EA045012B1 - LIMITED CONDITIONALLY ACTIVATE BINDING PROTEINS - Google Patents

Description

State of the art

Selective destruction of a single cell or a specific cell type is often desirable in a variety of clinical settings. For example, the primary goal of cancer therapy is to specifically destroy tumor cells while keeping healthy cells and tissues as intact and intact as possible. One such method is to induce an immune response against the tumor to induce immune effector cells, such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs), to attack and destroy tumor cells.

The use of intact monoclonal antibodies (mAbs), which provide excellent binding specificity and affinity for tumor-associated antigen, has been successfully applied in the field of cancer treatment and diagnosis. However, the large size of intact mAbs, their poor biodistribution, low activity, and long-term persistence in the blood pool have limited their clinical use. For example, intact antibodies may exhibit specific accumulation in the tumor region. Biodistribution studies with precise tumor examination show a heterogeneous distribution of antibodies with primary accumulation in peripheral areas. Due to tumor necrosis, heterogeneous antigen distribution, and increased intercellular tissue pressure, it is not possible to reach the central regions of the tumor using intact antibody constructs. In contrast, smaller antibody fragments demonstrate rapid localization to the tumor, penetrate deeper into the tumor, and are also relatively quickly cleared from the bloodstream. However, many antibodies, including scFv and other constructs, exhibit on-target/off-tumor effects, in which the molecule is active on non-tumor cells, causing side effects, some of which may be toxic. This invention relates to new constructs that are selectively activated in the presence of tumor proteases.

Brief description of the invention

The present invention provides a number of different protein compositions for the treatment of cancer. Accordingly, in one aspect, this invention relates to format 2 proteins containing, from N- to C-terminus: a first single domain antigen binding domain (sdABD) that binds to a human tumor target antigen (TTA). target antigens) (odASD-AMO); b) a first domain linker; c) a limited Fv domain containing: i) a first variable heavy domain containing vhCDR1, vhCDR2 and vhCDR3; ii) constrained non-cleavable linker (CNCL - constrained non-cleavable linker); and iii) a first variable light domain containing vlCDR1, vlCDR2 and vlCDR3; d) a second domain linker; e) second ODAS-AMO; f) cleavable linker (RL - CL - cleavable linker); g) a limited pseudo Fv domain comprising: i) a first pseudo variable light domain; ii) non-cleavable linker (NRL - NCL - non-cleavable linker); and iii) a first pseudo heavy variable domain; h) third domain linker; and i) a third odASD, which binds to human serum albumin; wherein said first variable heavy domain and said first variable light domain are capable of binding human CD3, but said limited Fv domain does not bind CD3; said first variable heavy domain and said first pseudo variable light domain are intramolecularly associated with the formation of an inactive Fv; and said first variable light domain and said first pseudo variable heavy domain are intramolecularly associated with the formation of an inactive Fv.

In an additional aspect, the present invention relates to format 1 proteins comprising, from N to the C-terminus: a) a first odASD-AMO; b) a first domain linker; c) a limited Fv domain containing: i) a first variable heavy domain containing vhCDR1, vhCDR2 and vhCDR3; ii) a limiting scissile linker (RLL); and iii) a first variable light domain containing vlCDR1, vlCDR2 and vlCDR3; d) a second domain linker; e) second ODAS-AMO; f) scissile linker (RL); g) a limited pseudo Fv domain comprising: i) a first pseudo variable light domain; ii) non-cutting linker (NRL); and iii) a first pseudo variable heavy domain; h) third domain linker; and i) a third odASD, which binds to human serum albumin; wherein said first variable heavy domain and said first variable light domain are capable of binding human CD3, but said limited Fv domain does not bind CD3; wherein said first variable heavy domain and said first pseudo variable light domain are intramolecular

- 1 045012 are associated with the formation of inactive Fv; and wherein said first variable light domain and said first pseudo variable heavy domain are intramolecularly associated with the formation of an inactive Fv.

In an additional aspect, the present invention relates to format 4 proteins comprising, from N to the C-terminus: a) a single domain antigen binding domain (sADB) that binds to a human tumor antigen (HTA) (sADA-AMO); b) a first domain linker; c) a limited Fv domain containing: i) a first variable heavy domain containing vhCDR1, vhCDR2 and vhCDR3; ii) restricted non-cutting linker (CNL); and iii) a first variable light domain containing vlCDR1, vlCDR2 and vlCDR3; d) scissile linker (RL); e) a second odASD that binds to human serum albumin; f) domain linker; g) a limited pseudo Fv domain comprising: i) a first pseudo variable light domain; ii) non-cutting linker (NRL); and iii) a first pseudo variable heavy domain; wherein said first variable heavy domain and said first variable light domain are capable of binding human CD3, but said limited Fv domain does not bind CD3; wherein said first variable heavy domain and said first pseudo variable light domain are intramolecularly associated with the formation of an inactive Fv; and wherein said first variable light domain and said first pseudo variable heavy domain are intramolecularly associated with the formation of an inactive Fv.

In a further aspect, for the format 1, format 2 and format 4 proteins listed above, said first variable heavy domain is N-terminal to said first variable light domain, and said pseudo variable light domain is N-terminal to said pseudo variable heavy domain. domain.

In a further aspect, for the format 1, format 2 and format 4 proteins listed above, said first variable heavy domain is N-terminal to said first variable light domain, and said pseudo variable heavy domain is N-terminal to said pseudo variable light domain. domain.

In a further aspect, for the format 1, format 2 and format 4 proteins listed above, said first variable light domain is N-terminal to the first variable heavy domain, and said pseudo variable light domain is N-terminal to said pseudo variable heavy domain.

In a further aspect, for the format 1, format 2 and format 4 proteins listed above, said first variable light domain is N-terminal to the first variable heavy domain, and said pseudo variable heavy domain is N-terminal to said pseudo variable light domain.

In a further aspect, the present invention provides format 1 and 2 proteins wherein said first and second AMOs are the same.

In a further aspect, the present invention provides format 1 and 2 proteins in which said first and second AMOs are different.

In a further aspect, the present invention provides format 1, 2 and 4 proteins, wherein said first and second AMOs are selected from EGFR, EpCAM, FOLR1 and B7H3. The sequence data may be selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29; SEQ ID NO: 33; SEQ ID NO: 37 and SEQ ID NO: 41.

In a further aspect, the present invention provides format 1, 2 and 4 proteins, wherein said half-life extension domain has SEQ ID NO: 45.

In a further aspect, the present invention relates to format 1, 2 and 4 proteins, wherein said cleavable linker is cut by a human protease selected from the group consisting of MMP2, MMP9, Meprin A, Meprin B, Cathepsin S, Cathepsin K, Cathepsin L, Granzyme B, UPA, Kallikrienin7, matriptase and thrombin.

In a further aspect, the present invention provides a protein selected from the group consisting of Pro186, Pro225, Pro226, Pro233, Pro311, Pro312, Pro313, Pro495, Pro246, Pro254, Pro255, Pro256, Pro420, Pro421, Pro432, Pro479, Pro480, Pro187, Pro221, Pro222, Pro223, Pro224, Pro393, Pro394, Pro395, Pro396, Pro429, Pro430 and Pro431.

In a further aspect, the present invention provides nucleic acids encoding a protein of format 1, format 2 or format 4, as described herein, as well as expression vectors and host cells containing nucleic acids encoding the protein.

In a further aspect, the present invention relates to methods for producing the proteins of the present invention and methods for treating patients in need thereof.

In a further aspect, the present invention relates to compositions containing 3A format prodrug protein pairs comprising: a) a first protein comprising N- to C-terminus: i) a first odASD-AMO; ii) first domain linker; iii) pseudo Fv domain containing from N- to C-terminus: 1) variable heavy chain containing vhCDR1, vhCDR2 and vhCDR3; 2) cutable linker; and 3) a first pseudo variable light domain containing iVLCDR1, iVLCDR2 and iVLCDR3; iv) second

- 2 045012 domain linker; v) odASD-HSA; a) a first second protein containing from the N- to the C-terminus: i) a third ODASD, which binds to a human tumor target antigen; ii) a third domain linker; iii) pseudo Fv domain containing from N- to C-terminus: 1) variable light chain containing VLCDR1, VLCDR2 and VLCDR3; 2) cutable linker; and 3) the first pseudo variable heavy domain containing iVHCDR1, iVHCDR2 and iVHCDR3; iv) a fourth domain linker; v) odASD-HSA; wherein said first variable heavy domain and said first variable light domain are capable of binding human CD3, in association; wherein said first variable heavy domain and said first pseudo variable light domain are intermolecularly associated with the formation of an inactive Fv; wherein said first variable light domain and said first pseudo variable heavy domain are intermolecularly associated with the formation of an inactive Fv; and wherein said first and third ODASD are selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29; SEQ ID NO: 33; SEQ ID NO: 37 and SEQ ID NO: 41.

