NANOBODIES AGAINST TUMOR NECROSIS FACTOR-ALPHA

Abstract

The present invention relates to improved Nanobodies™ against Tumor Necrosis Factor-alpha (TNF-alpha), as well as to polypeptides comprising or essentially consisting of one or more of such Nanobodies. The invention also relates to nucleic acids encoding such Nanobodies and polypeptides; to methods for preparing such Nanobodies and polypeptides; to host cells expressing or capable of expressing such Nanobodies or polypeptides; to compositions comprising such Nanobodies, polypeptides, nucleic acids or host cells; and to uses of such Nanobodies, such polypeptides, such nucleic acids, such host cells or such compositions, in particular for prophylactic, therapeutic or diagnostic purposes, such as the prophylactic, therapeutic or diagnostic purposes.

Claims

1.-111. (canceled)

112. A polypeptide comprising the amino acid sequence of: DVQLVESGGGLVQPGGSLKLSCAASGFDFSX.sup.31X.sup.32WMYWVRQAPGKELEWLSEINTNG LITX.sup.59YX.sup.61DSVKGRFTVSRNNAANKMYLELTRLEPEDTALYYCARX.sup.99X.sup.100X.sup.101GX.sup.103NK GQGTQVTVSS (SEQ ID NO: 477), wherein: the amino acid of X.sup.31 is a polar amino acid selected from the group consisting of G, S, T, C, N, Q, Y, K, R, H, D and E; the amino acid of X.sup.32 is a polar amino acid selected from the group consisting of G, S, T, C, N, Q, Y, K, R, H, D and E; the amino acid of X.sup.59 is a polar, positively charged amino acid selected from the group consisting of K, R, and H; the amino acid of X.sup.61 is a small aliphatic, nonpolar or slightly polar residue selected from the group consisting of A, S, T, P, and G; the amino acid of X.sup.99 is a polar, uncharged amino acid selected from the group consisting of G, S, T, C, N, Q, and Y; the amino acid of X.sup.100 is any amino acid; the amino acid of X.sup.101 is a polar amino acid selected from the group consisting of G, S, T, C, N, Q, Y, K, R, H, D, and E; and the amino acid of X.sup.103 is a nonpolar, uncharged amino acid selected from the group consisting of A, V, L, I, F, M, W, and P.

113. The polypeptide of claim 112, wherein the polypeptide binds human TNFα.

114. A pharmaceutical composition comprising the polypeptide of 112.

115. The pharmaceutical composition of claim 114, further comprising one or more excipients.

116. A method for treating a Tumor Necrosis Factor-α (TNF-α) related disorder comprising administering, to a subject in need thereof, an effective amount of the pharmaceutical composition of claim 114.

117. The method of claim 116, wherein the disorder is Crohn's disease, ulcerative colitis or inflammatory bowel syndrome.

Description

[1579] FIG. 1: Sequence alignment of human TNFα nanobodies

[1580] FIG. 2: Sequence alignment of serum albumin specific TNFα nanobodies

[1581] FIG. 3: Binding of albumin specific TNFα nanobodies to human serum albumin

[1582] FIG. 4: Binding of albumin specific TNFα nanobodies to rhesus serum albumin

[1583] FIG. 5: Binding of albumin specific TNFα nanobodies to mouse serum albumin

[1584] FIG. 6: Purity of TNFα and serum albumin nanobodies (SDS-PAGE)

[1585] FIG. 7: Western Blot analysis of TNFα and serum albumin nanobodies

[1586] FIG. 8: Binding of TNFα nanobodies to human TNFα (ELISA)

[1587] FIG. 9: Binding of TNFα nanobodies to rhesus TNFα (ELISA)

[1588] FIG. 10: Receptor-inhibition assay on Enbrel for human TNFα

[1589] FIG. 11: Receptor-inhibition assay on Enbrel for rhesus TNFα

[1590] FIG. 12: Binding of TNFα nanobodies to human TNFα (Biacore)

[1591] FIG. 13: Binding of TNFα nanobodies to rhesus TNFα (Biacore)

[1592] FIG. 14: Binding of TNFα nanobodies to Protein A (Biacore)

[1593] FIG. 15: Temperature treatment of TNFα and serum albumin nanobodies (Western Blot)

[1594] FIG. 16: Stability: temperature treatment of TNFα nanobodies (ELISA)

[1595] FIG. 17: Temperature treatment of serum albumin nanobodies (Biacore)

Bivalent TNFα Nanobodies

[1596] FIG. 18: Purity of bivalent TNFα nanobodies (SDS-PAGE)

[1597] FIG. 19: Western Blot analysis of bivalent TNFα nanobodies

[1598] FIG. 20: Receptor-inhibition assay on Enbrel for bivalent TNFα nanobodies

[1599] FIG. 21: Stability: temperature treatment of bivalent TNFα nanobodies (ELISA)

Humanised Monovalent TNFα Nanobodies

[1600] FIG. 22: Multiple sequence alignment of TNF1 humanised nanobodies; DP51 is SEQ ID NO: 472, DP53 is SEQ ID NO: 473.

[1601] FIG. 23: Multiple sequence alignment of TNF2 humanised nanobodies; DP54 is SEQ ID NO: 474.

[1602] FIG. 24: Multiple sequence alignment of TNF3 humanised nanobodies; DP29 is SEQ ID NO: 475.

[1603] FIG. 25: Multiple sequence alignment of ALB1 humanised nanobodies

[1604] FIG. 26: Purity of humanised TNFα and serum albumin nanobodies (SDS-PAGE)

[1605] FIG. 27: Western Blot analysis of humanised TNFα and serum albumin nanobodies

[1606] FIG. 28: Binding of humanised TNFα nanobodies to human TNFα

[1607] FIG. 29: Binding of humanised serum albumin nanobodies to human serum albumin

[1608] FIG. 30: Stability: temperature treatment of humanised TNFα nanobodies (ELISA)

Trivalent TNFα Nanobodies

[1609] FIG. 31: Purity of trivalent TNFα nanobodies (SDS-PAGE)

[1610] FIG. 32: Western Blot analysis of trivalent TNFα nanobodies

[1611] FIG. 33: Stability: temperature treatment of trivalent TNFα nanobodies (ELISA)

Humanised Monovalent TNFα Nanobodies (Second Round)

[1612] FIG. 34: Multiple sequence alignment of TNF1 humanised nanobodies

[1613] FIG. 35: Multiple sequence alignment of TNF2 humanised nanobodies

[1614] FIG. 36: Multiple sequence alignment of TNF3 humanised nanobodies

[1615] FIG. 37: Multiple sequence alignment of ALB1 humanised nanobodies

[1616] FIG. 38: Purity of humanised TNFα nanobodies (SDS-PAGE)

[1617] FIG. 39: Western Blot analysis of humanised TNFα nanobodies

[1618] FIG. 40: Binding of humanised TNFα nanobodies to human TNFα

[1619] FIG. 41: Stability: temperature treatment of humanised TNFα nanobodies (ELISA)

[1620] FIG. 42: Analysis of purified TNF60 on Silver stained SDS-PAGE gel (A) Coomassie stained SDS-PAGE gel (B) and in Western blot analysis using anti-NB (C) for detection

[1621] FIG. 43: Chromatogram of analytical size exclusion of TNF60 on Superdex HR75

[1622] FIG. 44: Binding of TNF60 to human TNF-alpha

[1623] FIG. 45: Dose response curve obtained in cytotoxicity assay with human TNF-alpha using Nanobody™ TNF60 in comparison with Enbrel (Etanercept), Humira (Adalimumab) and Remicade (Infliximab)

[1624] FIG. 46: Dose response curve obtained in cytotoxicity assay with rhesus TNFα using Nanobody™ TNF60 in comparison with Enbrel (Etanercept), Humira (Adalimumab) and Remicade (Infliximab)

[1625] FIG. 47: Pharmacokinetic profile of TNF60 in mice

[1626] FIG. 48: Immunogenicity profile of TNF60 in mice

[1627] FIG. 49: Analysis of purified TNF56-PEG40, TNF56-PEG60, TNF56-biotine, TNF55-PEG40, TNF55-PEG60 and TNF55-biotine on Coomassie stained SDS-PAGE gel

[1628] FIG. 50: Analysis of purified TNF56-PEG40 on SDS-PAGE gel using Silver stain (A) Coomassie stain (B) and in Western blot analysis using anti-NB (C) for detection

[1629] FIG. 51: Chromatogram of analytical size exclusion of TNF56-PEG40 on Superdex HR 75

[1630] FIG. 52: Chromatogram of analytical size exclusion of TNF56-PEG40 on Superdex HR 200

[1631] FIG. 53: Dose response curve obtained in cytotoxicity assay with human TNFα using Nanobody™ TNF56-PEG40 and the monovalent wild-type Nanobody™ TNF1 in comparison with Enbrel (Etanercept), Humira (Adalimumab) and Remicade (Infliximab)

[1632] FIG. 54: Dose response curve obtained in cytotoxicity assay with rhesus TNFα using Nanobody™ TNF56-PEG40 in comparison with Enbrel (Etanercept), Humira (Adalimumab) and Remicade (Infliximab)

[1633] FIG. 55: Pharmacokinetic analysis of pegylated bivalent Nanobody™ TNF56-PEG40 and TNF56-PEG60 after intravenous administration in mice

[1634] FIG. 56: Pharmacokinetic analysis of pegylated bivalent Nanobody™ 3E-3E-PEG20, pegylated bivalent Nanobody™ 3E-3E-PEG40 and bispecific Nanobody™ 3E-3E-AR1 after intravenous administration in mice

[1635] FIG. 57: Immunogenicity profile of TNF56-PEG40 and TNF56-PEG60 in mice

[1636] FIG. 58: Efficacy of TNF60 in the prevention of chronic polyarthritis in mice

[1637] FIG. 59: Efficacy of TNF60 in therapeutic treatment of chronic polyarthritis in mice

[1638] FIG. 60: Effect of TNF60 Nanobody™ formatting on efficacy in the prevention of chronic polyarthritis in mice

[1639] FIG. 61: Sequence alignment of Nanobodies™ PMP1C2, 3E, 1A and 3G

[1640] FIG. 62: Molecular model of TNF-60

[1641] The appended Tables form an integral part of the present specification and are as follows:

Monovalent TNFα Nanobodies

[1642] Table 8: Sequence listing of TNFα nanobodies

[1643] Table 9: K.sub.off values of human TNFα nanobodies

[1644] Table 10: Homology of TNFα and serum albumin nanobodies to human germline sequences

[1645] Table 11: Expression levels of TNFα and serum albumin nanobodies

[1646] Table 12: ELISA binding to human and rhesus TNFα

[1647] Table 13: Receptor-inhibition assay of TNFα nanobodies

[1648] Table 14: Biacore analysis of TNFα nanobodies

[1649] Table 15: Binding of TNFα nanobodies to TNFα (K.sub.D-values)

[1650] Table 16: Potency of TNFα nanobodies to neutralize human (a) and rhesus (b) TNFα

[1651] Table 17: OD280 nm of TNFα and serum albumin nanobodies after temperature treatment

[1652] Table 18: Potency of TNFα nanobodies after temperature treatment

Bivalent TNFα Nanobodies

[1653] Table 19: Sequence listing of bivalent TNFα nanobodies and linker sequences

[1654] Table 20: Bivalent TNFα nanobody constructs

[1655] Table 21: Expression levels of bivalent TNFα nanobodies

[1656] Table 22: Receptor-inhibition assay of bivalent TNFα nanobodies

[1657] Table 23: Potency of TNFα nanobodies to neutralize human (a) and rhesus (b) TNFα

[1658] Table 24: OD280 nm of bivalent TNFα nanobodies

Humanised Monovalent TNFα Nanobodies

[1659] Table 25: Sequence listing of humanised monovalent TNFα and serum albumin nanobodies

[1660] Table 26: Expression levels of humanised TNFα and serum albumin nanobodies

[1661] Table 27: Potency of TNFα nanobodies to neutralize human TNFα

[1662] Table 28: OD280 nm of humanised TNFα and serum albumin nanobodies

Trivalent TNFα Nanobodies

[1663] Table 29: Sequence listing of trivalent TNFα nanobodies

[1664] Table 30: Trivalent TNFα nanobody constructs

[1665] Table 31: Expression levels of trivalent TNFα nanobodies

[1666] Table 32: Potency of trivalent TNFα nanobodies to neutralize human TNFα

[1667] Table 33: Binding of trivalent nanobodies to serum albumin (K.sub.D-values)

[1668] Table 34: OD280 nm of trivalent TNFα nanobodies

Humanised Monovalent TNFα Nanobodies (Second Round)

[1669] Table 35: Sequence listing of second round humanised monovalent TNFα nanobodies

[1670] Table 36: Expression levels of humanised TNFα nanobodies

[1671] Table 37: Potency of TNFα nanobodies to neutralize human TNFα

[1672] Table 38: OD280 nm of humanised TNFα nanobodies

[1673] Table 39: Comparing bio-activity of nanobodies

Further Tables

[1674] Table 40: Overview of oligonucleotides used in formatting of trivalent Nanobodies™

[1675] Table 41: Overview of oligonucleotides used in cloning of trivalent Nanobodies™

[1676] Table 42: EC50 values obtained in cytotoxicity assay using trivalent Nanobody™ TNF60 in comparison with commercial controls (Enbrel, Remicade, Humira)

[1677] Table 43: Affinity determination of TNF60 and TNF24 on human serum albumin in Biacore. Nd, not determined.

[1678] Table 44: Overview of oligonucleotides used in formatting of bivalent Nanobodies™

[1679] Table 45: EC50 values obtained in cytotoxicity assay using bivalent Nanobodies™ in comparison with commercial controls (Enbrel, Remicade, Humira)

[1680] Table 46: Results of synovium derived fibroblast studies

[1681] Table 47: Results of murine air pouch studies

EXAMPLES

Example 1: Identification of TNFα and Serum Albumin Specific Nanobodies

[1682] Antagonistic nanobodies were identified using two llamas (Llama glama) immunized with human TNFα by 6 injections of 100 μg of the cytokine at weekly intervals. Screening was performed using a competition based assay, in which individual nanobodies were analyzed for their capability to inhibit binding of labeled TNFα to its receptor. The albumin specific nanobodies were identified from a llama immunized with human serum albumin. Screening of individual nanobodies was performed by ELISA using human, rhesus and mouse albumin, yielding a panel of nanobodies cross-reacting with the serum albumin of various species.

Example 2: Sequence Analysis of Isolated Nanobodies

[1683] Different classes of nanobodies were identified based on sequence analysis (FIG. 1) using a BLOSUM62 scoring matrix and a similarity significance value cut-off of ≥60%: Class I (PMP1 C2, PMP1 G11, PMP1 H6), Class II (PMP1 G5, PMP1 H2, PMP3 G2), Class IIb (PMP1 D2), Class III (PMP3 D10, PMP5 F10). Table 8 lists the sequences of these TNFα nanobodies (SEQ ID NOs: 52 to 60).

[1684] Based on sequence analysis (FIG. 2) different classes of serum albumin nanobodies were identified using the BLOSUM62 scoring matrix and a similarity significance value cut-off of ≥60%. Table 8 lists the sequences of these serum albumin nanobodies (SEQ ID NOs: 61 to 67).

Example 3: Biacore Analysis

TNFα

[1685] Binding of nanobodies to TNFα was characterised by surface plasmon resonance in a Biacore 3000 instrument. TNF from different species was covalently bound to CM5 sensor chips surface via amine coupling until an increase of 250 response units was reached. Remaining reactive groups were inactivated. Nanobody binding was assessed at one concentration (1 in 1,000 diluted). Each nanobody was injected for 4 minutes at a flow rate of 45 μl/min to allow for binding to chip-bound antigen. Binding buffer without nanobody was sent over the chip at the same flow rate to allow spontaneous dissociation of bound nanobody for 4 hours. K.sub.off-values were calculated from the sensorgrams obtained for the different nanobodies.

[1686] Of each class of nanobodies unpurified proteins were analyzed in Biacore. K.sub.off data is listed in Table 9.

