as with other aspects and embodiments of the invention described herein, when a specific ISVD (such as the ISVD against IL-23 described in this Example) or compound comprising the same is said to be “according to the invention” or “as further described herein”, the preferred aspects/embodiments and preferences that are generally described herein for the ISVD's or compounds of the invention also specifically apply to said specific ISVD or compound, respectively, unless explicitly indicated otherwise or unless the specific technical context requires otherwise.

(103) Thus, in a particular aspect, the present invention relates to a VH domain (and in particular an ISVD) that is directed against IL-23, in which (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). In particular, in such VH domains against IL-23: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

(104) The VH domains of the invention against IL-23 may further be as described herein and may again in particular be an ISVD (and more in particular a Nanobody) against IL-23 or a protein, polypeptide or other compound or construct that comprises as least one such ISVD. Such a protein, polypeptide or other compound or construct may also be as further described herein, and may for example have an increased half-life (i.e. as described herein, e.g. a half-life—expressed as t½ beta—in human subjects of in human subjects of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days), and for this purpose may for example comprise a serum-albumin binding Nanobody, which may also be a serum-albumin binding Nanobody of the invention (again, as described herein).

(105) Also, such an ISVD may suitably have a C-terminal extension (as further described herein and in WO 12/175741), in particular when said ISVD forms the C-terminal end of a protein, polypeptide or other compound or construct comprising the same (again, as further described herein).

(106) In one preferred aspect, the VH domains of the invention against IL-23 comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 173 (which is preferred) or that is an amino acid sequence that has only one amino acid difference with the sequence of SEQ ID NO: 173; (ii) a CDR2 sequence that is the sequence of SEQ ID NO: 174 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 174; and (iii) a CDR3 sequence that is the sequence of SEQ ID NO: 175 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 175.

(107) More preferably, in a VH domain of the invention against IL-23 according to this aspect: (i) CDR1 is SEQ ID NO:173; (ii) CDR2 is SEQ ID NO: 174; and (iii) CDR3 is SEQ ID NO: 175.

(108) In one specific aspect, a Nanobody of the invention against IL-23 is a variant of the Nanobody of SEQ ID NO:172 (with at least 90% sequence identity, such as at least 95% sequence identity, with SEQ ID NO:172), in which: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). The CDR's of such an ISV are preferably as defined in the preceding two paragraphs.

(109) In another preferred aspect, the VH domains of the invention against IL-23 comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 191 (which is preferred) or that is an amino acid sequence that has only one amino acid difference with the sequence of SEQ ID NO: 191; (ii) a CDR2 sequence that is the sequence of SEQ ID NO: 192 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 192; and (iii) a CDR3 sequence that is the sequence of SEQ ID NO: 193 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 193.

(110) More preferably, in a VH domain of the invention against IL-23 according to this aspect: (i) CDR1 is SEQ ID NO: 191; (ii) CDR2 is SEQ ID NO: 192; and (iii) CDR3 is SEQ ID NO: 193.

(111) In one specific aspect, a Nanobody of the invention against IL-23 is a variant of the Nanobody of SEQ ID NO:172 (with at least 90% sequence identity, such as at least 95% sequence identity, with SEQ ID NO:190), in which: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). Again, the CDR's of such an ISV are preferably as defined in the preceding two paragraphs.

(112) Some specifically preferred, but non-limiting examples of such Nanobodies of the invention against IL-23 are listed in FIG. 12A as SEQ ID NO's: 176 to 189 and FIG. 12B as SEQ ID NO's: 194 to 207, respectively; and each of these Nanobodies form a further aspect of the invention.

(113) The invention also relates to a compound of the invention against IL-23 that comprises at least one (such as one, two or three) of the Nanobodies of the invention of SEQ ID NO's: 176 to 189 and/or 194 to 207. Such compounds of the invention against IL-23 may again be as further described herein, and thus for example may comprise suitable linkers, may comprise a C-terminal extension as described herein, and may be half-life extended (for example because they comprise a Nanobody against human serum albumin, such as (preferably) a Nanobody of the invention against human serum albumin). Reference is made to Table R below.

(114) As described in for example WO 2009/068627, WO 2010/142534 and WO2011/135026, on particularly preferred class of Nanobody-based compounds against IL-23 are biparatopic compounds. Thus, in one aspect of the invention, a compound of the invention against IL-23 is a biparatopic construct that comprises one ISV that is either SEQ ID NO: 172 or (preferably) an ISV of the invention that has been derived from SEQ ID NO:172 (as described in this Example 8) and one ISV that is either SEQ ID NO: 190 or (preferably) an ISV of the invention that has been derived from SEQ ID NO: 190 (as described in this Example 8), provided that at least one (and preferably both) of these ISV's are ISV's of the invention. Such biparatopic constructs may also be half-life extended (i.e. by means of a serum albumin-binding ISV). Some specific examples of such biparatopic constructs are given in SEQ ID NO: 514 to 549.

(115) Some specifically preferred examples of compounds of the invention against IL-23 are given in FIG. 22 as SEQ ID NO's: 514 to 549; and each of these compounds form a further aspect of the invention. Thus, in another aspect, the invention relates to a polypeptide that is directed against IL-23 and that has an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 514 to 549. More generally, compounds of the invention against IL-23 may be as described in WO 2009/068627, WO 2010/142534 and WO2011/135026, but comprising ISV's of the invention. They may also be used for the purposes described in WO 2009/068627, WO 2010/142534 and WO2011/135026.

(116) TABLE-US-00026 TABLE R Examples of compounds of the invention against IL-23. Polypeptide/ construct(.sup.1) General formula ISV building blocks and linkers Monovalent [IL-23] [IL-23] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 Monovalent/ [IL-23]-X(n) [IL-23] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 C-terminal extension Monovalent/ [IL-23]-L.sub.1-[SA] [IL-23] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L.sub.1-[IL-23] [SA] = (see legend below) extended L.sub.1 = (see legend below) Monovalent/ [IL-23]-L.sub.1-[SA]-X(n) [IL-23] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L.sub.1-[IL-23]-X(n) [SA] = (see legend below) extended/ L.sub.1 = (see legend below) C-terminal X(n) = (see legend below) extension Bivalent(.sup.2) [IL-23]-L.sub.1-[IL-23] At least one [IL-23] present is = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 (.sup.4) L.sub.1 = (see legend below) Bivalent/ [IL-23]-L.sub.1-[IL-23]-X(n) At least one [IL-23] = one of SEQ ID NO's: 464 to 477 and/or C-terminal 482 to 495 (.sup.4) extension(.sup.2) L.sub.1 = (see legend below) X(n) = (see legend below) Bivalent/ [IL-23]-L.sub.1-[IL-23]-L.sub.2-[SA] At least one [IL-23] present is = one of SEQ ID NO's: 464 to half-life [IL-23]-L.sub.1-[SA]-L.sub.2-[IL-23] 477 and/or 482 to 495 (.sup.4) extended(.sup.2) [SA]-L.sub.1-[IL-23]-L.sub.2-[IL-23] [SA] = (see legend below) L.sub.1 = (see legend below) L.sub.2 = (see legend below) Bivalent/ [IL-23]-L.sub.1-[IL-23]-L.sub.2-[SA]-X(n) At least one [IL-23] present is = one of SEQ ID NO's: 464 to half-life [IL-23]-L.sub.1-[SA]-L.sub.2-[IL-23]-X(n) 477 and/or 482 to 495 (.sup.4) extended/ [SA]-L.sub.1-[IL-23]-L.sub.2-[IL-23]-X(n) [SA] = (see legend below) C-terminal L.sub.1 = (see legend below) extension(.sup.2) L.sub.2 = (see legend below) X(n) = (see legend below) Bispecific (.sup.3) [IL-23]-L.sub.1-[Nb] At least one [IL-23] present is = one of SEQ ID NO's: 464 to [Nb]-L.sub.1-[IL-23] 477 and/or 482 to 495 (.sup.4) [IL-23]-L.sub.1-[IL-23]-L.sub.2-[Nb] L.sub.1 = (see legend below) [IL-23]-L.sub.1-[Nb]-L.sub.2-[IL-23] L.sub.2 = (see legend below) [Nb]-L.sub.1-[IL-23]-L.sub.2-[IL-23] [Nb] = (see legend below) Bispecific/ [IL-23]-L.sub.1-[Nb]-X(n) At least one [IL-23] present is = one of SEQ ID NO's: 464 to C-terminal [Nb]-L.sub.1-[IL-23]-X(n) 477 and/or 482 to 495 (.sup.4) extension (.sup.3) [IL-23]-L.sub.1-[IL-23]-L.sub.2-[Nb]-X(n) L.sub.1 = (see legend below) [IL-23]-L.sub.1-[Nb]-L.sub.2-[IL-23]-X(n) L.sub.2 = (see legend below) [Nb]-L.sub.1-[IL-23]-L.sub.2-[IL-23]-X(n) X(n) = (see legend below) [Nb] = (see legend below) Bispecific/ [IL-23]-L.sub.1-[Nb]-L.sub.2-[SA] At least one [IL-23] present is = one of SEQ ID NO's: 464 to half-life [IL-23]-L.sub.1-[SA]-L.sub.2-[Nb] 477 and/or 482 to 495 (.sup.4) extended (.sup.3) [Nb]-L.sub.1-[SA]-L.sub.2-[IL-23] [SA] = (see legend below) [Nb]-L.sub.1-[IL-23]-L.sub.2-[SA] L.sub.1, L.sub.2, L.sub.3(see legend below) [SA]-L.sub.1-[IL-23]-L.sub.2-[Nb] [Nb] = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[IL-23] Bispecific/ [IL-23]-L.sub.1-[IL-23]-L.sub.2-[Nb]-L.sub.3-[SA] At least one [IL-23] present is = one of SEQ ID NO's: 464 to half-life [Nb]-L.sub.1-[IL-23]-L.sub.2-[IL-23]-L.sub.3-[SA] 477 and/or 482 to 495 (.sup.4) extended (.sup.3) [SA]-L.sub.1-[IL-23]-L.sub.2-[IL-23]-L.sub.3-[Nb] [SA] = (see legend below) [SA]-L.sub.1-[IL-23]-L.sub.2-[Nb]-L.sub.3-[IL-23] L.sub.1, L.sub.2, L.sub.3(see legend below) [IL-23]-L.sub.1-[Nb]-L.sub.2-[IL-23]-L.sub.3-[SA] [Nb] = (see legend below) Bispecific/ [IL-23]-L.sub.1-[Nb]-L.sub.2-[SA]-X(n) At least one [IL-23] present is = one of SEQ ID NO's: 464 to half-life [IL-23]-L.sub.1-[SA]-L.sub.2-[Nb]-X(n) 477 and/or 482 to 495 (.sup.4) extended/ [Nb]-L.sub.1-[SA]-L.sub.2-[IL-23]-X(n) [SA] = (see legend below) C-terminal [Nb]-L.sub.1-[IL-23]-L.sub.2-[SA]-X(n) L.sub.1, L.sub.2, L.sub.3(see legend below) extension (.sup.3) [SA]-L.sub.1-[IL-23]-L.sub.2-[Nb]-X(n) X(n) = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[IL-23]-X(n) [Nb] = (see legend below) [IL-23]-L.sub.1-[IL-23]-L.sub.2-[Nb]-L.sub.3-[SA]-X(n) [Nb]-L.sub.1-[IL-23]-L.sub.2-[IL-23]-L.sub.3-[SA]-X(n) [SA]-L.sub.1-[IL-23]-L.sub.2-[IL-23]-L.sub.3-[Nb]-X(n) [SA]-L.sub.1-[IL-23]-L.sub.2-[Nb]-L.sub.3-[IL-23]-X(n) [IL-23]-L.sub.1-[Nb]-L.sub.2-[IL-23]-L.sub.3-[SA]-X(n) Legend: [SA] is an ISV against (human) serum albumin, preferably an ISV of the invention against (human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 or even more preferably one of the ISVD's of the invention of SEQ ID NO's: 47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L.sub.1, L.sub.2 and L.sub.3 is (independently) a suitable linker. Each of L.sub.1, L.sub.2 and L.sub.3 may (independently) be present or not. Non-limiting examples of suitable linkers are the gly-ser linkers referred to herein, such as the 9GS, 30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminal extension as described in herein and/or in WO 12/175741 [Nb] is an ISV against another therapeutic target. Notes: (.sup.1)In this Table: “Monovalent” generally refers to polypeptides/constructs comprising a single ISV against IL-23. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bivalent” generally refers to polypeptides/constructs comprising two ISV's against IL-23 (which may be the same or different). These may again further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bispecific” generally refers to polypeptides/constructs comprising at least one (such as 1 or 2) ISV's against IL-23 and at least one (such as 1 or 2) other ISV against a therapeutic target. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). In the polypeptides/constructs described in this table, at least one of the ISV's against IL-23 present is an ISV of the invention, and preferably all of the ISV's against IL-23 present in such polypeptide/construct are ISV's of the invention. Also, when a half-life extending ISV and/or an ISV against another therapeutic target is present in such polypeptide/construct, each of these (and preferably all of these) may also be (and preferably are) ISV's of the invention (.sup.2)All “bivalent” constructs in this Table may also be biparatopic, meaning that they comprise at least two (such as two) ISV's against IL-23, which are directed against different epitopes on IL-23. (.sup.3) As will be clear to the skilled person, other/further bispecific constructs than those listed can be made using the building blocks and linkers mentioned. (.sup.4) Preferably, each [IL-23] present is independently chosen from SEQ ID NO's: 464 to 477 and/or 482 to 495. Also, the [IL-23] present may be the same or different; in a biparatopic polypeptide/construct they will be directed against different epitopes on IL-23. (.sup.5) Each of the polypeptides/constructs mentioned to in this column by means of reference to a SEQ ID forms an individual specific aspect of the invention.

Example 9: VH Domains (and in Particular Nanobodies) Against OX40-L, and Compounds of the Invention Comprising the Same

(117) In one specific aspect, the VH domains of the invention (and in particular ISVDs of the invention and more in particular Nanobodies of the invention) and compounds of the invention may be directed against OX40-L.

(118) Such a VH domain of the invention against OX40-L will generally comprise: (i) suitable framework sequences that suitably comprise the amino acid residues/mutations of the invention as described herein; as well as (ii) CDR sequences that allow the VH domain of the invention to specifically bind to OX40-L. In addition, such a VH domain of the invention against OX40-L may also suitably have a C-terminal extension as described herein, in particular when said VH domain is monovalent or forms the C-terminal end of the compound of the invention in which said VH domain is present (again, as further described herein). Such VH domains of the invention against OX40-L may further be as further described herein, and may in particular be ISVD's.

(119) Again, as with other aspects and embodiments of the invention described herein, when a specific ISVD (such as the ISVD against OX40-L described in this Example) or compound comprising the same is said to be “according to the invention” or “as further described herein”, the preferred aspects/embodiments and preferences that are generally described herein for the ISVD's or compounds of the invention also specifically apply to said specific ISVD or compound, respectively, unless explicitly indicated otherwise or unless the specific technical context requires otherwise.

(120) Thus, in a particular aspect, the present invention relates to a VH domain (and in particular an ISVD) that is directed against OX40-L, in which (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). In particular, in such VH domains against OX40-L: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

(121) The VH domains of the invention against OX40-L may further be as described herein and may again in particular be an ISVD (and more in particular a Nanobody) against OX40-L or a protein, polypeptide or other compound or construct that comprises as least one such ISVD. Such a protein, polypeptide or other compound or construct may also be as further described herein, and may for example have an increased half-life (i.e. as described herein, e.g. a half-life—expressed as t½ beta—in human subjects of in human subjects of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days), and for this purpose may for example comprise a serum-albumin binding Nanobody, which may also be a serum-albumin binding Nanobody of the invention (again, as described herein).

(122) Also, such an ISVD may suitably have a C-terminal extension (as further described herein and in WO 12/175741), in particular when said ISVD forms the C-terminal end of a protein, polypeptide or other compound or construct comprising the same (again, as further described herein).

(123) In one preferred aspect, the VH domains of the invention against OX40-L comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 209 (which is preferred) or that is an amino acid sequence that has only one amino acid difference with the sequence of SEQ ID NO: 209; (ii) a CDR2 sequence that is the sequence of SEQ ID NO: 210 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 210; and (iii) a CDR3 sequence that is the sequence of SEQ ID NO: 211 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 211.

(124) More preferably, in a VH domain of the invention against OX40-L according to this aspect: (i) CDR1 is SEQ ID NO:209; (ii) CDR2 is SEQ ID NO: 210; and (iii) CDR3 is SEQ ID NO: 211.

(125) In one specific aspect, a Nanobody of the invention against OX40-L is a variant of the Nanobody of SEQ ID NO:208 (with at least 90% sequence identity, such as at least 95% sequence identity, with SEQ ID NO:208), in which: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). The CDR's of such an ISV are preferably as defined in the preceding two paragraphs.

(126) Some specifically preferred, but non-limiting examples of Nanobodies of the invention against OX40-L are listed in FIG. 13 as SEQ ID NO's: 212 to 225; and each of these Nanobodies form a further aspect of the invention.

(127) The invention also relates to a compound of the invention against OX40-L that comprises at least one (such as one, two or three) of the Nanobodies of the invention of SEQ ID NO's: 212 to 225. Such compounds of the invention against OX40-L may again be as further described herein, and thus for example may comprise suitable linkers, may comprise a C-terminal extension as described herein, and may be half-life extended (for example because they comprise a Nanobody against human serum albumin, such as (preferably) a Nanobody of the invention against human serum albumin). Reference is made to Table S below.

(128) Some specifically preferred examples of compounds of the invention against OX40-L are given in FIG. 23 as SEQ ID NO's: 550 to 585; and each of these compounds form a further aspect of the invention. Thus, in another aspect, the invention relates to a polypeptide that is directed against OX40-L and that has an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 550 to 585. More generally, compounds of the invention against OX40-L may be as described in WO 2011/073180, but comprising ISV's of the invention. They may also be used for the purposes described in WO 2011/073180.