In a further aspect, the present invention relates to compositions containing 3B format prodrug protein pairs, comprising a) a first protein comprising N- to C-terminus: i) a first odASD-AMO; ii) first domain linker; iii) second ODASD-AMO; iv) a second domain linker; iii) pseudo Fv domain containing from N- to C-terminus: 1) variable heavy chain containing vhCDR1, vhCDR2 and vhCDR3; 2) cutable linker; and 3) a first pseudo variable light domain containing iVLCDR1, iVLCDR2 and iVLCDR3; iv) third domain linker; and v) odASD-HSA; a) a first second protein containing from the N- to the C-terminus: i) a third odASD-AMO; ii) a fourth linker domain; iii) fourth odASD-AMO; iv) fifth domain linker; iii) pseudo Fv domain containing from N- to C-terminus: 1) variable light chain containing VLCDR1, VLCDR2 and VLCDR3; 2) cutable linker; and 3) the first pseudo variable heavy domain containing iVHCDR1, iVHCDR2 and iVHCDR3; iv) a sixth domain linker; v) odASD-HSA; wherein said first variable heavy domain and said first variable light domain are capable of binding human CD3, in association; wherein said first variable heavy domain and said first pseudo variable light domain are intermolecularly associated with the formation of an inactive Fv; and wherein said first variable light domain and said first pseudo variable heavy domain associate intermolecularly with the formation of an inactive Fv.

In a further aspect, the format 3A and format 3B proteins have odASD-HSA, which have SEQ ID NO: 45.

In an additional aspect, the format 3A and format 3B proteins have an odASD-AMO that binds to an AMO selected from EGFR, EpCAM, FOLR1 and B7H3. ODASD-AMO may be selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO : 25, SEQ ID NO: 29; SEQ ID NO: 33; SEQ ID NO: 37 and SEQ ID NO: 41.

In a further aspect, the present invention provides nucleic acid compositions comprising first nucleic acids that encode the first protein members of a prodrug pair, and second nucleic acids that encode the second protein members of the pairs, as well as expression vectors and host cells containing the nucleic acids.

Brief description of graphic materials

In fig. 1 depicts format 1 of the protease activation type of the present invention, referred to herein as constrained, cut constructs, or op constructs. In this embodiment, the typical design is Pro140: there are ASDs for two AMOs (as depicted in FIG. 1, both are the same, although as described herein they may be different). After cutting, the prodrug construct is cut into three components, one of which contains an α-AMO domain linked through a domain linker to the active VH of aCD3, the second contains an α-AMO domain linked through a domain linker to the active VL of aCD3, and the remaining element contains an extension domain half-life associated with inactive VH and VL. The two active variable domains can then freely associate to form a functional anti-CD3 binding domain. It should be noted that in Format 1 embodiments, the resulting active component is trivalent: there is monovalent binding to CD3 and divalent binding to AMO, forming a bispecific binding protein, although in some cases this trivalency may be trispecific with monovalent binding to CD3, monovalent binding to the first AMO and monovalent binding to the second AMO. Fig. 1 also demonstrates the human serum albumin (HSA) domain as the half-life extension domain, in many embodiments of sADSA, as defined herein, although, as discussed herein, this is optional and/or may be replaced by another half-life extension domain. half-life In addition, the half-life extension domain may also be N-terminal to the construct or internal. Fig. 1 also has VH and VL Fv and iVH and iVL pseudo Fv in a particular order, such as N- to C-terminus, VH-linker-VL (and iVL-linker-iVH), although as those skilled in the art will appreciate that they can be reversed (VL-linker-VH and

- 3 045012 iVH-linker-iVL). Alternatively, one of these Fvs could be in one orientation and the other in a different orientation, although protein expression in the orientation demonstrated here was unexpectedly higher than in the other orientations.

In fig. 2 depicts format 2 protease activation type of the present invention, referred to herein as restricted, uncutable constructs or LRLC constructs, also sometimes referred to herein as dimerization constructs, as discussed herein. These constructs are not isomerized as discussed herein. Once cut, the two prodrug constructs are cut into four components, two half-life extension domains (in this case sdASD for HSA) associated with two pseudodomains (which may or may not be able to self-associate, depending on the length of the linkers and inactivating mutations), and two active moieties that self-assemble into a dimeric active group that contains four anti-AMO domains (which can be the same or two the same and the other two different). It should be noted that in Format 2 embodiments, the resulting active component is hexavalent: there is divalent binding to CD3 and tetravalent binding to AMO, forming a bispecific binding protein, although in some cases this hexavalency may be trispecific, with divalent binding to CD3, divalent binding with the first AMO and divalent binding to the second AMO. Fig. 2 also demonstrates the human serum albumin (HSA) domain as the half-life extension domain, in many embodiments of sADSA, as defined herein, although, as discussed herein, this is optional and/or may be replaced by another life extension domain. half-life In addition, the half-life extension domain may also be N-terminal to the construct or internal. Fig. 2 also has VH and VL Fv and iVH and iVL pseudo Fv in a specific order, such as N- to C-terminus, VH-linker-VL (and iVL-linkeriVH), although as those skilled in the art will appreciate, they can be reversed (VL-linkerVH and iVH-linker-iVL). Alternatively, one of these Fvs may be in one orientation and the other in a different orientation, although protein expression in the orientation, as demonstrated herein, was unexpectedly higher than in the other orientations.

Fig. 3A-3B depict type 3 format constructs, also sometimes referred to as hemiconstructs or hemi-COBRA™ (COBRA - Conditional Bispecific Redirected Activation construct), as defined herein, since they represent two different polypeptide chains that together constitute a MCE (multivalent conditionally effective) therapeutic agent, as further discussed herein. In this embodiment, the constructs are delivered in pairs, with intramolecular self-assembly prior to cleavage resulting in inactive anti-CD3 Fv domains. Once cut, the inert variable domains are released and the two active variable domains are then intermolecularly assembled to form the active anti-CD3 binding domain. Two sDASD-AMOs bind to the corresponding receptor on the surface of tumor cells, and cleavage is carried out by a protease. This enables intermolecular assembly as the molecules are physically held in place, facilitating the assembly of the anti-CD3 active domain. As above for formats 1 and 2, in this embodiment, the order from N- to C-terminus of the variable domains can be reversed or mixed. In addition, odASD (HSA) may be located at the N- or C-terminus of each half-construct. Pro16 has odASD (HSA) at the C-terminus, and Pro17 has it at the N-terminus (see Pro19, SEQ ID NO: XX, has odASD (HSA) at the C-terminus). In fig. 3A shows format 3 designs with one ODASDAMO domain per half-structure, and FIG. 3B demonstrates format 3 designs with two sDASD-AMOs per half-design in dual-targeting or hetero-targeting configurations. Note that in FIG. 3B uses FOLR1 and EGFR as the two AMOs, but other combinations may also be used as described herein.

In fig. 4 shows type 4 format designs, which are similar to type 2 designs, but have only one ODASD-AMO. This figure demonstrates odASD-AMO-EGFR, but as those skilled in the art will appreciate, other AMOs can also be used. After cutting, the prodrug construct is cut into two components: a half-life extension domain (in this case, dASD for HSA) associated with the pseudo Fv, and an active fragment, which, in the presence of a second active fragment from another cleaved molecule, self-assembles into a dimeric active fragment that contains two domain against AMO. It should be noted that in embodiments of Format 4, the resulting active component is tetravalent: there is divalent binding to CD3 and divalent binding to AMO, which forms a bispecific binding protein. Fig. 4 also demonstrates the human serum albumin (HSA) domain as a half-life extension domain, in many embodiments sASD(1/2) as defined herein, although as discussed herein, this is not required and/or it may be replaced by other half-life extension domains. In addition, the half-life extension domain may also be N-terminal or internal to the construct. Fig. 4 also has VH and VL Fv and iVH and iVL pseudo Fv in a specific order, for example N- to C

- 4 045012 end, VH-linker-VL (and iVL-linker-iVH), although those skilled in the art will understand that they can be reversed (VL-linker-VH and iVH-linker-iVL). Alternatively, one of these Fvs may be in one orientation and the other in a different orientation, although protein expression in the orientation, as demonstrated herein, was unexpectedly higher than in the other orientations.

Fig. 5A-5G depict a number of sequences according to this invention. CDRs are underlined for antigen binding domains. As described in more detail herein, these domains can be assembled in a wide variety of configurations in this invention, including format 1, format 2, format 3, and format 4 orientations. It should be noted that SEQ ID NO: 90 cuts slightly faster with MMP9 than SEQ ID NOs: 75 and 76, and SEQ ID NO: 91 cuts more slowly than SEQ ID NOs: 75 and 76.

In fig. 6A-6B depict several suitable protease cleavage sites. As those skilled in the art will appreciate, these cleavage sites can be used as cleavable linkers. In some embodiments, for example when more flexible cleavage linkers are required, additional amino acids (typically glycines and serines) may be present that are N- or C-terminal to these cleavage sites.

Fig. 7A-7D depict some data associated with format 3 or hemiCOBRA™ structures. This demonstrates that format 3 constructs bind cooperatively to CD3 upon cleavage by a protease (in this case EK protease, although any of the protease cleavage sites described herein and depicted in FIGS. 5 and 6 can be used) and form a CD3 binding site. as demonstrated by FACS sandwich analysis.

Fig. 8A-8D demonstrate that protease cleavage coactivates T cell killing of EGFR+ target cells via complementary hemi-COBRA™ pairs. Fig. 8A and 8B demonstrate that constructs in isolation but cut with different concentrations of protease do not affect the viability of target cells. However, FIG. 8C demonstrates that in combination, in the presence of protease, the viability of target cells is significantly reduced. Fig. 8D shows the general mechanism.