[1687] Representative nanobodies from each class were retained for further analysis based on k.sub.off value. For Class I PMP1C2 (TNF1) was selected; PMP1G5 (TNF2) was selected as representative of Class II; PMP5F10 (TNF3) was selected as representative of Class III.

Serum Albumin

[1688] Binding was assayed as described above except that 1 in 20 dilutions were used. FIGS. 3, 4 and 5 illustrate screening of albumin specific TNFα nanobodies versus human, rhesus and mouse serum albumin using unpurified protein.

[1689] The nanobodies are ranked according to k.sub.off-values, see Table III below:

TABLE-US-00031 TABLE III Class Human Rhesus Mouse C PMP6A8 PMP6A8 PMP6B4 C PMP6B4 PMP6B4 PMP6A8 B PMP6A6 PMP6A6 PMP6A6 B PMP6C1 PMP6C1 PMP6C1 A PMP6G8 PMP6G8 PMP6G8 A PMP6A5 PMP6A5 PMP6A5 D PMP6G7 PMP6G7 PMP6G7

[1690] The best k.sub.off were obtained for members of family C and family B. Cross-reactivity between mouse, human and rhesus serum albumin was also observed for members of those families. A representative nanobody from class B and C was defined for further analysis: PMP6A6 (ALB1) was selected as representative of Class B and PMP6A8 (ALB2) was selected as representative of Class C.

Example 4: Cloning of Monovalent Nanobodies in pAX051

[1691] Description of Escherichia coli Expression Vector

[1692] pAX051 is a derivative of pUC19. It contains the LacZ promoter which enables a controlled induction of expression using IPTG. The vector has a resistance gene for Ampicillin or Carbenicillin. The multicloning sites harbours several restriction sites of which SfiI and BstEII are frequently used for cloning of Nanobodies™. In frame with the NB coding sequence the vector codes for a C-terminal c-myc tag and a (His)6 tag. The signal peptide is the gen3 leader sequence which translocates the expressed Nanobody™ to the periplasm.

[1693] The DNA coding for the selected nanobodies TNF1 (PMP1C2), TNF2 (PMP1G5), TNF3 (PMP5F10), ALB1 (PMP6A6) and ALB2 (PMP6A8) was cloned in pAX051 and the construct was transformed to TG1 electrocompetent cells. Clones were analyzed for PCR insert and the nucleotide sequence was determined from 4 positive clones. Glycerol stocks were prepared from clones containing the correct sequence and stored at −80° C.

Example 5: Expression of Monovalent Nanobodies

[1694] A preculture was started by inoculating a single colony of the clone expressing the respective nanobodies at 37° C. in Luria Broth, Ampicillin/Carbenicillin (100 μg/ml) and 2% glucose overnight. This preculture was used to inoculate. Inoculum is 1% percent (v/v) of the production culture (TB medium+Ampicillin/Carbenicillin+0.1% Glucose). The production culture is grown at 37° C. until an OD600 nm of 5-10 is reached and nanobody expression is induced by adding IPTG (1 mM final concentration). Protein expression is allowed to continue either for 4 h at 37° C. or overnight at 28° C., at which point cells are collected by centrifugation and stored as wet cell paste at −20° C.

[1695] Preparative periplasmic extracts of the −20° C. stored wet cell paste are made by resuspending the pellet in Peri-buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, adjusted pH to 8.0), rotating the mixture for 30 min at 4° C. and centrifuging the mixture using a preparative centrifuge (Sorvall RC-3C Plus with H-6000 A rotor) to pellet the cells. Supernatant, representing a rough extract of the periplasmic space, is collected for further purification.

[1696] The His(6)-tagged nanobodies are purified on Immobilized Metal Affinity Chromatography (IMAC). The TALON resin (Clontech) is processed according to the manufacturer's instructions. The extracts are incubated with the resin for 30 min at RT on a rotator. The resin is washed with PBS and transferred to a column. The packed resin is washed with 15 mM Imidazole. The nanobodies are eluted from the column using 150 mM Imidazole. The eluated fractions are analyzed by spotting on Hybond Membrane and visualization with Ponceau. Fractions containing protein are pooled and dialysed against PBS. Dialysed proteins are collected, filter sterilized, concentration determined and stored in aliquots at −20° C.

Characterisation of Monovalent TNFα Nanobodies

Example 6: Homology to Human Germline Sequences

[1697] The nanobody amino acid sequences were compared to the human germline sequences as represented in Table 10. In order of homology to human sequences the nanobodies rank as follows: TNF1>TNF2>TNF3 for the TNFα nanobodies; ALB1>ALB2 for the serum albumin nanobodies.

Example 7: Expression Level

[1698] Expression levels were calculated and represented in Table 11. In order of yield the nanobodies rank as follows: TNF1>TNF2>TNF3 for the TNFα nanobodies; ALB1>ALB2 for the serum albumin nanobodies.

Example 8: SDS-Page Analysis

[1699] To determine the purity, protein samples were analyzed on a 15% SDS-PAGE gel. 10 μl Laemmli sample buffer was added to 10 μl (1 ug) purified protein, the sample was heated for 10 minutes at 95° C., cooled and loaded on a 15% SDS-PAGE gel. The gel was processed according to general procedures and stained with Coomassie Brilliant Blue (CBB). FIG. 6 represents the SDS-PAGE for the TNFα-specific and serum albumin-specific nanobodies.

Example 9: Western Blot Analysis

[1700] 100 ng of purified protein was loaded on the gel. Following SDS-PAGE, proteins were transferred to a nitrocellulose membrane using the Mini Trans-Blot® Electrophoretic Transfer Cell (Biorad). The membrane was blocked overnight in PBS, 1% casein at 4° C. As all constructs were fused to c-myc tag, mouse monoclonal anti-myc antibody was used as a detection tool. In addition, rabbit polyclonal anti-Nanobody (R23) was used as a detection tool. The blot was incubated for 1 h at room temperature with agitation in 1/2000 diluted anti-myc antibody in PBS or 1/2000 anti-Nanobody antibody in PBS, 1% casein. The membrane was washed 5 times in PBS before the secondary antibody was applied (rabbit-anti-mouse IgG alkaline phosphatase conjugate, Sigma, A1902, diluted 1/1000 in PBS or goat anti-rabbit IgG alkaline phosphatase conjugate, Sigma, A8025, 1% casein). After incubation with gentle agitation for 1 h at room temperature, the membrane was washed 5 times in PBS. Blots were developed using BCIP/NBT solutions and the reaction was stopped by washing the blot with milliQ water when bands were clearly visible. FIG. 7 represents the Western Blot analysis.

Example 10: ELISA Binding to Human and Rhesus TNFα

[1701] An ELISA was performed to examine binding to human and rhesus TNFα. A 96-well Maxisorp plate was coated with 2 μg/ml Neutravidin in PBS ON at 4° C. Plates were blocked with 1% casein for 2 hrs at RT. Biotinylated TNFα (400 ng/ml) was added to the wells and incubated for 1 hr at RT. Nanobody samples were diluted starting at 2 μg/ml and using 1 in 3 dilutions. Nanobodies were detected using mouse anti-myc (1/2000 diluted) and rabbit anti-mouse alkaline phosphatase (1/2000 diluted, Sigma, A1902) and pNPP (2 mg/ml) as substrate. FIGS. 9 and 10 represent the binding in ELISA to human and rhesus TNFα.

[1702] Results are summarized in Table 12. TNF1 and TNF3 show binding to both human and rhesus TNFα. TNF2 is binding to human TNFα but is only weakly reactive to rhesus TNFα.

Example 11: Receptor-Inhibition Assay

[1703] The ability to inhibit receptor-ligand interaction was analyzed for rhesus and human TNFα. A 96-well Maxisorp plate was coated with 2 μg/ml Enbrel in PBS ON at 4° C. Plates were blocked with 1% Casein for 2 hrs at RT. Nanobody samples were pre-incubated for 30 min at RT with biotinylated TNFα (10 ng/ml) starting at a concentration of 5 μg/ml and using 1 in 2 dilutions. Samples were added to the plates and incubated for 1 hr at RT. Biotinylated TNFα was detected using ExtrAvidin alkaline phosphatase (1/2000 diluted) and pNPP (2 mg/ml) as substrate. FIGS. 11 and 12 represent an inhibition ELISA for human and rhesus TNFα. Results are summarized in Table 13. Inhibition of ligand/receptor binding is observed for TNF1 and TNF3 for both human and rhesus TNF, while TNF2 is only inhibiting human TNFα.

Example 12: Biacore Analysis

TNFα Binding

[1704] The analysis was performed as described in Example 3. FIGS. 13 and 14 illustrate the binding to human and rhesus TNFα via Biacore analysis. Results are summarized in Table 14. Binding experiments in Biacore confirm the ELISA results: cross-reactive binding for TNF1 and TNF3, while TNF2 only significantly binds human TNFα.

Serum Albumin

[1705] Binding was assayed as described above except that series of different concentrations were used. Each concentration was injected for 4 minutes at a flow rate of 45 μl/min to allow for binding to chip-bound antigen. Binding buffer without analyte was sent over the chip at the same flow rate to allow for dissociation of bound nanobody. After 15 minutes, remaining bound analyte was removed by injection of the regeneration solution (25 mM NaOH).

[1706] From the sensorgrams obtained for the different concentrations of each analyte K.sub.D-values were calculated via steady state affinity when equilibrium was reached.

[1707] Results are summarized in Table 15. Cross-reactivity is observed for both ALB1 and ALB2. The highest affinity is observed for ALB2 on human and rhesus TNFα. However, the difference in affinity for human/rhesus versus mouse serum albumin is more pronounced for ALB2 (factor 400), while for ALB1 a difference of a factor 12 is observed.

Example 13: Bio-Assay

[1708] The TNFα sensitive mouse fibroblast cell line L929s was used for measuring the anti-TNFα activity of the selected nanobodies. At a sufficiently high concentration of TNFα in the medium, i.e. cytotoxic dose, L929s cells undergo necrosis. The inhibition of TNFα interaction with its receptor was determined by pre-incubating a series of antibody dilutions with a cytotoxic concentration of TNFα before adding the mixture to the cells. The presence of actinomycin D in the medium sensitises the cells further to TNFα, resulting in increased sensitivity of the bioassay for free TNFα.

[1709] The L929 cells were grown to nearly confluency, plated out in 96-well microtiter plates at 5000 cells per well and incubated overnight. Actinomycin D was added to the cells at a final concentration of 1 μg/ml. Serial dilutions of the nanobodies to be tested were mixed with a cytotoxic concentration of TNFα (final assay concentration is 0.5 ng/ml or 15 IU/ml). After at least 30 minutes of incubation at 37° C., this mixture was added to the plated cells. Plates were incubated for 24 hours at 37° C. and 5% CO.sub.2. Cell viability was determined by use of the tetrazolium salt WST-1. Dose-response curves and EC.sub.50 values were calculated with Graphpad Prism.

[1710] The results are summarized in Table 16 for human and rhesus TNFα. Based on their potency to neutralize cytotoxic activity, the molecules are ranked as follows: TNF3>TNF1>TNF2 for human TNFα, and TNF1=TNF3>TNF2 for rhesus TNFα.

Example 14: Protein a Binding

[1711] FIG. 14 represents Protein A binding analyzed in Biacore as described in Example 12. Positive binding was obtained for TNF1, TNF2, ALB1. No or weak binding was observed for TNF3 and ALB2.

Example 15: Temperature Stability

[1712] Samples were diluted at 200 μg/ml and divided in 8 aliquots containing 500 μl. The different vials were incubated each at a given temperature ranging from RT to 90° C. After treatment the samples were cooled down for 2 hrs at RT, they were kept at 4° C. Precipitates were removed by centrifugation for 30 min at 14,000 rpm. SN was carefully removed and further analysed.

OD280 nm

[1713] OD at 280 nm was measured and the concentration was calculated. Results are summarized in Table 17. A decrease in protein content was observed for TNF2 and TNF3 starting at 80° C., while for ALB2 a decrease is observed starting from 70° C.

Western Blot

[1714] 2 μg of treated protein was separated on a 15% SDS-PAGE and transferred to a nitrocellulose membrane and treated as described above. Detection was performed using polyclonal anti-Nanobody (R23, 1/2000 diluted) and anti-rabbit horse radish peroxidase (DAKO, P0448, 1/2000 diluted). FIG. 15 represents the Western Blot analysis. A clear drop in protein concentration was observed for ALB2 treated at 70, 80 and 90° C. Aggregation was still observed for TNF1 treated at 70, 80 and 90° C.; for TNF3 treated at 90° C.; for ALB1 treated at 90° C., meaning that the SN still contains traces of precipitates which result in a higher OD280 nm read-out. This explains why the protein concentration as measured at OD280 nm does not decrease for TNF1, TNF3 and ALB1 treated at these higher T.

ELISA

[1715] The ELISA to detect binding to human TNFα was essentially performed as described in Example 10. Results are presented in FIG. 16. Human TNFα binding is decreased for TNF1, TNF2, TNF3 starting at 80° C.

Bio-Assay

[1716] The bio-assay was performed as described in Example 13. The results are summarized in Table 18. Potency of the nanobodies is decreased for TNF1 starting at 70° C.; for TNF2 and TNF3 starting at 80° C.

Biacore

[1717] Binding to human serum albumin was determined as described in Example 12. A fixed concentration was used (1 in 50 diluted). Results are presented in FIG. 17. Temperature treatment is not influencing binding to serum albumin for ALB 1. The treatment has an effect on the k.sub.on for ALB2 starting from T=70° C.

Bivalent Nanobodies

Example 16: Formatting of Bivalent TNFα Specific Nanobodies

[1718] TNF1, TNF2 and TNF3 were formatted to bivalent nanobodies. As spacer between the two building blocks either a 9AA GlySer linker (Table 19 SEQ ID No: 68) or a 30 AA GlySer linker (Table 19 SEQ ID No: 69) was used. This generated the constructs represented by Table 20. Table 19 lists the sequences of these bivalent TNFα nanobodies (SEQ ID NOs: 70 to 75).

Example 17: Expression of Bivalent TNFα Specific Nanobodies

[1719] Expression was performed as described in Example 5. The His(6)-tagged nanobodies were purified on Immobilized Metal Affinity Chromatography (IMAC). The Ni-NTA resin (Qiagen) was processed according to the manufacturer's instructions. The extracts were incubated with the resin and incubated for 30 min at RT on a rotator. The resin was washed with PBS and transferred to a column. The packed resin was washed with PBS (1 in 10 diluted). The column was pre-eluted with 15 mM Imidazole. The nanobodies were eluted from the column using 25 mM Citric Acid pH=4. The eluated fractions were analyzed by spotting on Hybond Membrane and by visualization with Ponceau. Fractions containing protein were pooled and further purified on Cation exchange followed by size exclusion. Purified proteins were collected, filter sterilized, concentration determined and stored in aliquots at −20° C.

Characterisation of Bivalent TNFα Specific Nanobodies

Example 18: Expression Level

[1720] Expression levels of the bivalent TNFα nanobodies were calculated and represented in Table 21. The linker has no significant effect on the expression level of the nanobodies.

Example 19: SDS-PAGE

[1721] SDS-Page was performed as described in Example 8. FIG. 18 shows the result of the SDS-Page.

Example 20: Western Blot

[1722] Western Blot analysis was performed as described in Example 9. FIG. 19 represents the Western Blot results.

Example 21: Receptor-Inhibition Assay

[1723] The assay was performed as described in Example 11. FIG. 20 and Table 22 represent the results. Enhancement of inhibition of ligand/receptor binding was observed for all bivalent nanobodies compared to the monovalent format.

Example 22: Bio-Assay

[1724] The assay was performed as described in Example 13. Results are summarized in Table 23. Based on their potency to neutralize cytotoxic activity TNF8, TNF7, TNF9 and TNF5 have a potency in the range of Enbrel.

Example 23: Temperature Stability

[1725] Samples were analysed as described in Example 15.