(129) TABLE-US-00027 TABLE S Examples of compounds of the invention against OX40-L. Polypeptide/ construct(.sup.1) General formula ISV building blocks and linkers Monovalent [OX40-L] [OX40-L] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 Monovalent/ [OX40-L]-X(n) [OX40-L] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 C-terminal extension Monovalent/ [OX40-L]-L.sub.1-[SA] [OX40-L] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L.sub.1-[OX40-L] [SA] = (see legend below) extended L.sub.1 = (see legend below) Monovalent/ [OX40-L]-L.sub.1-[SA]-X(n) [OX40-L] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L.sub.1-[OX40-L]-X(n) [SA] = (see legend below) extended/ L.sub.1 = (see legend below) C-terminal X(n) = (see legend below) extension Bivalent(.sup.2) [OX40-L]-L.sub.1-[OX40-L] At least one [OX40-L] present is = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 (.sup.4) L.sub.1 = (see legend below) Bivalent/ [OX40-L]-L.sub.1-[OX40-L]-X(n) At least one [OX40-L] = one of SEQ ID NO's: 464 to 477 C-terminal and/or 482 to 495 (.sup.4) extension(.sup.2) L.sub.1 = (see legend below) X(n) = (see legend below) Bivalent/ [OX40-L]-L.sub.1-[OX40-L]-L.sub.2-[SA] At least one [OX40-L] present is = one of SEQ ID NO's: 464 to half-life [OX40-L]-L.sub.1-[SA]-L.sub.2-[OX40-L] 477 and/or 482 to 495 (.sup.4) extended(.sup.2) [SA]-L.sub.1-[OX40-L]-L.sub.2-[OX40-L] [SA] = (see legend below) L.sub.1 = (see legend below) L.sub.2 = (see legend below) Bivalent/ [OX40-L]-L.sub.1-[OX40-L]-L.sub.2-[SA]-X(n) At least one [OX40-L] present is = one of SEQ ID NO's: 464 to half-life [OX40-L]-L.sub.1-[SA]-L.sub.2-[OX40-L]-X(n) 477 and/or 482 to 495 (.sup.4) extended/ [SA]-L.sub.1-[OX40-L]-L.sub.2-[OX40-L]-X(n) [SA] = (see legend below) C-terminal L.sub.1 = (see legend below) extension(.sup.2) L.sub.2 = (see legend below) X(n) = (see legend below) Bispecific (.sup.3) [OX40-L]-L.sub.1-[Nb] At least one [OX40-L] present is = one of SEQ ID NO's: 464 to [Nb]-L.sub.1-[OX40-L] 477 and/or 482 to 495 (.sup.4) [OX40-L]-L.sub.1-[OX40-L]-L.sub.2-[Nb] L.sub.1 = (see legend below) [OX40-L]-L.sub.1-[Nb]-L.sub.2-[OX40-L] L.sub.2 = (see legend below) [Nb]-L.sub.1-[OX40-L]-L.sub.2-[OX40-L] [Nb] = (see legend below) Bispecific/ [OX40-L]-L.sub.1-[Nb]-X(n) At least one [OX40-L] present is = one of SEQ ID NO's: 464 to C-terminal [Nb]-L.sub.1-[OX40-L]-X(n) 477 and/or 482 to 495 (.sup.4) extension (.sup.3) [OX40-L]-L.sub.1-[OX40-L]-L.sub.2-[Nb]-X(n) L.sub.1 = (see legend below) [OX40-L]-L.sub.1-[Nb]-L.sub.2-[OX40-L]-X(n) L.sub.2 = (see legend below) [Nb]-L.sub.1-[OX40-L]-L.sub.2-[OX40-L]-X(n) X(n) = (see legend below) [Nb] = (see legend below) Bispecific/ [OX40-L]-L.sub.1-[Nb]-L.sub.2-[SA] At least one [OX40-L] present is = one of SEQ ID NO's: 464 to half-life [OX40-L]-L.sub.1-[SA]-L.sub.2-[Nb] 477 and/or 482 to 495 (.sup.4) extended (.sup.3) [Nb]-L.sub.1-[SA]-L.sub.2-[OX40-L] [SA] = (see legend below) [Nb]-L.sub.1-[OX40-L]-L.sub.2-[SA] L.sub.1, L.sub.2, L.sub.3(see legend below) [SA]-L.sub.1-[OX40-L]-L.sub.2-[Nb] [Nb] = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[OX40-L] Bispecific/ [OX40-L]-L.sub.1-[OX40-L]-L.sub.2-[Nb]-L.sub.3-[SA] At least one [OX40-L] present is = one of SEQ ID NO's: 464 to half-life [Nb]-L.sub.1-[OX40-L]-L.sub.2-[OX40-L]-L.sub.3-[SA] 477 and/or 482 to 495 (.sup.4) extended (.sup.3) [SA]-L.sub.1-[OX40-L]-L.sub.2-[OX40-L]-L.sub.3-[Nb] [SA] = (see legend below) [SA]-L.sub.1-[OX40-L]-L.sub.2-[Nb]-L.sub.3-[OX40-L] L.sub.1, L.sub.2, L.sub.3(see legend below) [OX40-L]-L.sub.1-[Nb]-L.sub.2-[OX40-L]-L.sub.3-[SA] [Nb] = (see legend below) Bispecific/ [OX40-L]-L.sub.1-[Nb]-L.sub.2-[SA]-X(n) At least one [OX40-L] present is = one of SEQ ID NO's: 464 to half-life [OX40-L]-L.sub.1-[SA]-L.sub.2-[Nb]-X(n) 477 and/or 482 to 495 (.sup.4) extended/ [Nb]-L.sub.1-[SA]-L.sub.2-[OX40-L]-X(n) [SA] = (see legend below) C-terminal [Nb]-L.sub.1-[OX40-L]-L.sub.2-[SA]-X(n) L.sub.1, L.sub.2, L.sub.3(see legend below) extension (.sup.3) [SA]-L.sub.1-[OX40-L]-L.sub.2-[Nb]-X(n) X(n) = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[OX40-L]-X(n) [Nb] = (see legend below) [OX40-L]-L.sub.1-[OX40-L]-L.sub.2-[Nb]-L.sub.3-[SA]-X(n) [Nb]-L.sub.1-[OX40-L]-L.sub.2-[OX40-L]-L.sub.3-[SA]-X(n) [SA]-L.sub.1-[OX40-L]-L.sub.2-[OX40-L]-L.sub.3-[Nb]-X(n) [SA]-L.sub.1-[OX40-L]-L.sub.2-[Nb]-L.sub.3-[OX40-L]-X(n) [OX40-L]-L.sub.1-[Nb]-L.sub.2-[OX40-L]-L.sub.3-[SA]-X(n) Legend: [SA] is an ISV against (human) serum albumin, preferably an ISV of the invention against (human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 or even more preferably one of the ISVD's of the invention of SEQ ID NO's: 47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L.sub.1, L.sub.2 and L.sub.3 is (independently) a suitable linker. Each of L.sub.1, L.sub.2 and L.sub.3 may (independently) be present or not. Non-limiting examples of suitable linkers are the gly-ser linkers referred to herein, such as the 9GS, 30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminal extension as described in herein and/or in WO 12/175741 [Nb] is an ISV against another therapeutic target. Notes: (.sup.1)In this Table: “Monovalent” generally refers to polypeptides/constructs comprising a single ISV against OX40-L. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bivalent” generally refers to polypeptides/constructs comprising two ISV's against OX40-L (which may be the same or different). These may again further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bispecific” generally refers to polypeptides/constructs comprising at least one (such as 1 or 2) ISV's against OX40-L and at least one (such as 1 or 2) other ISV against a therapeutic target. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). In the polypeptides/constructs described in this table, at least one of the ISV's against OX40-L present is an ISV of the invention, and preferably all of the ISV's against OX40-L present in such polypeptide/construct are ISV's of the invention. Also, when a half-life extending ISV and/or an ISV against another therapeutic target is present in such polypeptide/construct, each of these (and preferably all of these) may also be (and preferably are) ISV's of the invention (.sup.2)All “bivalent” constructs in this Table may also be biparatopic, meaning that they comprise at least two (such as two) ISV's against OX40-L, which are directed against different epitopes on OX40-L. (.sup.3) As will be clear to the skilled person, other/further bispecific constructs than those listed can be made using the building blocks and linkers mentioned. (.sup.4) Preferably, each [OX40-L] present is independently chosen from SEQ ID NO's: 464 to 477 and/or 482 to 495. Also, the [OX40-L] present may be the same or different; in a biparatopic polypeptide/construct they will be directed against different epitopes on OX40-L. (.sup.5) Each of the polypeptides/constructs mentioned to in this column by means of reference to a SEQ ID forms an individual specific aspect of the invention.

Example 10: VH Domains (and in Particular Nanobodies) Against IgE, and Compounds of the Invention Comprising the Same

(130) In one specific aspect, the VH domains of the invention (and in particular ISVDs of the invention and more in particular Nanobodies of the invention) and compounds of the invention may be directed against IgE.

(131) Such a VH domain of the invention against IgE will generally comprise: (i) suitable framework sequences that suitably comprise the amino acid residues/mutations of the invention as described herein; as well as (ii) CDR sequences that allow the VH domain of the invention to specifically bind to IgE. In addition, such a VH domain of the invention against IgE may also suitably have a C-terminal extension as described herein, in particular when said VH domain is monovalent or forms the C-terminal end of the compound of the invention in which said VH domain is present (again, as further described herein). Such VH domains of the invention against IgE may further be as further described herein, and may in particular be ISVD's.

(132) Again, as with other aspects and embodiments of the invention described herein, when a specific ISVD (such as the ISVD against IgE described in this Example) or compound comprising the same is said to be “according to the invention” or “as further described herein”, the preferred aspects/embodiments and preferences that are generally described herein for the ISVD's or compounds of the invention also specifically apply to said specific ISVD or compound, respectively, unless explicitly indicated otherwise or unless the specific technical context requires otherwise.

(133) Thus, in a particular aspect, the present invention relates to a VH domain (and in particular an ISVD) that is directed against IgE, in which (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). In particular, in such VH domains against IgE: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

(134) The VH domains of the invention against IgE may further be as described herein and may again in particular be an ISVD (and more in particular a Nanobody) against IgE or a protein, polypeptide or other compound or construct that comprises as least one such ISVD. Such a protein, polypeptide or other compound or construct may also be as further described herein, and may for example have an increased half-life (i.e. as described herein, e.g. a half-life—expressed as t½ beta—in human subjects of in human subjects of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days), and for this purpose may for example comprise a serum-albumin binding Nanobody, which may also be a serum-albumin binding Nanobody of the invention (again, as described herein).

(135) Also, such an ISVD may suitably have a C-terminal extension (as further described herein and in WO 12/175741), in particular when said ISVD forms the C-terminal end of a protein, polypeptide or other compound or construct comprising the same (again, as further described herein).

(136) In one preferred aspect, the VH domains of the invention against IgE comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 227 (which is preferred) or that is an amino acid sequence that has only one amino acid difference with the sequence of SEQ ID NO: 227; (ii) a CDR2 sequence that is the sequence of SEQ ID NO: 228 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 228; and (iii) a CDR3 sequence that is the sequence of SEQ ID NO: 229 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 229.

(137) More preferably, in a VH domain of the invention against IgE according to this aspect: (i) CDR1 is SEQ ID NO: 227; (ii) CDR2 is SEQ ID NO: 228; and (iii) CDR3 is SEQ ID NO: 229.

(138) In one specific aspect, a Nanobody of the invention against IgE is a variant of the Nanobody of SEQ ID NO: 226 (with at least 90% sequence identity, such as at least 95% sequence identity, with SEQ ID NO: 226), in which: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). The CDR's of such an ISV are preferably as defined in the preceding two paragraphs.

(139) Some specifically preferred, but non-limiting examples of Nanobodies of the invention against IgE are listed in FIG. 14 as SEQ ID NO's: 230 to 243; and each of these Nanobodies form a further aspect of the invention.

(140) The invention also relates to a compound of the invention against IgE that comprises at least one (such as one, two or three) of the Nanobodies of the invention of SEQ ID NO's: 230 to 243. Such compounds of the invention against IgE may again be as further described herein, and thus for example may comprise suitable linkers, may comprise a C-terminal extension as described herein, and may be half-life extended (for example because they comprise a Nanobody against human serum albumin, such as (preferably) a Nanobody of the invention against human serum albumin). Reference is made to Table T below.

(141) Some specifically preferred examples of compounds of the invention against IgE are given in FIG. 24 as SEQ ID NO's: 586 to 594; and each of these compounds form a further aspect of the invention. Thus, in another aspect, the invention relates to a polypeptide that is directed against IgE and that has an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 586 to 594.

(142) More generally, compounds of the invention against IgE may be as described in WO 2012/175740 and the relevant parts of WO2012/175400, but comprising ISV's of the invention. They may also be used for the purposes described in WO 2012/175740.

(143) TABLE-US-00028 TABLE T Examples of compounds of the invention against IgE. Polypeptide/ construct(.sup.1) General formula ISV building blocks and linkers Monovalent [IgE] [IgE] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 Monovalent/ [IgE]-X(n) [IgE] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 C-terminal extension Monovalent/ [IgE]-L.sub.1-[SA] [IgE] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L.sub.1-[IgE] [SA] = (see legend below) extended L.sub.1 = (see legend below) Monovalent/ [IgE]-L.sub.1-[SA]-X(n) [IgE] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L.sub.1-[IgE]-X(n) [SA] = (see legend below) extended/ L.sub.1 = (see legend below) C-terminal X(n) = (see legend below) extension Bivalent(.sup.2) [IgE]-L.sub.1-[IgE] At least one [IgE] present is = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 (.sup.4) L.sub.1 = (see legend below) Bivalent/ [IgE]-L.sub.1-[IgE]-X(n) At least one [IgE] = one of SEQ ID NO's: 464 to 477 and/or C-terminal 482 to 495 (.sup.4) extension(.sup.2) L.sub.1 = (see legend below) X(n) = (see legend below) Bivalent/ [IgE]-L.sub.1-[IgE]-L.sub.2-[SA] At least one [IgE] present is = one of SEQ ID NO's: 464 to half-life [IgE]-L.sub.1-[SA]-L.sub.2-[IgE] 477 and/or 482 to 495 (.sup.4) extended(.sup.2) [SA]-L.sub.1-[IgE]-L.sub.2-[IgE] [SA] = (see legend below) L.sub.1 = (see legend below) L.sub.2 = (see legend below) Bivalent/ [IgE]-L.sub.1-[IgE]-L.sub.2-[SA]-X(n) At least one [IgE] present is = one of SEQ ID NO's: 464 to half-life [IgE]-L.sub.1-[SA]-L.sub.2-[IgE]-X(n) 477 and/or 482 to 495 (.sup.4) extended/ [SA]-L.sub.1-[IgE]-L.sub.2-[IgE]-X(n) [SA] = (see legend below) C-terminal L.sub.1 = (see legend below) extension(.sup.2) L.sub.2 = (see legend below) X(n) = (see legend below) Bispecific (.sup.3) [IgE]-L.sub.1-[Nb] At least one [IgE] present is = one of SEQ ID NO's: 464 to [Nb]-L.sub.1-[IgE] 477 and/or 482 to 495 (.sup.4) [IgE]-L.sub.1-[IgE]-L.sub.2-[Nb] L.sub.1 = (see legend below) [IgE]-L.sub.1-[Nb]-L.sub.2-[IgE] L.sub.2 = (see legend below) [Nb]-L.sub.1-[IgE]-L.sub.2-[IgE] [Nb] = (see legend below) Bispecific/ [IgE]-L.sub.1-[Nb]-X(n) At least one [IgE] present is = one of SEQ ID NO's: 464 to C-terminal [Nb]-L.sub.1-[IgE]-X(n) 477 and/or 482 to 495 (.sup.4) extension (.sup.3) [IgE]-L.sub.1-[IgE]-L.sub.2-[Nb]-X(n) L.sub.1 = (see legend below) [IgE]-L.sub.1-[Nb]-L.sub.2-[IgE]-X(n) L.sub.2 = (see legend below) [Nb]-L.sub.1-[IgE]-L.sub.2-[IgE]-X(n) X(n) = (see legend below) [Nb] = (see legend below) Bispecific/ [IgE]-L.sub.1-[Nb]-L.sub.2-[SA] At least one [IgE] present is = one of SEQ ID NO's: 464 to half-life [IgE]-L.sub.1-[SA]-L.sub.2-[Nb] 477 and/or 482 to 495 (.sup.4) extended (.sup.3) [Nb]-L.sub.1-[SA]-L.sub.2-[IgE] [SA] = (see legend below) [Nb]-L.sub.1-[IgE]-L.sub.2-[SA] L.sub.1, L.sub.2, L.sub.3(see legend below) [SA]-L.sub.1-[IgE]-L.sub.2-[Nb] [Nb] = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[IgE] Bispecific/ [IgE]-L.sub.1-[IgE]-L.sub.2-[Nb]-L.sub.3-[SA] At least one [IgE] present is = one of SEQ ID NO's: 464 to half-life [Nb]-L.sub.1-[IgE]-L.sub.2-[IgE]-L.sub.3-[SA] 477 and/or 482 to 495 (.sup.4) extended (.sup.3) [SA]-L.sub.1-[IgE]-L.sub.2-[IgE]-L.sub.3-[Nb] [SA] = (see legend below) [SA]-L.sub.1-[IgE]-L.sub.2-[Nb]-L.sub.3-[IgE] L.sub.1, L.sub.2, L.sub.3(see legend below) [IgE]-L.sub.1-[Nb]-L.sub.2-[IgE]-L.sub.3-[SA] [Nb] = (see legend below) Bispecific/ [IgE]-L.sub.1-[Nb]-L.sub.2-[SA]-X(n) At least one [IgE] present is = one of SEQ ID NO's: 464 to half-life [IgE]-L.sub.1-[SA]-L.sub.2-[Nb]-X(n) 477 and/or 482 to 495 (.sup.4) extended/ [Nb]-L.sub.1-[SA]-L.sub.2-[IgE]-X(n) [SA] = (see legend below) C-terminal [Nb]-L.sub.1-[IgE]-L.sub.2-[SA]-X(n) L.sub.1, L.sub.2, L.sub.3(see legend below) extension (.sup.3) [SA]-L.sub.1-[IgE]-L.sub.2-[Nb]-X(n) X(n) = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[IgE]-X(n) [Nb] = (see legend below) [IgE]-L.sub.1-[IgE]-L.sub.2-[Nb]-L.sub.3-[SA]-X(n) [Nb]-L.sub.1-[IgE]-L.sub.2-[IgE]-L.sub.3-[SA]-X(n) [SA]-L.sub.1-[IgE]-L.sub.2-[IgE]-L.sub.3-[Nb]-X(n) [SA]-L.sub.1-[IgE]-L.sub.2-[Nb]-L.sub.3-[IgE]-X(n) [IgE]-L.sub.1-[Nb]-L.sub.2-[IgE]-L.sub.3-[SA]-X(n) Legend: [SA] is an ISV against (human) serum albumin, preferably an ISV of the invention against (human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 or even more preferably one of the ISVD's of the invention of SEQ ID NO's: 47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L.sub.1, L.sub.2 and L.sub.3 is (independently) a suitable linker. Each of L.sub.1, L.sub.2 and L.sub.3 may (independently) be present or not. Non-limiting examples of suitable linkers are the gly-ser linkers referred to herein, such as the 9GS, 30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminal extension as described in herein and/or in WO 12/175741 [Nb] is an ISV against another therapeutic target. Notes: (.sup.1)In this Table: “Monovalent” generally refers to polypeptides/constructs comprising a single ISV against IgE. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bivalent” generally refers to polypeptides/constructs comprising two ISV's against IgE (which may be the same or different). These may again further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bispecific” generally refers to polypeptides/constructs comprising at least one (such as 1 or 2) ISV's against IgE and at least one (such as 1 or 2) other ISV against a therapeutic target. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). In the polypeptides/constructs described in this table, at least one of the ISV's against IgE present is an ISV of the invention, and preferably all of the ISV's against IgE present in such polypeptide/construct are ISV's of the invention. Also, when a half-life extending ISV and/or an ISV against another therapeutic target is present in such polypeptide/construct, each of these (and preferably all of these) may also be (and preferably are) ISV's of the invention (.sup.2)All “bivalent” constructs in this Table may also be biparatopic, meaning that they comprise at least two (such as two) ISV's against IgE, which are directed against different epitopes on IgE. (.sup.3) As will be clear to the skilled person, other/further bispecific constructs than those listed can be made using the building blocks and linkers mentioned. (.sup.4) Preferably, each [IgE] present is independently chosen from SEQ ID NO's: 464 to 477 and/or 482 to 495. Also, the [IgE] present may be the same or different; in a biparatopic polypeptide/construct they will be directed against different epitopes on IgE. (.sup.5) Each of the polypeptides/constructs mentioned to in this column by means of reference to a SEQ ID forms an individual specific aspect of the invention.

Example 11: VH Domains (and in Particular Nanobodies) Against CXCR4, and Compounds of the Invention Comprising the Same

(144) In one specific aspect, the VH domains of the invention (and in particular ISVDs of the invention and more in particular Nanobodies of the invention) and compounds of the invention may be directed against CXCR4.

(145) Such a VH domain of the invention against CXCR4 will generally comprise: (i) suitable framework sequences that suitably comprise the amino acid residues/mutations of the invention as described herein; as well as (ii) CDR sequences that allow the VH domain of the invention to specifically bind to CXCR4. In addition, such a VH domain of the invention against CXCR4 may also suitably have a C-terminal extension as described herein, in particular when said VH domain is monovalent or forms the C-terminal end of the compound of the invention in which said VH domain is present (again, as further described herein). Such VH domains of the invention against CXCR4 may further be as further described herein, and may in particular be ISVD's.

(146) Again, as with other aspects and embodiments of the invention described herein, when a specific ISVD (such as the ISVD against CXCR4 described in this Example) or compound comprising the same is said to be “according to the invention” or “as further described herein”, the preferred aspects/embodiments and preferences that are generally described herein for the ISVD's or compounds of the invention also specifically apply to said specific ISVD or compound, respectively, unless explicitly indicated otherwise or unless the specific technical context requires otherwise.

(147) Thus, in a particular aspect, the present invention relates to a VH domain (and in particular an ISVD) that is directed against CXCR4, in which (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). In particular, in such VH domains against CXCR4: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

(148) The VH domains of the invention against CXCR4 may further be as described herein and may again in particular be an ISVD (and more in particular a Nanobody) against CXCR4 or a protein, polypeptide or other compound or construct that comprises as least one such ISVD. Such a protein, polypeptide or other compound or construct may also be as further described herein, and may for example have an increased half-life (i.e. as described herein, e.g. a half-life—expressed as t½ beta—in human subjects of in human subjects of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days), and for this purpose may for example comprise a serum-albumin binding Nanobody, which may also be a serum-albumin binding Nanobody of the invention (again, as described herein).

(149) Also, such an ISVD may suitably have a C-terminal extension (as further described herein and in WO 12/175741), in particular when said ISVD forms the C-terminal end of a protein, polypeptide or other compound or construct comprising the same (again, as further described herein).

(150) In one preferred aspect, the VH domains of the invention against CXCR4 comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 245 (which is preferred) or that is an amino acid sequence that has only one amino acid difference with the sequence of SEQ ID NO: 245; (ii) a CDR2 sequence that is the sequence of SEQ ID NO: 246 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 246; and (iii) a CDR3 sequence that is the sequence of SEQ ID NO: 247 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 247.