In fig. Figure 9 demonstrates some non-target controls for use in assays to test the effectiveness of format 1 constructs.

Fig. 10A-10F demonstrate that generation of an active CD3 binding domain is dependent on target binding of both arms, eg, the sDASD-AMO domains, one of which is found on each of the two constructs. The TDCC assay was performed as described in the Examples.

In fig. 11 shows a diagram of suitable hemi-COBRA™ pairs. Mep represents the meprin protease cleavage site, His-6 is a tag which is discussed in more detail herein, ST14 is the matriptase protease cleavage site, and Thb is the thrombin protease cleavage site.

Fig. 12A-12C illustrate TDCC data associated with the designs of FIGS. 11. In FIG. 12A demonstrates that addition of pre-cleaved hemi-COBRA pairs results in efficacy in OvCAR8 cells. FIG. 12B demonstrates that addition of pre-cleaved hemi-COBRA pairs results in efficacy in HCT116 cells, and FIG. 12C demonstrates that addition of pre-cleaved hemi-COBRA pairs results in efficacy on LoVo cells, which are all cancer cell lines.

Fig. 13A-13B demonstrate that the MMP9 linker is stable in vivo. NSG mice were administered a single intravenous bolus dose of either Pro40 (MMP9 cut), Pro74 (uncut) via the tail vein at a dose level of 0.5 mg/kg. A dose solution for each compound was prepared in a vehicle of 25 mM citric acid, 75 mM L-arginine, 75 mM NaCl and 4% sucrose, pH 7.0. Two blood samples were collected at preselected time points from each animal, one at the start of the study, collected by orbital hemorrhage or submandibular hemorrhage, and the other at the end time point by cardiac puncture. Blood collection time points were 0.083, 1, 6, 24, 72 and 168 hours. Plasma was prepared from each individual blood sample using K ₂ EDTA tubes. Concentrations were determined using the MSD assay with anti-HSA odASD-specific mAb and determined using the extracellular domain of EGFR.

In fig. 14 depicts schematics of the 3A format hemi-COBRA™ designs used in the experiments depicted in FIG. 15. Pro51 is a positive control because it is always included because it produces active anti-CD3 Fv. Pro98 is a negative control because its sADS is directed against chicken egg lysozyme, which is not expressed by the tumor. Pro77 and Pro53 are a 3A format prodrug pair using an anti-EGFR sDASD and an MMP9 cleavage site. Pro74 and Pro72 are a 3A format pair with negative controls because they do not have cutting sites.

In fig. 15 demonstrates that format 1 constructs work for tumor regression in vivo using two different tumor cell lines implanted into mice using the example protocols. Antitumor activity against hemi-COBRA constructs

- 5 045012 (Pro77 and Pro53) depended on the inclusion of both anti-EGFR sdASD and MMP9-cut linkers, along with active anti-CD3 Fv.

In fig. 16 shows a diagram of a next-generation format, full-length construct that has two pseudo Fv domains with cleavage sites in between, as generally described in US 2018/0134789, incorporated herein by reference. However, as demonstrated in the following figures, this first-generation full-length construct does not show very good conditionality since it can isomerize to form either an active or inactive construct.

In fig. 17 demonstrates that pairs of 3A designs actually exhibit better conditionality than the first generation full length Pro100 designs.

In fig. 18 shows additional first-generation full-length designs tested in FIG. 19.

In fig. 19 demonstrates that first generation designs exhibit high activity even in the uncut format, such as poor conditionality.

In fig. 20 demonstrated that first-generation full-length constructs exhibit two monomer peaks on analytical SEC.

In fig. 21 shows a diagram of the reason for the uncut activity, which is that the full-length first generation of constructs isomerizes to form two conformations, one of which is inactive because no active anti-CD3 Fv (bivalent scFv) is produced and another that is active in the absence of protease, type configuration - single-chain diabody. See PEDS 23 (8): 667-677 (2010).

In fig. 22 demonstrates the results of a TDCC assay performed at 37°C for 2 days with first-generation single-chain constructs. The results demonstrate that non-cleaved constructs exhibit strong killing. These results led to the creation of format 1 designs.

23A - 23G illustrate format 1 designs used in the present invention. As will be appreciated by those skilled in the art and described herein, they are depicted with the sDASD-EGFR targeting moiety, although sDASD may be used for other AMOs.

In fig. 24 demonstrates that format 1 constructs (in this case Pro140) form a single isomer that is stable at 37°C.

In fig. 25 demonstrates that format 1 constructs have very low binding to human CD3 in uncut format as measured by octet analysis. The top line is Pro120, the middle line is Pro51 (positive control), and the bottom lines are Pro140 maintained at 4°C or 37°C for 3 days.

In fig. 26 also demonstrated that format 1 constructs have very low TDCC activity in the uncut form.

In fig. 27 depicts a specific format 1 construct, Pro140, used in in vivo testing using sDASD-EGFR as the target fragments and MMP9 cleavage site.

Fig. 28A-28B demonstrate tumor regression using a format 1 design.

In fig. 29 demonstrated that due to the cutting site in a limited Fv, several different fragments can be generated: a partially cut fragment and a fully cut fragment. Surprisingly, the partially cut format is more active than the fully cut format, resulting in the generation of format 2.

In fig. 30 demonstrates a number of format 2 schemes, all of which use domains targeting sdASD-EGFR, although, as described herein and listed in the sequences, sdASDs for other AMOs may be used. Pro51 and Pro201 are positive controls (in the active hemi configuration), and Pro214 is a full-length negative control since there is no cut site.

In fig. 31 demonstrates the TDCC activity of the Pro187 format 2 construct, which utilizes the meprin cleavage site. Pro187 in the TDCC assay was 1200-fold more active when added precut than when added without cutting. Precut Pro187 showed activity that fell between the positive controls Pro51 and Pro201. Uncut Pro187 exhibited activity similar to Pro214, which does not contain a protease-cut linker.

In fig. 32 demonstrates the TDCC activity of the format 2 construct Pro186, which utilizes the MMP9 cleavage site. Pro186 in the TDCC assay was 18-fold more active when added precut than when added without cutting. Precut Pro186 showed activity that fell between the positive controls Pro51 and Pro201. Uncut Pro186 showed greater activity than Pro214, which does not contain a protease-cut linker.

In fig. 33 demonstrated that the Pro186 construct binds to cells that have varying levels of EGFR receptors, while CHO cells do not express EGFR on the cell surface.

- 6 045012 details. Pro186 saturates cells expressing varying levels of EGFR at similar concentrations of COBRA.

In fig. 34 shows a diagram of the format 2 constructs used in the in vivo studies of FIG. 35, all of which use domains targeting sADS-EGFR.

In fig. 35 demonstrated that the Pro186 format 2 design was highly effective at both concentrations and was superior to the Pro140 format 1 design at lower concentrations.

In fig. 36 demonstrates a number of format 2 constructs based on Pro186 but with different protease cleavage sites. Although all of these constructs use sDASD-EGFR for both targeting domains, other sDASDs for different AMOs may be used, which may be the same or different. Thus, it is possible to perform both homo-targeting (both target odASD to the same AMO) and hetero-targeting (one odASD to the first AMO and another to the other AMO).

In fig. 37 shows designs for various format 2 constructs that change the length of the linker between the Fv domains. These are demonstrated using the MMP9 cutting site, although others can be used as described herein. Similarly, although all of these constructs use sDASD-EGFR for both target domains, other sDASDs for different AMOs may be used, and these may be the same or different.

In fig. 38 demonstrated that the linker length for a pseudo Fv can vary, for example, that a format 2 construct with a short linker between an active Fv (short active) and a longer linker between a pseudo Fv (long inactive) exhibits activity similar to a short active with a short inactive. Thus, the conventionality of the COBRA construct does not depend on the restriction of active and inactive scFv linkers; as long as one is limited, single-chain diabody folding appears to be favored over divalent scFv folding.

In fig. 39 demonstrated that the linker length for the active Fv can vary, for example, format 2 constructs with long active and short inactive behave similarly to short active and short inactive constructs. Thus, the conventionality of the COBRA construct does not depend on the restriction of active and inactive scFv linkers; as long as one is limited, single-chain diabody folding appears to be favored over divalent scFv folding.

Fig. 40A-40C show schematics for a number of different designs. The Pro188 is a format 1 design that is similar to the Pro140 except for the long linker (16-mer) in the pseudo Fv. Pro189 and Pro190 (format 2 constructs) are similar to Pro186 and Pro187, except for a long linker (16-mer) in the pseudo Fv domain. Pro191 and Pro192 (also format 2 designs) are similar to Pro189 and Pro190 except that they have an additional cutting site in front of the sADS (1/2). Pro193 (format 4) has one EGFR targeting domain, iVH and iVL are reversed and an additional cutting site is located upstream of sADS (1/2). Pro195 is a format 2 construct similar to Pro186 with targeting domains that bind to the same AMO, EGFR, but different epitopes. Pro196, Pro197 and Pro198 are format 2 constructs with rearranged variable domains.

Fig. 41 depicts the fact that different sADS clones targeting human FOLR1 exhibit differential killing. A Pro22 type construct (Pro51 with a FLAG sequence instead of NRL) that binds to human FOLR1 was compared with the Pro22EGFR construct against a number of cell line families.