OD280 nm

[1726] OD at 280 nm was measured and the concentration was calculated. Results are summarized in Table 24. A decrease in protein content was observed for TNF4 and TNF7 starting at 70° C., while for TNF5, TNF6, TNF8 and TNF9 a decrease was observed starting from 80° C.

Western Blot

[1727] Samples were analyzed for the presence of aggregates as described in Example 15.

ELISA

[1728] The ELISA to detect binding to human TNFα was essentially performed as described above. Results are presented in FIG. 21. Human TNFα binding was decreased for TNF5, TNF6, TNF8 and TNF9 starting at 80° C., for TNF4 and TNF7 starting from 70° C.

Humanised Monovalent Nanobodies

Example 24: Identification of Non-Human Amino Acid Positions in TNFα and Serum Albumin Specific Nanobodies

[1729] FIG. 22 (TNF1), FIG. 23 (TNF2), FIG. 24 (TNF3) and FIG. 25 (ALB1) represent multiple sequence alignments (Clustal W 1.7) with DP51, DP53, DP54 and DP29 sequences.

[1730] In addition to the amino acid mutations, codon optimization was performed yielding the sequences of Table 25 SEQ ID NOs: 76 to 89 (Nanobodies against TNF-alpha and human serum albumin, respectively).

Example 25: Generation of Codon Optimised Mutants

[1731] Oligonucleotides were synthesised spanning the entire sequence of the nanobodies.

Example 26: Expression of Bivalent TNFα Specific Nanobodies

[1732] Expression was performed as described in Example 5.

Characterisation of Humanised Nanobody

Example 27: Expression Level

[1733] Table 26 represents calculated expression levels. Expression was achieved with yields in the range of 3.5-11.7 mg/ml. Induction time did not influence the yield.

Example 28: SDS-PAGE

[1734] SDS-PAGE was performed as described in Example 8. FIG. 26 represents the SDS-PAGE gel.

Example 29: Western Blot

[1735] Western Blot analysis was performed as described in Example 9. FIG. 27 represents the Western Blot results.

Example 30: Bio-Assay

[1736] The assay was performed as described in Example 13.

[1737] The results of the humanised nanobodies are summarized in Table 27. The wildtype nanobodies are included as reference.

Example 31: Biacore

[1738] The analysis was performed as described in Example 12. FIGS. 28 and 31 shows Biacore results.

Example 32: Temperature Stability

[1739] Samples were analysed as described in Example 15.

OD280 nm

[1740] OD at 280 nm was measured and the concentration was calculated. Results are summarized in Table 28.

[1741] No significant decrease in protein concentration is observed for the humanised TNF1 nanobodies (TNF13-14). A decrease in protein concentration is observed for humanised TNF2 (TNF15-19) and TNF3 (TNF20-23) starting at 80° C. A decrease in protein concentration is observed for humanized ALB1 (ALB4-5) starting at 70° C. and for ALB3 starting at 60° C.

Western Blot

[1742] Samples were analyzed for the presence of aggregates as described in Example 15.

ELISA

[1743] The ELISA to detect binding to human TNFα was essentially performed as described in Example 15. Results are presented in FIG. 30.

[1744] Human TNFα binding is comparable for temperature treated WT TNF1 and the humanized TNF13 and 14; for temperature treated WT TNF2 and the humanized TNF15-19; human TNFα binding is decreased for TNF21 and 22, and to a less extent for TNF23, while no effect is observed for TNF20 compared to the temperature treated WT TNF3.

Trivalent TNFα Nanobodies

Example 33: Formatting of Trivalent TNFα Specific Nanobodies

[1745] TNF1, TNF2, TNF3 and ALB1 were formatted to trivalent nanobodies. As spacer between 2 building blocks either a 9AA GlySer linker (Table 19 SEQ ID No 68) or a 30 AA GlySer linker (Table 19 SEQ ID No 69) was used. This generated the constructs of Table 30. Table 29 lists the sequences of trivalent TNFα nanobodies (SEQ ID NOs: 91 to 94).

Example 34: Expression of Trivalent TNFα Specific Nanobody

[1746] Expression was performed as described in Example 5. The His(6)-tagged nanobodies are purified on Immobilized Metal Affinity Chromatography (IMAC). The Ni-NTA resin (Qiagen) is processed according to the manufacturer's instructions. The extracts are incubated with the resin and incubated for 30 min at RT on a rotator. The resin is washed with PBS and transferred to a column. The packed resin is washed with PBS (1 in 10 diluted). Pre-elute with 15 mM Imidazole. The nanobodies are eluted from the column using 25 mM Citric Acid pH=4. The eluated fractions are analyzed by spotting on Hybond Membrane and visualization with Ponceau. Fractions containing protein are pooled and further purified on Cation exchange followed by size exclusion. Purified proteins are collected, filter sterilized, concentration determined and stored in aliquots at −20° C.

Characterization of Trivalent TNFα/SA Specific Nanobodies

Example 35: Expression Level

[1747] Expression levels were calculated and represented in Table 31.

Example 36: SDS-PAGE Analysis

[1748] SDS-PAGE was performed as described in Example 8. FIG. 31 represents the SDS-PAGE gel.

Example 37: Western Blot Analysis

[1749] Western Blot analysis was performed as described in Example 9. FIG. 32 represents the Western Blot analysis.

Example 38: Bio-Assay

[1750] The assay was performed as described in Example 13.

[1751] The results of the bivalent nanobodies are summarized in Table 32. Based on their potency to neutralize cytotoxic activity, the molecules are equally potent and comparable to their potency as bivalent molecules.

Example 39: Binding to Human Serum Albumin

[1752] Binding was assayed as described above except that series of different concentrations were used. Each concentration was injected for 4 minutes at a flow rate of 45 μl/min to allow for binding to chip-bound antigen. Next, binding buffer without analyte was sent over the chip at the same flow rate to allow for dissociation of bound nanobody. After 15 minutes, remaining bound analyte was removed by injection of the regeneration solution (25 mM NaOH).

[1753] From the sensorgrams obtained for the different concentrations of each analyte K.sub.D-values were calculated via steady state affinity when equilibrium was reached.

[1754] Results are summarized in Table 33. A decrease in affinity was observed for the formatted ALB1 binder compared to the wild type ALB1. The affinity however is still in the range of 7.2-14 nM.

Example 40: Temperature Stability

[1755] Samples were analysed as described in Example 15.

OD280 nm

[1756] OD at 280 nm was measured and the concentration was calculated. Results are summarized in Table 34. A decrease in protein content is observed for TNF24, TNF27 and TNF28 starting at 60° C., while for TNF25 and TNF26 starting from 70° C.

Western Blot

[1757] Samples were analyzed for the presence of aggregates as described in Example 15.

ELISA

[1758] The ELISA to detect binding to human TNFα was essentially performed as described above. Results are presented in FIG. 33. Human TNFα binding is decreased for TNF24 and TNF27, starting from 60° C. and for TNF25, TNF26 and TNF28 starting at 70° C.

Humanised Monovalent Nanobodies (Second Round)

Example 41: Identification of Non-Human Amino Acid Positions in TNFα and Serum Albumin Specific Nanobodies

[1759] FIG. 34 (TNF1), FIG. 35 (TNF2), FIG. 36 (TNF3) and FIG. 37 (ALB1) represent multiple sequence alignments (Clustal W 1.7) with DP51, DP53, DP54 and DP29 sequences. The mutated molecules were expressed and purified as described above, yielding the sequences of Table 35 SEQ ID NOs: 95 to 104 (against TNF-alpha and human serum albumin, respectively).

Characterisation of Humanised Nanobody

Example 42: Expression Level

[1760] Table 36 represents calculated expression levels. Expression was achieved with yields in the range of 0.5-2.7 mg/ml.

Example 43: SDS-PAGE

[1761] SDS-Page was performed as described in Example 8. FIG. 38 represents the SDS-Page gel.

Example 44: Western Blot

[1762] Western Blot analysis was performed as described in Example 9. FIG. 39 represents the Western Blot results.

Example 45: Bio-Assay

[1763] The assay was performed as described in Example 13.

[1764] The results of the humanised nanobodies are summarized in Table 37. The wildtype nanobodies and first round of humanised nanobodies are included as reference.

Example 46: Biacore

[1765] The analysis was performed as described in Example 12. FIG. 40 shows Biacore results.

Example 47: Temperature Stability

[1766] Samples were analysed as described in Example 15.

OD280 nm

[1767] OD at 280 nm was measured and the concentration was calculated. Results are summarized in Table 38.

[1768] No significant decrease in protein concentration is observed for the humanised TNF1 nanobodies (TNF29-30). A decrease in protein concentration is observed for humanised TNF2 (TNF31-32) and TNF3 (TNF33) starting at 80° C.

Western Blot

[1769] Samples were analyzed for the presence of aggregates as described in Example 15.

ELISA

[1770] The ELISA to detect binding to human TNFα was essentially performed as described in Example 15. Results are presented in FIG. 41.

[1771] Human TNFα binding is comparable for WT TNF1 and the humanised TNF29 and TNF30; comparable for WT TNF2 and the humanised TNF31 and TNF32; and also for WT TNF3 and humanised TNF33.

Comparative Example

[1772] In this Comparative Example, nine Nanobodies of the invention were compared with three Nanobodies from WO 04/041862, called “V.sub.HH#1A” or “1A”, “V.sub.HH#3E” or “3E” and “V.sub.HH#3G” or “3G” respectively (SEQ ID NOS:1, 4 and 5 in WO 04/041862). The assay used was the cell based assay using KYM-cells referred to in WO 04/41862 (see for example Example 1, under 3)). The results are mentioned in Table 39 below. As can be seen, the Nanobodies of the invention have an EC50 value in this assay that is 18-fold better than the EC50 value of 3E, the best performing Nanobody according to WO 04/041862.

Example 48: Generation of Trivalent Bispecific Humanized Nanobodies™

[1773] Trivalent bispecific Nanobodies were formatted and cloned in the E. coli expression vector pAX054 first and then rescued through PCR and cloned in the pPICZαA expression vector.

Description of Escherichia coli Expression Vector

[1774] pAX54 is a derivative of pUC19. It contains the LacZ promoter which enables a controlled induction of expression using IPTG. The vector has a resistance gene for Ampicillin or Carbenicillin. The multicloning sites harbours several restriction sites of which SfiI and BstEII are frequently used for cloning of Nanobodies™. The signal peptide is the gen3 leader sequence which translocates the expressed Nanobody™ to the periplasm.

Description of Pichia pastoris Expression Vector

[1775] pPICZαA contains a pUC-derived origin of replication allowing propagation in E coli. It contains the promoter of the Pichia pastoris AOX1 (alcohol oxidase 1) gene. This 942 bp promoter region (i) allows methanol-inducible, high-level expression of the gene of interest, and (ii) targets plasmid integration to the AOX1 locus following transformation of Pichia with vector DNA that is linearized within the 5′ AOX1 promoter region. Note that pPICZα vectors do not contain a yeast origin of replication and that, consequently, transformants can only be isolated if recombination occurs between the plasmid and the Pichia genome. The vector specifies resistance to the antibiotic Zeocin in both E. coli and Pichia pastoris host cells. The vector incorporates the secretion signal of the Saccharomyces cerevisiae α-mating factor allowing for efficient secretion of most proteins to the culture medium. The initiation ATG in the α-factor signal sequence corresponds to the native initiation ATG of the AOX1 gene. The multicloning site harbours several restriction sites of which Xho1/EcoR1 or Xho1/Not1 are typically used for fusion of the Nanobody™ coding sequences to the secretion signal. The multicloning site is followed by the AOX1 transcription termination region. More details on this expression vector can be found on the website of Invitrogen (http://www.invitrogen.com/content/sfs/manuals/ppiczalpha_man.pdf).

Formatting Trivalent Nanobodies

[1776] Three separate PCR reactions were set up to amplify the N-terminal, the middle and the C-terminal Nanobody™ subunit using the oligo combinations indicated in the WPA-0012. The N-terminal Nanobody™ was amplified using M13_rev/Rev_9GlySer_L108; the middle Nanobody™ was amplified using For_GlySer/Short and Rev_15BspEI_L108; the C-terminal Nanobody™ was amplified using For_BspEI/M13_for. A PCR reaction of 1 μl plasmid DNA (50-100 ng), 1.5 μl forward primer (10 μM.fwdarw.300 nM), 1.5 μl reverse primer (10 μM.fwdarw.300 nM), 1 μl dNTPs (10 mM.fwdarw.0.2 mM), 5 μl buffer (10×.fwdarw.1×), 0.75 μl enzyme (3.5 U/μl.fwdarw.2.6 U/μl) and 39.25 μl H.sub.2O with a total volume of 50 μl was prepared. Primer sequences are given in Table 40. A PCR program was started with 2 minutes at 94° C. A cycle of 30 seconds at 94° C., 30 seconds at 50° C. and 1 minute at 72° C. was repeated 30 times and followed by 10 minutes at 72° C. Amplification was checked by separating 5 μl of the PCR reaction on a 2% agarose gel. The PCR product was purified using the QIAquick PCR Purification Kit according to the manufacturer's instructions. One column was used and eluted with 50 μl EB buffer. The N-terminal V.sub.HH fragment was prepared by incubating 50 μl DNA and 2 μl BamHI (10 U/μl) in the appropriate buffer recommended by the manufacturer at 37° C. for 2 hours. Subsequently, 2 μl SfiI (10 U/μl) was added and the mixture was incubated at 55° C. for 2 hours. The middle VHH fragment was prepared by incubating 50 μl DNA and 2 μl BamHI (10 U/μl) and 2 μl BspEI (10 U/μl) in the appropriate buffer recommended by the manufacturer at 37° C. for 2 hours. The C-terminal V.sub.HH fragment was prepared by incubating 50 μl DNA with 2 μl BspEI (10 U/μl) in the appropriate buffer recommended by the manufacturer at 37° C. for 2 hours. Subsequently, 2 μl BstEII (10 U/μl) was added and the mixture was incubated at 60° C. for 1 hours. The previous digestion reactions were separated on a 2% agarose gel. The V.sub.HH bands (350-450 bp) were cut out of the gel and the DNA was purified using the QIAquick Gel Extraction Kit according to the manufacturer's instructions. One column (with a maximum of 400 mg agarose gel per column) was used and the bound DNA was eluted with 50 μl EB buffer. DNA concentration was determined by measuring OD.sub.260 (1 OD unit=50 μg/ml). A ligation mixture with a final volume of 10 μl containing 100 ng vector pAX54, 12 ng N-terminal V.sub.HH, 12 ng middle V.sub.HH fragment, 12 ng C-terminal V.sub.HH fragment, 1 μl ligation buffer and 1 μl ligase (3 U) was prepared and incubated for 2 hours at room temperature. Transformation of E. coli, TG1 was performed by using 2 μl of ligation mixture. Colonies are analysed using PCT as described in WPA-0010. Sequence analysis is performed on positive clones. Plasmid preparation was performed using the Qiaprep spin Miniprep kit (Qiagen) according to the manufacturer's instructions and described above. Sequencing was performed at the VIB sequence facility, Antwerp, Belgium.

Amplification of Coding DNA

[1777] The Nanobody™ coding region cloned in the pAX054 E. coli expression vector is rescued through PCR using an appropriate primer pair. To ensure that the Nanobody™ is expressed with a native N-terminus, the coding region is cloned in frame with the Kex2 cleavage site of the secretion signal. The forward primer fuses the C-terminal part of the secretion signal, up to the Xho1 recognition site, to the Nanobody™ coding region. A PCR reaction of 1 μl plasmid DNA (50-100 ng), 1.5 μl forward primer (10 μM.fwdarw.300 nM), 1.5 μl reverse primer (10 μM.fwdarw.300 nM), 1 μl dNTPs (10 mM.fwdarw.0.2 mM), 5 μl buffer (10×.fwdarw.1×), 0.75 μl enzyme (3.5 U/μl.fwdarw.2.6 U) and 39.25 μl H.sub.2O with a total volume of 50 μl was prepared. Primer sequences are given in Table 41. A PCR program was started with 2 minutes at 94° C. A cycle of 30 seconds at 94° C., 30 seconds at 50° C. and 2 minutes at 72° C. was repeated 20 times and followed by 10 minutes at 72° C. Amplification was checked by separating 5 μl of the PCR reaction on a 2% agarose gel. The PCR product was purified using the QIAquick PCR Purification Kit according to the manufacturer's instructions. One column was used and the bound DNA was eluted with 50 μl EB buffer.