(151) More preferably, in a VH domain of the invention against CXCR4 according to this aspect: (i) CDR1 is SEQ ID NO: 245; (ii) CDR2 is SEQ ID NO: 246; and (iii) CDR3 is SEQ ID NO: 247.

(152) In one specific aspect, a Nanobody of the invention against CXCR4 is a variant of the Nanobody of SEQ ID NO: 244 (with at least 90% sequence identity, such as at least 95% sequence identity, with SEQ ID NO: 244), in which: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). The CDR's of such an ISV are preferably as defined in the preceding two paragraphs.

(153) In another preferred aspect, the VH domains of the invention against CXCR4 comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 263 (which is preferred) or that is an amino acid sequence that has only one amino acid difference with the sequence of SEQ ID NO: 263; (ii) a CDR2 sequence that is the sequence of SEQ ID NO: 264 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 264; and (iii) a CDR3 sequence that is the sequence of SEQ ID NO: 265 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 265.

(154) More preferably, in a VH domain of the invention against CXCR-4 according to this aspect: (i) CDR1 is SEQ ID NO: 263; (ii) CDR2 is SEQ ID NO: 264; and (iii) CDR3 is SEQ ID NO: 265.

(155) In one specific aspect, a Nanobody of the invention against IL-23 is a variant of the Nanobody of SEQ ID NO: 262 (with at least 90% sequence identity, such as at least 95% sequence identity, with SEQ ID NO: 262), in which: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). Again, the CDR's of such an ISV are preferably as defined in the preceding two paragraphs.

(156) Some specifically preferred, but non-limiting examples of Nanobodies of the invention against CXCR4 are listed in FIG. 15A as SEQ ID NO's: 248 to 261 and in FIG. 15B as SEQ ID NO's: 266 to 279; and each of these Nanobodies form a further aspect of the invention.

(157) The invention also relates to a compound of the invention against CXCR4 that comprises at least one (such as one, two or three) of the Nanobodies of the invention of SEQ ID NO's: 248 to 261 and/or 266 to 279. Such compounds of the invention against CXCR4 may again be as further described herein, and thus for example may comprise suitable linkers, may comprise a C-terminal extension as described herein, and may be half-life extended (for example because they comprise a Nanobody against human serum albumin, such as (preferably) a Nanobody of the invention against human serum albumin). Reference is made to Table U below.

(158) As described in for example WO 2009/138519, WO 2011/042398 and WO 2011/161266, one particularly preferred class of Nanobody-based compounds against CXCR4 are biparatopic compounds. Thus, in one aspect of the invention, a compound of the invention against CXCR4 is a biparatopic construct that comprises one ISV that is either SEQ ID NO: 244 or (preferably) an ISV of the invention that has been derived from SEQ ID NO: 244 (as described in this Example 11) and one ISV that is either SEQ ID NO: 262 or (preferably) an ISV of the invention that has been derived from SEQ ID NO: 262 (as described in this Example 11), provided that at least one (and preferably both) of these ISV's are ISV's of the invention. Such biparatopic constructs may also be half-life extended (i.e. by means of a serum albumin-binding ISV). Some specific examples of such biparatopic constructs are given in SEQ ID NO: 595 to 603.

(159) Some specifically preferred examples of compounds of the invention against CXCR-4 are given in FIG. 25 as SEQ ID NO's: 595 to 603; and each of these compounds form a further aspect of the invention. Thus, in another aspect, the invention relates to a polypeptide that is directed against CXCR-4 and that has an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 595 to 603. More generally, compounds of the invention against CXCR-4 may be as described in WO 2009/138519, WO 2011/042398 and WO 2011/161266WO 2011/144749, but comprising ISV's of the invention. They may also be used for the purposes described in WO 2009/138519, WO 2011/042398 and WO 2011/161266.

(160) TABLE-US-00029 TABLE U Examples of compounds of the invention against CXCR-4. Polypeptide/ construct(.sup.1) General formula ISV building blocks and linkers Monovalent [CXCR-4] [CXCR-4] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 Monovalent/ [CXCR-4]-X(n) [CXCR-4] = one of SEQ ID NO's: 464 to 477 and/or 482 to C-terminal 495 extension Monovalent/ [CXCR-4]-L.sub.1-[SA] [CXCR-4] = one of SEQ ID NO's: 464 to 477 and/or 482 to half-life [SA]-L.sub.1-[CXCR-4] 495 extended [SA] = (see legend below) L.sub.1 = (see legend below) Monovalent/ [CXCR-4]-L.sub.1-[SA]-X(n) [CXCR-4] = one of SEQ ID NO's: 464 to 477 and/or 482 to half-life [SA]-L.sub.1-[CXCR-4]-X(n) 495 extended/ [SA] = (see legend below) C-terminal L.sub.1 = (see legend below) extension X(n) = (see legend below) Bivalent(.sup.2) [CXCR-4]-L.sub.1-[CXCR-4] At least one [CXCR-4] present is = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 (.sup.4) L.sub.1 = (see legend below) Bivalent/ [CXCR-4]-L.sub.1-[CXCR-4]-X(n) At least one [CXCR-4] = one of SEQ ID NO's: 464 to 477 C-terminal and/or 482 to 495 (.sup.4) extension(.sup.2) L.sub.1 = (see legend below) X(n) = (see legend below) Bivalent/ [CXCR-4]-L.sub.1-[CXCR-4]-L.sub.2-[SA] At least one [CXCR-4] present is = one of SEQ ID NO's: 464 half-life [CXCR-4]-L.sub.1-[SA]-L.sub.2-[CXCR-4] to 477 and/or 482 to 495 (.sup.4) extended(.sup.2) [SA]-L.sub.1-[CXCR-4]-L.sub.2-[CXCR-4] [SA] = (see legend below) L.sub.1 = (see legend below) L.sub.2 = (see legend below) Bivalent/ [CXCR-4]-L.sub.1-[CXCR-4]-L.sub.2-[SA]-X(n) At least one [CXCR-4] present is = one of SEQ ID NO's: 464 half-life [CXCR-4]-L.sub.1-[SA]-L.sub.2-[CXCR-4]-X(n) to 477 and/or 482 to 495 (.sup.4) extended/ [SA]-L.sub.1-[CXCR-4]-L.sub.2-[CXCR-4]-X(n) [SA] = (see legend below) C-terminal L.sub.1 = (see legend below) extension(.sup.2) L.sub.2 = (see legend below) X(n) = (see legend below) Bispecific (.sup.3) [CXCR-4]-L.sub.1-[Nb] At least one [CXCR-4] present is = one of SEQ ID NO's: 464 [Nb]-L.sub.1-[CXCR-4] to 477 and/or 482 to 495 (.sup.4) [CXCR-4]-L.sub.1-[CXCR-4]-L.sub.2-[Nb] L.sub.1 = (see legend below) [CXCR-4]-L.sub.1-[Nb]-L.sub.2-[CXCR-4] L.sub.2 = (see legend below) [Nb]-L.sub.1-[CXCR-4]-L.sub.2-[CXCR-4] [Nb] = (see legend below) Bispecific/ [CXCR-4]-L.sub.1-[Nb]-X(n) At least one [CXCR-4] present is = one of SEQ ID NO's: 464 C-terminal [Nb]-L.sub.1-[CXCR-4]-X(n) to 477 and/or 482 to 495 (.sup.4) extension (.sup.3) [CXCR-4]-L.sub.1-[CXCR-4]-L.sub.2-[Nb]-X(n) L.sub.1 = (see legend below) [CXCR-4]-L.sub.1-[Nb]-L.sub.2-[CXCR-4]-X(n) L.sub.2 = (see legend below) [Nb]-L.sub.1-[CXCR-4]-L.sub.2-[CXCR-4]-X(n) X(n) = (see legend below) [Nb] = (see legend below) Bispecific/ [CXCR-4]-L.sub.1-[Nb]-L.sub.2-[SA] At least one [CXCR-4] present is = one of SEQ ID NO's: 464 half-life [CXCR-4]-L.sub.1-[SA]-L.sub.2-[Nb] to 477 and/or 482 to 495 (.sup.4) extended (.sup.3) [Nb]-L.sub.1-[SA]-L.sub.2-[CXCR-4] [SA] = (see legend below) [Nb]-L.sub.1-[CXCR-4]-L.sub.2-[SA] L.sub.1, L.sub.2, L.sub.3(see legend below) [SA]-L.sub.1-[CXCR-4]-L.sub.2-[Nb] [Nb] = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[CXCR-4] Bispecific/ [CXCR-4]-L.sub.1-[CXCR-4]-L.sub.2-[Nb]-L.sub.3-[SA] At least one [CXCR-4] present is = one of SEQ ID NO's: 464 half-life [Nb]-L.sub.1-[CXCR-4]-L.sub.2-[CXCR-4]-L.sub.3-[SA] to 477 and/or 482 to 495 (.sup.4) extended (.sup.3) [SA]-L.sub.1-[CXCR-4]-L.sub.2-[CXCR-4]-L.sub.3-[Nb] [SA] = (see legend below) [SA]-L.sub.1-[CXCR-4]-L.sub.2-[Nb]-L.sub.3-[CXCR-4] L.sub.1, L.sub.2, L.sub.3(see legend below) [CXCR-4]-L.sub.1-[Nb]-L.sub.2-[CXCR-4]-L.sub.3-[SA] [Nb] = (see legend below) Bispecific/ [CXCR-4]-L.sub.1-[Nb]-L.sub.2-[SA]-X(n) At least one [CXCR-4] present is = one of SEQ ID NO's: 464 half-life [CXCR-4]-L.sub.1-[SA]-L.sub.2-[Nb]-X(n) to 477 and/or 482 to 495 (.sup.4) extended/ [Nb]-L.sub.1-[SA]-L.sub.2-[CXCR-4]-X(n) [SA] = (see legend below) C-terminal [Nb]-L.sub.1-[CXCR-4]-L.sub.2-[SA]-X(n) L.sub.1, L.sub.2, L.sub.3(see legend below) extension (.sup.3) [SA]-L.sub.1-[CXCR-4]-L.sub.2-[Nb]-X(n) X(n) = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[CXCR-4]-X(n) [Nb] = (see legend below) [CXCR-4]-L.sub.1-[CXCR-4]-L.sub.2-[Nb]-L.sub.3-[SA]-X(n) [Nb]-L.sub.1-[CXCR-4]-L.sub.2-[CXCR-4]-L.sub.3-[SA]-X(n) [SA]-L.sub.1-[CXCR-4]-L.sub.2-[CXCR-4]-L.sub.3-[Nb]-X(n) [SA]-L.sub.1-[CXCR-4]-L.sub.2-[Nb]-L.sub.3-[CXCR-4]-X(n) [CXCR-4]-L.sub.1-[Nb]-L.sub.2-[CXCR-4]-L.sub.3-[SA]-X(n) Legend: [SA] is an ISV against (human) serum albumin, preferably an ISV of the invention against (human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 or even more preferably one of the ISVD's of the invention of SEQ ID NO's: 47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L.sub.1, L.sub.2 and L.sub.3 is (independently) a suitable linker. Each of L.sub.1, L.sub.2 and L.sub.3 may (independently) be present or not. Non-limiting examples of suitable linkers are the gly-ser linkers referred to herein, such as the 9GS, 30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminal extension as described in herein and/or in WO 12/175741 [Nb] is an ISV against another therapeutic target. Notes: (.sup.1)In this Table: “Monovalent” generally refers to polypeptides/constructs comprising a single ISV against CXCR-4. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bivalent” generally refers to polypeptides/constructs comprising two ISV's against CXCR-4 (which may be the same or different). These may again further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bispecific” generally refers to polypeptides/constructs comprising at least one (such as 1 or 2) ISV's against CXCR-4 and at least one (such as 1 or 2) other ISV against a therapeutic target. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). In the polypeptides/constructs described in this table, at least one of the ISV's against CXCR-4 present is an ISV of the invention, and preferably all of the ISV's against CXCR-4 present in such polypeptide/construct are ISV's of the invention. Also, when a half-life extending ISV and/or an ISV against another therapeutic target is present in such polypeptide/construct, each of these (and preferably all of these) may also be (and preferably are) ISV's of the invention (.sup.2)All “bivalent” constructs in this Table may also be biparatopic, meaning that they comprise at least two (such as two) ISV's against CXCR-4, which are directed against different epitopes on CXCR-4. (.sup.3) As will be clear to the skilled person, other/further bispecific constructs than those listed can be made using the building blocks and linkers mentioned. (.sup.4) Preferably, each [CXCR-4] present is independently chosen from SEQ ID NO's: 464 to 477 and/or 482 to 495. Also, the [CXCR-4] present may be the same or different; in a biparatopic polypeptide/construct they will be directed against different epitopes on CXCR-4. (.sup.5) Each of the polypeptides/constructs mentioned to in this column by means of reference to a SEQ ID forms an individual specific aspect of the invention.

Example 12: VH Domains (and in Particular Nanobodies) Against HER-3, and Compounds of the Invention Comprising the Same

(161) In one specific aspect, the VH domains of the invention (and in particular ISVDs of the invention and more in particular Nanobodies of the invention) and compounds of the invention may be directed against HER-3.

(162) Such a VH domain of the invention against HER-3 will generally comprise: (i) suitable framework sequences that suitably comprise the amino acid residues/mutations of the invention as described herein; as well as (ii) CDR sequences that allow the VH domain of the invention to specifically bind to HER-3. In addition, such a VH domain of the invention against HER-3 may also suitably have a C-terminal extension as described herein, in particular when said VH domain is monovalent or forms the C-terminal end of the compound of the invention in which said VH domain is present (again, as further described herein). Such VH domains of the invention against HER-3 may further be as further described herein, and may in particular be ISVD's.

(163) Again, as with other aspects and embodiments of the invention described herein, when a specific ISVD (such as the ISVD against HER-3 described in this Example) or compound comprising the same is said to be “according to the invention” or “as further described herein”, the preferred aspects/embodiments and preferences that are generally described herein for the ISVD's or compounds of the invention also specifically apply to said specific ISVD or compound, respectively, unless explicitly indicated otherwise or unless the specific technical context requires otherwise.

(164) Thus, in a particular aspect, the present invention relates to a VH domain (and in particular an ISVD) that is directed against HER-3, in which (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). In particular, in such VH domains against HER-3: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

(165) The VH domains of the invention against HER-3 may further be as described herein and may again in particular be an ISVD (and more in particular a Nanobody) against HER-3 or a protein, polypeptide or other compound or construct that comprises as least one such ISVD. Such a protein, polypeptide or other compound or construct may also be as further described herein, and may for example have an increased half-life (i.e. as described herein, e.g. a half-life—expressed as t½ beta—in human subjects of in human subjects of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days), and for this purpose may for example comprise a serum-albumin binding Nanobody, which may also be a serum-albumin binding Nanobody of the invention (again, as described herein).

(166) Also, such an ISVD may suitably have a C-terminal extension (as further described herein and in WO 12/175741), in particular when said ISVD forms the C-terminal end of a protein, polypeptide or other compound or construct comprising the same (again, as further described herein).

(167) In one preferred aspect, the VH domains of the invention against HER-3 comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 281 (which is preferred) or that is an amino acid sequence that has only one amino acid difference with the sequence of SEQ ID NO: 281; (ii) a CDR2 sequence that is the sequence of SEQ ID NO: 282 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 282; and (iii) a CDR3 sequence that is the sequence of SEQ ID NO: 283 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 283.

(168) More preferably, in a VH domain of the invention against HER-3 according to this aspect: (i) CDR1 is SEQ ID NO: 281; (ii) CDR2 is SEQ ID NO: 282; and (iii) CDR3 is SEQ ID NO: 283.

(169) In one specific aspect, a Nanobody of the invention against HER-3 is a variant of the Nanobody of SEQ ID NO: 280 (with at least 90% sequence identity, such as at least 95% sequence identity, with SEQ ID NO: 280), in which: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). The CDR's of such an ISV are preferably as defined in the preceding two paragraphs.

(170) In another preferred aspect, the VH domains of the invention against HER-3 comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 299 (which is preferred) or that is an amino acid sequence that has only one amino acid difference with the sequence of SEQ ID NO: 299; (ii) a CDR2 sequence that is the sequence of SEQ ID NO: 300 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 300; and (iii) a CDR3 sequence that is the sequence of SEQ ID NO: 301 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 301.

(171) More preferably, in a VH domain of the invention against HER-3 according to this aspect: (i) CDR1 is SEQ ID NO: 299; (ii) CDR2 is SEQ ID NO: 300; and (iii) CDR3 is SEQ ID NO: 301.

(172) In one specific aspect, a Nanobody of the invention against HER-3 is a variant of the Nanobody of SEQ ID NO: 298 (with at least 90% sequence identity, such as at least 95% sequence identity, with SEQ ID NO: 298), in which: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). Again, the CDR's of such an ISV are preferably as defined in the preceding two paragraphs.

(173) Some specifically preferred, but non-limiting examples of Nanobodies of the invention against HER-3 are listed in FIG. 16A as SEQ ID NO's: 284 to 297 and FIG. 16B as SEQ ID NO's: 302 to 315, respectively; and each of these Nanobodies form a further aspect of the invention.

(174) The invention also relates to a compound of the invention against HER-3 that comprises at least one (such as one, two or three) of the Nanobodies of the invention of SEQ ID NO's: 284 to 297 and/or 302 to 315. Such compounds of the invention against HER-3 may again be as further described herein, and thus for example may comprise suitable linkers, may comprise a C-terminal extension as described herein, and may be half-life extended (for example because they comprise a Nanobody against human serum albumin, such as (preferably) a Nanobody of the invention against human serum albumin). Reference is made to Table V below.

(175) As described in for example WO 2011/144749, one particularly preferred class of Nanobody-based compounds against HER-3 are biparatopic compounds. Thus, in one aspect of the invention, a compound of the invention against HER-3 is a biparatopic construct that comprises one ISV that is either SEQ ID NO: 280 or (preferably) an ISV of the invention that has been derived from SEQ ID NO: 280 (as described in this Example 12) and one ISV that is either SEQ ID NO: 298 or (preferably) an ISV of the invention that has been derived from SEQ ID NO: 298 (as described in this Example 12), provided that at least one (and preferably both) of these ISV's are ISV's of the invention. Such biparatopic constructs may also be half-life extended (i.e. by means of a serum albumin-binding ISV). Some specific examples of such biparatopic constructs are given in SEQ ID NO: 604 to 639.

(176) Some specifically preferred examples of compounds of the invention against HER-3 are given in FIG. 26 as SEQ ID NO's: 604 to 639; and each of these compounds form a further aspect of the invention. Thus, in another aspect, the invention relates to a polypeptide that is directed against HER-3 and that has an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 604 to 639. More generally, compounds of the invention against HER-3 may be as described in WO 2011/144749, but comprising ISV's of the invention. They may also be used for the purposes described in WO 2011/144749.