In fig. 42 demonstrates designs for four sdASD-FOLRI constructs, including the use of the positive control Pro201 using sdASD-EGFR2 (with two molecules intermolecularly linked to form two active anti-CD3 Fvs) and two format 2 test constructs, Pro311, using h77.2 sdASD and Pro312 using h59.3 odASD, as well as two negative controls, Pro299 using h77.2 odASD and Pro303 using h59.3 odASD.

Fig. 43 depicts format 2 design diagrams for FOLR/MMP9 in vivo design.

In fig. 44 demonstrated the in vivo efficacy of the Pro312 construct and demonstrated that the MMP9 scissile linker is required for antitumor activity.

In fig. 45 shows schematics of some formats using sADS for human B7H3 (sADS-B7H3), including Pro244, a positive control (using sDSD-B7H3 (hF7) (with two molecules intermolecularly associated to form two active anti-CD3 Fvs), and two test format 2 constructs, Pro225, format 2 construct, and Pro295, negative control lacking the cutting site.

Fig. 46 demonstrates that the Pro225 has greater conditionality compared to the control Pro295.

In fig. 47 demonstrated that the format 2 design using the meprine linker, Pro373, exhibits greater conventionality compared to Pro295.

In fig. 48 demonstrated a series of constructs odASD-B7NZ (using the hF12 sequence), which demonstrated the positive control of Pro51 using odASD-EGFR,

- 7 045012 positive control Pro244 using odASD-hF12 B7H3, test construct, Pro226 and negative control Pro296 without cutting site.

In fig. 49 demonstrated good conditionality of the Pro226 design in the TDCC analysis.

In fig. 50 demonstrated the humanization of sADS for human EpCAM.

In fig. 51 shows a design of several formats: Pro22hVIB13 and Pro205 are positive controls, Pro199 is a format 2 design, and Pro175 is a negative control.

In fig. 52 demonstrated the TDCC activity of sdASD-EpCAM constructs, demonstrating good conditionality.

Fig. 53A-53B demonstrate the TDCC activity of the sDASD-EpCAM Pro199 construct, demonstrating good conditionality in HT29 and LoVo cell models.

Fig. 54A-54B demonstrate the TDCC activity of the sDASD-EpCAM Pro200 construct, which shows good conditionality in HT29 and LoVo cell models.

In fig. 55 shows the Pro255 design, which uses two different odASD-AMOs, one for EGFR (odASD-EGFR) and the other for EpCAM (odASD-EpCAM), compared to Pro199, with dual EpCAM odASDs. These are sometimes referred to in this document as heterotargeting constructs, in this case the format 2 construct.

In fig. 56 demonstrated that the dual targeting molecule Pro255 with the MMP9 cleavage site exhibited good conditionality.

In fig. 57A-57D show the results of experiments with three different cell types. First, Raji transfectants were generated with equal expression levels of EpCAM, EGFR, and EpCAM+EGFR (data not shown). Pro255, which targets both EpCAM and EGFR, was then tested in TDCC assays using each cell type. Fig. A demonstrates the Raji parental line, which does not express either receptor. Fig. 57B demonstrates the convention on the EpCAM line. Fig. 57C demonstrates the convention on the EGRF line. Fig. 57D demonstrates convention on the EpCAM/EGFR line.

In fig. Figure 58 shows a design diagram of the format 4 Pro258.

Fig. 59A-59B demonstrate that Pro258 is conditional in both FBS and human serum. MMP9 linker status is underestimated due to MMP9 activity in culture. Interestingly, the TDCC activity of Pro51 is inhibited by HSA binding, whereas the TDCC activity of Pro258 is similar to that of Pro51 in the presence of HSA. Finally, the conditionality of Pro258 is slightly increased in the presence of HSA by a factor of 6.

Fig. 60A-60C demonstrate cleavage of the MMP9 substrate by other MMPs.

Fig. 61A-61B show some illustrative designs and their formats.

Fig. 62A-62U show a number of sequences according to this invention, although many additional sequences are also found in the sequence listing. CDRs are underlined and in bold, linkers are double underlined (cut linkers are italic and double underlined), and domain separations are indicated by /. All His6 tags are optional as they can be used to reduce immunogenicity in humans and also as purification tags.

Detailed description of the invention

Introduction.

This invention is directed to methods for reducing the toxicity and side effects of bispecific antibodies (including antibody-like functional proteins) that bind to important physiological targets such as CD3 and tumor antigens. Many antigen-binding proteins, such as antibodies, can have significant off-target side effects, and thus there is a need to activate only the binding capabilities of the therapeutic molecule in close proximity to the disease tissue to avoid off-target interactions. Accordingly, the present invention is directed to multivalent conditionally effective (MCE) proteins that have a number of functional protein domains. In general, one of these domains is an antigen binding domain (ABD), which will bind a tumor target antigen (TAM), and the other is an ABD, which will bind a T cell antigen, such as CD3, under certain conditions. In addition, MCE proteins also include one or more protease cleavage sites. That is, therapeutic molecules are manufactured in a prodrug-like format where the CD3-binding domain is inactive until exposed to the tumor environment. The tumor environment contains proteases, so when exposed to a protease, the prodrug is cut and becomes active.

This is typically achieved by proteins that include a pseudo variable heavy domain and a pseudo variable light domain directed to a T cell antigen, such as CD3, that keep the CD3 MCE Fv in an inactive format as discussed herein. Because AMO targets MCE to the tumor environment, MCE is thus exposed to the protease. After cutting, the active variable heavy domain and the active light domain can now

- 8 045012 couple to form one or more CD3-active ASDs and thus recruit T cells to the tumor, leading to treatment.

Typically, the CD3 binding domain (Fv) has a limited format in which the linker between the active variable heavy domain and the active variable light domain that traditionally forms the Fv is too short to allow the two active variable domains to bind to each other; it is called a limiting linker; they may be limited and cutable (ORL, as used in format 1) or limited and uncut (ONRL, as used in format 2). More likely, in a prodrug (eg, uncut) format, the prodrug polypeptide also contains a pseudo Fv domain. The pseudo-Fv domain contains a variable heavy and light domain with standard framework regions but inert or inactive CDRs. The pseudo-Fv domain also has a limiting linker between the inactive variable heavy and inactive variable light domains. Because neither the Fv nor the pseudo Fv domains can self-assemble due to steric constraints, there is an intramolecular assembly that links aVL to iVH and aVH to iVL due to the affinity of the framework regions of each. However, due to the inert pseudodomains of the CDRs, the resulting ASDs will not bind CD3, thus preventing unwanted toxicity. However, in the presence of proteases that reside in or near the tumor, the prodrug construct is cut so that the pseudo Fv domain is released from the surface and thus allows the real variable heavy and variable light domains to bind intramolecularly (e.g., the two cut constructs are combined) , thus triggering active CD3 binding and, as a consequence, antitumor efficacy. These constructs are commonly referred to herein as conditional bispecific redirected activation constructs, or COBRA™ (Conditional Bispecific Redirected Activation constructs). The stability of intramolecular assembly is demonstrated by conditionality experiments, whereby in the absence of protease, uncleaved constructs have no activity (e.g., no active CD3 binding domain is formed).

Interestingly, for ease of description, although all of these constructs are referred to herein as constrained, additional work demonstrates that intramolecular assembly is favored even if one of the Fv domains is not constrained, for example, one of the domains may have a longer, flexible linker. That is, as shown in FIG. 37, fig. 38 and fig. 39. Intramolecular assembly still occurs (eg, uncleaved constructs are inactive in the absence of protease cutting) unless one of the Fv domains, either one with active VL and VH, or a pseudo Fv domain, is restricted. However, in modern systems, when both linkers are restrictive, the protein has better expression. However, as those skilled in the art will appreciate, any of the format 1, format 2, or format 4 constructs herein may have one of these Fv domains with a non-limiting or flexible linker. For ease of reference, the designs are demonstrated with both Fv domains in a limited format.

The designs and formats of the invention are variations from those described in WO2017/156178, which is incorporated herein by reference in its entirety. As shown in FIG. 21, previous constructs have the ability to isomerize due to the presence of two sets of VH and VL domains in a single polypeptide, forming both a divalent scFv and a single-chain diabody. Even after purification of each isoform, the divalent construct can still reach equilibrium with the diabody isoform. Since the single-chain diabody has the ability to bind to CD3 in the absence of protease cutting, the utility of the construct is reduced.

To solve this problem, the present invention provides four separate types of designs for performing this conditional activation. Activation of a prodrug can occur in one of four general ways, as generally demonstrated in the figures. In fig. 1 demonstrates the mechanism of format 1. In this embodiment, the prodrug construct has two cutting sites: one between the VH and VL domains of the restricted Fv, which releases the two variable domains for binding, and a second at a site that releases the pseudo Fv domain from the prodrug construct, leaving two molecules that associate due to the intrinsic ability to self-assemble variable heavy and variable light domains, each of which also has an antigen binding domain directed to the tumor antigen, allowing the recruitment of T cells to the tumor site.

In an alternative embodiment, the prodrug design is shown in FIG. 2, format 2 mechanism. In this embodiment, the domain linker between the active variable heavy chain and the active light chain is a limiting but non-cutting linker (LCCL). In the prodrug format, the inactive VH and VL of the limited pseudo Fv domain are associated with the VH and VL of the limited Fv domain, so that CD3 binding is absent. However, once cleavage occurs in the tumor environment, two different activated proteins, each containing an active variable heavy and light domain, bind to form two anti-CD3 binding domains.