Cloning Strategy

[1778] The DNA fragment coding for the NB as well as the pPICZαA expression vector is digested with the appropriate restriction enzymes (XhoI+NotI). The insert is obtained by incubating 50 μl PCR product with 2 μl XhoI (10 U/μl) and 2 μl NotI (10 U/μl) in the appropriate buffer recommended by the manufacturer for 3 hrs at 37° C. Vector is obtained similarly, adapting the amount of restriction enzymes to the amount of plasmid. Both the vector and the NB coding fragment are purified and the DNA concentration is quantified using the BioPhotometer (Eppendorf). The fragment and the acceptor vector are ligated in equimolar ratio's using 1 Unit T4 ligase (Promega) for 30 minutes at room temperature or overnight at 16° C. The DNA (20-30 ng) is transformed to TG1 cells. Colonies are analysed through PCR using the 3′AOX1 R and 5′AOX1 F primers. Sequence analysis is performed on positive clones. TNF30, TNF33 and ALB8 were formatted to trivalent bispecific Nanobodies™. As spacer between the building blocks a 9AA GlySer linker was used.

Transformation P. pastoris

[1779] To isolate plasmid DNA, a preculture is started by inoculating a single colony of the clone in 50 ml Luria Broth+Ampicillin or Carbenicillin (100 μg/ml)+2% glucose and incubation at 37° C. overnight. Plasmid DNA is prepared using the Plasmid Midi kit (Qiagen) according to the manufacturer's instructions. The DNA is linearized by incubating 30 μg plasmid DNA with 6 μl BstX1 (10 U/μl) in the appropriate buffer according to the manufacturer's instructions for 3 hrs at 45° C. Digested DNA is purified using the PCR Purification kit (Qiagen) according to the manufacturer's instructions. The DNA is concentrated using EtOH precipitation according to standard procedures. X-33 electrocompetent cells are transformed with 10 μg linearized DNA and cells are allowed to grow for 48 hrs on a selective YPD agar plate containing Zeocin (100/250/500 μg/ml). X-33 is a wild type Pichia pastoris strain; the strain itself as well as the derived recombinant strains contain the native AOX1 gene and are able to metabolize methanol (Mut.sup.+).

[1780] Clones are screened for expression level by incubating single colonies in 1 ml BGCM in a 24-well plate and growing them for 48 hrs at 30° C. at 120 rpm. Cells are centrifuged and fresh BGCM is added to the cells for growth at 30° C. at 120 rpm during 48 hrs. Next, MeOH is added to a final concentration of 0.5% and cells are grown at 30° C. at 120 rpm during 8 hrs, after which MeOH is added again to a final concentration of 0.5%. Cells are grown overnight at 30° C. at 120 rpm. Cells are centrifuged and the supernatant is harvested and analysed in ELISA as described in example 10.

Example 49: Expression and Purification of Trivalent Bispecific Humanized Nanobodies™

[1781] Production in Pichia pastoris

[1782] Composition of buffers, solutions and others can be found on the website of Invitrogen (http://www.invitrogen.com/content/sfs/manuals/ppiczalpha_man.pdf). A preculture was started by inoculating a single colony from plate in 5 ml YPD. The culture was grown overnight at 180 rpm and 30° C. The next day, the pre-culture was diluted to 50 ml of YPD and grown overnight at 180 rpm and 30° C. Production cultures were started by inoculating the pre-culture to a final OD600 nm=0.04-0.08. Cultures were grown in BGCM for 24 hrs at 30° C. at 180 rpm and centrifuged at 4,500 rpm for 30 minutes. Cells were resuspended in ⅓ of the original volume in BGCM medium with a final OD600 nm=15-20. Cells were induced with MeOH at regular time points, typically 3 times/day, never exceeding the 1% MeOH content. After 50 hours of induction the supernatant is harvested.

Purification of Nanobody Expressed in Pichia pastoris

[1783] Culture supernatant is filtered over a 0.22 μm filtration membrane Micro filtration (Hydrosart, Sartorius). Sample is concentrated using diafiltration on 10 kDa ultra filtration membrane (HydroSart, Sartorius) and concentrated to 0.5-1 L.

Nanobodies™ are purified using Protein A affinity chromatography (MabSelect Xtra, GE Healthcare) using PBS as running buffer and Glycine [100 mM pH=2.5] for elution. Samples are neutralized using 1.5 M Tris pH=8.8. Nanobodies™ are further processed in Anion Exchange Chromatography (Source 30Q, GE Healthcare). Samples are diluted 10-fold with 10 mM Piperazine pH=10.2 and adjusted to pH=10.2 with 1 M NaOH and a conductivity of <2 mS/cm with MilliQ water.
Nanobodies are processed in Size Exclusion chromatography (Superdex 75 μg, Hiload XK26/60, GE Healthcare) and LPS is removed via Anion Exchange Chromatography (Source 30Q, GE Healthcare) by passage through 5 ml column, which is sanitized with 1 M NaOH and equilibrated in Dulbecco PBS.

[1784] To determine the purity, protein samples were analyzed on a 15% SDS-PAGE gel as described in example 8. The gel is processed using the SilverQuest™ according to general procedures described by the manufacturer (Invitrogen). Alternatively, gel is processed using coomassie brilliant blue or in western blot as described in example 8 and 9. Results are given in FIG. 42.

Example 50: Characterization of Trivalent Bispecific Humanized Nanobodies™

[1785] TNF60 consists of 363 amino acids. The protein has a molecular weight of 38,441 Da. The pI is 8.71. The extinction coefficient at 280 nm is 1.736.

Mass Spectrophotometry

[1786] The mass of the protein was determined in ESI-MS according to standard procedures. The theoretical mass of TNF60 is 38,441 Da. The protein has 2 S—S bridges which should result in a mass of 38,435 Da in ESI-MS. The mass that was experimentally determined for TNF60 derived from 3 different batches ranges from 38,433 Da to 38,435 Da, differing maximally 0.005% with the theoretical mass.

N-Terminal Sequencing

[1787] N-terminal sequencing was performed by Edman degradation according to standard procedures. N-terminal sequencing showed that the protein sequence for the first 7 amino acids is as follows: EVQLVES (SEQ ID NO: 487). This is consistent with the theoretical protein sequence, which indicates proper N-terminal processing.

Analytical Sizing

[1788] Samples (100 μg) were analysed on the high resolution Superdex75 column, to characterize the different batches of Nanobody™. Size exclusion chromatography of the Nanobody™ typically yields a symmetrical peak, with a retention time of 11.5 min on Superdex75. The absorbance is typically recorded at 280, 254 and 214 nm. The 214 nm measurement permits higher detection sensitivity. Analytical sizing in PBS provides a symmetrical peak. No contaminants were observed. The retention time observed for 3 different batches is 11.5-11.55 min. A representative profile is shown in FIG. 43.

Example 51: Binding of TNF60 to Human TNFα in ELISA

[1789] The functionality of TNF60, i.e. binding to human TNFα was analyzed in ELISA as described in example 10. The results are summarized in FIG. 44 and clearly demonstrate a dose-dependent and saturable binding of 2 batches of TNF60 to human TNFα.

Example 52: Functionality in Cell-Based Assay

[1790] The potency to neutralize the cytotoxic activity of TNFα was analyzed in a cell-based assay as described in example 13. The results are summarized in Table 42 and in FIGS. 45 and 46.

[1791] The data show that TNF60 has potency in the range of Enbrel/Etanercept and a 10-fold better potency than Humira/Adalimumab and Remicade/Infliximab.

Example 53: Binding of TNF60 to Serum Albumin

[1792] Binding to human and rhesus serum albumin was analyzed in Biacore as described in example 12. KD, kon and koff values are represented in Table 43. TNF60 is compared to TNF24, which is the trivalent bispecific parent Nanobody™ with wild-type building blocks. Affinity of TNF60 for human and rhesus serum albumin is similar. Affinity is 2-fold lower as compared to the affinity observed for TNF24 which is the wild type analogue of TNF60. K.sub.on is identical for both molecules, but the k.sub.off is 2-fold higher for TNF60.

Example 54: Pharmacokinetic and Immunogenicity Analysis of Trivalent Bispecific Humanized Nanobodies in Mice

Animals

[1793] DBA1 or BALBc mice were warmed up under an infrared lamp and 200 μl Nanobody™ (100 μg per mouse) was injected intravenously in the tail. Blood samples were obtained at different time points by making a small incision in the tail and collecting the blood in a microtube. Typically, blood was sampled at t=15 min, 2 hrs, 4 hrs, 6 hrs, 1 day, 2 days, 3 days, 4 days, 7 days, 14 days. Serum was prepared according to standard procedures.

Determination of Nanobody™ Concentration in Mouse Serum

[1794] A microtiterplate (NUNC, Maxisorb) was coated with 2 μg/ml neutravidin overnight at 4° C. The plate was washed 5 times with PBS/0.05% Tween-20 and blocked for 2 hours at RT with PBS/1% casein. Biotinylated human TNFα (1/2000 in PBS/0.2% casein; 400 ng/ml) was applied to the wells and incubated for 1 hr at RT. The standard reference Nanobody™ was applied starting at a concentration of 5 μg/ml and using 5-fold dilutions in PBS containing 1% mouse plasma. The Nanobodies™ were allowed to bind for 2 hours at RT. The plate was washed 5 times and rabbit polyclonal anti-Nanobody (R23) was applied at a 2000-fold dilution for one hour at RT. After washing of the plate, binding was detected with goat-polyclonal-anti-rabbit-HRP (DAKO) at a 3000-fold dilution for one hour at RT, and stained with ABTS/H.sub.2O.sub.2. The OD405 nm was measured.

[1795] This first ELISA was used to determine the linear range of the standard reference. In a second ELISA, the standard reference was used at concentrations in this linear range and typically using 2-fold dilutions. In this second ELISA, serum test samples were diluted 100-fold and further 5-fold dilutions were made in 1% mouse plasma, to determine the dilution at which the serum samples provide a read-out in the linear range of the standard curve. In a third ELISA, serum samples are diluted at an appropriate concentration determined in the second ELISA and using 2-fold dilutions for accurate determination of the Nanobody™ concentration in the serum samples.

[1796] Experiments were performed to determine the pharmacokinetic profile of TNF60 in mice (n=3). A Cmax value of 103.84±31 μg/ml was reached 15 minutes after administration. The half-life (t1/2β) was determined to be 1.9 days, similar to the half-life of mouse serum albumin, indicating that TNF60 adopts the half-life of serum albumin. Data are presented in FIG. 47.

Determination of Anti-Nanobody Antibodies in Mice

[1797] Nanobody™ was coated at 5 μg/ml in PBS at 4° C. overnight. The plate was washed 5 times with PBS/0.05% Tween-20 and blocked for 2 hours at RT with PBS/1% casein. Serum samples were diluted 100-fold and applied to the wells for incubation during 1 hr at room temperature. Detection was performed using 1000-fold diluted polyclonal rabbit anti-mouse-HRP (DAKO, P0260) and using ABTS as substrate.

Serum samples were diluted 50-fold and analyzed for the presence of mouse anti-TNF60 antibodies. Lack of immunogenicity was demonstrated for TNF60. Data are presented in FIG. 48.

Example 55: Generation of Bivalent Long Half-Lived Humanized Nanobodies™

[1798] Description of Pichia pastoris Expression Vector

[1799] See example 48

Formatting Bivalent Nanobodies™

[1800] Two separate PCR reactions were set up to amplify the N-terminal and the C-terminal Nanobody™ subunit using the procedures as indicated in the WPA-0011. For the amplification of the N-terminal Nanobody™ PiForLong and Rev_30GiySer_L108 were used as primer combination; for the amplification of the C-terminal Nanobody™ For_GlySer and PiRevCyslhum were used or alternatively For_GlySer and PiRevCys2hum, introducing the restriction sites required for formatting and free cysteine residues required for C-terminal modifications.

[1801] PCR reaction of 1 μl plasmid DNA (50-100 ng), 1.5 μl forward primer (10 μM.fwdarw.300 nM), 1.5 μl reverse primer (10 μM.fwdarw.300 nM), 1 μl dNTPs (10 mM.fwdarw.0.2 mM), 5 μl buffer (10×.fwdarw.1×), 0.75 μl enzyme (3.5 U/μl.fwdarw.2.6 U/μl) and 39.25 μl H.sub.2O with a total volume of 50 μl was prepared. Primer sequences are given in Table 44. A PCR program was started with 2 minutes at 94° C. A cycle of 30 seconds at 94° C., 30 seconds at 50° C. and 1 minute at 72° C. was repeated 30 times and followed by 10 minutes at 72° C. Amplification was checked by separating 5 μl of the PCR reaction on a 2% agarose gel. The PCR product was purified using the QIAquick PCR Purification Kit according to the manufacturer's instructions. One column was used and eluted with 50 μl EB buffer. The N-terminal V.sub.HH fragment was prepared by incubating 50 μl DNA and 2 μl BamHI (10 U/μl) and 2 μl XhoI (10 U/μl) in the appropriate buffer recommended by the manufacturer at 37° C. for 1.5 hours. The C-terminal V.sub.HH fragment was prepared by incubating 50 μl DNA and 2 μl BamHI (10 U/pI) and 2 μl EcoRI (10 U/μl) in the appropriate buffer recommended by the manufacturer at 37° C. for 1 hour. The previous digestion reactions were separated on a 2% agarose gel. The V.sub.HH bands (350-450 bp) were cut out of the gel and the DNA was purified using the QIAquick Gel Extraction Kit according to the manufacturer's instructions. One column (with a maximum of 400 mg agarose gel per column) was used and the bound DNA was eluted with 50 μl EB buffer. DNA concentration was determined by measuring OD.sub.260 (1 OD unit=50 μg/ml). A ligation mixture with a final volume of 10 μl containing 100 ng vector pPICZαA, linearized with Xho1/EcoRI, 30 ng N-terminal VHH, 30 ng C-terminal V.sub.HH fragment, 1 μl ligation buffer and 1 μl ligase (3 U) was prepared and incubated for 1 hour at RT. Transformation of E. coli, TG1 was performed by using 2 μl of ligation mixture. Colonies are analysed using PCR as described in WPA-0010 but using the AOXIFor/AOXIRev primer combination. Sequence analysis is performed on positive clones. Plasmid preparation was performed using the Qiaprep spin Miniprep kit (Qiagen) according to the manufacturer's instructions and described above. Sequencing was performed at the VIB sequence facility, Antwerp, Belgium.

Transformation of P. pastoris

[1802] See example 48.

[1803] TNF30 was formatted to bivalent Nanobodies™. As spacer between the 2 building blocks a 30 AA GlySer linker was used. To allow for C-terminal site-specific modifications a free cysteine was introduced, either as the last AA of the Nanobody™ or with an extra spacer consisting of GlyGlyGlyCys (SEQ ID NO: 471).

Example 56: Expression and Purification of Bivalent Long Half-Lived Humanized Nanobodies™

[1804] Production in Pichia pastoris

[1805] See example 49

Purification of Bivalent Nanobodies

[1806] The culture medium was made cell-free via centrifugation and 0.22 μm filtration. The sterile medium was stored at 4° C. until further processed. Low molecular weight contaminants were reduced via ultra filtration on a 10 kDa ultra filtration (UF) membrane (HydroSart Sartocon Slice Cassette, Sartorius) as follows: four liter medium was concentrated to 0.5-1 lit, then diluted with 5 lit PBS and again concentrated to 0.5 lit. This action was carried out twice.

The retentate of the UF was filtered through nylon 47 mm membranes 0.45 μm (Alltech #2024).