(177) TABLE-US-00030 TABLE V Examples of compounds of the invention against HER-3. Polypeptide/ construct(.sup.1) General formula ISV building blocks and linkers Monovalent [HER-3] [HER-3] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 Monovalent/ [HER-3]-X(n) [HER-3] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 C-terminal extension Monovalent/ [HER-3]-L.sub.1-[SA] [HER-3] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L.sub.1-[HER-3] [SA] = (see legend below) extended L.sub.1 = (see legend below) Monovalent/ [HER-3]-L.sub.1-[SA]-X(n) [HER-3] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L.sub.1-[HER-3]-X(n) [SA] = (see legend below) extended/ L.sub.1 = (see legend below) C-terminal X(n) = (see legend below) extension Bivalent(.sup.2) [HER-3]-L.sub.1-[HER-3] At least one [HER-3] present is = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 (.sup.4) L.sub.1 = (see legend below) Bivalent/ [HER-3]-L.sub.1-[HER-3]-X(n) At least one [HER-3] = one of SEQ ID NO's: 464 to 477 C-terminal and/or 482 to 495 (.sup.4) extension(.sup.2) L.sub.1 = (see legend below) X(n) = (see legend below) Bivalent/ [HER-3]-L.sub.1-[HER-3]-L.sub.2-[SA] At least one [HER-3] present is = one of SEQ ID NO's: 464 to half-life [HER-3]-L.sub.1-[SA]-L.sub.2-[HER-3] 477 and/or 482 to 495 (.sup.4) extended(.sup.2) [SA]-L.sub.1-[HER-3]-L.sub.2-[HER-3] [SA] = (see legend below) L.sub.1 = (see legend below) L.sub.2 = (see legend below) Bivalent/ [HER-3]-L.sub.1-[HER-3]-L.sub.2-[SA]-X(n) At least one [HER-3] present is = one of SEQ ID NO's: 464 to half-life [HER-3]-L.sub.1-[SA]-L.sub.2-[HER-3]-X(n) 477 and/or 482 to 495 (.sup.4) extended/ [SA]-L.sub.1-[HER-3]-L.sub.2-[HER-3]-X(n) [SA] = (see legend below) C-terminal L.sub.1 = (see legend below) extension(.sup.2) L.sub.2 = (see legend below) X(n) = (see legend below) Bispecific (.sup.3) [HER-3]-L.sub.1-[Nb] At least one [HER-3] present is = one of SEQ ID NO's: 464 to [Nb]-L.sub.1-[HER-3] 477 and/or 482 to 495 (.sup.4) [HER-3]-L.sub.1-[HER-3]-L.sub.2-[Nb] L.sub.1 = (see legend below) [HER-3]-L.sub.1-[Nb]-L.sub.2-[HER-3] L.sub.2 = (see legend below) [Nb]-L.sub.1-[HER-3]-L.sub.2-[HER-3] [Nb] = (see legend below) Bispecific/ [HER-3]-L.sub.1-[Nb]-X(n) At least one [HER-3] present is = one of SEQ ID NO's: 464 to C-terminal [Nb]-L.sub.1-[HER-3]-X(n) 477 and/or 482 to 495 (.sup.4) extension (.sup.3) [HER-3]-L.sub.1-[HER-3]-L.sub.2-[Nb]-X(n) L.sub.1 = (see legend below) [HER-3]-L.sub.1-[Nb]-L.sub.2-[HER-3]-X(n) L.sub.2 = (see legend below) [Nb]-L.sub.1-[HER-3]-L.sub.2-[HER-3]-X(n) X(n) = (see legend below) [Nb] = (see legend below) Bispecific/ [HER-3]-L.sub.1-[Nb]-L.sub.2-[SA] At least one [HER-3] present is = one of SEQ ID NO's: 464 to half-life [HER-3]-L.sub.1-[SA]-L.sub.2-[Nb] 477 and/or 482 to 495 (.sup.4) extended (.sup.3) [Nb]-L.sub.1-[SA]-L.sub.2-[HER-3] [SA] = (see legend below) [Nb]-L.sub.1-[HER-3]-L.sub.2-[SA] L.sub.1, L.sub.2, L.sub.3(see legend below) [SA]-L.sub.1-[HER-3]-L.sub.2-[Nb] [Nb] = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[HER-3] Bispecific/ [HER-3]-L.sub.1-[HER-3]-L.sub.2-[Nb]-L.sub.3-[SA] At least one [HER-3] present is = one of SEQ ID NO's: 464 to half-life [Nb]-L.sub.1-[HER-3]-L.sub.2-[HER-3]-L.sub.3-[SA] 477 and/or 482 to 495 (.sup.4) extended (.sup.3) [SA]-L.sub.1-[HER-3]-L.sub.2-[HER-3]-L.sub.3-[Nb] [SA] = (see legend below) [SA]-L.sub.1-[HER-3]-L.sub.2-[Nb]-L.sub.3-[HER-3] L.sub.1, L.sub.2, L.sub.3(see legend below) [HER-3]-L.sub.1-[Nb]-L.sub.2-[HER-3]-L.sub.3-[SA] [Nb] = (see legend below) Bispecific/ [HER-3]-L.sub.1-[Nb]-L.sub.2-[SA]-X(n) At least one [HER-3] present is = one of SEQ ID NO's: 464 to half-life [HER-3]-L.sub.1-[SA]-L.sub.2-[Nb]-X(n) 477 and/or 482 to 495 (.sup.4) extended/ [Nb]-L.sub.1-[SA]-L.sub.2-[HER-3]-X(n) [SA] = (see legend below) C-terminal [Nb]-L.sub.1-[HER-3]-L.sub.2-[SA]-X(n) L.sub.1, L.sub.2, L.sub.3(see legend below) extension (.sup.3) [SA]-L.sub.1-[HER-3]-L.sub.2-[Nb]-X(n) X(n) = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[HER-3]-X(n) [Nb] = (see legend below) [HER-3]-L.sub.1-[HER-3]-L.sub.2-[Nb]-L.sub.3-[SA]-X(n) [Nb]-L.sub.1-[HER-3]-L.sub.2-[HER-3]-L.sub.3-[SA]-X(n) [SA]-L.sub.1-[HER-3]-L.sub.2-[HER-3]-L.sub.3-[Nb]-X(n) [SA]-L.sub.1-[HER-3]-L.sub.2-[Nb]-L.sub.3-[HER-3]-X(n) [HER-3]-L.sub.1-[Nb]-L.sub.2-[HER-3]-L.sub.3-[SA]-X(n) Legend: [SA] is an ISV against (human) serum albumin, preferably an ISV of the invention against (human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 or even more preferably one of the ISVD's of the invention of SEQ ID NO's: 47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L.sub.1, L.sub.2 and L.sub.3 is (independently) a suitable linker. Each of L.sub.1, L.sub.2 and L.sub.3 may (independently) be present or not. Non-limiting examples of suitable linkers are the gly-ser linkers referred to herein, such as the 9GS, 30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminal extension as described in herein and/or in WO 12/175741 [Nb] is an ISV against another therapeutic target. Notes: (.sup.1)In this Table: “Monovalent” generally refers to polypeptides/constructs comprising a single ISV against HER-3. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bivalent” generally refers to polypeptides/constructs comprising two ISV's against HER-3 (which may be the same or different). These may again further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bispecific” generally refers to polypeptides/constructs comprising at least one (such as 1 or 2) ISV's against HER-3 and at least one (such as 1 or 2) other ISV against a therapeutic target. These may farther comprise a half-life extending ISV (such as an ISV against serum albumin). In the polypeptides/constructs described in this table, at least one of the ISV's against HER-3 present is an ISV of the invention, and preferably all of the ISV's against HER-3 present in such polypeptide/construct are ISV's of the invention. Also, when a half-life extending ISV and/or an ISV against another therapeutic target is present in such polypeptide/construct, each of these (and preferably all of these) may also be (and preferably are) ISV's of the invention (.sup.2)All “bivalent” constructs in this Table may also be biparatopic, meaning that they comprise at least two (such as two) ISV's against HER-3, which are directed against different epitopes on HER-3. (.sup.3) As will be clear to the skilled person, other/further bispecific constructs than those listed can be made using the building blocks and linkers mentioned. (.sup.4) Preferably, each [HER-3] present is independently chosen from SEQ ID NO's: 464 to 477 and/or 482 to 495. Also, the [HER-3] present may be the same or different; in a biparatopic polypeptide/construct they will be directed against different epitopes on HER-3. (.sup.5) Each of the polypeptides/constructs mentioned to in this column by means of reference to a SEQ ID forms an individual specific aspect of the invention.

Example 13: VH Domains (and in Particular Nanobodies) Against TNF, and Compounds of the Invention Comprising the Same

(178) In one specific aspect, the VH domains of the invention (and in particular ISVDs of the invention and more in particular Nanobodies of the invention) and compounds of the invention may be directed against TNF.

(179) Such a VH domain of the invention against TNF will generally comprise: (i) suitable framework sequences that suitably comprise the amino acid residues/mutations of the invention as described herein; as well as (ii) CDR sequences that allow the VH domain of the invention to specifically bind to TNF. In addition, such a VH domain of the invention against TNF may also suitably have a C-terminal extension as described herein, in particular when said VH domain is monovalent or forms the C-terminal end of the compound of the invention in which said VH domain is present (again, as further described herein). Such VH domains of the invention against TNF may further be as further described herein, and may in particular be ISVD's.

(180) Again, as with other aspects and embodiments of the invention described herein, when a specific ISVD (such as the ISVD against TNF described in this Example) or compound comprising the same is said to be “according to the invention” or “as further described herein”, the preferred aspects/embodiments and preferences that are generally described herein for the ISVD's or compounds of the invention also specifically apply to said specific ISVD or compound, respectively, unless explicitly indicated otherwise or unless the specific technical context requires otherwise.

(181) Thus, in a particular aspect, the present invention relates to a VH domain (and in particular an ISVD) that is directed against TNF, in which (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). In particular, in such VH domains against TNF: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

(182) The VH domains of the invention against TNF may further be as described herein and may again in particular be an ISVD (and more in particular a Nanobody) against TNF or a protein, polypeptide or other compound or construct that comprises as least one such ISVD. Such a protein, polypeptide or other compound or construct may also be as further described herein, and may for example have an increased half-life (i.e. as described herein, e.g. a half-life—expressed as t½ beta—in human subjects of in human subjects of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days), and for this purpose may for example comprise a serum-albumin binding Nanobody, which may also be a serum-albumin binding Nanobody of the invention (again, as described herein).

(183) Also, such an ISVD may suitably have a C-terminal extension (as further described herein and in WO 12/175741), in particular when said ISVD forms the C-terminal end of a protein, polypeptide or other compound or construct comprising the same (again, as further described herein).

(184) In one preferred aspect, the VH domains of the invention against TNF comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 317 (which is preferred) or that is an amino acid sequence that has only one amino acid difference with the sequence of SEQ ID NO: 317; (ii) a CDR2 sequence that is the sequence of SEQ ID NO: 318 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 318; and (iii) a CDR3 sequence that is the sequence of SEQ ID NO: 319 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 319.

(185) More preferably, in a VH domain of the invention against TNF according to this aspect: (i) CDR1 is SEQ ID NO: 317; (ii) CDR2 is SEQ ID NO: 318; and (iii) CDR3 is SEQ ID NO: 319.

(186) In one specific aspect, a Nanobody of the invention against TNF is a variant of the Nanobody of SEQ ID NO: 316 (with at least 90% sequence identity, such as at least 95% sequence identity, with SEQ ID NO: 316), in which: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). The CDR's of such an ISV are preferably as defined in the preceding two paragraphs.

(187) Some specifically preferred, but non-limiting examples of Nanobodies of the invention against TNF are listed in FIG. 17A as SEQ ID NO's: 320 to 333; and each of these Nanobodies form a further aspect of the invention.

(188) The invention also relates to a compound of the invention against TNF that comprises at least one (such as one, two or three) of the Nanobodies of the invention of SEQ ID NO's: 320 to 333. Such compounds of the invention against TNF may again be as further described herein, and thus for example may comprise suitable linkers, may comprise a C-terminal extension as described herein, and may be half-life extended (for example because they comprise a Nanobody against human serum albumin, such as (preferably) a Nanobody of the invention against human serum albumin). Reference is made to Table W below. Generally, as TNF is a multivalent target, compounds of the invention comprising two or three anti-TNF ISV's (and linkers of suitable length, see WO 06/122786) are preferred)

(189) Some specifically preferred examples of compounds of the invention against TNF are given in FIG. 27 as SEQ ID NO's: 640 to 675; and each of these compounds form a further aspect of the invention. Thus, in another aspect, the invention relates to a polypeptide that is directed against TNF and that has an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 640 to 675. More generally, compounds of the invention against TNF may be as described in WO 2006/122786, but comprising ISV's of the invention. They may also be used for the purposes described in WO 2006/122786.

(190) TABLE-US-00031 TABLE W Examples of compounds of the invention against TNF. Polypeptide/ construct(.sup.1) General formula ISV building blocks and linkers Monovalent [TNF] [TNF] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 Monovalent/ [TNF]-X(n) [TNF] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 C-terminal extension Monovalent/ [TNF]-L.sub.1-[SA] [TNF] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L.sub.1-[TNF] [SA] = (see legend below) extended L.sub.1 = (see legend below) Monovalent/ [TNF]-L.sub.1-[SA]-X(n) [TNF] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L.sub.1-[TNF]-X(n) [SA] = (see legend below) extended/ L.sub.1 = (see legend below) C-terminal X(n) = (see legend below) extension Bivalent(.sup.2) [TNF]-L.sub.1-[TNF] At least one [TNF] present is = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 (.sup.4) L.sub.1 = (see legend below) Bivalent/ [TNF]-L.sub.1-[TNF]-X(n) At least one [TNF] = one of SEQ ID NO's: 464 to 477 and/or C-terminal 482 to 495 (.sup.4) extension(.sup.2) L.sub.1 = (see legend below) X(n) = (see legend below) Bivalent/ [TNF]-L.sub.1-[TNF]-L.sub.2-[SA] At least one [TNF] present is = one of SEQ ID NO's: 464 to half-life [TNF]-L.sub.1-[SA]-L.sub.2-[TNF] 477 and/or 482 to 495 (.sup.4) extended(.sup.2) [SA]-L.sub.1-[TNF]-L.sub.2-[TNF] [SA] = (see legend below) L.sub.1 = (see legend below) L.sub.2 = (see legend below) Bivalent/ [TNF]-L.sub.1-[TNF]-L.sub.2-[SA]-X(n) At least one [TNF] present is = one of SEQ ID NO's: 464 to half-life [TNF]-L.sub.1-[SA]-L.sub.2-[TNF]-X(n) 477 and/or 482 to 495 (.sup.4) extended/ [SA]-L.sub.1-[TNF]-L.sub.2-[TNF]-X(n) [SA] = (see legend below) C-terminal L.sub.1 = (see legend below) extension(.sup.2) L.sub.2 = (see legend below) X(n) = (see legend below) Bispecific (.sup.3) [TNF]-L.sub.1-[Nb] At least one [TNF] present is = one of SEQ ID NO's: 464 to [Nb]-L.sub.1-[TNF] 477 and/or 482 to 495 (.sup.4) [TNF]-L.sub.1-[TNF]-L.sub.2-[Nb] L.sub.1 = (see legend below) [TNF]-L.sub.1-[Nb]-L.sub.2-[TNF] L.sub.2 = (see legend below) [Nb]-L.sub.1-[TNF]-L.sub.2-[TNF] [Nb] = (see legend below) Bispecific/ [TNF]-L.sub.1-[Nb]-X(n) At least one [TNF] present is = one of SEQ ID NO's: 464 to C-terminal [Nb]-L.sub.1-[TNF]-X(n) 477 and/or 482 to 495 (.sup.4) extension (.sup.3) [TNF]-L.sub.1-[TNF]-L.sub.2-[Nb]-X(n) L.sub.1 = (see legend below) [TNF]-L.sub.1-[Nb]-L.sub.2-[TNF]-X(n) L.sub.2 = (see legend below) [Nb]-L.sub.1-[TNF]-L.sub.2-[TNF]-X(n) X(n) = (see legend below) [Nb] = (see legend below) Bispecific/ [TNF]-L.sub.1-[Nb]-L.sub.2-[SA] At least one [TNF] present is = one of SEQ ID NO's: 464 to half-life [TNF]-L.sub.1-[SA]-L.sub.2-[Nb] 477 and/or 482 to 495 (.sup.4) extended (.sup.3) [Nb]-L.sub.1-[SA]-L.sub.2-[TNF] [SA] = (see legend below) [Nb]-L.sub.1-[TNF]-L.sub.2-[SA] L.sub.1, L.sub.2, L.sub.3(see legend below) [SA]-L.sub.1-[TNF]-L.sub.2-[Nb] [Nb] = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[TNF] Bispecific/ [TNF]-L.sub.1-[TNF]-L.sub.2-[Nb]-L.sub.3-[SA] At least one [TNF] present is = one of SEQ ID NO's: 464 to half-life [Nb]-L.sub.1-[TNF]-L.sub.2-[TNF]-L.sub.3-[SA] 477 and/or 482 to 495 (.sup.4) extended (.sup.3) [SA]-L.sub.1-[TNF]-L.sub.2-[TNF]-L.sub.3-[Nb] [SA] = (see legend below) [SA]-L.sub.1-[TNF]-L.sub.2-[Nb]-L.sub.3-[TNF] L.sub.1, L.sub.2, L.sub.3(see legend below) [TNF]-L.sub.1-[Nb]-L.sub.2-[TNF]-L.sub.3-[SA] [Nb] = (see legend below) Bispecific/ [TNF]-L.sub.1-[Nb]-L.sub.2-[SA]-X(n) At least one [TNF] present is = one of SEQ ID NO's: 464 to half-life [TNF]-L.sub.1-[SA]-L.sub.2-[Nb]-X(n) 477 and/or 482 to 495 (.sup.4) extended/ [Nb]-L.sub.1-[SA]-L.sub.2-[TNF]-X(n) [SA] = (see legend below) C-terminal [Nb]-L.sub.1-[TNF]-L.sub.2-[SA]-X(n) L.sub.1, L.sub.2, L.sub.3(see legend below) extension (.sup.3) [SA]-L.sub.1-[TNF]-L.sub.2-[Nb]-X(n) X(n) = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[TNF]-X(n) [Nb] = (see legend below) [TNF]-L.sub.1-[TNF]-L.sub.2-[Nb]-L.sub.3-[SA]-X(n) [Nb]-L.sub.1-[TNF]-L.sub.2-[TNF]-L.sub.3-[SA]-X(n) [SA]-L.sub.1-[TNF]-L.sub.2-[TNF]-L.sub.3-[Nb]-X(n) [SA]-L.sub.1-[TNF]-L.sub.2-[Nb]-L.sub.3-[TNF]-X(n) [TNF]-L.sub.1-[Nb]-L.sub.2-[TNF]-L.sub.3-[SA]-X(n) Legend: [SA] is an ISV against (human) serum albumin, preferably an ISV of the invention against (human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 or even more preferably one of the ISVD's of the invention of SEQ ID NO's: 47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L.sub.1, L.sub.2 and L.sub.3 is (independently) a suitable linker. Each of L.sub.1, L.sub.2 and L.sub.3 may (independently) be present or not. Non-limiting examples of suitable linkers are the gly-ser linkers referred to herein, such as the 9GS, 30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminal extension as described in herein and/or in WO 12/175741 [Nb] is an ISV against another therapeutic target. Notes: (.sup.1)In this Table: “Monovalent” generally refers to polypeptides/constructs comprising a single ISV against TNF. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bivalent” generally refers to polypeptides/constructs comprising two ISV's against TNF (which may be the same or different). These may again further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bispecific” generally refers to polypeptides/constructs comprising at least one (such as 1 or 2) ISV's against TNF and at least one (such as 1 or 2) other ISV against a therapeutic target. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). In the polypeptides/constructs described in this table, at least one of the ISV's against TNF present is an ISV of the invention, and preferably all of the ISV's against TNF present in such polypeptide/construct are ISV's of the invention. Also, when a half-life extending ISV and/or an ISV against another therapeutic target is present in such polypeptide/construct, each of these (and preferably all of these) may also be (and preferably are) ISV's of the invention (.sup.2)All “bivalent” constructs in this Table may also be biparatopic, meaning that they comprise at least two (such as two) ISV's against TNF, which are directed against different epitopes on TNF. (.sup.3) As will be clear to the skilled person, other/further bispecific constructs than those listed can be made using the building blocks and linkers mentioned. (.sup.4) Preferably, each [TNF] present is independently chosen from SEQ ID NO's: 464 to 477 and/or 482 to 495. Also, the [TNF] present may be the same or different; in a biparatopic polypeptide/construct they will be directed against different epitopes on TNF. (.sup.5) Each of the polypeptides/constructs mentioned to in this column by means of reference to a SEQ ID forms an individual specific aspect of the invention.

Example 14: Further VH Domains (and in Particular Nanobodies) Against TNF, and Compounds of the Invention Comprising the Same

(191) In one specific aspect, the VH domains of the invention (and in particular ISVDs of the invention and more in particular Nanobodies of the invention) and compounds of the invention may be directed against TNF.

(192) Such a VH domain of the invention against TNF will generally comprise: (i) suitable framework sequences that suitably comprise the amino acid residues/mutations of the invention as described herein; as well as (ii) CDR sequences that allow the VH domain of the invention to specifically bind to TNF. In addition, such a VH domain of the invention against TNF may also suitably have a C-terminal extension as described herein, in particular when said VH domain is monovalent or forms the C-terminal end of the compound of the invention in which said VH domain is present (again, as further described herein). Such VH domains of the invention against TNF may further be as further described herein, and may in particular be ISVD's.

(193) Again, as with other aspects and embodiments of the invention described herein, when a specific ISVD (such as the ISVD against TNF described in this Example) or compound comprising the same is said to be “according to the invention” or “as further described herein”, the preferred aspects/embodiments and preferences that are generally described herein for the ISVD's or compounds of the invention also specifically apply to said specific ISVD or compound, respectively, unless explicitly indicated otherwise or unless the specific technical context requires otherwise.

(194) Thus, in a particular aspect, the present invention relates to a VH domain (and in particular an ISVD) that is directed against TNF, in which (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). In particular, in such VH domains against TNF: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

(195) The VH domains of the invention against TNF may further be as described herein and may again in particular be an ISVD (and more in particular a Nanobody) against TNF or a protein, polypeptide or other compound or construct that comprises as least one such ISVD. Such a protein, polypeptide or other compound or construct may also be as further described herein, and may for example have an increased half-life (i.e. as described herein, e.g. a half-life—expressed as t½ beta—in human subjects of in human subjects of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days), and for this purpose may for example comprise a serum-albumin binding Nanobody, which may also be a serum-albumin binding Nanobody of the invention (again, as described herein).