- 9 045012

In addition to the single-chain protein COBRA formats discussed above, where all components are contained in a single amino acid sequence, there are also designs based on two hemi-COBRA proteins that act in pairs, as demonstrated in FIG. 3. In this embodiment, each protein has one active and one inert variable domain separated by a protease cleavage site. Each molecule contains an AMO-binding domain, so that when the molecules bind to AMO and are exposed to a tumor protease, the inert domains are cleaved off and the two active variable domains self-assemble to form the anti-CD3 binding domain.

In addition, the invention also provides format 4 designs, as shown in FIG. 4. These are similar to format 2 constructs, except that a single ASD is used for the AMO, so that after cutting, the two prodrug molecules now form a tetravalent bispecific construct containing two anti-CD3 active domains, as further described below.

Accordingly, the formats and designs of the present invention find use in the treatment of diseases.

Definitions.

In order that the application may be more fully understood, several definitions are given below. Such definitions are intended to cover grammatical equivalents.

By amino acid and amino acid identity, as used herein, is meant one of the 20 naturally occurring amino acids or any non-natural analogues that may be present at a specific defined position. In many embodiments, an amino acid means one of the 20 naturally occurring amino acids. By protein, as used herein, is meant at least two covalently linked amino acids, which includes proteins, polypeptides, oligopeptides and peptides.

By amino acid modification, as used herein, is meant an amino acid substitution, insertion and/or deletion in a polypeptide sequence, or a change in a moiety chemically linked to a protein. For example, the modification may be an altered carbohydrate or PEG structure attached to the protein. For clarity, unless otherwise noted, an amino acid modification always refers to the amino acid encoded by DNA, for example, the 20 amino acids that have codons in DNA and RNA. The preferred amino acid modification herein is substitution.

By amino acid substitution or substitution as used herein is meant the replacement of an amino acid at a specific position in the original polypeptide sequence with another amino acid. Specifically, in some embodiments, the substitution is an amino acid that does not occur naturally at the particular position, or does not occur naturally in the body, or in any organism. To be clear, a protein that has been engineered to change the nucleic acid coding sequence but not to replace the original amino acid (for example, exchanging CGG (encoding arginine) for CGA (still encoding arginine) to increase expression level in the host) is not amino acid replacement; that is, despite the creation of a new gene encoding the same protein, if the protein has the same amino acid in the specific Position at which it began, it is not an amino acid replacement.

By insertion or insertion of an amino acid, as used herein, is meant the addition of an amino acid sequence at a specific position in the original polypeptide sequence.

By deletion or removal of an amino acid, as used herein, is meant the removal of an amino acid sequence at a specific Position in the original polypeptide sequence.

The polypeptides of this invention specifically bind to CD3 and tumor target antigens (TATs), such as target cell receptors, as described herein. Specific binding or specifically binds or is specific for a particular antigen or epitope means binding that is measurably different from a nonspecific interaction. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is typically a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

Specific binding for a particular antigen or epitope may be exhibited, for example, by an antibody having a KD to the antigen or epitope of at least about 10 ^-4 M, at least 10 ^-5 M, at least about 10 ^-6 M, at least about 10 ^-7 M, at least about 10 ^-8 M, at least about 10 ^-9 M, alternatively at least about 10 ^-10 M, with at least about 10 ^{- 11} M, at least about 10 ^-12 M or more, wherein KD refers to the rate of dissociation of a particular antibody-antigen interaction. Typically, an antibody that specifically binds an antigen will have a KD that is 20, 50, 100, 500, 1000, 5000, 10,000 or more times higher for the control molecule relative to the antigen or epitope.

- 10 045012

Additionally, specific binding for a particular antigen or epitope may be exhibited, for example, by an antibody having a KA or Ka for the antigen or epitope that is at least 20, 50, 100, 500, 1000, 5000, 10,000 or more times greater for that antigen or epitope. epitope compared to a control, with KA or Ka referring to the rate of association of a particular antibody-antigen interaction. Binding affinity is typically measured using Biacore or Octet assays, as is known in the art.

By parent or precursor polypeptide (including parent Fc or Fc precursors), as used herein, is meant a polypeptide that is subsequently modified to produce a variant. Said parent polypeptide may be a naturally occurring polypeptide or a variant or engineered variant of a naturally occurring polypeptide. The parent polypeptide may refer to the polypeptide itself, compositions that contain the parent polypeptide, or the amino acid sequence that encodes it. Accordingly, by a parent Fc polypeptide, as used herein, is meant an unmodified Fc polypeptide that is modified to produce a variant antibody, and by a parent antibody, as used herein, is meant an unmodified antibody that is modified to produce a variant antibody.

By position, as used herein, is meant a location in the protein sequence. The provisions may be numbered sequentially or according to a specified format, such as the EC suffix for antibody numbering.

As used herein, the term target antigen/target antigen means a molecule that is specifically bound to the variable region of a given antibody. The target antigen may be a protein, carbohydrate, lipid, or other chemical compound. A number of suitable exemplary target antigens are described herein.

The term target cell as used herein means a cell that expresses the target antigen. Typically, for the purposes of the invention, the target cells are either tumor cells that express AMO or T cells that express CD3 antigen.

By "Fv or Fv domain" or Fv region, as used herein, is meant a polypeptide that contains the VL and VH domains of an antigen binding domain, typically from an antibody. Fv domains typically form an antigen binding domain or ABD, as discussed herein, if they contain active VH and VL domains (although in some cases an Fv containing a limiting linker is used so that an active ABD is not formed prior to cleavage). As discussed below, Fv domains can be organized in several ways in the present invention and can be active or inactive, such as in scFv format, limited Fv format, pseudo Fv format, etc. It should be understood that in the present invention, in some cases, the Fv domain consists of a VH and a VL domain on the same polypeptide chain, for example, as demonstrated in FIG. 1 and fig. 2, but with a limiting linker such that an intramolecular ASD cannot be formed. In these embodiments, it is after cutting that two active ASDs are formed. In some cases, the Fv domain consists of VH and VL domains, one of which is inert, so that only after cutting is an intermolecular ASD formed. As discussed below, Fv domains can be organized in several ways in the present invention and can be active or inactive, such as in scFv format, limited Fv format, pseudo Fv format, etc. In addition, as discussed herein, Fv domains containing VH and VL can constitute/form ASDs, and other ASDs that do not contain VH and VL domains can be formed using sdASDs.

By variable domain, as used herein, is meant a region of an immunoglobulin that contains one or more Ig domains substantially encoded by any of the Υκ, Υλ, and/or VH genes that constitute the kappa, lambda, and immunoglobulin heavy chain genetic loci, respectively. In some cases a single variable domain such as sdFv (also referred to herein as sdASD) may be used.

In embodiments using both variable heavy (VH) and variable light (VL) domains, each VH and VL consists of three hypervariable regions (complementarity determining regions, CDRs) and four framework regions or FRs, located from the amino terminus to carboxy terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Thus, the VH domain has the structure vhPR1-vhCDR1-vhFR2-vhCDR2-vhPR3-vhCDR3-vhFR4, and the VL domain has the structure vlFR1-vlCDR1-vlFR2-vlCDR2-vlFR3-vlCDR3-vlFR4-. As more fully described herein, vhFR regions and vlFR regions self-assemble to form Fv domains. In general, in the prodrug formats of the present invention, there are limited Fv domains, in which the VH and VL domains cannot self-associate, and pseudo Fv domains, in which the CDRs do not form antigen binding domains when self-associating.

Hypervariable regions confer antigen-binding specificity and typically span amino acid residues from approximately amino acid residues 24-34 (LCDR1; L is light chain), 50-56 (LCDR2) and up to 89-97 (LCDR3) in and around the light chain variable region approximately

- 11 045012

31-35B (HCDR1; H is heavy chain), 50-65 (HCDR2) and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and/or residues forming a hypervariable loop (for example, residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53- 55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region Chothia and Lesk (1987) J Mol Biol 196:901-917 Specific CDRs of the present invention are described below.

As those skilled in the art will appreciate, the exact numbering and placement of CDRs may vary between numbering systems. However, it should be understood that disclosure of the variable severe and/or variable mild sequence includes disclosure of the associated CDRs. Accordingly, the disclosure of each variable heavy region is a vhCDR disclosure (eg, vhCDR1, vhCDR2 and vhCDR3), and the disclosure of each variable light region is a vlCDR disclosure (eg, vlCDR1, vlCDR2 and vlCDR3).

A useful comparison of CDR numbering is provided below, see Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003):

Table 1

Kabat + Chotia IMGT Kabat AbM Chotia Contact vhCDRl 26-35 27-38 31-35 26-35 26-32 30-35 vhCDR2 50-65 56-65 50-65 50-58 52-56 47-58 vhCDR3 95-102 105-117 95-102 95-102 95-102 93-101 vlCDRl 24-34 27-38 24-34 24-34 24-34 30-36 vlCDR2 50-56 56-65 50-56 50-56 50-56 46-55 vlCDR3 89-97 105-117 89-97 89-97 89-97 89-96

As used herein, the Kabat numbering system is generally used to designate a residue in the variable domain (approximately light chain variable region residues 1-107 and heavy chain variable region residues 1-113) and the EC numbering system for Fc regions (e.g., Kabat et al. , supra (1991)).