[1807] In a next step bivalent Nanobody™ was captured from the concentrated medium via Protein A affinity purification (using MabSelectXtra™, GE Healthcare). The column [35×100 mm] was equilibrated in PBS and after sample application washed extensively with PBS. TNF56 was eluted with Glycine [100 mM, pH=2.5].

[1808] The eluted fractions of MabSelectXtra™ were neutralized with Tris [1.5 M, pH 8,8] and stored at 4° C. TNF56 was concentrated and purified via AEX (A=10 mM piperazin, pH 10.8 and B=1 M NaCl in 50 mM Tris, pH 7.5) using Source 30Q (GE Healthcare). To this end the Nanobody™ fractions were diluted with A buffer (10 mM piperazin, pH 10.8) to a conductivity of 5 mS/cm and the pH was adjusted to 10.8. The column [25×100 mm] was equilibrated in A buffer before loading the sample onto the column. TNF56 was eluted with a 5 Column Volume (CV) gradient. The pH of the collected fractions was adjusted to 7.8 using 1 M Tris pH=7.8.

Pegylation of Bivalent Nanobodies™ Expressed in Pichia pastoris

Reduction of C-Terminal Cysteines

[1809] Dithiotreitol (DTT, Aldrich Cat 15,046-0) was added to the neutralized fractions to reduce potential disulfide bridges that formed between the carboxy terminal cysteines of the Nanobodies™ (usually around 20%). A final concentration of 10 mM DTT and incubation overnight at 4° C. was found to be optimal. The reduction was evaluated by analytical size exclusion chromatography (SEC). Therefore 25p1 of the reduced Nanobody™ was added to 75 μl D-PBS and injected on a Sup75 10/300 GL column equilibrated in Dulbecco's PBS (D-PBS, Gibco™ REF 14190-094).

[1810] Non reduced Nanobody™ and DTT was removed by preparative SEC on a Hiload 26/60 Superdex75 prep grade column equilibrated in D-PBS.

[1811] The concentration of the reduced Nanobody™ was measured by measuring the Absorbance at 280 nm. A Uvikon 943 Double Beam UV/VIS Spectrophotometer (method: see SOP ABL-0038) was used. The absorption was measured in a wavelength scan 245-330 nm. Two Precision cells made of Quartz Suprasil® cells were used (Hellma type No.: 104-QS; light path: 10 mm). First the absorption of the blank was measured at 280 nm by placing two cells filled by 900 μl D-PBS. The sample was diluted (1/10) by adding 100 μl of the sample to the first cell. The absorption of the sample was measured at 280 nm. The concentration was calculated with following formula:

[00001]$\frac{{\mathrm{OD}}_{280}\mathrm{Sample}-{\mathrm{OD}}_{280}\mathrm{blank}}{\epsilon \times 1}\times 10$

For TNF55: ε=1.85.

For TNF56: ε=1.83.

PEGylation

[1812] To PEGylate Nanobody™ a 5× molar excess of freshly made 1 mM PEG40 solution was added to the reduced Nanobody™ solution. (MPEG2-MAL-40K of NEKTAR™

[1813] Transforming Therapeutics (2D3YOTO1) Mw=40,000 g/mol; MPEG2-MAL-60K of NEKTAR™ Transforming Therapeutics (2D3YOVO1) Mw=60,000 g/mol).

[1814] The Nanobody™-PEG mixture was incubated for 1 h at room temperature (RT) with gentle agitation and then transferred to 4° C. The PEGylation was evaluated via analytical SEC. Therefore 25 μl of the Nanobody™ was added to 75 μl D-PBS and injected on a Sup75HR 10/300 column equilibrated in D-PBS. Pegylated Nanobody™ eluted in the range of the exclusion volume of the column (>75 kDa).

[1815] The PEGylated and non PEGylated Nanobody™ were separated via cation exchange chromatography (CEX, using Source30S, GE Healthcare; A buffer=25 mM citric acid pH=4 and B=1 M NaCl in PBS). The sample was diluted to a conductivity of <5 mS/cm and the pH was adjusted to 4.0. The column [25×100 mm] was equilibrated and after sample application washed extensively with A-buffer. Pegylated Nanobody™ was eluted with a 3 CV gradient. The collected Nanobody™ was buffer exchanged to D-PBS by SEC on a Hiload 26/60 Superdex 75 prep grade column equilibrated in D-PBS.

[1816] Finally the Nanobody™ was made LPS-free via passage over an anion exchange column (Source30Q). The column (10×100 mm) was sanitized overnight in NaOH [1 M] and afterwards equilibrated in endotoxin free D-PBS.

Biotinylation

[1817] To biotinylate Nanobody™ a 5× Molar excess of biotin (EZ-Link® Maleimide-PO2-Biotin, Pierce #21901) from a 10 mM stock solution was added to the reduced Nanobody™ (see 5.5.1). The biotin-Nanobody™ mixture was incubated for 1 h at RT with gentle agitation and then stored at 4° C.

[1818] The purity of biotinylated Nanobody™ was controlled via analytical SEC. Therefore 25 μl of biotinylated Nanobody™ was added to 75 μl D-PBS and injected on a Sup75HR 10/300 column equilibrated in D-PBS. From the obtained chromatogram could be concluded that the Nanobody™-biotin needs no further purification: no dimerization of Nanobody™ via an oxidation of free sulfidrils could be detected. A buffer change to D-PBS was done by a passage over a desalting column Sephadex G25 fine (90 ml) column.

[1819] Finally the Nanobody™-biotin was made LPS-free by passage over an anion exchange column (Source30Q, GE Healthcare). The column (1×10 cm) was sanitized overnight in 1 M NaOH and then equilibrated in D-PBS.

[1820] To determine the purity, protein samples were analyzed on a 15% SDS-PAGE gel as described in example 8 and 49. Results are presented in FIGS. 49 and 50.

Example 57: Characterization of Bivalent Long Half-Lived Humanized Nanobodies™

Biochemical Characterization

[1821] TNF55 consists of 260 amino acids. The protein has a molecular weight of 27,106 Da. The pI is 8.67. The extinction coefficient at 280 nm is 1.850.

[1822] TNF56 consists of 264 amino acids. The protein has a molecular weight of 27,365 Da. The pI is 8.67. The extinction coefficient at 280 nm is 1.830.

Mass Spectrophotometry

[1823] The theoretical mass of TNF55 is 27,106 Da. The TNF55-Biotine protein has 2 S—S bridges and a biotine modification which should result in a mass of 27,627 Da in ESI-MS. The mass that was experimentally determined for TNF55-biotine is 27,627 Da.

[1824] The theoretical mass of TNF56 is 27,365 Da. The TNF55-Biotine protein has 2 S—S bridges and a biotine modification which should result in a mass of 27,886 Da in ESI-MS. The mass that was experimentally determined for TNF55-biotine is 27,886 Da.

N-Terminal Sequencing

[1825] N-terminal sequencing of TNF56-PEG40 showed that the protein sequence for the first 7 amino acids is as follows: EVQLVES (SEQ ID NO: 487). This is consistent with the theoretical protein sequence, which indicates proper N-terminal processing.

Analytical Sizing

[1826] Analytical sizing of TNF56-PEG40 in PBS provides a symmetrical peak. No contaminants were observed. The retention time observed is 8.5 ml on Superdex HR 75 and 10.32 ml on Superdex HR 200. A representative profile is shown in FIGS. 51 and 52.

Example 58: Functionality in Cell-Based Assay

[1827] The potency to neutralize the cytotoxic activity of TNFα was analyzed in a cell-based assay. Potency was examined at different concentrations of Nanobody™ as well as of the commercially available Enbrel, Humira and Remicade on a molar base. The higher the EC50 observed the lower the activity of the compound to neutralize TNFα.

[1828] The results are summarized in Table 45 and FIGS. 53 and 54.

[1829] The data show an increase in potency for the bivalent Nanobodies™ when compared to the monovalent Nanobody™ TNF1. Potency of TNF55 derivatives is similar to TNF56 derivatives, which is in the range of Enbrel and 10-fold better than Humira and Remicade.

Example 59: Pharmacokinetic and Immunogenicity Analysis of Bivalent Long Half-Lived Humanized Nanobodies in Mice

[1830] See example 54

[1831] Experiments were performed in order to examine the half-life of pegylated Nanobodies™ in mice. The half-life of bivalent TNF56-PEG40 was compared to the half-life of TNF56-PEG60. Both Nanobodies™ have comparable half-life of ˜2 days. The results are presented in FIG. 55.

[1832] In addition, the half-life of pegylated bivalent 3E-3E was explored. The half-life of 3E-3E-PEG20 was compared to the half-life of 3E-3E-PEG40 after intravenous administration of 100 μg of the Nanobodies™. 3E-3E-PEG20 has a half-life of 17 hrs, while 3E-3E-PEG40 has a half-life of 2.1 days, comparable to the half-life of 3E-3E-MSA21. The results are presented in FIG. 56.

[1833] Serum samples were diluted 100-fold and analyzed for the presence of mouse anti-TNF56-PEG40 or anti-TNF56-PEG60 antibodies. Lack of immunogenicity was demonstrated for both molecules. Data are presented in FIG. 57.

Example 60: Efficacy of Anti-TNF-α Nanobody TNF60 (TNF60) in Prevention of Chronic Polyarthritis

[1834] Transgenic mouse lines carrying and expressing a 3′-modified human tumour necrosis factor (hTNF-alpha, cachectin) transgene were used as a model to study the efficacy of TNF60 (TNF60) in preventing the development of arthritis (EMBO J. 10, 4025-4031). These mice have been shown to develop chronic polyarthritis with 100% incidence at four to seven weeks of age.

[1835] From the third week of age, litters of transgenic mice were divided into groups of eight animals. Before initializing the study, the average body weight was calculated for each group. From then on, during the whole study animal weights were recorded once a week for each group.

[1836] To test the efficacy of TNF60 in the prevention of chronic polyarthritis, intraperitoneal injections were given twice a week to each animal of a particular group according to the following scheme: [1837] Group 1 (negative control): phosphate buffered saline (PBS) (formulation buffer) [1838] Group 2 (Nanobody treatment): TNF60 at a final dose of 30 mg/kg [1839] Group 3 (Nanobody treatment): TNF60 at a final dose of 10 mg/kg [1840] Group 4 (Nanobody treatment): TNF60 at a final dose of 3 mg/kg [1841] Group 5 (1.sup.st positive control): Enbrel at a final dose of 30 mg/kg [1842] Group 6 (1.sup.st positive control): Enbrel at a final dose of 10 mg/kg [1843] Group 7 (2.sup.nd positive control): Remicade at a final dose of 30 mg/kg [1844] Group 8 (2.sup.nd positive control): Remicade at a final dose of 10 mg/kg [1845] Group 9 (2.sup.nd positive control): Remicade at a final dose of 3 mg/kg
For each group, dates of injection and injection volumes were noted.

[1846] Injections continued for seven weeks. During this period, clinical scores were recorded by observing macroscopic changes in joint morphology for each animal.

[1847] At 10 weeks of age, all mice were sacrificed and sera and joints were collected. Sera were stored at −70° C. and ankle joints were conserved in formalin.

[1848] For selected groups, ankle joints were embedded in paraffin and sectioned. Ankle joint sections were subsequently used for histopathological evaluation of disease progression.

[1849] Results are depicted in FIG. 58.

Example 61: Efficacy of Anti-TNF-α Nanobody TNF60 (TNF60) in Therapeutic Treatment of Chronic Polyarthritis

[1850] Transgenic mouse lines carrying and expressing a 3′-modified human tumour necrosis factor (hTNF-alpha, cachectin) transgene were used as a model to study the efficacy of TNF60 (TNF60) in therapeutic treatment of arthritis (EMBO J. 10, 4025-4031). These mice have been shown to develop chronic polyarthritis with 100% incidence at four to seven weeks of age.

[1851] From the sixth week of age, litters of transgenic mice were divided into groups of eight animals. Before initializing the study, the average body weight was calculated for each group. From then on, during the whole study animal weights were recorded once a week for each group.

[1852] To test the efficacy of TNF60 in the therapeutic treatment of chronic polyarthritis, intraperitoneal injections were given twice a week to each animal of a particular group according to the following scheme: [1853] Group 1 (negative control): phosphate buffered saline (PBS) (formulation buffer) [1854] Group 2 (Nanobody treatment): TNF60 at a final dose of 30 mg/kg [1855] Group 3 (Nanobody treatment): TNF60 at a final dose of 10 mg/kg [1856] Group 4 (1.sup.st positive control): Enbrel at a final dose of 30 mg/kg [1857] Group 5 (2.sup.nd positive control): Remicade at a final dose of 30 mg/kg
For each group, dates of injection and injection volumes were noted.

[1858] Injections continued for seven weeks. During this period, clinical scores were recorded by observing macroscopic changes in joint morphology for each animal.

[1859] At 13 weeks of age, all mice were sacrificed and sera and joints were collected. Sera were stored at −70° C. and ankle joints were conserved in formalin.

[1860] For selected groups, ankle joints were embedded in paraffin and sectioned. Ankle joint sections were subsequently used for histopathological evaluation of disease progression.

[1861] Results are depicted in FIG. 59.

Example 62: Effect of Formatting on Efficacy of an Anti-TNF-α Nanobody in Prevention of Chronic Polyarthritis

[1862] Transgenic mouse lines carrying and expressing a 3′-modified human tumour necrosis factor (hTNF-alpha, cachectin) transgene were used as a model to study the efficacy of an anti-TNF-α Nanobody formatted in different ways in the prevention of chronic polyarthritis (EMBO J. 10, 4025-4031). These mice have been shown to develop chronic polyarthritis with 100% incidence at four to seven weeks of age.

[1863] From the third week of age, litters of transgenic mice were divided into groups of eight animals. Before initializing the study, the average body weight was calculated for each group. From then on, during the whole study animal weights were recorded once a week for each group.

[1864] To study the efficacy of an anti-TNF-αc Nanobody in different formats for prevention of chronic polyarthritis, intraperitoneal injections were given twice a week to each animal of a particular group according to the following scheme: [1865] Group 1 (negative control): phosphate buffered saline (PBS) (formulation buffer) [1866] Group 2 (Nanobody format 1): TNF60 at a final dose of 10 mg/kg [1867] Group 3 (Nanobody format 1): TNF60 at a final dose of 2.5 mg/kg [1868] Group 4 (Nanobody format 1): TNF60 at a final dose of 1 mg/kg [1869] Group 5 (Nanobody format 2): TNF56-PEG40 at a final dose of 10 mg/kg [1870] Group 6 (Nanobody format 2): TNF56-PEG40 at a final dose of 1.8 mg/kg [1871] Group 7 (Nanobody format 2): TNF56-PEG40 at a final dose of 0.7 mg/kg [1872] Group 8 (Nanobody format 3): TNF56-biot at a final dose of 1.8 mg/kg [1873] Group 9 (Nanobody format 4): TNF30 at a final dose of 1 mg/kg [1874] Group 10 (Nanobody format 5): TNF1 at a final dose of 1 mg/kg [1875] Group 11 (1.sup.st positive control): Enbrel at a final dose of 10 mg/kg [1876] Group 12 (2.sup.nd positive control): Remicade at a final dose of 10 mg/kg

[1877] For each group, dates of injection and injection volumes were noted.

[1878] Injections continued for seven weeks. During this period, clinical scores were recorded by observing macroscopic changes in joint morphology for each animal.

[1879] At 10 weeks of age, all mice were sacrificed and sera and joints were collected. Sera were stored at −70° C. and ankle joints were conserved in formalin.

[1880] For selected groups, ankle joints were embedded in paraffin and sectioned. Ankle joint sections were subsequently used for histopathological evaluation of disease progression.

[1881] Results are depicted in FIG. 60.

Example 63: Pharmacokinetic Study of Anti-TNF-α Nanobodies TNF60 (TNF60) and TNF56-PEG40 in Rhesus Monkey

[1882] Captive-bred rhesus monkeys (Macaca mulatta) are used to determine the pharmacokinetic profile of TNF60 and TNF56-PEG40.