(196) Also, such an ISVD may suitably have a C-terminal extension (as further described herein and in WO 12/175741), in particular when said ISVD forms the C-terminal end of a protein, polypeptide or other compound or construct comprising the same (again, as further described herein).

(197) In one preferred aspect, the VH domains of the invention against TNF comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 335 (which is preferred) or that is an amino acid sequence that has only one amino acid difference with the sequence of SEQ ID NO: 335; (ii) a CDR2 sequence that is the sequence of SEQ ID NO: 336 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 336; and (iii) a CDR3 sequence that is the sequence of SEQ ID NO: 337 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 337.

(198) More preferably, in a VH domain of the invention against TNF according to this aspect: (i) CDR1 is SEQ ID NO: 335; (ii) CDR2 is SEQ ID NO: 336; and (iii) CDR3 is SEQ ID NO: 337.

(199) In one specific aspect, a Nanobody of the invention against TNF is a variant of the Nanobody of SEQ ID NO: 334 (with at least 90% sequence identity, such as at least 95% sequence identity, with SEQ ID NO: 334), in which: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). The CDR's of such an ISV are preferably as defined in the preceding two paragraphs.

(200) Some specifically preferred, but non-limiting examples of Nanobodies of the invention against TNF are listed in FIG. 17B as SEQ ID NO's: 338 to 351; and each of these Nanobodies form a further aspect of the invention.

(201) The invention also relates to a compound of the invention against TNF that comprises at least one (such as one, two or three) of the Nanobodies of the invention of SEQ ID NO's: 338 to 251. Such compounds of the invention against TNF may again be as further described herein, and thus for example may comprise suitable linkers, may comprise a C-terminal extension as described herein, and may be half-life extended (for example because they comprise a Nanobody against human serum albumin, such as (preferably) a Nanobody of the invention against human serum albumin). Reference is made to Table X below. Generally, as TNF is a multivalent target, compounds of the invention comprising two or three anti-TNF ISV's are preferred.

(202) TABLE-US-00032 TABLE X Examples of compounds of the invention against TNF. Polypeptide/ construct(.sup.1) General formula ISV building blocks and linkers Monovalent [TNF] [TNF] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 Monovalent/ [TNF]-X(n) [TNF] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 C-terminal extension Monovalent/ [TNF]-L.sub.1-[SA] [TNF] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L.sub.1-[TNF] [SA] = (see legend below) extended L.sub.1 = (see legend below) Monovalent/ [TNF]-L.sub.1-[SA]-X(n) [TNF] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L.sub.1-[TNF]-X(n) [SA] = (see legend below) extended/ L.sub.1 = (see legend below) C-terminal X(n) = (see legend below) extension Bivalent(.sup.2) [TNF]-L.sub.1-[TNF] At least one [TNF] present is = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 (.sup.4) L.sub.1 = (see legend below) Bivalent/ [TNF]-L.sub.1-[TNF]-X(n) At least one [TNF] = one of SEQ ID NO's: 464 to 477 and/or C-terminal 482 to 495 (.sup.4) extension(.sup.2) L.sub.1 = (see legend below) X(n) = (see legend below) Bivalent/ [TNF]-L.sub.1-[TNF]-L.sub.2-[SA] At least one [TNF] present is = one of SEQ ID NO's: 464 to 477 half-life [TNF]-L.sub.1-[SA]-L.sub.2-[TNF] and/or 482 to 495 (.sup.4) extended(.sup.2) [SA]-L.sub.1-[TNF]-L.sub.2-[TNF] [SA] = (see legend below) L.sub.1 = (see legend below) L.sub.2 = (see legend below) Bivalent/ [TNF]-L.sub.1-[TNF]-L.sub.2-[SA]-X(n) At least one [TNF] present is = one of SEQ ID NO's: 464 to 477 half-life [TNF]-L.sub.1-[SA]-L.sub.2-[TNF]-X(n) and/or 482 to 495 (.sup.4) extended/ [SA]-L.sub.1-[TNF]-L.sub.2-[TNF]-X(n) [SA] = (see legend below) C-terminal L.sub.1 = (see legend below) extension(.sup.2) L.sub.2 = (see legend below) X(n) = (see legend below) Bispecific (.sup.3) [TNF]-L.sub.1-[Nb] At least one [TNF] present is = one of SEQ ID NO's: 464 to 477 [Nb]-L.sub.1-[TNF] and/or 482 to 495 (.sup.4) [TNF]-L.sub.1-[TNF]-L.sub.2-[Nb] L.sub.1 = (see legend below) [TNF]-L.sub.1-[Nb]-L.sub.2-[TNF] L.sub.2 = (see legend below) [Nb]-L.sub.1-[TNF]-L.sub.2-[TNF] [Nb] = (see legend below) Bispecific/ [TNF]-L.sub.1-[Nb]-X(n) At least one [TNF] present is = one of SEQ ID NO's: 464 to 477 C-terminal [Nb]-L.sub.1-[TNF]-X(n) and/or 482 to 495 (.sup.4) extension (.sup.3) [TNF]-L.sub.1-[TNF]-L.sub.2-[Nb]-X(n) L.sub.1 = (see legend below) [TNF]-L.sub.1-[Nb]-L.sub.2-[TNF]-X(n) L.sub.2 = (see legend below) [Nb]-L.sub.1-[TNF]-L.sub.2-[TNF]-X(n) X(n) = (see legend below) [Nb] = (see legend below) Bispecific/ [TNF]-L.sub.1-[Nb]-L.sub.2-[SA] At least one [TNF] present is = one of SEQ ID NO's: 464 to 477 half-life [TNF]-L.sub.1-[SA]-L.sub.2-[Nb] and/or 482 to 495 (.sup.4) extended (.sup.3) [Nb]-L.sub.1-[SA]-L.sub.2-[TNF] [SA] = (see legend below) [Nb]-L.sub.1-[TNF]-L.sub.2-[SA] L.sub.1, L.sub.2, L.sub.3(see legend below) [SA]-L.sub.1-[TNF]-L.sub.2-[Nb] [Nb] = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[TNF] Bispecific/ [TNF]-L.sub.1-[TNF]-L.sub.2-[Nb]-L.sub.3-[SA] At least one [TNF] present is = one of SEQ ID NO's: 464 to 477 half-life [Nb]-L.sub.1-[TNF]-L.sub.2-[TNF]-L.sub.3-[SA] and/or 482 to 495 (.sup.4) extended (.sup.3) [SA]-L.sub.1-[TNF]-L.sub.2-[TNF]-L.sub.3-[Nb] [SA] = (see legend below) [SA]-L.sub.1-[TNF]-L.sub.2-[Nb]-L.sub.3-[TNF] L.sub.1, L.sub.2, L.sub.3(see legend below) [TNF]-L.sub.1-[Nb]-L.sub.2-[TNF]-L.sub.3-[SA] [Nb] = (see legend below) Bispecific/ [TNF]-L.sub.1-[Nb]-L.sub.2-[SA]-X(n) At least one [TNF] present is = one of SEQ ID NO's: 464 to 477 half-life [TNF]-L.sub.1-[SA]-L.sub.2-[Nb]-X(n) and/or 482 to 495 (.sup.4) extended/ [Nb]-L.sub.1-[SA]-L.sub.2-[TNF]-X(n) [SA] = (see legend below) C-terminal [Nb]-L.sub.1-[TNF]-L.sub.2-[SA]-X(n) L.sub.1, L.sub.2, L.sub.3(see legend below) extension (.sup.3) [SA]-L.sub.1-[TNF]-L.sub.2-[Nb]-X(n) X(n) = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[TNF]-X(n) [Nb] = (see legend below) [TNF]-L.sub.1-[TNF]-L.sub.2-[Nb]-L.sub.3-[SA]-X(n) [Nb]-L.sub.1-[TNF]-L.sub.2-[TNF]-L.sub.3-[SA]-X(n) [SA]-L.sub.1-[TNF]-L.sub.2-[TNF]-L.sub.3-[Nb]-X(n) [SA]-L.sub.1-[TNF]-L.sub.2-[Nb]-L.sub.3-[TNF]-X(n) [TNF]-L.sub.1-[Nb]-L.sub.2-[TNF]-L.sub.3-[SA]-X(n) Legend: [SA] is an ISV against (human) serum albumin, preferably an ISV of the invention against (human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 or even more preferably one of the ISVD's of the invention of SEQ ID NO's: 47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L.sub.1, L.sub.2 and L.sub.3 is (independently) a suitable linker. Each of L.sub.1, L.sub.2 and L.sub.3 may (independently) be present or not. Non-limiting examples of suitable linkers are the gly-ser linkers referred to herein, such as the 9GS, 30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminal extension as described in herein and/or in WO 12/175741 [Nb] is an ISV against another therapeutic target. Notes: (.sup.1)In this Table: “Monovalent” generally refers to polypeptides/constructs comprising a single ISV against TNF. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bivalent” generally refers to polypeptides/constructs comprising two ISV's against TNF (which may be the same or different). These may again further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bispecific” generally refers to polypeptides/constructs comprising at least one (such as 1 or 2) ISV's against TNF and at least one (such as 1 or 2) other ISV against a therapeutic target. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). In the polypeptides/constructs described in this table, at least one of the ISV's against TNF present is an ISV of the invention, and preferably all of the ISV's against TNF present in such polypeptide/construct are ISV's of the invention. Also, when a half-life extending ISV and/or an ISV against another therapeutic target is present in such polypeptide/construct, each of these (and preferably all of these) may also be (and preferably are) ISV's of the invention (.sup.2)All “bivalent” constructs in this Table may also be biparatopic, meaning that they comprise at least two (such as two) ISV's against TNF, which are directed against different epitopes on TNF. (.sup.3) As will be clear to the skilled person, other/further bispecific constructs than those listed can be made using the building blocks and linkers mentioned. (.sup.4) Preferably, each [TNF] present is independently chosen from SEQ ID NO's: 464 to 477 and/or 482 to 495. Also, the [TNF] present may be the same or different; in a biparatopic polypeptide/construct they will be directed against different epitopes on TNF. (.sup.5) Each of the polypeptides/constructs mentioned to in this column by means of reference to a SEQ ID forms an individual specific aspect of the invention.

Example 15: VH Domains (and in Particular Nanobodies) Against c-Met, and Compounds of the Invention Comprising the Same

(203) In one specific aspect, the VH domains of the invention (and in particular ISVDs of the invention and more in particular Nanobodies of the invention) and compounds of the invention may be directed against c-Met.

(204) Such a VH domain of the invention against c-Met will generally comprise: (i) suitable framework sequences that suitably comprise the amino acid residues/mutations of the invention as described herein; as well as (ii) CDR sequences that allow the VH domain of the invention to specifically bind to c-Met. In addition, such a VH domain of the invention against c-Met may also suitably have a C-terminal extension as described herein, in particular when said VH domain is monovalent or forms the C-terminal end of the compound of the invention in which said VH domain is present (again, as further described herein). Such VH domains of the invention against c-Met may further be as further described herein, and may in particular be ISVD's.

(205) Again, as with other aspects and embodiments of the invention described herein, when a specific ISVD (such as the ISVD against c-Met described in this Example) or compound comprising the same is said to be “according to the invention” or “as further described herein”, the preferred aspects/embodiments and preferences that are generally described herein for the ISVD's or compounds of the invention also specifically apply to said specific ISVD or compound, respectively, unless explicitly indicated otherwise or unless the specific technical context requires otherwise.

(206) Thus, in a particular aspect, the present invention relates to a VH domain (and in particular an ISVD) that is directed against c-Met, in which (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). In particular, in such VH domains against c-Met: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

(207) The VH domains of the invention against c-Met may further be as described herein and may again in particular be an ISVD (and more in particular a Nanobody) against c-Met or a protein, polypeptide or other compound or construct that comprises as least one such ISVD. Such a protein, polypeptide or other compound or construct may also be as further described herein, and may for example have an increased half-life (i.e. as described herein, e.g. a half-life—expressed as t½ beta—in human subjects of in human subjects of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days), and for this purpose may for example comprise a serum-albumin binding Nanobody, which may also be a serum-albumin binding Nanobody of the invention (again, as described herein).

(208) Also, such an ISVD may suitably have a C-terminal extension (as further described herein and in WO 12/175741), in particular when said ISVD forms the C-terminal end of a protein, polypeptide or other compound or construct comprising the same (again, as further described herein).

(209) In one preferred aspect, the VH domains of the invention against c-Met comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 353 (which is preferred) or that is an amino acid sequence that has only one amino acid difference with the sequence of SEQ ID NO: 353; (ii) a CDR2 sequence that is the sequence of SEQ ID NO: 354 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 354; and (iii) a CDR3 sequence that is the sequence of SEQ ID NO: 355 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 355.

(210) More preferably, in a VH domain of the invention against c-Met according to this aspect: (i) CDR1 is SEQ ID NO: 353; (ii) CDR2 is SEQ ID NO: 354; and (iii) CDR3 is SEQ ID NO: 355.

(211) In one specific aspect, a Nanobody of the invention against c-Met is a variant of the Nanobody of SEQ ID NO: 352 (with at least 90% sequence identity, such as at least 95% sequence identity, with SEQ ID NO: 352), in which: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). The CDR's of such an ISV are preferably as defined in the preceding two paragraphs.

(212) In another preferred aspect, the VH domains of the invention against c-Met comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 371 (which is preferred) or that is an amino acid sequence that has only one amino acid difference with the sequence of SEQ ID NO: 371; (ii) a CDR2 sequence that is the sequence of SEQ ID NO: 372 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 372; and (iii) a CDR3 sequence that is the sequence of SEQ ID NO: 373 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 373.

(213) More preferably, in such a VH domain of the invention against c-Met according to this aspect: (i) CDR1 is SEQ ID NO: 371; (ii) CDR2 is SEQ ID NO: 372; and (iii) CDR3 is SEQ ID NO: 373.

(214) In one specific aspect, a Nanobody of the invention against c-Met is a variant of the Nanobody of SEQ ID NO: 370 (with at least 90% sequence identity, such as at least 95% sequence identity, with SEQ ID NO: 370), in which: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). Again, the CDR's of such an ISV are preferably as defined in the preceding two paragraphs.

(215) Some specifically preferred, but non-limiting examples of Nanobodies of the invention against c-Met are listed in FIG. 18A as SEQ ID NO's: 356 to 369 and in FIG. 18B as SEQ ID NO's: 374 to 387, respectively; and each of these Nanobodies form a further aspect of the invention.

(216) The invention also relates to a compound of the invention against c-Met that comprises at least one (such as one, two or three) of the Nanobodies of the invention of SEQ ID NO's: 356 to 369 and/or 374 to 387. Such compounds of the invention against c-Met may again be as further described herein, and thus for example may comprise suitable linkers, may comprise a C-terminal extension as described herein, and may be half-life extended (for example because they comprise a Nanobody against human serum albumin, such as (preferably) a Nanobody of the invention against human serum albumin). Reference is made to Table Y below.

(217) As described in for example WO 2013/045707, one particularly preferred class of Nanobody-based compounds against c-Met are biparatopic compounds. Thus, in one aspect of the invention, a compound of the invention against c-Met is a biparatopic construct that comprises one ISV that is either SEQ ID NO: 352 or (preferably) an ISV of the invention that has been derived from SEQ ID NO: 352 (as described in this Example 15) and one ISV that is either SEQ ID NO: 370 or (preferably) an ISV of the invention that has been derived from SEQ ID NO: 370 (as described in this Example 15), provided that at least one (and preferably both) of these ISV's are ISV's of the invention. Such biparatopic constructs may also be half-life extended (i.e. by means of a serum albumin-binding ISV). Some specific examples of such biparatopic constructs are given in SEQ ID NO: 676 to 693. Also, bispecific constructs against c-Met may also comprise an ISV against VEGF or EGFR. Reference is again made to WO 2014/341309.

(218) Some specifically preferred examples of compounds of the invention against c-Met are given in FIGS. 28A and 28B SEQ ID NO's: 676 to 694; and each of these compounds form a further aspect of the invention. Thus, in another aspect, the invention relates to a polypeptide that is directed against c-Met and that has an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 676 to 694. More generally, compounds of the invention against c-Met may be as described in WO 2013/045707, but comprising ISV's of the invention. They may also be used for the purposes described in WO 2013/045707.

(219) TABLE-US-00033 TABLE Y Examples of compounds of the invention against c-Met. Polypeptide/ construct(.sup.1) General formula ISV building blocks and linkers Monovalent [c-Met] [c-Met] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 Monovalent/ [c-Met]-X(n) [c-Met] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 C-terminal extension Monovalent/ [c-Met]-L.sub.1-[SA] [c-Met] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L.sub.1-[c-Met] [SA] = (see legend below) extended L.sub.1 = (see legend below) Monovalent/ [c-Met]-L.sub.1-[SA]-X(n) [c-Met] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L.sub.1-[c-Met]-X(n) [SA] = (see legend below) extended/C- L.sub.1 = (see legend below) terminal X(n) = (see legend below) extension Bivalent(.sup.2) [c-Met]-L.sub.1-[c-Met] At least one [c-Met] present is = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 (.sup.4) L.sub.1 = (see legend below) Bivalent/ [c-Met]-L.sub.1-[c-Met]-X(n) At least one [c-Met] = one of SEQ ID NO's: 464 to 477 and/or 482 C-terminal to 495 (.sup.4) extension(.sup.2) L.sub.1 = (see legend below) X(n) = (see legend below) Bivalent/ [c-Met]-L.sub.1-[c-Met]-L.sub.2-[SA] At least one [c-Met] present is = one of SEQ ID NO's: 464 to 477 half-life [c-Met]-L.sub.1-[SA]-L.sub.2-[c-Met] and/or 482 to 495 (.sup.4) extended(.sup.2) [SA]-L.sub.1-[c-Met]-L.sub.2-[c-Met] [SA] = (see legend below) L.sub.1 = (see legend below) L.sub.2 = (see legend below) Bivalent/ [c-Met]-L.sub.1-[c-Met]-L.sub.2-[SA]-X(n) At least one [c-Met] present is = one of SEQ ID NO's: 464 to 477 half-life [c-Met]-L.sub.1-[SA]-L.sub.2-[c-Met]-X(n) and/or 482 to 495 (.sup.4) extended/ [SA]-L.sub.1-[c-Met]-L.sub.2-[c-Met]-X(n) [SA] = (see legend below) C-terminal L.sub.1 = (see legend below) extension(.sup.2) L.sub.2 = (see legend below) X(n) = (see legend below) Bispecific (.sup.3) [c-Met]-L.sub.1-[Nb] At least one [c-Met] present is = one of SEQ ID NO's: 464 to 477 [Nb]-L.sub.1-[c-Met] and/or 482 to 495 (.sup.4) [c-Met]-L.sub.1-[c-Met]-L.sub.2-[Nb] L.sub.1 = (see legend below) [c-Met]-L.sub.1-[Nb]-L.sub.2-[c-Met] L.sub.2 = (see legend below) [Nb]-L.sub.1-[c-Met]-L.sub.2-[c-Met] [Nb] = (see legend below) Bispecific/ [c-Met]-L.sub.1-[Nb]-X(n) At least one [c-Met] present is = one of SEQ ID NO's: 464 to 477 C-terminal [Nb]-L.sub.1-[c-Met]-X(n) and/or 482 to 495 (.sup.4) extension (.sup.3) [c-Met]-L.sub.1-[c-Met]-L.sub.2-[Nb]-X(n) L.sub.1 = (see legend below) [c-Met]-L.sub.1-[Nb]-L.sub.2-[c-Met]-X(n) L.sub.2 = (see legend below) [Nb]-L.sub.1-[c-Met]-L.sub.2-[c-Met]-X(n) X(n) = (see legend below) [Nb] = (see legend below) Bispecific/ [c-Met]-L.sub.1-[Nb]-L.sub.2-[SA] At least one [c-Met] present is = one of SEQ ID NO's: 464 to 477 half-life [c-Met]-L.sub.1-[SA]-L.sub.2-[Nb] and/or 482 to 495 (.sup.4) extended (.sup.3) [Nb]-L.sub.1-[SA]-L.sub.2-[c-Met] [SA] = (see legend below) [Nb]-L.sub.1-[c-Met]-L.sub.2-[SA] L.sub.1, L.sub.2, L.sub.3(see legend below) [SA]-L.sub.1-[c-Met]-L.sub.2-[Nb] [Nb] = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[c-Met] Bispecific/ [c-Met]-L.sub.1-[c-Met]-L.sub.2-[Nb]-L.sub.3-[SA] At least one [c-Met] present is = one of SEQ ID NO's: 464 to 477 half-life [Nb]-L.sub.1-[c-Met]-L.sub.2-[c-Met]-L.sub.3-[SA] and/or 482 to 495 (.sup.4) extended (.sup.3) [SA]-L.sub.1-[c-Met]-L.sub.2-[c-Met]-L.sub.3-[Nb] [SA] = (see legend below) [SA]-L.sub.1-[c-Met]-L.sub.2-[Nb]-L.sub.3-[c-Met] L.sub.1, L.sub.2, L.sub.3(see legend below) [c-Met]-L.sub.1-[Nb]-L.sub.2-[c-Met]-L.sub.3-[SA] [Nb] = (see legend below) Bispecific/ [c-Met]-L.sub.1-[Nb]-L.sub.2-[SA]-X(n) At least one [c-Met] present is = one of SEQ ID NO's: 464 to 477 half-life [c-Met]-L.sub.1-[SA]-L.sub.2-[Nb]-X(n) and/or 482 to 495 (.sup.4) extended/ [Nb]-L.sub.1-[SA]-L.sub.2-[c-Met]-X(n) [SA] = (see legend below) C-terminal [Nb]-L.sub.1-[c-Met]-L.sub.2-[SA]-X(n) L.sub.1, L.sub.2, L.sub.3(see legend below) extension (.sup.3) [SA]-L.sub.1-[c-Met]-L.sub.2-[Nb]-X(n) X(n) = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[c-Met]-X(n) [Nb] = (see legend below) [c-Met]-L.sub.1-[c-Met]-L.sub.2-[Nb]-L.sub.3-[SA]-X(n) [Nb]-L.sub.1-[c-Met]-L.sub.2-[c-Met]-L.sub.3-[SA]-X(n) [SA]-L.sub.1-[c-Met]-L.sub.2-[c-Met]-L.sub.3-[Nb]-X(n) [SA]-L.sub.1-[c-Met]-L.sub.2-[Nb]-L.sub.3-[c-Met]-X(n) [c-Met]-L.sub.1-[Nb]-L.sub.2-[c-Met]-L.sub.3-[SA]-X(n) Legend: [SA] is an ISV against (human) serum albumin, preferably an ISV of the invention against (human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 or even more preferably one of the ISVD's of the invention of SEQ ID NO's: 47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L.sub.1, L.sub.2 and L.sub.3 is (independently) a suitable linker. Each of L.sub.1, L.sub.2 and L.sub.3 may (independently) be present or not. Non-limiting examples of suitable linkers are the gly-ser linkers referred to herein, such as the 9GS, 30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminal extension as described in herein and/or in WO 12/175741 [Nb] is an ISV against another therapeutic target. Notes: (.sup.1)In this Table: “Monovalent” generally refers to polypeptides/constructs comprising a single ISV against c-Met. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bivalent” generally refers to polypeptides/constructs comprising two ISV's against c-Met (which may be the same or different). These may again further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bispecific” generally refers to polypeptides/constructs comprising at least one (such as 1 or 2) ISV's against c-Met and at least one (such as 1 or 2) other ISV against a therapeutic target. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). In the polypeptides/constructs described in this table, at least one of the ISV's against c-Met present is an ISV of the invention, and preferably all of the ISV's against c-Met present in such polypeptide/construct are ISV's of the invention. Also, when a half-life extending ISV and/or an ISV against another therapeutic target is present in such polypeptide/construct, each of these (and preferably all of these) may also be (and preferably are) ISV's of the invention (.sup.2)All “bivalent” constructs in this Table may also be biparatopic, meaning that they comprise at least two (such as two) ISV's against c-Met, which are directed against different epitopes on c-Met. (.sup.3) As will be clear to the skilled person, other/further bispecific constructs than those listed can be made using the building blocks and linkers mentioned. (.sup.4) Preferably, each [c-Met] present is independently chosen from SEQ ID NO's: 464 to 477 and/or 482 to 495. Also, the [c-Met] present may be the same or different; in a biparatopic polypeptide/construct they will be directed against different epitopes on c-Met. (.sup.5) Each of the polypeptides/constructs mentioned to in this column by means of reference to a SEQ ID forms an individual specific aspect of the invention.