The present invention provides a large number of different sets of CDRs. In this case, the complete set of CDRs in the context of the anti-CD3 component includes three variable light and three variable heavy CDRs, for example, vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2 and vhCDR3. As will be appreciated by those skilled in the art, each set of CDRs, CDR VH and VL, can bind to antigens either individually or as a set. For example, in restricted Fv domains, vhCDRs can bind, for example, CD3, and vlCDRs can bind CD3, but in a restricted format they cannot bind CD3.

In the context of single domain ASD (sADD), which is typically used herein for binding to tumor target antigens (TATs), the set of CDRs is only three CDRs; they are sometimes also referred to in the art as VHH domains.

These CDRs may be part of a larger variable light or variable heavy domain, respectively. In addition, as more fully described herein, the variable heavy and variable light domains may be on separate polypeptide chains or on a single polypeptide chain in the case of scFv sequences, depending on the format and configuration of the fragments herein.

CDRs promote the formation of antigen-binding or, more specifically, epitope-binding sites. An epitope refers to a determinant that interacts with a specific antigen binding site in variable regions known as paratope. Epitopes are groups of molecules, such as the side chains of amino acids or sugars, and usually have specific structural characteristics as well as specific charge characteristics. One antigen can have more than one epitope.

An epitope may contain amino acid residues directly involved in binding (also called the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by a specific antigen binding peptide; in other words, the amino acid residue is within the range of action of the specific antigen-binding peptide.

Epitopes can be conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. Conformational and non-conformational epitopes may differ in that binding to the former, but not the latter, is lost in the presence of denaturing solvents.

- 12 045012 lei.

An epitope typically comprises at least 3, and more typically at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be tested using a simple immunoassay demonstrating the ability of one antibody to block the binding of another antibody to a target antigen, such as binning. As stated below, the present invention includes not only the listed antigen binding domains and antibodies, but also those that compete for binding to epitopes associated with the listed antigen binding domains.

The variable heavy and variable light domains of the present invention may be active or inactive.

As used herein, inactive VH (iVH) and inactive VL (iVL) refer to components of the pseudo Fv domain that, when paired with their cognate partners VL or VH, respectively, form a resulting VH/VL pair that does not specifically bind with the antigen that an active VH or active VL would bind to if it were associated with a similar VL or VH that was not inactive. Typical inactive VH and inactive VL domains are formed by mutation of the wild-type VH or VL sequence, as described in more detail below. Typical mutations are within CDR1, CDR2 or CDR3 of VH or VL. A typical mutation involves placement of a linker domain in CDR2, thereby creating an inactive VH or inactive VL domain. In contrast, an active VH or active VL is one that, after mating with its active sibling partner, i.e. VL or VH, respectively, is able to specifically bind to its target antigen. Thus, it will be understood that the pseudo Fv may be a VH/iVL pair, an iVH/VL pair, or an iVH/iVL pair.

In contrast, as used herein, the term active refers to a CD3 binding domain that is capable of specifically binding to CD3. The term is used in two contexts: (a) when referring to one member of an Fv binding pair (ie VH or VL) that has a sequence capable of pairing with its cognate partner and, in particular, binding to CD3; and (b) by referring to a pair of related (ie, VH and VL) sequences capable of specifically binding to CD3. An exemplary active VH, VL, or VH/VL pair is a wild-type or parental sequence.

CD-x belongs to the cluster of differentiation (CD) proteins. In illustrative embodiments, CD-x is selected from those CD proteins that play a role in T cell recruitment or activation in a subject administered a polypeptide construct of the present invention. In an exemplary embodiment, CD-x is CD3, the sequence of which is shown in FIG. 5.

The term binding/binding domain characterizes, in connection with this invention, a domain that (specifically) binds/interacts with/recognizes a given target epitope or a given target site on target molecules (antigens), for example: EGFR and CD3, respectively. The structure and function of the target antigen binding domain (EGFR recognition) and, preferably, also the structure and/or function of the CD3 binding domain (CD3 recognition) is/are based on the structure and/or function of an antibody, e.g., a complete or whole immunoglobulin molecule, including odASD. In accordance with the present invention, the target antigen-binding domain is typically characterized by the presence of three CDRs that bind the tumor target antigen (commonly referred to in the art as variable heavy domains, although there are no corresponding light chain CDRs). Alternatively, AMO ASDs may include three light chain CDRs (ie, CDR1, CDR2 and CDR3 VL regions) and/or three heavy chain CDRs (ie, CDR1, CDR2 and CDR3 VH regions). The CD3 binding domain preferably also contains at least the minimum structural requirements of the antibody that allow binding to the target. More preferably, the CD3 binding domain contains at least three light chain CDRs (ie, CDR1, CDR2 and CDR3 VL regions) and/or three heavy chain CDRs (ie, CDR1, CDR2 and CDR3 VH regions). In illustrative embodiments, the target antigen and/or CD3 binding domain is contemplated to be, or may be, obtained by phage display or library screening methods.

The term domain as used herein means a protein sequence with a function as described herein. The domains of this invention include tumor antigen binding domains (AMO domains), variable heavy domains, variable light domains, linker domains and half-life extension domains.

By domain linker, as used herein, is meant an amino acid sequence that connects two domains, as defined herein. Domain linkers can be severable linkers, restricted severable linkers, non-cutable linkers, restricted non-cutable linkers, scFv linkers, etc.

By cutable linker (SL), as used herein, is meant an amino acid sequence that can be cut by a protease, preferably a human protease, in pathological tissue, as described herein. Cutable linkers typically have a length of

- 13 045012 at least 3 amino acids, with 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids being used in this invention, depending on the required flexibility. A number of cut linker sequences can be found in FIG. 6 and fig. 5.

By non-cutting linker (NCL) as used herein is meant an amino acid sequence that cannot be cut by a human protease under normal physiological conditions.

By limiting cleavage linker (RLL), as used herein, is meant a short polypeptide that contains a protease cleavage site (as defined herein) that connects two domains, as described herein, such that the two domains cannot significantly interact with each other. each other until they are on different polypeptide chains, for example, after cutting. When ORL is attached to a VH and VL domain, as defined herein, VH and VL cannot independently assemble to form a functional Fv prior to scission due to steric intramolecular constraints (although they can assemble into pseudo Fv domains in an intermolecular junction). Once cut by an appropriate protease, VH and VL can be assembled to form an active antigen-binding domain in an intermolecular manner. Typically, ORLs are less than 10 amino acids in length, with 9, 8, 7, 6, 5 and 4 amino acids being used in this invention. In general, protease cleavage sites are typically at least 4+ amino acids in length to provide sufficient specificity, as demonstrated in FIG. 6.

By limiting uncutting linker (UCRL), as used herein, is meant a short polypeptide that connects two domains, as described herein, such that the two domains cannot significantly interact with each other, and which is not cut by human proteases to a significant extent under physiological conditions. conditions.

By restricted Fv domain, as used herein, is meant an Fv domain that contains an active variable heavy domain and an active variable light domain covalently linked to a limiting linker as described herein, such that the active heavy and light variable domains cannot be intramolecularly interact to form an active Fv that will bind an antigen such as CD3. Thus, a restricted Fv domain is a domain that is similar to scFv but is unable to bind antigen due to the presence of a restricting linker (although they can assemble intramolecularly with inert variable domains to form pseudo Fv domains).

By pseudo Fv domain, as used herein, is meant a domain that contains a pseudo or inactive variable heavy domain or a pseudo or inactive variable light domain, or both, linked by a domain linker (which may be cleavable, limiting, non-cutting, non-limiting, etc. .). The iVH and iVL domains of the pseudo Fv domain do not bind human antigen, either when associated with each other (iVH/iVL) or when associated with active VH or VL; thus, the Fv domains iVH/iVL, iVH/VL and iVL/VH do not bind appreciably to human protein, so these domains are inert in humans.

By single chain Fv or scFv as used herein is meant a variable heavy domain (VH) covalently attached to a variable light domain (VL), typically using a domain linker as discussed herein, to form an scFv or scFv domain. The scFv domain can have any orientation from N to C-terminus (VH-linker-VL or VL-linker-VH).

By single domain Fv, sdFv or sdASD, as used herein, we mean an antigen binding domain that has only three CDRs, typically based on camel antibody technology. See: Protein Engineering 9 (7): 1129-35 (1994); Rev Mol Biotech 74: 277-302 (2001); Ann Rev Biochem 82: 775–97 (2013). As stated herein, there are two main types of sdASDs used in this document: sdASDs that bind to AMOs and are annotated as such (sdASD-AMO for the generic term, or sdASD-EGFR for the one that binds to EGFR, sdASD-FOLRI for the one that binds to FOLR1, etc.) and odASD, which bind to HSA (odASD-HSA or odASD (1/2)).

The term protease cleavage site refers to the amino acid sequence recognized and cut by a protease. Suitable protease cleavage sites are outlined below and demonstrated in FIG. 5 and 6.