[1883] Sixteen animals are used in this study (eight males and eight females) and are divided into four groups (two males and two females per group). All animals weighed approximately 5 kg and are disease-free for at least six weeks prior to use. Sniff® Pri vegetarisch V3994 serves as food. Sixty g/kg b.w. are offered to each monkey. The residue is removed. At regular intervals (at least twice a year) the food is analyzed based on EPA/USA for contaminants by LUFA-ITL. Tap water is offered ad libitum. The animals in each treatment group are housed in a block of several adjacent cages within the monkey unit. The monkeys are kept singly in V.sub.2A steel cages with a size of 90 cm×82 cm×96 cm. The room temperature is maintained at 23° C.±3° C. (maximum range) and the relative humidity at 60%±20% (maximum range). Deviations from the maximum range caused for example during cleaning procedure are dealt with in SOPs. The rooms are lit and darkened for periods of 12 hours each.

[1884] Two groups are infused with TNF60 and two groups are infused with TNF56-PEG40. Intravenous infusions of TNF60 and TNF56-PEG40 (dissolved in PBS) into the vena cephalica of the right or the left arm using indwelling catheters and a TSE infusion pump (see below) are given at a fixed dose of 2 mg/kg.

[1885] Four single administrations are performed, separated by a wash-out period of at least fourteen days. After the last administration the follow-up period is at least eight weeks. Two out of the four groups are treated with TNF60 or TNF56-PEG40 in combination with methotrexate (MTX) (dissolved in PBS). Group 2 is treated with TNF60 and MTX; group 4 is treated with TNF56-PEG40 and MTX. MTX is dosed weekly intramuscularly at 0.2 mg/kg. On the administration days, MTX is given approximately 30 minutes prior to administration. Dosing starts at the first Nanobody administration and will continue throughout the eight week wash-out after the fourth dose. There are fourteen single MTX administrations, separated by a wash-out period of at least one week starting at the first test item administration.

Example 64: Synovium-Derived Fibroblast Studies

[1886] In this study the ability of the anti-TNF biologicals, ALX0071 and Etanercept, to attenuate TNFα-induced IL-6 production by RA-synovium derived fibroblasts was assessed.

Isolation of Synovial Fibroblasts

[1887] Synovial joint tissue from consenting RA patients was stored in DMEM-based medium with antibiotics at 4° C. for up to 96 hours after joint replacement surgery. Synovial cells were isolated from dissected synovium by collagenase digestion at 37° C. for 2 hours. The resultant cell suspension was then washed by a series of centrifugation and resuspension steps and the resultant cells then cultured at 37° C. in DMEM-based culture medium supplemented with 10% FCS (v/v). The resultant fibroblasts were used for the following experiment at either the second or third passage. Cells from four donors were used in individual experiments. Fibroblasts were seeded into 96-well flat bottom polystyrene plates at 1.5×10.sup.4 cells in a final volume of 250 μL of DMEM-based culture medium supplemented with 10% FCS (v/v) per well and cultured overnight.

Stimulation of Synovial Fibroblasts

[1888] Cells were then incubated for 72 hours in DMEM-based culture medium supplemented with TNFα at 50 ng per mL (3 nM (R&D Systems 210-TA/CF) alone or in the presence of increasing doses of ALX0071 (0.575 to 1920 ng per mL; 0.015 to 50 nM) or Etanercept (Wyeth Labs; 3.75 to 11250 ng per mL; 0.025 to 75 nM). The final volume in each well was 250 μL and each assessment was performed in triplicate. After 72 hours, the supernatant media was removed and stored at −40° C. prior to analysis by IL-6 ELISA (R&D Systems). The inhibition of TNFα-induced IL-6 production was determined and IC.sub.50 values were calculated for both ALX0071 and Etanercept.

Summary of Results

[1889] Both ALX10071 and Etanercept dose-dependently reduced TNF□-induced IL-6 production by RA synovium derived fibroblasts from all four donors. There was a similar potency between the two reagents under these assay conditions.

Example 65: Murine Air Pouch Studies

[1890] In this study the ability of the anti-TNF biologicals, ALX0071 and Etanercept, to attenuate TNFα-induced cell infiltration in to a murine air pouch was assessed.

Creation of Air Pouch

[1891] Air pouches were formed by the sub-cutaneous (s.c.) injection of 2.5 mL of sterile air in to the dorsal surface of anaesthetised male C57Bl/6/J mice (25-30 g, Harlan). The pouch was re-inflated by injecting 2.5 ml of sterile air 3 days later.

TNFα Stimulation

[1892] Six days after the initial creation of the air pouch, the animals were anaesthetised and the pouch injected with 1 ml of 0.5% CarboxyMethylCellulose (CMC) vehicle containing 0.1 μg recombinant human TNFα (R & D Systems, 210-TA-050/CF). In three other groups of animals, ALX0071 (0.0625, 0.125 and 0.25 mg/kg) was injected s.c. 19 hours prior to the injection of TNFα. A second three groups of animals were injected (s.c.) with Etanercept (Wyeth Labs, 0.125, 0.25 and 0.5 mg/kg) immediately prior to the injection of TNFα.

[1893] 24 hours following TNFα □injection, mice were culled with a rising concentration of CO.sub.2. Pouches were lavaged with 2 ml of ice cold endotoxin free sterile PBS containing 5 IU/ml heparin. Volumes were recorded and 0.5 ml aliquots were separated for counting of the total white blood cell (WBC) population on a Sysmex XT-Vet cell counter. The mean and standard error of the mean (SEM) total WBC counts for each group were calculated per ml of lavage fluid withdrawn. Statistical analysis was by ANOVA with Kruskal-Wallis post-test on untransformed data.

Summary of Results

[1894] Both ALX0071 and Etanercept attenuated the TNFα-induced WBC infiltration in to the air pouches (Table). While this attenuation reached statistical significance at both the 0.125 (P<0.01) and 0.25 mg/kg (P<0.05) ALX0071 dose groups, statistical significance was not observed with any Etanercept dose group.

TABLE-US-00032 TABLE 8 SEQ Name ID NO Sequence PMP1C2 (TNF1) 52 QVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLE WVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTA LYYCARSPSGFNRGQGTQVTVSS PMP1G11 53 QVQLQESGGGMVQPGGSLRLSCAASGFDFGVSWMYWVRQAPGKGLE WVSEINTNGLITKYPDSVKGRFTISRDNAKTTLYLQMNSLKPEDTA LYYCARSPSGSFRGQGTQVTVSS PMP1H6 54 EVQLVESGGGLVQPGGSLRLSCATSGFDFSVSWMYWVRQAPGKGLE WVSEINTNGLITKYVDSVKGRFTISRDNAKNTLYLQMDLIP EDTA LYYCARSPSGSFRGQGTQVTVSS PMP1G5 (TNF2) 55 QVQLVESGGGLVQAGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAP GKEREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLLMNSLK PEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSS PMP1H2 56 QVKLEESGGGLVQPGDSLRLSCAASGRTFSDYSGYTYTVGWFRQAP GKEREFVARIYWSSGNTYYADSVKGRFTISRDIAKNTVDLLMNNLE PEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSS PMP3G2 57 AVQLVESGGGLVQPGDSLRLSCAASGRTFSDYSGYTYTVGWFRQAP GKEREFVARIYWSSGNTYYADSVKGRFTISRDIAKNTVDLLMNNLE PEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSS PMP1D2 58 AVQLVDSGGGLVQAGGSLRLSCAASGRTFSAHSVYTMGWFRQAPGK EREFVARIYWSSANTYYADSVKGRFTISRDNAKNTVDLLMNCLKPE DTAVYYCAARDGIPTSRSVEAYNYWGQGTQVTVSS PMP3D10 59 QVQLVESGGGLVQAGGSLSLSCAASGRSFTGYYMGWFRQAPGKERQ LLASISWRGDNTYYKESVKGRFTISRDDAKNTIYLQMNSLKPEDTA VYYCAASILPLSDDPGWNTNWGQGTQVTVSS PMP5F10 (TNF3) 60 EVQLVESGGGLVQAGGSLSLSCSASGRSLSNYYMGWFRQAPGKERE LLGNISWRGYNIYYKDSVKGRFTISRDDAKNTIYLQMNRLKPEDTA VYYCAASILPLSDDPGWNTYWGQGTQVTVSS PMP6A8 (ALB2) 61 AVQLVESGGGLVQGGGSLRLACAASERIFDLNLMGWYRQGPGNERE LVATCITVG.DSTNYADSVKGRFTISMDYTKQTVYLHMNSLRPEDT GLYYCKIRRTWHSELWGQGTQVTVSS PMP6B4 62 EVQLVESGGGLVQEGGSLRLACAASERIWDINLLGWYRQGPGNERE LVATITVG.DSTSYADSVKGRFTISRDYDKNTLYLQMNSLRPEDTG LYYCKIRRTWHSELWGQGTQVTVSS PMP6A6 (ALB1) 63 AVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTA VYYCTIGGSLSRSSQGTQVTVSS PMP6C1 64 AVQLVDSGGGLVQPGGSLRLSCAASGFSFGSFGMSWVRQYPGKEPE WVSSINGRGDDTRYADSVKGRFSISRDNAKNTLYLQMNSLKPEDTA EYYCTIGRSVSRSRTQGTQVTVSS PMP6G8 65 AVQLVESGGGLVQPGGSLRLTCTASGFTFRSFGMSWVRQAPGKDQE WVSAISADSSTKNYADSVKGRFTISRDNAKKMLYLEMNSLKPEDTA VYYCVIGRGSPSSPGTQVTVSS PMP6A5 66 QVQLAESGGGLVQPGGSLRLTCTASGFTFGSFGMSWVRQAPGEGLE WVSAISADSSDKRYADSVKGRFTISRDNAKKMLYLEMNSLKSEDTA VYYCVIGRGSPASQGTQVTVSS PMP6G7 67 QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMYWVRVAPGKGLE RISRDISTGGGYSYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDT ALYYCAKDREAQVDTLDFDYRGQGTQVTVSS NC55TNF_S1C4 105 EVQLVESGGGLVQAGDSLRLSCAASQIIFGSHVAAWFRQAPGRERE FVAEIRPSGDFGPEGEFEHVTASLKGRFTIAKNSVDNTVYLQMNSL KPEDTAVYYCAAAPYRGGRDYRWEYEYEYWGQGTQVTV NC55TNF_S1C3 106 EVQLVESGGGLVQPGGSLRLSCKNAGSTSNAYATGWFRRAPGKERE FVAGIQWSGGDAFYRNSVKGRFRITRDPDNTVYLQMNDLKPEDTAI YYCAQKLSPYYNDFDSSNYEYWGQGTQVTV NC55TNF_S2C1 107 EVQLVESGGDLVQPGGSLRLSCAVSGQLFSTNDVGWYRRAPGKQRE LVATITDDGTTDYGDDVKGRFVISREGEMVYLEMNSLKPEDTAVYY CNINRLRSTWGIRYDVWGQGTQVTVSS NC55TNF_S2C5 108 EVQLVESGGGLVQPGGSLRLSCVVSGFTFSTTSMTWVRQAPGKFEE WVSFINSDGSSTTYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTA MYYCGRRGYGRDRSKGIQVTVAS NC55TNF_S3C7 109 EVQLVESGGGTVQAGDSLRLSCAASGRSFSSVAMGWFRQAPGKQRE FLAGVGYDGSSIRYAESVKGRFTIARGNRESTVFLQMENLKPEDTA VYFCTAEPIGAYEGLWTYWGQGTQVTVSS NC55TNF_S3C1 110 XXXXVESGGGLMQPGGSLKLSCAASGFMFSDSAMGWFRQAPGKERE FVATISWNGGSSSYADFVKGRFTISRDNAKNTVYLQMNGLTPQDTA IYYCAGSYSNGNPHRFSQYQYWGQGTQVTVSS NC55TNF_BMP1B2 111 EVQLVESGGGLVQAGGSLRLSCAASGRTFGTYAMGWFRQAPGKERE FVAAISWGGGSIVYAESAKGRFTISRDNAKXTMYLQMDSLKPEDTA VYYCAAANNIATLRQGSWGQGTQVTVSS NC55TNF_BMP1D2 112 EVQLVESGGELVQAGGSLKLSCTASGRNFVTYAMSWFRRAPGKERE FVASISWSGDTTYYSNSVKGRFTVSRDNGKNTAYLRMNSLKPEDTA DYYCAWQVIDPSWSGVNLDDYDYLGSGTQVTVSS NC55TNF_BMP1E2 113 EVQLVESGGRLVQPGGSLRLSCKNAGSTSNAYATGWFRRAPGKERE FVAGIQWSGGDAFYRNSVKGRFRITRDPDNTVYLQMNDLKPEDTAI YYCAQKLSPYYNDFDSSNYEYWGQGTQVTVSS NC55TNF_BMP1G2 114 EVQLVESGGGLVQPGGSLRLSCAASATISSIVMLGWYRQAPGKQRE WVASITIGSRTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV YFCNAVPPRDDYWGQGTQVTVSS NC55TNF_BMP2A2 115 EVQLVESGGGLVQAGGSLRLSCAASGQTSSSYDMGWFRQAPGEGRE FVARISGSDGSTYYSDRAKDRFTISRDNTKNMVYLQMDRLKPDDTA VYYCRVPRYENQWSSYDYWGQGTQVTVSS NC55TNF_BMP2C2 116 EVQLVESGGGLVQPGGSLRLSCAASGSTFSTYDMSWVRQAPGKGLE WVSGIDSGGGSPMYVDSVKGRFTVSRDNAKNTLYLQMNSLKPEDTA VYYCAKFSTGADGGSWYWSYGMDSWGKGTQVTVSS NC55TNF_BMP2F2 117 EVQLVESGGGLVQAGDSLRLSCEASERSSNRYNMAWFRQAPGKERE FLARVDVSGGNTLYGDSVKDRFTVSRINGKNAMYLQMNNLKPEDTA IYYCAAGGWGTTQYDYDYWGQGTQVTVSS NC55TNF_NC10 118 EVQLVESGGGLVQPGGSLRLSCVCVSSGCTFSAYSMTWVRQAPGKA EEWVSFINSDGSSTTYADSVNGRFKISRDNAKKTLYLQMNSLGPED TAMYYCQRRGYALDRGQGTQVTVSS NC55TNF_NC11 119 EVQLVESGGGLVQAGDSLTLSCASSGRGFYKNAMGWFRQPPGKERE FVASIKWNGNNTYYADSVRGRFTISRGNAKNTENTVSLQMNSLKPE DTADYYCAADSSHYSYVYSKAYEYDYWGQGTQVTVSS NC55TNF_NC1 120 EVQLVESGGGLVQPGGSLRLSCVFSGFAFSASSMAWVRQAPGKYEE WVSFINSDGSSTTYADSVQGRFTISRDNAKNTLYLQMNSLKSEDTA MYYCGRRGYGRDRSQGIQVTVSS NC55TNF_NC2 121 EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKERE FVAAISWSGTITNYADSVKGRFTISRDNGKNTVHLQMNSLKPEDTA VYHCAVVQPYSGGDYYTGVEEYDYWGXGTQVTVSS NC55TNF_NC3 122 EVQLVESGGGLVQPGGSLRLSCWSGFTFSATSMTWVRQAPGKAEE WVSFINSDGSSTTYADSVKGRFTISRDNAKNTLYLQMDDLQSEDTA MYYCGRRGYGRDRSRGIQVTVSS NC55TNF_NC5 123 EVQLVESGGGLVQAGGSLRLSCAASGGAFSNYDVGWFRQAPGEGRE IVARISGSGDSTYSSNRAKGRFTISRDNAKNTVYLQMNSLKREDTA VYYCRAARYNGTWSSNDYWGQGTQVTVSS NC55TNF_NC6 124 EVQLVESGGGLVQPGGSLRLSCECVSSGCTFSAYSMTWVRQAPGKA EEFVSFINSDGSSTTYANSVNGRFKISRDNAKKTLYLQMNSLGPED TAMYYCQRRGYALDRGQGTQVTVSS NC55TNF_NC7 125 QVQLVESGGGLVQAGGSLRLSCTASGQTSSTADMGWFRQPPGKGRE FVARISGIDGTTYYDEPVKGRFTISRDKAQNTVYLQMDSLKPEDTA VYYCRSPRYADQWSAYDYWGQGTQVTVSS NC55TNF_NC8 126 EVQLVESGGGLVQPGGSLRLSCVVSGFTFSTTSMTWVRQAPGKFEE WVSFINSDGSSTTYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTA MYYCGRRGYGRDRSKGIQVTVSS NC55TNF_S2C2 127 EVQLVESGGGLVQPGGSLRLSCVASASGVKVNDMGWYRQAPGKERE LVATITDDGRTNYEDFAKGRFTISRDNAKNTVYLQMNSLLPEDTAV YYCNARTYWAHLPTYWGQGTQVTVSS NC55TNF_S1C6 128 EVQLVESGGGLVQAGGSLRLSCAASGRSFGSVAMGWFRQAPGKERE FVAAIGYDGNSIRYGDSVKGRFTISRDNIKNTMYLEMENLNADDTA RYLCAAEPLARYEGLWTYWGQGTQVTVSS NC55TNF_S3C2 129 EVQLVESGGGLVQAGASLRLSCTTSTRTNDRFNMAWFHQAPGKDRE FVSRIDVAGYNTAYGDFVKGRFTVSRDSAENTWLQMNSLRPEDTG VYYCAAGGWGISQSDYDLWGQGTQVTVSS