Example 16: VH Domains (and in Particular Nanobodies) Against RANK-L, and Compounds of the Invention Comprising the Same

(220) In one specific aspect, the VH domains of the invention (and in particular ISVDs of the invention and more in particular Nanobodies of the invention) and compounds of the invention may be directed against RANK-L.

(221) Such a VH domain of the invention against RANK-L will generally comprise: (i) suitable framework sequences that suitably comprise the amino acid residues/mutations of the invention as described herein; as well as (ii) CDR sequences that allow the VH domain of the invention to specifically bind to RANK-L. In addition, such a VH domain of the invention against RANK-L may also suitably have a C-terminal extension as described herein, in particular when said VH domain is monovalent or forms the C-terminal end of the compound of the invention in which said VH domain is present (again, as further described herein). Such VH domains of the invention against RANK-L may further be as further described herein, and may in particular be ISVD's.

(222) Again, as with other aspects and embodiments of the invention described herein, when a specific ISVD (such as the ISVD against RANK-L described in this Example) or compound comprising the same is said to be “according to the invention” or “as further described herein”, the preferred aspects/embodiments and preferences that are generally described herein for the ISVD's or compounds of the invention also specifically apply to said specific ISVD or compound, respectively, unless explicitly indicated otherwise or unless the specific technical context requires otherwise.

(223) Thus, in a particular aspect, the present invention relates to a VH domain (and in particular an ISVD) that is directed against RANK-L, in which (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). In particular, in such VH domains against RANK-L: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

(224) The VH domains of the invention against RANK-L may further be as described herein and may again in particular be an ISVD (and more in particular a Nanobody) against RANK-L or a protein, polypeptide or other compound or construct that comprises as least one such ISVD. Such a protein, polypeptide or other compound or construct may also be as further described herein, and may for example have an increased half-life (i.e. as described herein, e.g. a half-life—expressed as t½ beta—in human subjects of in human subjects of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days), and for this purpose may for example comprise a serum-albumin binding Nanobody, which may also be a serum-albumin binding Nanobody of the invention (again, as described herein).

(225) Also, such an ISVD may suitably have a C-terminal extension (as further described herein and in WO 12/175741), in particular when said ISVD forms the C-terminal end of a protein, polypeptide or other compound or construct comprising the same (again, as further described herein).

(226) In one preferred aspect, the VH domains of the invention against RANK-L comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 389 (which is preferred) or that is an amino acid sequence that has only one amino acid difference with the sequence of SEQ ID NO: 389; (ii) a CDR2 sequence that is the sequence of SEQ ID NO: 390 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 390; and (iii) a CDR3 sequence that is the sequence of SEQ ID NO: 391 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 391.

(227) More preferably, in a VH domain of the invention against RANK-L according to this aspect: (i) CDR1 is SEQ ID NO: 389; (ii) CDR2 is SEQ ID NO: 390; and (iii) CDR3 is SEQ ID NO: 391.

(228) In one specific aspect, a Nanobody of the invention against RANK-L is a variant of the Nanobody of SEQ ID NO: 388 (with at least 90% sequence identity, such as at least 95% sequence identity, with SEQ ID NO: 388), in which: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). The CDR's of such an ISV are preferably as defined in the preceding two paragraphs.

(229) Some specifically preferred, but non-limiting examples of Nanobodies of the invention against RANK-L are listed in FIG. 19 as SEQ ID NO's: 392 to 405; and each of these Nanobodies form a further aspect of the invention.

(230) The invention also relates to a compound of the invention against RANK-L that comprises at least one (such as one, two or three) of the Nanobodies of the invention of SEQ ID NO's: 392 to 405. Such compounds of the invention against RANK-L may again be as further described herein, and thus for example may comprise suitable linkers, may comprise a C-terminal extension as described herein, and may be half-life extended (for example because they comprise a Nanobody against human serum albumin, such as (preferably) a Nanobody of the invention against human serum albumin). Reference is made to Table Z below.

(231) Some specifically preferred examples of compounds of the invention against RANK-L are given in FIG. 29 as SEQ ID NO's: 694 to 729; and each of these compounds form a further aspect of the invention. Thus, in another aspect, the invention relates to a polypeptide that is directed against RANK-L and that has an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 694 to 729.

(232) More generally, compounds of the invention against RANK-L may be as described in WO 2008/142164, but comprising ISV's of the invention. They may also be used for the purposes described in WO 2008/142164.

(233) TABLE-US-00034 TABLE Z Examples of compounds of the invention against RANK-L. Polypeptide/ construct(.sup.1) General formula ISV building blocks and linkers Monovalent [RANK-L] [RANK-L] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 Monovalent/ [RANK-L]-X(n) [RANK-L] = one of SEQ ID NO's: 464 to 477 and/or 482 to C-terminal 495 extension Monovalent/ [RANK-L]-L.sub.1-[SA] [RANK-L] = one of SEQ ID NO's: 464 to 477 and/or 482 to half-life [SA]-L.sub.1-[RANK-L] 495 extended [SA] = (see legend below) L.sub.1 = (see legend below) Monovalent/ [RANK-L]-L.sub.1-[SA]-X(n) [RANK-L] = one of SEQ ID NO's: 464 to 477 and/or 482 to half-life [SA]-L.sub.1-[RANK-L]-X(n) 495 extended/C- [SA] = (see legend below) terminal L.sub.1 = (see legend below) extension X(n) = (see legend below) Bivalent(.sup.2) [RANK-L]-L.sub.1-[RANK-L] At least one [RANK-L] present is = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 (.sup.4) L.sub.1 = (see legend below) Bivalent/ [RANK-L]-L.sub.1-[RANK-L]-X(n) At least one [RANK-L] = one of SEQ ID NO's: 464 to 477 C-terminal and/or 482 to 495 (.sup.4) extension(.sup.2) L.sub.1 = (see legend below) X(n) = (see legend below) Bivalent/ [RANK-L]-L.sub.1-[RANK-L]-L.sub.2-[SA] At least one [RANK-L] present is = one of SEQ ID NO's: 464 half-life [RANK-L]-L.sub.1-[SA]-L.sub.2-[RANK-L] to 477 and/or 482 to 495 (.sup.4) extended(.sup.2) [SA]-L.sub.1-[RANK-L]-L.sub.2-[RANK-L] [SA] = (see legend below) L.sub.1 = (see legend below) L.sub.2 = (see legend below) Bivalent/ [RANK-L]-L.sub.1-[RANK-L]-L.sub.2-[SA]-X(n) At least one [RANK-L] present is = one of SEQ ID NO's: 464 half-life [RANK-L]-L.sub.1-[SA]-L.sub.2-[RANK-L]-X(n) to 477 and/or 482 to 495 (.sup.4) extended/ [SA]-L.sub.1-[RANK-L]-L.sub.2-[RANK-L]-X(n) [SA] = (see legend below) C-terminal L.sub.1 = (see legend below) extension(.sup.2) L.sub.2 = (see legend below) X(n) = (see legend below) Bispecific (.sup.3) [RANK-L]-L.sub.1-[Nb] At least one [RANK-L] present is = one of SEQ ID NO's: 464 [Nb]-L.sub.1-[RANK-L] to 477 and/or 482 to 495 (.sup.4) [RANK-L]-L.sub.1-[RANK-L]-L.sub.2-[Nb] L.sub.1 = (see legend below) [RANK-L]-L.sub.1-[Nb]-L.sub.2-[RANK-L] L.sub.2 = (see legend below) [Nb]-L.sub.1-[RANK-L]-L.sub.2-[RANK-L] [Nb] = (see legend below) Bispecific/ [RANK-L]-L.sub.1-[Nb]-X(n) At least one [RANK-L] present is = one of SEQ ID NO's: 464 C-terminal [Nb]-L.sub.1-[RANK-L]-X(n) to 477 and/or 482 to 495 (.sup.4) extension (.sup.3) [RANK-L]-L.sub.1-[RANK-L]-L.sub.2-[Nb]-X(n) L.sub.1 = (see legend below) [RANK-L]-L.sub.1-[Nb]-L.sub.2-[RANK-L]-X(n) L.sub.2 = (see legend below) [Nb]-L.sub.1-[RANK-L]-L.sub.2-[RANK-L]-X(n) X(n) = (see legend below) [Nb] = (see legend below) Bispecific/ [RANK-L]-L.sub.1-[Nb]-L.sub.2-[SA] At least one [RANK-L] present is = one of SEQ ID NO's: 464 half-life [RANK-L]-L.sub.1-[SA]-L.sub.2-[Nb] to 477 and/or 482 to 495 (.sup.4) extended (.sup.3) [Nb]-L.sub.1-[SA]-L.sub.2-[RANK-L] [SA] = (see legend below) [Nb]-L.sub.1-[RANK-L]-L.sub.2-[SA] L.sub.1, L.sub.2, L.sub.3(see legend below) [SA]-L.sub.1-[RANK-L]-L.sub.2-[Nb] [Nb] = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[RANK-L] Bispecific/ [RANK-L]-L.sub.1-[RANK-L]-L.sub.2-[Nb]-L.sub.3-[SA] At least one [RANK-L] present is = one of SEQ ID NO's: 464 half-life [Nb]-L.sub.1-[RANK-L]-L.sub.2-[RANK-L]-L.sub.3-[SA] to 477 and/or 482 to 495 (.sup.4) extended (.sup.3) [SA]-L.sub.1-[RANK-L]-L.sub.2-[RANK-L]-L.sub.3-[Nb] [SA] = (see legend below) [SA]-L.sub.1-[RANK-L]-L.sub.2-[Nb]-L.sub.3-[RANK-L] L.sub.1, L.sub.2, L.sub.3(see legend below) [RANK-L]-L.sub.1-[Nb]-L.sub.2-[RANK-L]-L.sub.3-[SA] [Nb] = (see legend below) Bispecific/ [RANK-L]-L.sub.1-[Nb]-L.sub.2-[SA]-X(n) At least one [RANK-L] present is = one of SEQ ID NO's: 464 half-life [RANK-L]-L.sub.1-[SA]-L.sub.2-[Nb]-X(n) to 477 and/or 482 to 495 (.sup.4) extended/ [Nb]-L.sub.1-[SA]-L.sub.2-[RANK-L]-X(n) [SA] = (see legend below) C-terminal [Nb]-L.sub.1-[RANK-L]-L.sub.2-[SA]-X(n) L.sub.1, L.sub.2, L.sub.3(see legend below) extension (.sup.3) [SA]-L.sub.1-[RANK-L]-L.sub.2-[Nb]-X(n) X(n) = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[RANK-L]-X(n) [Nb] = (see legend below) [RANK-L]-L.sub.1-[RANK-L]-L.sub.2-[Nb]-L.sub.3-[SA]-X(n) [Nb]-L.sub.1-[RANK-L]-L.sub.2-[RANK-L]-L.sub.3-[SA]-X(n) [SA]-L.sub.1-[RANK-L]-L.sub.2-[RANK-L]-L.sub.3-[Nb]-X(n) [SA]-L.sub.1-[RANK-L]-L.sub.2-[Nb]-L.sub.3-[RANK-L]-X(n) [RANK-L]-L.sub.1-[Nb]-L.sub.2-[RANK-L]-L.sub.3-[SA]-X(n) Legend: [SA] is an ISV against (human) serum albumin, preferably an ISV of the invention against (human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 or even more preferably one of the ISVD's of the invention of SEQ ID NO's: 47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L.sub.1, L.sub.2 and L.sub.3 is (independently) a suitable linker. Each of L.sub.1, L.sub.2 and L.sub.3 may (independently) be present or not. Non-limiting examples of suitable linkers are the gly-ser linkers referred to herein, such as the 9GS, 30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminal extension as described in herein and/or in WO 12/175741 [Nb] is an ISV against another therapeutic target. Notes: (.sup.1)In this Table: “Monovalent” generally refers to polypeptides/constructs comprising a single ISV against RANK-L. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bivalent” generally refers to polypeptides/constructs comprising two ISV's against RANK-L (which may be the same or different). These may again further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bispecific” generally refers to polypeptides/constructs comprising at least one (such as 1 or 2) ISV's against RANK-L and at least one (such as 1 or 2) other ISV against a therapeutic target. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). In the polypeptides/constructs described in this table, at least one of the ISV's against RANK-L present is an ISV of the invention, and preferably all of the ISV's against RANK-L present in such polypeptide/construct are ISV's of the invention. Also, when a half-life extending ISV and/or an ISV against another therapeutic target is present in such polypeptide/construct, each of these (and preferably all of these) may also be (and preferably are) ISV's of the invention (.sup.2)All “bivalent” constructs in this Table may also be biparatopic, meaning that they comprise at least two (such as two) ISV's against RANK-L, which are directed against different epitopes on RANK-L. (.sup.3) As will be clear to the skilled person, other/further bispecific constructs than those listed can be made using the building blocks and linkers mentioned. (.sup.4) Preferably, each [RANK-L] present is independently chosen from SEQ ID NO's: 464 to 477 and/or 482 to 495. Also, the [RANK-L] present may be the same or different; in a biparatopic polypeptide/construct they will be directed against different epitopes on RANK-L. (.sup.5) Each of the polypeptides/constructs mentioned to in this column by means of reference to a SEQ ID forms an individual specific aspect of the invention.

Example 17: VH Domains (and in Particular Nanobodies) Against CXCR-7, and Compounds of the Invention Comprising the Same

(234) In one specific aspect, the VH domains of the invention (and in particular ISVDs of the invention and more in particular Nanobodies of the invention) and compounds of the invention may be directed against CXCR-7.

(235) Such a VH domain of the invention against CXCR-7 will generally comprise: (i) suitable framework sequences that suitably comprise the amino acid residues/mutations of the invention as described herein; as well as (ii) CDR sequences that allow the VH domain of the invention to specifically bind to CXCR-7. In addition, such a VH domain of the invention against CXCR-7 may also suitably have a C-terminal extension as described herein, in particular when said VH domain is monovalent or forms the C-terminal end of the compound of the invention in which said VH domain is present (again, as further described herein). Such VH domains of the invention against CXCR-7 may further be as further described herein, and may in particular be ISVD's.

(236) Again, as with other aspects and embodiments of the invention described herein, when a specific ISVD (such as the ISVD against CXCR-7 described in this Example) or compound comprising the same is said to be “according to the invention” or “as further described herein”, the preferred aspects/embodiments and preferences that are generally described herein for the ISVD's or compounds of the invention also specifically apply to said specific ISVD or compound, respectively, unless explicitly indicated otherwise or unless the specific technical context requires otherwise.

(237) Thus, in a particular aspect, the present invention relates to a VH domain (and in particular an ISVD) that is directed against CXCR-7, in which (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). In particular, in such VH domains against CXCR-7: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

(238) The VH domains of the invention against CXCR-7 may further be as described herein and may again in particular be an ISVD (and more in particular a Nanobody) against CXCR-7 or a protein, polypeptide or other compound or construct that comprises as least one such ISVD. Such a protein, polypeptide or other compound or construct may also be as further described herein, and may for example have an increased half-life (i.e. as described herein, e.g. a half-life—expressed as t½ beta—in human subjects of in human subjects of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days), and for this purpose may for example comprise a serum-albumin binding Nanobody, which may also be a serum-albumin binding Nanobody of the invention (again, as described herein).

(239) Also, such an ISVD may suitably have a C-terminal extension (as further described herein and in WO 12/175741), in particular when said ISVD forms the C-terminal end of a protein, polypeptide or other compound or construct comprising the same (again, as further described herein).

(240) In one preferred aspect, the VH domains of the invention against CXCR-7 comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 407 (which is preferred) or that is an amino acid sequence that has only one amino acid difference with the sequence of SEQ ID NO: 407; (ii) a CDR2 sequence that is the sequence of SEQ ID NO: 408 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 408; and (iii) a CDR3 sequence that is the sequence of SEQ ID NO: 409 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 409.

(241) More preferably, in a VH domain of the invention against CXCR-7 according to this aspect: (i) CDR1 is SEQ ID NO: 407; (ii) CDR2 is SEQ ID NO: 408; and (iii) CDR3 is SEQ ID NO: 409.

(242) In one specific aspect, a Nanobody of the invention against CXCR-7 is a variant of the Nanobody of SEQ ID NO: 406 (with at least 90% sequence identity, such as at least 95% sequence identity, with SEQ ID NO: 406), in which: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). The CDR's of such an ISV are preferably as defined in the preceding two paragraphs.

(243) In another preferred aspect, the VH domains of the invention against CXCR-7 comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 425 (which is preferred) or that is an amino acid sequence that has only one amino acid difference with the sequence of SEQ ID NO: 425; (ii) a CDR2 sequence that is the sequence of SEQ ID NO: 426 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 426; and (iii) a CDR3 sequence that is the sequence of SEQ ID NO: 427 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 427.