As used herein, the term protease cleavage domain refers to a peptide sequence including a protease cleavage site and any linkers between individual protease cleavage sites and between the protease cleavage site(s) and other functional components of the constructs of this invention (e.g., VH, VL, iVH, iVL, antigen target binding domain(s), half-life extension domain(s), etc.). As discussed herein, the protease cleavage domain may also include additional amino acids if necessary, for example, to impart flexibility.

The term COBRA™ and conditional bispecific redirection activation refers to a bispecific conditionally effective protein that has a number of functional protein domains. In some embodiments, one of the functional domains is an antigen-binding

- 14 045012 calling domain (ASD), which binds the tumor target antigen (AMO). In certain embodiments, the other domain is an ASD that binds to a T cell antigen under certain conditions. T cell antigen includes, but is not limited to, CD3. The term hemi-COBRA™ refers to a conditionally effective protein that can bind a T cell antigen, while a hemi-COBRA variable heavy chain can associate with the variable light chain of another hemi-COBRA™ (complementary hemi-COBRA™) due to for the inherent ability to self-assemble when concentrated on the surface of a cell expressing the target.

Fusion proteins of this invention.

The fusion proteins of this invention have a number of different components, commonly referred to herein as domains, that are linked to each other in various ways. Some of the domains are binding domains, each of which binds to a target antigen (eg, AMO or CD3). Because they bind to more than one antigen, they are referred to herein as multispecific; for example, the prodrug construct of this invention can bind to AMO and CD3 and is thus bispecific. The protein may also have higher specificity; for example, if the first aAMO binds to EGFR, the second to EpCAM, and there is an anti-CD3 binding domain, it will be a trispecific molecule. Likewise, adding an anti-HSA binding domain to this construct would make it tetraspecific, as demonstrated in FIG. 3B.

As will be appreciated by those skilled in the art, the proteins of this invention may have different valencies and may also be multispecific. That is, the proteins of this invention can bind a target with more than one binding site; for example, Pro140 is divalent for EGFR.

Proteins of the present invention may contain CD3 antigen binding domains arranged in various ways as described herein, tumor target antigen binding domains, half-life extension domains, linkers, etc.

CD3 antigen binding domains.

The specificity of the T cell response is mediated by antigen recognition (shown in the context of the major histocompatibility complex, MHC) by the T cell receptor complex. As part of the T cell receptor complex, CD3 is a protein complex that contains a CD3y (gamma) chain, a CD3δ (delta) chain, two CD3ε (epsilon) chains, and two CD3Z (zeta) chains, which are present on the cell surface. CD3 molecules bind to the α (alpha) and β (beta) chains of the T cell receptor (TCR), forming the TCR complex. Clustering of CD3 on T cells, for example by Fv domains that bind to CD3, results in T cell activation similar to that of the T cell receptor but independent of its lineage-specific specificity.

However, as is known in the art, activation of CD3 can cause a number of toxic side effects, and accordingly, the present invention is directed to ensuring active binding of the CD3 polypeptides of this invention only in the presence of tumor cells where specific proteases are found, which then cleave the prodrug polypeptides at the present invention to provide an active CD3 binding domain. Thus, in the present invention, the binding of the anti-CD3 Fv domain to CD3 is regulated by a protease cleavage domain, which limits the binding of the CD3 Fv domain to CD3 only in the microenvironment of the pathological cell or tissue with elevated levels of proteases, for example, in the tumor microenvironment, as described in this document.

Accordingly, the present invention provides two sets of VH and VL domains, an active set (VH and VL) and an inactive set (iVH and iVL), all four present in the prodrug construct. The design is formatted such that a set of VH and VL cannot be independently associated, but rather associated with an inactive partner, such as iVH and VL and iVL and VH, as demonstrated herein.

1. Active anti-CD3 variable heavy and variable light domains.

There are a number of suitable active sets of CDRs and/or VH and VL domains that are known in the art and find use in this invention. For example, the CDR and/or VH and VL domains are derived from known anti-CD3 antibodies, such as, for example, muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34 or I2C, TR- 66 or X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII -141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1 and WT-31.

In one embodiment, the VH and VL sequences that form the active Fv domain that binds human CD3 are shown in FIG. 5. As demonstrated herein, these active VH (aVH) and active VL (aVL) domains can be used in various configurations and formats 1, 2, 3 and 4.

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2. Inactive variable heavy and variable light domains against CD3.

The inactive iVH and iVL domains contain regular framework regions (FRs) that permit association, such that the inactive variable domain will associate with the active variable domain, rendering the pair inactive, eg, unable to bind CD3.

As will be appreciated by those skilled in the art, there are a number of inactive variable domains that find use in the present invention. Essentially, any human framework variable domain that allows self-assembly with another variable domain can be used, regardless of which amino acids are at the CDR position in the variable region. For clarity, inactive domains are said to contain CDRs, although technically inactive variable domains do not provide binding capabilities.

As will be appreciated in the art, inactive VH or VL domains are typically simply generated, and this can be done in a variety of ways. In some embodiments, generation of inactive variable domains is typically accomplished by changing one or more CDRs of the active Fv, including making changes to one or more of the three CDRs of the active variable domain. This can be done by making one or more amino acid substitutions at functionally important residues in one or more CDRs, replacing some or all CDR residues with random sequences, replacing one or more CDRs with tags or flag sequences, and/or swapping CDRs and/or variable regions with those of an irrelevant antibody (for example, one that is directed to a protein of another organism).

In some cases, only one of the CDRs in a variable region may be changed to render it inactive, although other embodiments include changes in one, two, three, four, five or six CDRs.

In some cases, inactive domains can be designed to promote selective binding in a prodrug format to promote the formation of intramolecular iVH-VL and VH-iVL domains prior to scission (eg, through intermolecular pairing). See, for example, Igawa et al., Protein Eng. Des. Selection 23 (8): 667-677 (2010), which is incorporated herein by reference in its entirety, and specifically for amino acid residue substitutions at the interaction surface.

In some embodiments, the CD3 binding domain of the polypeptide constructs described herein exhibits not only a strong CD3 binding affinity for human CD3, but also exhibits excellent cross-reactivity with the corresponding Cynomolgus monkey CD3 proteins. In some cases, the CD3-binding domain of the polypeptide constructs is cross-reactive with CD3 from the cynomolgus monkey. In some cases, human:cynomolgus KD to CD3 ratios range from 5 to 0.2.

In some embodiments, the CD3 binding domain of an antigen binding protein can be any domain that binds to CD3, including, but not limited to, domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody. In some cases, it is useful for the CD3 binding domain to be from the same species in which the antigen binding protein will ultimately be used. For example, for use in humans, it may be useful for the CD3 binding domain of the antigen binding protein to contain human or humanized residues from the antigen binding domain of an antibody or antibody fragment.

Thus, in one aspect, the antigen binding domain comprises a humanized or human binding domain. In one embodiment, the humanized or human anti-CD3 binding domain comprises one or more (e.g., all three) light chain complementarity determining region 1 (CDR1 LC), light chain complementarity determining region 2 (CDR2 LC), and light chain complementarity determining region 3 (CDR3 LC) of the humanized or human anti-CD3 binding domain described herein, and/or one or more (e.g., all three) heavy chain complementarity determining region 1 (CDR1 HC), heavy chain complementarity determining region 2 (CDR2 HC) and heavy chain complementarity determining region 3 (CDR3 HC) of the humanized or human anti-CD3 binding domain described herein, e.g., humanized or human anti-CD3. A CD3 binding domain comprising one or more, eg all three, LC CDRs and one or more, eg all three HC CDRs.

In some embodiments, the humanized or human anti-CD3 binding domain comprises a humanized or human light chain variable region specific for CD3, wherein the light chain variable region specific for CD3 includes a human or non-human light chain CDR in a human light chain framework region. In some cases, the light chain framework region is a light chain framework (lambda). In other cases, the light chain framework region is a light chain framework (kappa).

In some embodiments, one or more CD3 binding domains are humanized or fully human. In some embodiments, one or

- 16 045012 several activated CD3 binding domains have a binding KD of 1000 nM or less to CD3 on cells expressing CD3. In some embodiments, one or more activated CD3 binding domains have a KD binding of 100 nM or less to CD3 on cells expressing CD3. In some embodiments, one or more activated CD3 binding domains have a binding KD of 10 nM or less to CD3 on cells expressing CD3. In some embodiments, one or more CD3-binding domains are cross-reactive with cynomolgus CD3. In some embodiments, one or more CD3 binding domains comprise an amino acid sequence provided herein.

In some embodiments, the humanized or human anti-CD3 binding domain comprises a humanized or human CD3-specific heavy chain variable region, wherein the CD3-specific heavy chain variable region includes a human or non-human heavy chain CDR in a human heavy chain framework region.

In one embodiment, the anti-CD3 binding domain is an Fv comprising a light chain and a heavy chain of the amino acid sequence provided herein. In one embodiment, the anti-CD3 binding domain comprises a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but no more than 30, 20, or 10 modifications (e.g., substitutions) the amino acid sequence of the light chain variable region presented herein, or a sequence with 95-99% identity to the amino acid sequence presented herein; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions), but no more than 30, 20, or 10 modifications (e.g., substitutions) of the heavy chain variable region amino acid sequence represented by herein, or a sequence with 95-99% identity to the amino acid sequence presented herein. In one embodiment, the humanized or human anti-CD3 binding domain is a scFv, and a light chain variable region containing an amino acid sequence described herein is linked to a heavy chain variable region containing an amino acid sequence described herein by the scFv linker. The light chain variable region and the heavy chain variable region of the scFv may be, for example, in any of the following orientations: light chain variable region - scFv linker heavy chain variable region or heavy chain variable region - scFv linker - light chain variable region.