TABLE-US-00033 TABLE 9 nanobody Class Estimated koff (1/s) PMP5 F10 III 2.63E−04 PMP1 G5 II 3.59E−04 PMP1 C2 I 4.39E−04 PMP1 G11 I 1.15E−03 PMP1 H6 I 2.14E−03 PMP1 H2 II 3.65E−03 PMP3 G2 II 1.09E−02

TABLE-US-00034 TABLE 10 Germline #AA differences/ % AA Nanobody sequence total #AA identity ALB1 DP51/DP53 13/87 85.1 ALB2 DP54 26/87 70.2 TNF1 DP51/DP53  6/87 93.2 TNF2 DP54 16/87 81.7 TNF3 DP29 18/87 79.4

TABLE-US-00035 TABLE 11 Nanobody Induction time Yield (mg/L) ALB1 short/37° C. 18 ALB2 short/37° C. 4 TNF1 short/37° C. 8.3 TNF2 short/37° C. 5 TNF3 short/37° C. 0.8

TABLE-US-00036 TABLE 12 50% binding (ng/ml) ID Human TNFα Rhesus TNFα TNF1 12 12 TNF2 20 >3000 TNF3 18 16

TABLE-US-00037 TABLE 13 50% inhibition (ng/ml) ID Human TNFα Rhesus TNFα TNF1 530 220 TNF2 3500 >5000 TNF3 100 100

TABLE-US-00038 TABLE 14 Human TNFα Kd (1/s) TNF1 1.05E−03 TNF2 1.33E−03 TNF3 3.02E−04

TABLE-US-00039 TABLE 15 Human Rhesus Mouse albumin albumin albumin ALB1 KD (nM) 0.57 0.52 6.5 ka (1/Ms) 1.11E+06 1.05E+06 1.11E+06 kd (1/s) 6.30E−04 5.46E−04 7.25E−03 ALB2 KD (nM) 0.092 0.036 15.7 ka (1/Ms) 8.15E+05 1.94E+06 1.95E+05 kd (1/s) 7.52E−05 7.12E−05 3.07E−03

TABLE-US-00040 TABLE 16 assay: L929s + Act D (5000 c/w) TNF: human TNFa @ 0.5 ng/ml EC.sub.50 in nM relative potency Nanobody mean stdev # mean stdev TNF1 1C2 0.707 0.265 14 0.015 0.007 TNF2 1G5 1.412 0.622 14 0.007 0.002 TNF3 5F10 0.224 0.133 14 0.048 0.019 Enbrel 0.009 0.005 45 1.002 0.011 Humira 0.079 0.043 39 0.097 0.069 Remicade 0.083 0.037 45 0.103 0.058 assay: L929s + Act D (5000 c/w) TNF: rhesus TNFa @ 0.5 ng/ml EC.sub.50 in nM relative potency Nanobody mean stdev # mean stdev TNF1 1C2 0.693 0.305 9 0.015 0.009 TNF2 1G5 >50 9 TNF3 5F10 0.602 0.283 9 0.017 0.010 Enbrel 0.009 0.003 7 1 0.000 Humira 0.071 0.025 8 0.103 0.059 Remicade >6.7 7

TABLE-US-00041 TABLE 17 37° 50° 60° 70° 80° 90° % Untreated RT C. C. C. C. C. C. TNF1 100  98  98  98  98 95 92 90 TNF2 100  99 100  99  97 96 63 50 TNF3 100  96  97  98  96 94 75 70 ALB1 100 101 102 101 100 64 94 90 ALB2 100 100 102 100 100 28 8 17

TABLE-US-00042 TABLE 18 EC.sub.50 in relative nanobody temp in ° C. nM # potency TNF1 control 0.916 1 0.013 92#2302nr1.TNF1 RT 0.873 1 0.014 37 0.901 1 0.013 50 0.908 1 0.013 60 0.891 1 0.013 70 1.218 1 0.010 80 2.655 1 0.004 90 5.797 1 0.002 TNF2 control 2.500 1 0.005 92#2302nr2.TNF2 RT 2.165 1 0.005 37 2.212 1 0.005 50 2.241 1 0.005 60 1.782 1 0.007 70 2.487 1 0.005 80 2.818 1 0.004 90 6.135 1 0.002 TNF3 control 0.278 1 0.043 92#2302nr3.TNF3 RT 0.289 1 0.041 37 0.295 1 0.040 50 0.290 1 0.041 60 0.281 1 0.042 70 0.293 1 0.040 80 0.576 1 0.021 90 0.861 1 0.014

TABLE-US-00043 TABLE 19 SEQ Name ID NO Sequence GS9 68 GGGGSGGGS GS30 69 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS TNF1-GS9- 70 QVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLE TNF1(TNF4) WVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTA LYYCARSPSGFNRGQGTQVTVSSGGGGSGGGSQVQLVESGGGLVQP GGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKY PDSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCARSPSGFNRG QGTQVTVSS TNF2-GS9- 71 QVQLVESGGGLVQAGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAP TNF2(TNF5) GKEREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLLMNSLK PEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSSGGGGSGGGS QVQLVESGGGLVQAGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAP GKEREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLLMNSLK PEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSS TNF3-GS9- 72 EVQLVESGGGLVQAGGSLSLSCSASGRSLSNYYMGWFRQAPGKERE TNF3(TNF6) LLGNISWRGYNIYYKDSVKGRFTISRDDAKNTIYLQMNRLKPEDTA VYYCAASILPLSDDPGWNTYWGQGTQVTVSSGGGGSGGGSEVQLVE SGGGLVQAGGSLSLSCSASGRSLSNYYMGWFRQAPGKERELLGNIS WRGYNIYYKDSVKGRFTISRDDAKNTIYLQMNRLKPEDTAVYYCAA SILPLSDDPGWNTYWGQGTQVTVSS TNF1-GS30- 73 QVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLE TNF1(TNF7) WVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTA LYYCARSPSGFNRGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQ APGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNS LKPEDTALYYCARSPSGFNRGQGTQVTVSS TNF2-GS30- 74 QVQLVESGGGLVQAGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAP TNF2(TNF8) GKEREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLLMNSLK PEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSSGGGGSGGGG SGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVQAGGSLRLSCAAS GRTFSEPSGYTYTIGWFRQAPGKEREFVARIYWSSGLTYYADSVKG RFTISRDIAKNTVDLLMNSLKPEDTAVYYCAARDGIPTSRSVGSYN YWGQGTQVTVSS TNF3-GS30- 75 EVQLVESGGGLVQAGGSLSLSCSASGRSLSNYYMGWFRQAPGKERE TNF3(TNF9) LLGNISWRGYNIYYKDSVKGRFTISRDDAKNTIYLQMNRLKPEDTA VYYCAASILPLSDDPGWNTYWGQGTQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLSLSCSASGRSLSN YYMGWFRQAPGKERELLGNISWRGYNIYYKDSVKGRFTISRDDAKN TIYLQMNRLKPEDTAVYYCAASILPLSDDPGWNTYWGQGTQVTVSS TNF30-30GS- 419 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLE TNF30-C  WVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTA (TNF55) VYYCARSPSGFNRGQGTLVTVSSggggsggggsggggsggggsggg gsggggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQ APGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNS LRPEDTAVYYCARSPSGFNRGQGTLVTVSC TNF30-30GS- 420 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLE TNF30-gggC WVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTA (TNF56) VYYCARSPSGFNRGQGTLVTVSSggggsggggsggggsggggsggg gsggggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQ APGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNS LRPEDTAVYYCARSPSGFNRGQGTLVTVSSgggC

TABLE-US-00044 TABLE 20 ID Format Linker TNF4 TNF1-TNF1  9 AA GlySer TNF5 TNF2-TNF2  9 AA GlySer TNF6 TNF3-TNF3  9 AA GlySer TNF7 TNF1-TNF1 30 AA GlySer TNF8 TNF2-TNF2 30 AA GlySer TNF9 TNF3-TNF3 30 AA GlySer

TABLE-US-00045 TABLE 21 Nanobody ™ Induction time Yield (mg/L) TNF4 ON/28° C. 3.2 TNF5 short/37° C. 5.5 TNF6 short/37° C. 1.19 TNF7 ON/28° C. 2.7 TNF8 short/37° C. 6.6 TNF9 ON/28° C. 1.3

TABLE-US-00046 TABLE 22 50% inhibition (ng/ml) ID Human TNFα TNF4 13 TNF5 6.3 TNF6 30 TNF7 16 TNF8 23 TNF9 18

TABLE-US-00047 TABLE 23 assay: L929s + Act D (5000 c/w) TNF: human TNFa @ 0.5 ng/ml EC.sub.50 in nM relative potency Nanobody mean stdev # mean stdev TNF4 0.236 0.049 4 0.033 0.012 TNF5 0.020 0.010 9 0.566 0.275 TNF6 0.078 0.047 8 0.179 0.168 TNF7 0.013 0.005 8 0.673 0.211 TNF8 0.007 0.002 2 1.240 0.137 TNF9 0.012 0.005 6 0.729 0.242 Enbrel 0.009 0.005 45 1.002 0.011 Humira 0.079 0.043 39 0.097 0.069 Remicade 0.083 0.037 45 0.103 0.058 assay: L929s + Act D (5000 c/w) TNF: rhesus TNFa @ 0.5 ng/ml EC.sub.50 in nM relative potency Nanobody mean stdev # mean stdev TNF4 0.141 0.025 4 0.065 0.015 TNF5 35.000 16.000 5 0.000 0.000 TNF6 0.398 0.074 6 0.024 0.003 TNF7 0.011 0.005 4 0.860 0.142 TNF8 1.026 0.444 2 0.010 0.001 TNF9 0.038 0.012 4 0.249 0.032 Enbrel 0.009 0.003 7 1.000 0.000 Humira 0.071 0.025 8 0.103 0.059 Remicade >6.7 7

TABLE-US-00048 TABLE 24 37° 50° 60° 70° 80° 90° % untreated RT C. C. C. C. C. C. TNF4 100 99 99 99 98 55 34 17 TNF5 100 99 101 99 98 92 26 22 TNF6 100 103 104 103 105 99 7 7 TNF7 100 100 100 98 96 66 33 40 TNF8 100 99 100 99 100 89 11 8 TNF9 100 101 101 101 101 99 17 18

TABLE-US-00049 TABLE 25 SEQ ID Name NO Sequence TNF13 (TNF1 HUM1) 76 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLE WVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTA VYYCARSPSGFNRGQGTQVTVSS TNF14 (TNF1 HUM2) 77 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLE WVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTA LYYCARSPSGFNRGQGTLVTVSS TNF15 (TNF2 HUM1) 78 EVQLVESGGGLVQPGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAP GKGREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLQMNSLK PEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSS TNF16 (TNF2 HUM2) 79 EVQLVESGGGLVQPGGSLRLSCAASGFTFSEPSGYTYTIGWFRQAP GKGREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLQMNSLK PEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSS TNF17 (TNF2 HUM3) 80 EVQLVESGGGLVQPGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAP GKGREFVARIYWSSGLTYYADSVKGRFTISRDNAKNTVDLQMNSLK PEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSS TNF18 (TNF2 HUM4) 81 EVQLVESGGGLVQPGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAP GKGREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLQMNSLR PEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSS TNF19 (TNF2 HUM5) 82 EVQLVESGGGLVQPGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAP GKGREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLQMNSLK PEDTAVYYCAARDGIPTSRSVGSYNYWGQGTLVTVSS TNF20 (TNF3 HUM1) 83 EVQLVESGGGLVQPGGSLRLSCAASGRSLSNYYMGWFRQAPGKGRE LLGNISWRGYNIYYKDSVKGRFTISRDDSKNTIYLQMNSLKPEDTA VYYCAASILPLSDDPGWNTYWGQGTQVTVSS TNF21 (TNF3 HUM2) 84 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMGWFRQAPGKGRE LLGNISWRGYNIYYKDSVKGRFTISRDDSKNTIYLQMNSLKPEDTA VYYCAASILPLSDDPGWNTYWGQGTQVTVSS TNF22 (TNF3 HUM3) 85 EVQLVESGGGLVQPGGSLRLSCAASGRSLSNYYMGWFRQAPGKGRE LLGNISWRGYNIYYKDSVKGRFTISRDDSKNTIYLQMNSLKTEDTA VYYCAASILPLSDDPGWNTYWGQGTQVTVSS TNF23 (TNF3 HUM4) 86 EVQLVESGGGLVQPGGSLRLSCAASGRSLSNYYMGWFRQAPGKGRE LLGNISWRGYNIYYKDSVKGRFTISRDDSKNTIYLQMNSLKPEDTA VYYCAASILPLSDDPGWNTYWGQGTLVTVSS ALB3 (ALB1 HUM1) 87 EVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTA VYYCTIGGSLSRSSQGTQVTVSS ALB4 (ALB1 HUM2) 88 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMSWVRQAPGKEPE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTA VYYCTIGGSLSRSSQGTQVTVSS ALB5 (ALB1 HUM3) 89 EVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTA VYYCTIGGSLSRSSQGTQVTVSS

TABLE-US-00050 TABLE 26 Nanobody Induction time Yield (mg/L) TNF13 ON/28° C. 8.8 TNF14 ON/28° C. 7 TNF15 Short/37° C. 7.6 TNF16 Short/37° C. 8.7 TNF17 Short/37° C. 7.2 TNF18 Short/37° C. 4.8 TNF19 Short/37° C. 8 TNF20 ON/28° C. 3.5 TNF21 ON/28° C. 7.5 TNF22 ON/28° C. 6 TNF23 ON/28° C. 2.8 ALB3 ON/28° C. 11.8 ALB4 ON/28° C. 9 ALB5 ON/28° C. 11.7

TABLE-US-00051 TABLE 27 assay: L929s + Act D (5000 c/w) TNF: human TNFa @ 0.5 ng/ml EC.sub.50 in nM relative potency Nanobody mean stdev # mean stdev TNF1 0.707 0.265 14 0.015 0.007 TNF13 0.988 0.014 3 0.014 0.003 TNF14 0.981 0.007 3 0.014 0.003 TNF2 1.412 0.622 14 0.007 0.002 TNF15 1.669 1.253 4 0.002 0.000 TNF16 1.898 0.192 4 0.005 0.001 TNF17 3.023 0.562 4 0.001 0.001 TNF18 1.508 0.481 4 0.004 0.001 TNF19 2.191 0.941 4 0.001 0.001 TNF3 0.224 0.133 14 0.048 0.019 TNF20 0.380 0.080 3 0.035 0.005 TNF21 0.889 0.019 3 0.015 0.003 TNF22 0.303 0.005 3 0.044 0.011 TNF23 0.3 0.011 3 0.044 0.011 Enbrel 0.009 0.005 45 1.002 0.011 Humira 0.079 0.043 39 0.097 0.069 Remicade 0.083 0.037 45 0.103 0.058