(244) More preferably, in a VH domain of the invention against CXCR-7 according to this aspect: (i) CDR1 is SEQ ID NO: 425; (ii) CDR2 is SEQ ID NO: 426; and (iii) CDR3 is SEQ ID NO: 427.

(245) In one specific aspect, a Nanobody of the invention against CXCR-7 is a variant of the Nanobody of SEQ ID NO: 424 (with at least 90% sequence identity, such as at least 95% sequence identity, with SEQ ID NO: 424), in which: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). Again, the CDR's of such an ISV are preferably as defined in the preceding two paragraphs.

(246) Some specifically preferred, but non-limiting examples of Nanobodies of the invention against CXCR-7 are listed in FIG. 20A as SEQ ID NO's: 410 to 423 and in FIG. 20B as SEQ ID NO's: 428 to 441; and each of these Nanobodies form a further aspect of the invention.

(247) The invention also relates to a compound of the invention against CXCR-7 that comprises at least one (such as one, two or three) of the Nanobodies of the invention of SEQ ID NO's: 410 to 423 and/or 428 to 441. Such compounds of the invention against CXCR-7 may again be as further described herein, and thus for example may comprise suitable linkers, may comprise a C-terminal extension as described herein, and may be half-life extended (for example because they comprise a Nanobody against human serum albumin, such as (preferably) a Nanobody of the invention against human serum albumin). Reference is made to Table AA below.

(248) As described in for example WO2012/130874, one particularly preferred class of Nanobody-based compounds against CXCR7 are biparatopic compounds. Thus, in one aspect of the invention, a compound of the invention against CXCR-7 is a biparatopic construct that comprises one ISV that is either SEQ ID NO: 406 or (preferably) an ISV of the invention that has been derived from SEQ ID NO: 406 (as described in this Example 17) and one ISV that is either SEQ ID NO: 424 or (preferably) an ISV of the invention that has been derived from SEQ ID NO: 424 (as described in this Example 17), provided that at least one (and preferably both) of these ISV's are ISV's of the invention. Such biparatopic constructs may also be half-life extended (i.e. by means of a serum albumin-binding ISV).

(249) More generally, compounds of the invention against CXCR-7 may be as described in WO2012/130874, but comprising ISV's of the invention. They may also be used for the purposes described in WO2012/130874.

(250) TABLE-US-00035 TABLE AA Examples of compounds of the invention against CXCR-7. Polypeptide/ construct(.sup.1) General formula ISV building blocks and linkers Monovalent [CXCR-7] [CXCR-7] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 Monovalent/ [CXCR-7]-X(n) [CXCR-7] = one of SEQ ID NO's: 464 to 477 and/or 482 to C-terminal 495 extension Monovalent/ [CXCR-7]-L.sub.1-[SA] [CXCR-7] = one of SEQ ID NO's: 464 to 477 and/or 482 to half-life [SA]-L.sub.1-[CXCR-7] 495 extended [SA] = (see legend below) L.sub.1 = (see legend below) Monovalent/ [CXCR-7]-L.sub.1-[SA]-X(n) [CXCR-7] = one of SEQ ID NO's: 464 to 477 and/or 482 to half-life [SA]-L.sub.1-[CXCR-7]-X(n) 495 extended/C- [SA] = (see legend below) terminal L.sub.1 = (see legend below) extension X(n) = (see legend below) Bivalent(.sup.2) [CXCR-7]-L.sub.1-[CXCR-7] At least one [CXCR-7] present is = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 (.sup.4) L.sub.1 = (see legend below) Bivalent/ [CXCR-7]-L.sub.1-[CXCR-7]-X(n) At least one [CXCR-7] = one of SEQ ID NO's: 464 to 477 C-terminal and/or 482 to 495 (.sup.4) extension(.sup.2) L.sub.1 = (see legend below) X(n) = (see legend below) Bivalent/ [CXCR-7]-L.sub.1-[CXCR-7]-L.sub.2-[SA] At least one [CXCR-7] present is = one of SEQ ID NO's: 464 half-life [CXCR-7]-L.sub.1-[SA]-L.sub.2-[CXCR-7] to 477 and/or 482 to 495 (.sup.4) extended(.sup.2) [SA]-L.sub.1-[CXCR-7]-L.sub.2-[CXCR-7] [SA] = (see legend below) L.sub.1 = (see legend below) L.sub.2 = (see legend below) Bivalent/ [CXCR-7]-L.sub.1-[CXCR-7]-L.sub.2-[SA]-X(n) At least one [CXCR-7] present is = one of SEQ ID NO's: 464 half-life [CXCR-7]-L.sub.1-[SA]-L.sub.2-[CXCR-7]-X(n) to 477 and/or 482 to 495 (.sup.4) extended/ [SA]-L.sub.1-[CXCR-7]-L.sub.2-[CXCR-7]-X(n) [SA] = (see legend below) C-terminal L.sub.1 = (see legend below) extension(.sup.2) L.sub.2 = (see legend below) X(n) = (see legend below) Bispecific (.sup.3) [CXCR-7]-L.sub.1-[Nb] At least one [CXCR-7] present is = one of SEQ ID NO's: 464 [Nb]-L.sub.1-[CXCR-7] to 477 and/or 482 to 495 (.sup.4) [CXCR-7]-L.sub.1-[CXCR-7]-L.sub.2-[Nb] L.sub.1 = (see legend below) [CXCR-7]-L.sub.1-[Nb]-L.sub.2-[CXCR-7] L.sub.2 = (see legend below) [Nb]-L.sub.1-[CXCR-7]-L.sub.2-[CXCR-7] [Nb] = (see legend below) Bispecific/ [CXCR-7]-L.sub.1-[Nb]-X(n) At least one [CXCR-7] present is = one of SEQ ID NO's: 464 C-terminal [Nb]-L.sub.1-[CXCR-7]-X(n) to 477 and/or 482 to 495 (.sup.4) extension (.sup.3) [CXCR-7]-L.sub.1-[CXCR-7]-L.sub.2-[Nb]-X(n) L.sub.1 = (see legend below) [CXCR-7]-L.sub.1-[Nb]-L.sub.2-[CXCR-7]-X(n) L.sub.2 = (see legend below) [Nb]-L.sub.1-[CXCR-7]-L.sub.2-[CXCR-7]-X(n) X(n) = (see legend below) [Nb] = (see legend below) Bispecific/ [CXCR-7]-L.sub.1-[Nb]-L.sub.2-[SA] At least one [CXCR-7] present is = one of SEQ ID NO's: 464 half-life [CXCR-7]-L.sub.1-[SA]-L.sub.2-[Nb] to 477 and/or 482 to 495 (.sup.4) extended (.sup.3) [Nb]-L.sub.1-[SA]-L.sub.2-[CXCR-7] [SA] = (see legend below) [Nb]-L.sub.1-[CXCR-7]-L.sub.2-[SA] L.sub.1, L.sub.2, L.sub.3(see legend below) [SA]-L.sub.1-[CXCR-7]-L.sub.2-[Nb] [Nb] = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[CXCR-7] Bispecific/ [CXCR-7]-L.sub.1-[CXCR-7]-L.sub.2-[Nb]-L.sub.3-[SA] At least one [CXCR-7] present is = one of SEQ ID NO's: 464 half-life [Nb]-L.sub.1-[CXCR-7]-L.sub.2-[CXCR-7]-L.sub.3-[SA] to 477 and/or 482 to 495 (.sup.4) extended (.sup.3) [SA]-L.sub.1-[CXCR-7]-L.sub.2-[CXCR-7]-L.sub.3-[Nb] [SA] = (see legend below) [SA]-L.sub.1-[CXCR-7]-L.sub.2-[Nb]-L.sub.3-[CXCR-7] L.sub.1, L.sub.2, L.sub.3(see legend below) [CXCR-7]-L.sub.1-[Nb]-L.sub.2-[CXCR-7]-L.sub.3-[SA] [Nb] = (see legend below) Bispecific/ [CXCR-7]-L.sub.1-[Nb]-L.sub.2-[SA]-X(n) At least one [CXCR-7] present is = one of SEQ ID NO's: 464 half-life [CXCR-7]-L.sub.1-[SA]-L.sub.2-[Nb]-X(n) to 477 and/or 482 to 495 (.sup.4) extended/ [Nb]-L.sub.1-[SA]-L.sub.2-[CXCR-7]-X(n) [SA] = (see legend below) C-terminal [Nb]-L.sub.1-[CXCR-7]-L.sub.2-[SA]-X(n) L.sub.1, L.sub.2, L.sub.3(see legend below) extension (.sup.3) [SA]-L.sub.1-[CXCR-7]-L.sub.2-[Nb]-X(n) X(n) = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[CXCR-7]-X(n) [Nb] = (see legend below) [CXCR-7]-L.sub.1-[CXCR-7]-L.sub.2-[Nb]-L.sub.3-[SA]-X(n) [Nb]-L.sub.1-[CXCR-7]-L.sub.2-[CXCR-7]-L.sub.3-[SA]-X(n) [SA]-L.sub.1-[CXCR-7]-L.sub.2-[CXCR-7]-L.sub.3-[Nb]-X(n) [SA]-L.sub.1-[CXCR-7]-L.sub.2-[Nb]-L.sub.3-[CXCR-7]-X(n) [CXCR-7]-L.sub.1-[Nb]-L.sub.2-[CXCR-7]-L.sub.3-[SA]-X(n) Legend: [SA] is an ISV against (human) serum albumin, preferably an ISV of the invention against (human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 or even more preferably one of the ISVD's of the invention of SEQ ID NO's: 47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L.sub.1, L.sub.2 and L.sub.3 is (independently) a suitable linker. Each of L.sub.1, L.sub.2 and L.sub.3 may (independently) be present or not. Non-limiting examples of suitable linkers are the gly-ser linkers referred to herein, such as the 9GS, 30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminal extension as described in herein and/or in WO 12/175741 [Nb] is an ISV against another therapeutic target. Notes: (.sup.1)In this Table: “Monovalent” generally refers to polypeptides/constructs comprising a single ISV against CXCR-7. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bivalent” generally refers to polypeptides/constructs comprising two ISV's against CXCR-7 (which may be the same or different). These may again further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bispecific” generally refers to polypeptides/constructs comprising at least one (such as 1 or 2) ISV's against CXCR-7 and at least one (such as 1 or 2) other ISV against a therapeutic target. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). In the polypeptides/constructs described in this table, at least one of the ISV's against CXCR-7 present is an ISV of the invention, and preferably all of the ISV's against CXCR-7 present in such polypeptide/construct are ISV's of the invention. Also, when a half-life extending ISV and/or an ISV against another therapeutic target is present in such polypeptide/construct, each of these (and preferably all of these) may also be (and preferably are) ISV's of the invention (.sup.2)All “bivalent” constructs in this Table may also be biparatopic, meaning that they comprise at least two (such as two) ISV's against CXCR-7, which are directed against different epitopes on CXCR-7. (.sup.3) As will be clear to the skilled person, other/further bispecific constructs than those listed can be made using the building blocks and linkers mentioned. (.sup.4) Preferably, each [CXCR-7] present is independently chosen from SEQ ID NO's: 464 to 477 and/or 482 to 495. Also, the [CXCR-7] present may be the same or different; in a biparatopic polypeptide/construct they will be directed against different epitopes on CXCR-7. (.sup.5) Each of the polypeptides/constructs mentioned to in this column by means of reference to a SEQ ID forms an individual specific aspect of the invention.

Example 18: VH Domains (and in Particular Nanobodies) Against A-Beta, and Compounds of the Invention Comprising the Same

(251) In one specific aspect, the VH domains of the invention (and in particular ISVDs of the invention and more in particular Nanobodies of the invention) and compounds of the invention may be directed against A-beta.

(252) Such a VH domain of the invention against A-beta will generally comprise: (i) suitable framework sequences that suitably comprise the amino acid residues/mutations of the invention as described herein; as well as (ii) CDR sequences that allow the VH domain of the invention to specifically bind to A-beta. In addition, such a VH domain of the invention against A-beta may also suitably have a C-terminal extension as described herein, in particular when said VH domain is monovalent or forms the C-terminal end of the compound of the invention in which said VH domain is present (again, as further described herein). Such VH domains of the invention against A-beta may further be as further described herein, and may in particular be ISVD's.

(253) Again, as with other aspects and embodiments of the invention described herein, when a specific ISVD (such as the ISVD against A-beta described in this Example) or compound comprising the same is said to be “according to the invention” or “as further described herein”, the preferred aspects/embodiments and preferences that are generally described herein for the ISVD's or compounds of the invention also specifically apply to said specific ISVD or compound, respectively, unless explicitly indicated otherwise or unless the specific technical context requires otherwise.

(254) Thus, in a particular aspect, the present invention relates to a VH domain (and in particular an ISVD) that is directed against A-beta, in which (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). In particular, in such VH domains against A-beta: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

(255) The VH domains of the invention against A-beta may further be as described herein and may again in particular be an ISVD (and more in particular a Nanobody) against A-beta or a protein, polypeptide or other compound or construct that comprises as least one such ISVD. Such a protein, polypeptide or other compound or construct may also be as further described herein, and may for example have an increased half-life (i.e. as described herein, e.g. a half-life—expressed as t½ beta—in human subjects of in human subjects of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days), and for this purpose may for example comprise a serum-albumin binding Nanobody, which may also be a serum-albumin binding Nanobody of the invention (again, as described herein).

(256) Also, such an ISVD may suitably have a C-terminal extension (as further described herein and in WO 12/175741), in particular when said ISVD forms the C-terminal end of a protein, polypeptide or other compound or construct comprising the same (again, as further described herein).

(257) In one preferred aspect, the VH domains of the invention against A-beta comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 461 (which is preferred) or that is an amino acid sequence that has only one amino acid difference with the sequence of SEQ ID NO: 461; (ii) a CDR2 sequence that is the sequence of SEQ ID NO: 462 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 462; and (iii) a CDR3 sequence that is the sequence of SEQ ID NO: 463 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 463.

(258) More preferably, in a VH domain of the invention against A-beta according to this aspect: (i) CDR1 is SEQ ID NO: 461; (ii) CDR2 is SEQ ID NO: 462; and (iii) CDR3 is SEQ ID NO: 463.

(259) In one specific aspect, a Nanobody of the invention against A-beta is a variant of the Nanobody of SEQ ID NO: 460 (with at least 90% sequence identity, such as at least 95% sequence identity, with SEQ ID NO: 460), in which: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). The CDR's of such an ISV are preferably as defined in the preceding two paragraphs.

(260) In another preferred aspect, the VH domains of the invention against A-beta comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 479 (which is preferred) or that is an amino acid sequence that has only one amino acid difference with the sequence of SEQ ID NO: 479; (ii) a CDR2 sequence that is the sequence of SEQ ID NO: 480 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 480; and (iii) a CDR3 sequence that is the sequence of SEQ ID NO: 481 (which is preferred) or that is an amino acid sequence that has only one or two amino acid differences with the sequence of SEQ ID NO: 481.

(261) More preferably, in a VH domain of the invention against A-beta according to this aspect: (i) CDR1 is SEQ ID NO: 479; (ii) CDR2 is SEQ ID NO: 480; and (iii) CDR3 is SEQ ID NO: 481.

(262) In one specific aspect, a Nanobody of the invention against A-beta is a variant of the Nanobody of SEQ ID NO: 478 (with at least 90% sequence identity, such as at least 95% sequence identity, with SEQ ID NO: 478), in which: the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and the amino acid residue at position 14 is preferably suitably chosen from A or P; and the amino acid residue at position 41 is preferably suitably chosen from A or P; and the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and the amino acid residue at position 108 is preferably suitably chosen from Q or L; and the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and the amino acid residue at position 112 is preferably suitably chosen from S, K or Q;
such that either (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). Again, the CDR's of such an ISV are preferably as defined in the preceding two paragraphs.

(263) Some specifically preferred, but non-limiting examples of Nanobodies of the invention against A-beta are listed in FIG. 21A as SEQ ID NO's: 464 to 477 and in FIG. 21B as SEQ ID NO's: 482 to 495; and each of these Nanobodies form a further aspect of the invention.

(264) The invention also relates to a compound of the invention against A-beta that comprises at least one (such as one, two or three) of the Nanobodies of the invention of SEQ ID NO's: 464 to 477 and/or 482 to 495. Such compounds of the invention against A-beta may again be as further described herein, and thus for example may comprise suitable linkers, may comprise a C-terminal extension as described herein, and may be half-life extended (for example because they comprise a Nanobody against human serum albumin, such as (preferably) a Nanobody of the invention against human serum albumin). Reference is made to Table BB below.

(265) As described in for example WO 2006/040153 and in particular as described in EP2542579, one particularly preferred class of Nanobody-based compounds against A-beta are biparatopic compounds. Thus, in one aspect of the invention, a compound of the invention against A-beta is a biparatopic construct that comprises one ISV that is either SEQ ID NO: 460 or (preferably) an ISV of the invention that has been derived from SEQ ID NO: 460 (as described in this Example 17) and one ISV that is either SEQ ID NO: 478 or (preferably) an ISV of the invention that has been derived from SEQ ID NO: 478 (as described in this Example 17), provided that at least one (and preferably both) of these ISV's are ISV's of the invention. Such biparatopic constructs may also be half-life extended (i.e. by means of a serum albumin-binding ISV). Some specific examples of such biparatopic constructs are given in SEQ ID NO: 730 to 766.

(266) Some specifically preferred examples of compounds of the invention against A-beta are given in FIG. 30 as SEQ ID NO's: 730 to 766; and each of these compounds form a further aspect of the invention. Thus, in another aspect, the invention relates to a polypeptide that is directed against A-beta and that has an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 730 to 766. More generally, compounds of the invention against A-beta may be as described in WO 2006/040153 and in particular as described in EP2542579, but comprising ISV's of the invention. They may also be used for the purposes described in WO 2006/040153 and in particular EP2542579.