TABLE-US-00052 TABLE 28 % Untreated RT 37° C. 50° C. 60° C. 70° C. 80° C. 90° C. TNF13 100 104 99 98 99 84 93 93 TNF14 100 98 101 95 99 96 99 90 TNF15 100 100 91 99 95 90 59 46 TNF16 100 97 102 101 94 101 58 48 TNF17 100 102 98 100 90 90 69 59 TNF18 100 100 101 97 91 93 63 50 TNF19 100 102 111 98 92 91 60 49 TNF20 100 94 93 93 93 92 85 67 TNF21 100 98 99 101 98 96 36 40 TNF22 100 102 101 105 99 93 25 31 TNF23 100 98 97 99 97 98 87 55 ALB3 100 100 99 98 25 18 60 62 ALB4 100 100 100 100 99 29 61 55 ALB5 100 100 100 99 94 32 61 48

TABLE-US-00053 TABLE 29 SEQ ID Name NO Sequence TNF1-9GS-ALB1- 90 QVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLE 9GS-TNF1 (TNF24) WVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTA LYYCARSPSGFNRGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQP GNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSS QGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFT FSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDN AKNTLYLQMNSLKPEDTALYYCARSPSGFNRGQGTQVTVSS TNF2-9GS-TNF2- 91 QVQLVESGGGLVQAGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAP 9GS-ALB1 (TNF25) GKEREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLLMNSLK PEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSSGGGGSGGGS EVQLVESGGGLVQAGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAP GKEREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLLMNSLK PEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTA VYYCTIGGSLSRSSQGTQVTVSS TNF3-9GS-ALB1- 92 EVQLVESGGGLVQAGGSLSLSCSASGRSLSNYYMGWFRQAPGKERE 9GS-TNF3 (TNF26) LLGNISWRGYNIYYKDSVKGRFTISRDDAKNTIYLQMNRLKPEDTA VYYCAASILPLSDDPGWNTYWGQGTQVTVSSGGGGSGGGSEVQLVE SGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSIS GSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTI GGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLSL SCSASGRSLSNYYMGWFRQAPGKERELLGNISWRGYNIYYKDSVKG RFTISRDDAKNTIYLQMNRLKPEDTAVYYCAASILPLSDDPGWNTY WGQGTQVTVSS TNF1-30GS-TNF1- 93 QVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLE 9GS-ALB1 (TNF27) WVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTA LYYCARSPSGFNRGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQ APGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNS LKPEDTALYYCARSPSGFNRGQGTQVTVSSGGGGSGGGSEVQLVES GGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISG SGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIG GSLSRSSQGTQVTVSS TNF3-30GS-TNF3- 94 EVQLVESGGGLVQAGGSLSLSCSASGRSLSNYYMGWFRQAPGKERE 9GS-ALB1 (TNF28) LLGNISWRGYNIYYKDSVKGRFTISRDDAKNTIYLQMNRLKPEDTA VYYCAASILPLSDDPGWNTYWGQGTQVTVSSGGGGSGGGGSGGGGS GGGGSGGGSSGGGGSEVQWESGGGLVQAGGSLSLSCSASGRSLSN YYMGWFRQAPGKERELLGNISWRGYNIYYKDSVKGRFTISRDDAKN TIYLQMNRLKPEDTAVYYCAASILPLSDDPGWNTYWGQGTQVTVSS GGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWV RQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQM NSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS TNF30-9GS-ALB8- 417 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLE 9GS-TNF30 (TNF60) WVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTA VYYCARSPSGFNRGQGTLVTVSSggggsgggsEVQLVESGGGLVQP GNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSS QGTLVTVSSggggsgggsEVQLVESGGGLVQPGGSLRLSCAASGFT FSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDN AKNTLYLQMNSLRPEDTAVYYCARSPSGFNRGQGTLVTVSS TNF33-9GS-ALB8- 418 EVQLVESGGGLVQPGGSLRLSCAASGRSLSNYYMGWFRQAPGKGRE 9GS-TNF33 (TNF62) LLGNISWRGYNIYYKDSVKGRFTISRDDSKNTIYLQMNSLRPEDTA VYYCAASILPLSDDPGWNTYWGQGTLVTVSSggggsgggsEVQLVE SGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS GSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI GGSLSRSSQGTLVTVSSggggsgggsEVQLVESGGGLVQPGGSLRL SCAASGRSLSNYYMGWFRQAPGKGRELLGNISWRGYNIYYKDSVKG RFTISRDDSKNTIYLQMNSLRPEDTAVYYCAASILPLSDDPGWNTY WGQGTLVTVSS

TABLE-US-00054 TABLE 30 ID Format TNF24 TNF1-9GS-ALB1-9GS-TNF1 TNF25 TNF2-9GS-TNF2-9GS-ALB1 TNF26 TNF3-9GS-ALB1-9GS-TNF3 TNF27 TNF1-30GS-TNF1-9GS-ALB1 TNF28 TNF3-30GS-TNF3-9GS-ALB1

TABLE-US-00055 TABLE 31 Nanobody Induction time Yield (mg/L) TNF24 ON/28° C. 1.7 TNF25 short/37° C. 0.445 TNF26 short/37° C. 0.167 TNF27 ON/28° C. 2.2 TNF28 short/37° C. 1

TABLE-US-00056 TABLE 32 assay: L929s + Act D (5000 c/w) TNF: human TNFa @ 0.5 ng/ml EC.sub.50 in nM relative potency Nanobody mean stdev # mean stdev TNF24 0.011 0.003 11 0.878 0.248 TNF25 0.018 0.008 14 0.603 0.243 TNF26 0.020 0.009 14 0.583 0.210 TNF27 0.012 0.003 3 0.810 0.037 TNF28 0.021 0.008 6 0.548 0.360 Enbrel 0.009 0.005 45 1.002 0.011 Humira 0.079 0.043 39 0.097 0.069 Remicade 0.083 0.037 45 0.103 0.058

TABLE-US-00057 TABLE 33 Human albumin KD (nM) ka (1/Ms) kd (1/s) 6A6 (ALB1) 0.57 1.11E+6  6.30E−4  1C2-GS-6A6-GS-1C2 (TNF24) 11 2.26E+05 2.48E−03 1G5-GS-1G5-GS-6A6 (TNF25) 7.2 2.91E+05 2.10E−03 5F10-GS-6A6-GS-5F10 (TNF26) 7.3 2.81E+05 2.05E−03 1C2-GS6-1C2-GS-6A6 (TNF27) 8.9 3.19E+05 2.84E−03 5F10-GS6-5F10-GS-6A6 (TNF28) 14 1.55E+05 2.13E−03

TABLE-US-00058 TABLE 34 37° 50° 60° 70° 80° 90° % untreated RT C. C. C. C. C. C. TNF24 100 100 99 98 5 3 8 18 TNF25 100 nd 103 102 95 5 4 6 TNF26 100 109 115 112 107 10 8 10 TNF27 100 102 103 102 22 9 26 34 TNF28 100  97 99 99 66 3 6 10

TABLE-US-00059 TABLE 35 SEQ ID Name NO Sequence TNF29(TNF1 HUM1) 95 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLE WVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTA VYYCARSPSGFNRGQGTLVTVSS TNF30(TNF1 HUM2) 96 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLE WVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTA VYYCARSPSGFNRGQGTLVTVSS TNF31(TNF2 HUM1) 97 EVQLVESGGGLVQPGGSLRLSCAASGFTFSEPSGYTYTIGWFRQAP GKGREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLQMNSLR PEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSS TNF32(TNF2 HUM2) 98 EVQLVESGGGLVQPGGSLRLSCAASGFTFSEPSGYTYTIGWFRQAP GKGREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLQMNSLR PEDTAVYYCAARDGIPTSRSVGSYNYWGQGTLVTVSS TNF33(TNF3 HUM1) 99 EVQLVESGGGLVQPGGSLRLSCAASGRSLSNYYMGWFRQAPGKGRE LLGNISWRGYNIYYKDSVKGRFTISRDDSKNTIYLQMNSLRPEDTA VYYCAASILPLSDDPGWNTYWGQGTLVTVSS ALB6(ALB1 HUM1) 100 EVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSRSSQGTLVTVSS ALB7(ALB1 HUM2) 101 EVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSRSSQGTLVTVSS ALB8 (ALB1 HUM3) 102 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSRSSQGTLVTVSS ALB9(ALB1 HUM4) 103 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTA VYYCTIGGSLSRSSQGTLVTVSS ALB10(ALB1 HUM5) 104 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTA VYYCTIGGSLSRSGQGTLVTVSS

TABLE-US-00060 TABLE 36 Nanobody ™ Induction time yield TNF29 ON/28° C. 2.1 mg/L TNF30 ON/28° C. 2.7 mg/L TNF31 ON/28° C. 2 mg/L TNF32 ON/28° C. 1.5 mg/L TNF33 ON/28° C. 0.5 mg/L

TABLE-US-00061 TABLE 37 assay: L929s + Act D (5000 c/w) TNF: human TNFa @ 0.5 ng/ml EC.sub.50 in nM relative potency V.sub.HH mean stdev # mean stdev TNF1 0.707 0.265 14 0.015 0.007 TNF13 0.988 0.014 3 0.014 0.003 TNF14 0.981 0.007 3 0.014 0.003 TNF29 1.336 1 0.013 TNF30 0.985 1 0.017 TNF2 1.412 0.622 14 0.007 0.002 TNF15 5.896 1.253 4 0.002 0.000 TNF16 2.422 0.192 4 0.005 0.001 TNF17 7.555 0.562 4 0.001 0.001 TNF18 3.134 0.481 4 0.004 0.001 TNF19 7.372 0.941 4 0.001 0.001 TNF31 2.195 1 0.008 TNF32 2.506 1 0.007 TNF3 0.224 0.133 14 0.048 0.019 TNF20 0.380 0.080 3 0.035 0.005 TNF21 0.889 0.019 3 0.015 0.003 TNF22 0.303 0.005 3 0.004 0.011 TNF23 0.3 0.011 3 0.04 0.011 TNF33 0.3 1 0.057 Enbrel 0.009 0.005 45 1.002 0.011 Humira 0.079 0.043 39 0.097 0.069 Remicade 0.083 0.037 45 0.103 0.058

TABLE-US-00062 TABLE 38 37° 50° 60° 70° 80° 90° % untreated RT C. C. C. C. C. C. TNF29 100 100 100 100 100 96 91 89 TNF30 100 100 100  99 100 96 92 89 TNF31 100 100 100  98  91 84 56 43 TNF32 100  99  98  97  87 78 45 39 TNF33 100  98  97  97  94 91 79 49

TABLE-US-00063 TABLE 39 assay: alphaKYM (10000 c/w) TNF: human TNFa @ 1 ng/ml Nanobody EC.sub.50 in nM TNF1 2.466 TNF2 4.236 TNF3 0.655 TNF4 0.069 TNF5 0.008 TNF6 0.121 TNF7 0.009 TNF8 0.013 TNF9 0.020 Enbrel 0.040 Humira 0.103 Remicade 0.100 Results from WO 04/41862 Nanobody SEQ ID No EC.sub.50 in nM 1A 1 100 3E 4 12 3G 5 20 Remicade 0.080

TABLE-US-00064 TABLE 40 M13_rev SEQ ID GGATAACAATTTCACACAGG NO: 421 Rev_9GlySer_ SEQ ID TCAGTAACCTGGATCCGCCACCGCTGCCT L108 NO: 422 CCACCGCCTGAGGAGACGGTGACCAG For_GS/Short SEQ ID AGGTTACTGAGGATCCGAGGTGCAGCTGG NO: 423 TGGAGTCTGG Rev_15BspEI_ SEQ ID TCAGTAACCTTCCGGAACCGCCACCGCCT L108 NO: 424 GAGGAGACGGTGACAAG For_BspEI SEQ ID AGGTTACTGATCCGGAGGCGGTAGCGAGG NO: 425 TGCAGCTGGTGGAGTCTGG M13_for SEQ ID CACGACGTTGTAAAACGAC NO: 426

TABLE-US-00065 TABLE 41 Sequence Reverse primer PiRevhumNot/a40c SEQ ID ATGGTGGTGTGCGGCCGCCTATTAT (Not1) NO: 427 GAGGAGACGGTGACCAGG Forward primer Pi2for (Xho1) SEQ ID AGGGGTATCTCTCGAGAAAAGAGAG NO: 428 GTGCAGCTGGTGGAGTCTGG

TABLE-US-00066 TABLE 42 Human TNFα EC.sub.50 in nM VHH mean stdev Number of assays TNF60 0.010 0.002 6 Enbrel 0.014 0.009 33 Humira 0.141 0.074 33 Remicade 0.120 0.037 33

TABLE-US-00067 TABLE 43 human albumin rhesus albumin TNF60 K.sub.D (nM) 24.4 24.1 k.sub.on (1/Ms) 2.05E+05 2.09E+05 k.sub.off (1/s) 5.02E−03 5.04E−03 TNF24 K.sub.D (nM) 11 Nd k.sub.on (1/Ms) 2.26E+05 Nd k.sub.off (1/s) 2.48E−03 Nd

TABLE-US-00068 TABLE 44 PiForLong SEQ ID GCTAAAGAAGAAGGGGTATCTCTCGAGAAA NO: 429 AGAGAGGTGCAGCTGGTGGAGTCTGG Rev_30GlySer_ SEQ ID TCAGTAACCTGGATCCCCCGCCACCGCTGC L108 NO: 430 CTCCACCGCCGCTACCCCCGCCACCGCTGC CTCCACCGCCTGAGGAGACGGTGACAAG For_GlySer SEQ ID AGGTTACTGAGGATCCGGCGGTGGAGGCAG NO: 431 CGGTGGCGGGGGTAGCGAGGTGCAGCTGGT GGAGTCTGG PiRevCys1hum SEQ ID ATGGTGGTGTGAATTCTTATTAGCAGGAGA NO: 432 CGGTGACAAGG PiRevCys2hum SEQ ID ATGGTGGTGTGAATTCTTATTAGCAACCTC NO: 433 CACCTGAGGAGACGGTGACAAGG AOXIFor SEQ ID GACTGGTTCCAATTGACAAGC NO: 434 AOXIRev SEQ ID GCAAATGGCATTCTGACATCC NO: 435

TABLE-US-00069 TABLE 45 Human TNFα EC.sub.50 in nM VHH mean stdev Number of assays TNF1 0.748 0.153 27 TNF55-PEG40 0.004 0.001 8 TNF55-PEG60 0.004 0.002 6 TNF55-Biotine 0.012 0.003 5 TNF56-PEG40 0.006 0.003 57 TNF56-PEG60 0.005 0.003 7 TNF56-Biotine 0.017 0.009 13 Enbrel 0.013 0.006 71 Humira 0.127 0.058 67 Remicade 0.144 0.061 67

TABLE-US-00070 TABLE 46 DS534 P4 DS592 P4 DS605 P3 KM05-179 P4 p[IC]50 Etanercept 9.55 9.49 9.51 9.51 Accipiter 9.88 9.44 9.37 9.27 pM Etanercept 282 324 309 309 Accipiter 132 363 427 537

TABLE-US-00071 TABLE 47 Total WBC count (×10.sup.6/mL) Dose Group ALX0071 Etanercept CMC vehicle 0.86 ± 0.09 0.1 μg human TNFα 3.72 ± 0.21 0.0625 mg/kg 3.03 ± 0.6  — 0.125 mg/kg 1.23 ± 0.3** 2.40 ± 0.39 0.25 mg/kg 1.37 ± 0.17* 2.47 ± 0.54 0.5 mg/kg — 2.19 ± 0.10 *P < 0.05, **P < 0.01 vs 0.1 μg human TNFα

[1895] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[1896] All references disclosed herein are incorporated by reference in their entirety.