(267) TABLE-US-00036 TABLE BB Examples of compounds of the invention against A-beta. Polypeptide/ construct(.sup.1) General formula ISV building blocks and linkers Monovalent [A-beta] [A-beta] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 Monovalent/ [A-beta]-X(n) [A-beta] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 C-terminal extension Monovalent/ [A-beta]-L.sub.1-[SA] [A-beta] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L.sub.1-[A-beta] [SA] = (see legend below) extended L.sub.1 = (see legend below) Monovalent/ [A-beta]-L.sub.1-[SA]-X(n) [A-beta] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L.sub.1-[A-beta]-X(n) [SA] = (see legend below) extended/C- L.sub.1 = (see legend below) terminal X(n) = (see legend below) extension Bivalent(.sup.2) [A-beta]-L.sub.1-[A-beta] At least one [A-beta] present is = one of SEQ ID NO's: 464 to 477 and/or 482 to 495 (.sup.4) L.sub.1 = (see legend below) Bivalent/ [A-beta]-L.sub.1-[A-beta]-X(n) At least one [A-beta] = one of SEQ ID NO's: 464 to 477 and/or C-terminal 482 to 495 (.sup.4) extension(.sup.2) L.sub.1 = (see legend below) X(n) = (see legend below) Bivalent/ [A-beta]-L.sub.1-[A-beta]-L.sub.2-[SA] At least one [A-beta] present is = one of SEQ ID NO's: 464 to half-life [A-beta]-L.sub.1-[SA]-L.sub.2-[A-beta] 477 and/or 482 to 495 (.sup.4) extended(.sup.2) [SA]-L.sub.1-[A-beta]-L.sub.2-[A-beta] [SA] = (see legend below) L.sub.1 = (see legend below) L.sub.2 = (see legend below) Bivalent/ [A-beta]-L.sub.1-[A-beta]-L.sub.2-[SA]-X(n) At least one [A-beta] present is = one of SEQ ID NO's: 464 to half-life [A-beta]-L.sub.1-[SA]-L.sub.2-[A-beta]-X(n) 477 and/or 482 to 495 (.sup.4) extended/ [SA]-L.sub.1-[A-beta]-L.sub.2-[A-beta]-X(n) [SA] = (see legend below) C-terminal L.sub.1 = (see legend below) extension(.sup.2) L.sub.2 = (see legend below) X(n) = (see legend below) Bispecific (.sup.3) [A-beta]-L.sub.1-[Nb] At least one [A-beta] present is = one of SEQ ID NO's: 464 to [Nb]-L.sub.1-[A-beta] 477 and/or 482 to 495 (.sup.4) [A-beta]-L.sub.1-[A-beta]-L.sub.2-[Nb] L.sub.1 = (see legend below) [A-beta]-L.sub.1-[Nb]-L.sub.2-[A-beta] L.sub.2 = (see legend below) [Nb]-L.sub.1-[A-beta]-L.sub.2-[A-beta] [Nb] = (see legend below) Bispecific/ [A-beta]-L.sub.1-[Nb]-X(n) At least one [A-beta] present is = one of SEQ ID NO's: 464 to C-terminal [Nb]-L.sub.1-[A-beta]-X(n) 477 and/or 482 to 495 (.sup.4) extension (.sup.3) [A-beta]-L.sub.1-[A-beta]-L.sub.2-[Nb]-X(n) L.sub.1 = (see legend below) [A-beta]-L.sub.1-[Nb]-L.sub.2-[A-beta]-X(n) L.sub.2 = (see legend below) [Nb]-L.sub.1-[A-beta]-L.sub.2-[A-beta]-X(n) X(n) = (see legend below) [Nb] = (see legend below) Bispecific/ [A-beta]-L.sub.1-[Nb]-L.sub.2-[SA] At least one [A-beta] present is = one of SEQ ID NO's: 464 to half-life [A-beta]-L.sub.1-[SA]-L.sub.2-[Nb] 477 and/or 482 to 495 (.sup.4) extended (.sup.3) [Nb]-L.sub.1-[SA]-L.sub.2-[A-beta] [SA] = (see legend below) [Nb]-L.sub.1-[A-beta]-L.sub.2-[SA] L.sub.1, L.sub.2, L.sub.3(see legend below) [SA]-L.sub.1-[A-beta]-L.sub.2-[Nb] [Nb] = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[A-beta] Bispecific/ [A-beta]-L.sub.1-[A-beta]-L.sub.2-[Nb]-L.sub.3-[SA] At least one [A-beta] present is = one of SEQ ID NO's: 464 to half-life [Nb]-L.sub.1-[A-beta]-L.sub.2-[A-beta]-L.sub.3-[SA] 477 and/or 482 to 495 (.sup.4) extended (.sup.3) [SA]-L.sub.1-[A-beta]-L.sub.2-[A-beta]-L.sub.3-[Nb] [SA] = (see legend below) [SA]-L.sub.1-[A-beta]-L.sub.2-[Nb]-L.sub.3-[A-beta] L.sub.1, L.sub.2, L.sub.3(see legend below) [A-beta]-L.sub.1-[Nb]-L.sub.2-[A-beta]-L.sub.3-[SA] [Nb] = (see legend below) Bispecific/ [A-beta]-L.sub.1-[Nb]-L.sub.2-[SA]-X(n) At least one [A-beta] present is = one of SEQ ID NO's: 464 to half-life [A-beta]-L.sub.1-[SA]-L.sub.2-[Nb]-X(n) 477 and/or 482 to 495 (.sup.4) extended/ [Nb]-L.sub.1-[SA]-L.sub.2-[A-beta]-X(n) [SA] = (see legend below) C-terminal [Nb]-L.sub.1-[A-beta]-L.sub.2-[SA]-X(n) L.sub.1, L.sub.2, L.sub.3(see legend below) extension (.sup.3) [SA]-L.sub.1-[A-beta]-L.sub.2-[Nb]-X(n) X(n) = (see legend below) [SA]-L.sub.1-[Nb]-L.sub.2-[12345]-X(n) [Nb] = (see legend below) [A-beta]-L.sub.1-[A-beta]-L.sub.2-[Nb]-L.sub.3-[SA]-X(n) [Nb]-L.sub.1-[A-beta]-L.sub.2-[A-beta]-L.sub.3-[SA]-X(n) [SA]-L.sub.1-[A-beta]-L.sub.2-[A-beta]-L.sub.3-[Nb]-X(n) [SA]-L.sub.1-[A-beta]-L.sub.2-[Nb]-L.sub.3-[A-beta]-X(n) [A-beta]-L.sub.1-[Nb]-L.sub.2-[A-beta]-L.sub.3-[SA]-X(n) Legend: [SA] is an ISV against (human) serum albumin, preferably an ISV of the invention against (human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 or even more preferably one of the ISVD's of the invention of SEQ ID NO's: 47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L.sub.1, L.sub.2 and L.sub.3 is (independently) a suitable linker. Each of L.sub.1, L.sub.2 and L.sub.3 may (independently) be present or not. Non-limiting examples of suitable linkers are the gly-ser linkers referred to herein, such as the 9GS, 30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminal extension as described in herein and/or in WO 12/175741 [Nb] is an ISV against another therapeutic target. Notes: (.sup.1)In this Table: “Monovalent” generally refers to polypeptides/constructs comprising a single ISV against A-beta. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bivalent” generally refers to polypeptides/constructs comprising two ISV's against A-beta (which may be the same or different). These may again further comprise a half-life extending ISV (such as an ISV against serum albumin). “Bispecific” generally refers to polypeptides/constructs comprising at least one (such as 1 or 2) ISV's against A-beta and at least one (such as 1 or 2) other ISV against a therapeutic target. These may further comprise a half-life extending ISV (such as an ISV against serum albumin). In the polypeptides/constructs described in this table, at least one of the ISV's against A-beta present is an ISV of the invention, and preferably all of the ISV's against A-beta present in such polypeptide/construct are ISV's of the invention. Also, when a half-life extending ISV and/or an ISV against another therapeutic target is present in such polypeptide/construct, each of these (and preferably all of these) may also be (and preferably are) ISV's of the invention (.sup.2)All “bivalent” constructs in this Table may also be biparatopic, meaning that they comprise at least two (such as two) ISV's against A-beta, which are directed against different epitopes on A-beta. (.sup.3) As will be clear to the skilled person, other/further bispecific constructs than those listed can be made using the building blocks and linkers mentioned. (.sup.4) Preferably, each [A-beta] present is independently chosen from SEQ ID NO's: 464 to 477 and/or 482 to 495. Also, the [A-beta] present may be the same or different; in a biparatopic polypeptide/construct they will be directed against different epitopes on A-beta. (.sup.5) Each of the polypeptides/constructs mentioned to in this column by means of reference to a SEQ ID forms an individual specific aspect of the invention.

Example 19: Testing of Anti-A-Beta Constructs for Binding by Pre-Existing Antibodies

(268) Three half-life extended anti-A-beta constructs (with the general formula [A-beta-1]-9GS-ALB8-9GS-O-beta-2J-A) were tested and compared for binding by pre-existing antibodies, using the general protocol described herein. The two constructs according to the invention had the indicated mutations of the invention in all three building blocks (i.e. in the two anti-A-beta Nanobodies and in the serum albumin-binding Nanobody). The reference construct (SEQ ID NO:766) had no mutations of the invention in any of the building blocks. All constructs tested has a C-terminal alanine. The results are given in Table CC-1 and FIG. 31A

(269) TABLE-US-00037 TABLE CC-1 testing of anti-A-beta constructs for binding by pre-existing antibodies Nanobodies tested Binding Binding Binding on 92 samples Ref. no. in Level at 125 Level at 125 Level at 125 (healthy subjects) FIG. 30A seconds <10 RU seconds <20 RU seconds >20 RU SEQ ID NO: 766 (1) 0 1 91 (reference) SEQ ID NO: 733 (2) 11 41 51 (invention: L11V + V89L) SEQ ID NO: 749 (3) 16 56 36 (invention: L11V + V89L + T110K)

(270) The anti-A-beta Nanobodies that were present at the C-terminal end of the constructs were also tested separately as monovalent constructs (with a C-terminal alanine). The results are given in Table CC-2 and FIG. 31A.

(271) TABLE-US-00038 TABLE CC-2 testing of monovalent anti-A-beta Nanobodies for binding by pre-existing antibodies Nanobodies tested Binding Binding Binding on 145 samples Ref. no. in Level at 125 Level at 125 Level at 125 (healthy subjects) FIG. 30A seconds <10 RU seconds <20 RU seconds >20 RU SEQ ID NO: 478 (1) 67 87 58 (reference) (*) SEQ ID NO: 489 (2) 74 103 42 (invention: L11V + V89L) (*) SEQ ID NO: 490 (3) 116 138 7 (invention: L11V + V89L + T110K) (*) (*) all three monovalent Nanobodies tested had a C-terminal alanine added

Example 20: Overview of Serum Albumin Binders of the Invention

(272) Tables DD and EE below give some preferred, but non-limiting examples of serum albumin binding Nanobodies of the invention, based on Alb-8 (Table DD) and Alb-23 (Table EE), respectively.

(273) As mentioned, the invention also relates to a polypeptide, protein, compound or construct (and in particular a compound of the invention) that comprises one of the serum albumin-binding Nanobodies listed in Table DD or EE below. Such a polypeptide, protein, compound or construct (and in particular a compound of the invention) may further comprise at least one (such as one, two or three) binding domain or binding unit (such as an ISVD, and in particular an ISV of the invention) that is directed against at least one therapeutic target. Such a polypeptide, protein, compound or construct may again suitably be monospecific, bispecific or trispecific with respect to the therapeutic target(s), and may be bivalent, trivalent, tetravalent or of higher valency. It will usually also contain suitable linkers, and may comprise a C-terminal extension as described herein.

(274) In particular, such a polypeptide, protein, compound or construct may be a compound of the invention (as described herein) and/or may be as further described herein for the compounds of the invention (including preferred embodiments for compounds of the invention. Thus compounds comprising ISVD's and in particular Nanobodies are particularly preferred). Accordingly compounds of the invention comprising one of the serum albumin binding Nanobodies listed in Table DD or EE form further aspects of the invention.

(275) TABLE-US-00039 TABLE DD Alb-8 (reference) and Alb-8 variants according to the invention SEQ ID NO: Variant Sequence 46 Alb-8 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE (WO WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAV 06/122787) YYCTIGGSLSRSSQGTLVTVSS 496 89L + 110K EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAL YYCTIGGSLSRSSQGTLVKVSS 497 89L + 110Q EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAL YYCTIGGSLSRSSQGTLVQVSS 498 110K EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAV YYCTIGGSLSRSSQGTLVKVSS 499 110Q EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAV YYCTIGGSLSRSSQGTLVQVSS 47 112K EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAV YYCTIGGSLSRSSQGTLVTVKS 54 112Q EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAV YYCTIGGSLSRSSQGTLVTVQS 78 89T EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAT YYCTIGGSLSRSSQGTLVTVSS 109 11V + 89L EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAL YYCTIGGSLSRSSQGTLVTVSS 123 11V + 89L + EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE 110K WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAL YYCTIGGSLSRSSQGTLVKVSS 500 11V + 89L + EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE 110Q WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAL YYCTIGGSLSRSSQGTLVQVSS 501 11V + 110K EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAV YYCTIGGSLSRSSQGTLVKVSS 502 11V + 110Q EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAV YYCTIGGSLSRSSQGTLVQVSS 503 11V + 112K EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAV YYCTIGGSLSRSSQGTLVTVKS 504 11V + 112Q EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAV YYCTIGGSLSRSSQGTLVTVQS

(276) TABLE-US-00040 TABLE EE Alb-23 (reference) and Alb-23 variants according to the invention SEQ ID NO: Variant Sequence 61 A1b-23 EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPE (WO WVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAV 12/175400) YYCTIGGSLSRSSQGTLVTVSS 505 89L + 110K EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPE WVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALY YCTIGGSLSRSSQGTLVKVSS 506 89L + 110Q EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPE WVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALY YCTIGGSLSRSSQGTLVQVSS 507 110K EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPE WVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAV YYCTIGGSLSRSSQGTLVKVSS 508 110Q EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPE WVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAV YYCTIGGSLSRSSQGTLVQVSS 62 112K EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPE WVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAV YYCTIGGSLSRSSQGTLVTVKS 69 112Q EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPE WVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAV YYCTIGGSLSRSSQGTLVTVQS 86 89T EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPE WVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTATY YCTIGGSLSRSSQGTLVTVSS 116 11V + 89L EVQLLESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPE WVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALY YCTIGGSLSRSSQGTLVTVSS 130 11V + 89L + EVQLLESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPE 110K WVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALY YCTIGGSLSRSSQGTLVKVSS 509 11V + 89L + EVQLLESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPE 110Q WVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALY YCTIGGSLSRSSQGTLVQVSS 510 11V + 110K EVQLLESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPE WVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAV YYCTIGGSLSRSSQGTLVKVSS 511 11V + 110Q EVQLLESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPE WVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAV YYCTIGGSLSRSSQGTLVQVSS 512 11V + 112K EVQLLESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPE WVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAV YYCTIGGSLSRSSQGTLVTVKS 513 11V + 112Q EVQLLESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPE WVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAV YYCTIGGSLSRSSQGTLVTVQS

EXEMPLARY EMBODIMENTS

(277) 1. VH domain, in which: (i) the amino acid residue at position 112 is one of K or Q; and/or (ii) the amino acid residue at position 89 is T; and/or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q; and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V; and in which said VH domain contains a C-terminal extension (X).sub.n, in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an (preferably naturally occurring) amino acid residue that is independently chosen, and preferably independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I). rf
2. VH domain according to embodiment 1, which is an immunoglobulin single variable domain.
3. VH domain according to embodiment 2, which is a nanobody.
4. VH domain, in which the amino acid residue at position 112 is one of K or Q.
5. VH domain according to embodiment 4, which contains a C-terminal extension (X)n, in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an (preferably naturally occurring) amino acid residue that is independently chosen, and preferably independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).
6. VH domain according to embodiment 4 or embodiment 5, which is an immunoglobulin single variable domain.
7. VH domain according to embodiment 6, which is a nanobody.
8. VH domain, in which the amino acid residue at position 89 is T, and in which said VH domain contains a C-terminal extension (X).sub.n, in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an (preferably naturally occurring) amino acid residue that is independently chosen, and preferably independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).
9. VH domain according to embodiment 8, which is an immunoglobulin single variable domain.
10. VH domain according to embodiment 9, which is a nanobody.
11. VH domain, in which the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q.
12. VH domain according to embodiment 11, in which said VH domain contains a C-terminal extension (X)n, in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an (preferably naturally occurring) amino acid residue that is independently chosen, and preferably independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).
13. VH domain according to embodiment 11 or embodiment 12, which is an immunoglobulin single variable domain.
14. VH domain according to embodiment 13, which is a nanobody.
15. VH domain, in which the amino acid residue at position 11 is V and the amino acid residue at position 110 is one of K or Q.
16. VH domain according to embodiment 15, in which said VH domain contains a C-terminal extension (X)n, in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an (preferably naturally occurring) amino acid residue that is independently chosen, and preferably independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).
17. VH domain according to embodiment 15 or 16, which is an immunoglobulin single variable domain.
18. VH domain according to embodiment 17, which is a nanobody.
19. VH domain, in which the amino acid residue at position 89 is L and the amino acid residue at position 11 is V.
20. VH domain according to embodiment 19, in which said VH domain contains a C-terminal extension (X)n, in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an (preferably naturally occurring) amino acid residue that is independently chosen, and preferably independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).
21. VH domain according to embodiment 19 or embodiment 20, which is an immunoglobulin single variable domain.
22. VH domain according to embodiment 21, which is a nanobody.
23. VH domain, in which: the amino acid residue at position 11 is one of L, V or K; and the amino acid residue at position 14 is one of A or P; and the amino acid residue at position 41 is one of A or P; and the amino acid residue at position 89 is one of T, V or L; and the amino acid residue at position 108 is one of Q or L; and the amino acid residue at position 110 is one of T, K or Q; and the amino acid residue at position 112 is one of S, K or Q;
in which either (i) the amino acid residue at position 112 is one of K or Q; and/or (ii) the amino acid residue at position 89 is T; and/or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q; and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V; and
in which said VH domain optionally contains a C-terminal extension (X)n, in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an (preferably naturally occurring) amino acid residue that is independently chosen, and preferably independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).
24. VH domain according to embodiment 23, which is an immunoglobulin single variable domain.
25. VH domain according to embodiment 23 or embodiment 24, which is a nanobody.
26. VH domain according to any of embodiments 23 to 25, in which the amino acid residue at position 112 is one of K or Q.
27. VH domain according to any of embodiments 23 to 25, in which the amino acid residue at position 89 is T.
28. VH domain according to any of embodiments 23 to 25, in which the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q.
29. VH domain according to any of embodiments 23 to 25, in which the amino acid residue at position 89 is L and the amino acid residue at position 11 is V.
30. VH domain according to any of embodiments 23 to 29, in which said VH domain contains a C-terminal extension (X)n, in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an (preferably naturally occurring) amino acid residue that is independently chosen, and preferably independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).
31. VH domain, in which: the amino acid residue at position 11 is L; and the amino acid residue at position 14 is one of A or P; and the amino acid residue at position 41 is one of A or P; and the amino acid residue at position 89 is one of T, V or L; and the amino acid residue at position 108 is one of Q or L; and the amino acid residue at position 110 is one of K or Q; and the amino acid residue at position 112 is one of S, K or Q;
and in which said VH domain optionally contains a C-terminal extension (X)n, in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an (preferably naturally occurring) amino acid residue that is independently chosen, and preferably independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).
32. VH domain according to embodiment 31, which is an immunoglobulin single variable domain.
33. VH domain according to embodiment 30 or 31, which is a nanobody.
34. VH domain according to any of embodiments 30 to 32, in which said VH domain contains a C-terminal extension (X)n, in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an (preferably naturally occurring) amino acid residue that is independently chosen, and preferably independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).
35. VH domain according to any of the preceding embodiments, which can specifically bind to human serum albumin.
36. VH domain according to embodiment 35, which is an immunoglobulin single variable domain.
37. VH domain according to embodiment 36, which is a nanobody.
38. Library of VH domains according to any of embodiments 1 to 7.
39. Library of nucleic acid residues encoding VH domains according to any of embodiments 1 to 7.
40. Library according to embodiment 39, which is an expression library.
41. Library according to any of embodiments 38-40, which is a synthetic library.
42. Library according to any of embodiments 38-41, which contains at least at least 100 different sequences, such as at least 1000 different sequences, in particular more than 105 different sequences, more in particular more than 106 different sequences, such as 108 to 1010 or more different sequences.
43. Library of VH domains according to any of embodiments 8 to 10.
44. Library of nucleic acid residues encoding VH domains according to any of embodiments 8 to 10.
45. Library according to embodiment 44, which is an expression library.
46. Library according to any of embodiments 43-45, which is a synthetic library.
47. Library according to any of embodiments 43-46, which contains at least at least 100 different sequences, such as at least 1000 different sequences, in particular more than 105 different sequences, more in particular more than 106 different sequences, such as 108 to 1010 or more different sequences.
48. Library of VH domains according to any of embodiments 11 to 14.
49. Library of nucleic acid residues encoding VH domains according to any of embodiments 11 to 14.
50. Library according to embodiment 48, which is an expression library.
51. Library according to any of embodiments 48-50, which is a synthetic library.
52. Library according to any of embodiments 48-51, which contains at least at least 100 different sequences, such as at least 1000 different sequences, in particular more than 105 different sequences, more in particular more than 106 different sequences, such as 108 to 1010 or more different sequences.
53. Library of VH domains according to any of embodiments 15-18.
54. Library of nucleic acid residues encoding VH domains according to any of embodiments 15-18.
55. Library according to embodiment 54, which is an expression library.
56. Library according to any of embodiments 53-55, which is a synthetic library.
57. Library according to any of embodiments 53-56, which contains at least at least 100 different sequences, such as at least 1000 different sequences, in particular more than 105 different sequences, more in particular more than 106 different sequences, such as 108 to 1010 or more different sequences.
58. Library of VH domains according to any of embodiments 19-22.
59. Library of nucleic acid residues encoding VH domains according to any of embodiments 19-22.
60. Library according to embodiment 59, which is an expression library.
61. Library according to any of embodiments 58-60, which is a synthetic library.
62. Library according to any of embodiments 58-61, which contains at least at least 100 different sequences, such as at least 1000 different sequences, in particular more than 105 different sequences, more in particular more than 106 different sequences, such as 108 to 1010 or more different sequences.
63. Library of VH domains according to any of embodiments 23-30.
64. Library of nucleic acid residues encoding VH domains according to any of embodiments 23-30.
65. Library according to embodiment 64, which is an expression library.
66. Library according to any of embodiments 63-65, which is a synthetic library.
67. Library according to any of embodiments 63-66, which contains at least at least 100 different sequences, such as at least 1000 different sequences, in particular more than 105 different sequences, more in particular more than 106 different sequences, such as 108 to 1010 or more different sequences.
68. Library of VH domains according to any of embodiments 31 to 37.
69. Library of nucleic acid residues encoding VH domains according to any of embodiments 31 to 37.
70. Library according to embodiment 69, which is an expression library.
71. Library according to any of embodiments 68-70, which is a synthetic library.
72. Library according to any of embodiments 68-71, which contains at least at least 100 different sequences, such as at least 1000 different sequences, in particular more than 105 different sequences, more in particular more than 106 different sequences, such as 108 to 1010 or more different sequences.