In some embodiments, the CD3 binding domain of the antigen binding protein has an affinity for CD3 on cells expressing CD3 with a KD of 1000 nM or less, 100 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less , 1 nM or less or 0.5 nM or less. In some embodiments, the CD3 binding domain of the antigen binding protein has an affinity for CD3ε with a KD of 1000 nM or less, 100 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. In further embodiments, the CD3 binding domain of the antigen binding protein has low affinity for CD3, i.e. about 100 nM or more.

CD3 binding affinity can be determined, for example, by the ability of the antigen binding protein itself or its CD3 binding domain to bind CD3 coated on an assay plate; presented on the surface of the microbial cell; in solution; etc., as is known in the art, typically using Biacore or Octet assays. The binding activity of the antigen binding protein itself, or the CD3 binding domain thereof, of the present invention to CD3 can be assayed by immobilizing a ligand (e.g., CD3) or the antigen binding protein itself or the CD3 binding domain thereof on a bead, support, cell, etc. The agents can be added to an appropriate buffer and the binding partners can be incubated for some time at a given temperature. After washing to remove unbound material, the bound protein can be released, for example, using SDS, high pH buffers, etc., and analyzed, for example, using surface plasmon resonance (SPR).

In many embodiments, the preferred active and inert binding domains are those shown in FIG. 5.

Antigen-binding domains directed to tumor target antigens.

In addition to the described CD3 and half-life extension domains, the polypeptide constructs described herein also contain targeting domains that bind to one or more target antigens or one or more sites on one target antigen. It is contemplated herein that the polypeptide construct of the invention is cut, for example, in a disease-specific microenvironment or in the blood of a subject at a protease cutting domain, and that each antigen binding target domain will bind to a target antigen on the target cell, thereby activating CD3 -binding domain for T binding

- 17 045012 cells. Typically, AMO-binding domains can bind to their targets before being cut by a protease, so they can wait for activation on the target cell as a decoy for T cells. At least one target antigen is involved in and/or associated with a disease, disorder or condition. Typical target antigens include antigens associated with a proliferative disease, a neoplastic disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, graft-versus-host disease, or host-versus-graft disease. In some embodiments, the target antigen is a tumor antigen expressed on a tumor cell. Alternatively, in some embodiments, the target antigen is associated with a pathogen, such as a virus or bacterium. The at least one target antigen may also be directed against healthy tissue.

In some embodiments, the target antigen is a cell surface molecule, such as a protein, lipid, or polysaccharide. In some embodiments, the target antigen is a tumor cell, a virally infected cell, a bacterially infected cell, a damaged red blood cell, an arterial plaque cell, or a fibrous tissue cell.

Preferred embodiments of the invention use odASDs as targeting domains. They are preferred over scFv ASDs since the addition of other VH and VL domains to the construct of this invention may complicate the formation of pseudo Fv domains.

In some embodiments, the prodrug constructs of this invention utilize a single AMO binding domain, typically as depicted in FIG. 3A, as ODASDAMO pairs, and FIG. 4, in a format 4 configuration. In FIG. 4 demonstrates the use of a single ASD against EGFR, although other AMO binding domains can be used.

In some embodiments, particularly in the Format 1 and Format 2 constructs, the prodrug constructs of this invention utilize two AMO ASDs, again preferably in the ODASD-AMO format. When dual targeting domains are used, they can bind to the same epitope of the same AMO. For example, as discussed herein, many of the constructs herein use two identical targeting domains. In some embodiments, two targeting domains that bind to different epitopes of the same AMO may be used, for example, as demonstrated in FIG. 5, two EGFR sDASDs bind to different epitopes on human EGFR. In some embodiments, the two targeting domains bind to different AMOs, see, for example, FIG. 55.

The polypeptide constructs contemplated herein comprise at least one antigen binding domain, wherein said antigen binding domain binds to at least one target antigen. In some embodiments, the target antigen binding domains specifically bind to a cell surface molecule. In some embodiments, the target antigen binding domains specifically bind to a tumor antigen. In some embodiments, the target antigen binding domains specifically and independently bind to a tumor target antigen (TAA) selected from at least one of EpCAM, EGFR, HER-2, HER-3, cMet, LyPD3, B7H3, CEA and FOLR1.

Of particular use in the present invention are human EGFR sDASDs, as demonstrated in FIG. 5.

Additional uses in this invention are sDASD for human FOLR1, as demonstrated in FIG. 5.

Further applications in this invention are sDASD for human B7H3, as demonstrated in FIG. 5.

Additional uses in this invention are sDASD for human EpCAM, as demonstrated in FIG. 5.

In some embodiments, the protein before cutting by the protease cutting domain is less than about 100 kDa. In some embodiments, the protein after cutting by the protease cutting domain is from about 25 to about 75 kDa. In some embodiments, the protein, before being cut by the protease, is of a size that is above the renal threshold for first-pass clearance. In some embodiments, the protein has a half-life of at least about 50 hours before being cut by a protease. In some embodiments, the protein has a half-life of at least about 100 hours before being cut by a protease. In some embodiments, the protein has increased tissue penetration Compared to IgG, it is also a target antigen. In some embodiments, the protein has increased tissue distribution compared to IgG to the same target antigen.

Half-life extension domains.

The MCE proteins of this invention (again, also referred to herein as COBRA™ proteins or constructs) optionally contain half-life extension domains. Such domains are expected to include, but are not limited to, HSA-binding domains,

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Fc domains, small molecules and other half-life extension domains known in the art.

Human serum albumin (HSA) (molecular weight ~67 kDa) is the most abundant protein in plasma, present at concentrations of about 50 mg/mL (600 μM) and having a half-life in humans of about 20 days. HSA serves to maintain plasma pH, promotes colloidal blood pressure, functions as a transporter for many metabolites and fatty acids, and serves as a major transport protein for drugs in plasma.

Noncovalent association with albumin prolongs the half-life of short-lived proteins. For example, recombinant fusion of the albumin-binding domain to a Fab fragment resulted in a 25- and 58-fold reduction in in vivo clearance and a 26- and 37-fold increase in half-life when administered intravenously to mice and rabbits, respectively, compared with administration of the Fab fragment alone. In another example, when insulin is acylated with fatty acids to stimulate association with albumin, a long-lasting effect is observed when injected subcutaneously into rabbits or pigs. Together, these studies demonstrate a relationship between albumin binding and prolonged action.

In one aspect, the antigen binding proteins described herein contain a half-life extension domain, for example a domain that specifically binds to HSA. In other embodiments, the HSA-binding domain is a peptide. In other embodiments, the HSA-binding domain is a small molecule. The HSA binding domain of the antigen binding protein is expected to be quite small, being no more than 25 kDa, no more than 20 kDa, no more than 15 kDa, or no more than 10 kDa in some embodiments. In some cases, the HSA-binding domain is 5 kDa or less if it is a peptide or small molecule.

In many embodiments, the half-life extension domain is a single domain antigen binding domain from a single domain antibody that binds to HSA. This domain is commonly referred to herein as odASD for human HSA (odASD-HSA) or, alternatively, odASD( _1/2 ) to distinguish these binding domains from odASD for AMO. A particularly useful odASD ( _1/2 ) is demonstrated in FIG. 5.

The antigen binding protein half-life extension domain provides altered pharmacodynamics and pharmacokinetics of the antigen binding protein itself. As stated above, the half-life extension domain prolongs the half-life. The half-life extension domain also alters pharmacodynamic properties, including changes in tissue distribution, penetration, and diffusion of the antigen-binding protein. In some embodiments, the half-life extension domain provides improved tissue (including tumor) targeting, tissue penetration, tissue distribution, intratissue diffusion, and increased potency compared to a protein without the extended half-life binding domain. In one embodiment, therapeutic methods effectively and efficiently use a reduced amount of antigen binding protein, resulting in reduced side effects such as reduced cytotoxicity of non-tumor cells.

In addition, characteristics of a half-life extension domain, such as an HSA binding domain, include the binding affinity of the HSA binding domain for HSA. The affinity of the specified HSA-binding domain can be selected to target a specific half-life in a particular polypeptide construct. Thus, in some embodiments, the HSA-binding domain has high binding affinity. In other embodiments, the HSA binding domain has intermediate binding affinity. In still other embodiments, the HSA-binding domain has low or negligible binding affinity. Typical binding affinities include KD concentrations of 10 nM or less (high), 10 to 100 nM (medium), and greater than 100 nM (low). As stated above, binding affinity to HSA is determined by known methods such as surface plasmon resonance (SPR).

Protease cutting sites.

The protein compositions of this invention, and in particular the prodrug constructs, contain one or more protease cleavage sites typically present in cleavage linkers as described herein.

As described herein, the prodrug constructs of this invention contain at least one protease cleavage site containing an amino acid sequence that is cut by at least one protease. In some cases, the MCE proteins described herein contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more protease cutting sites that are cut by at least one protease. As discussed more fully herein, when more than one protease cleavage site is used in a prodrug design, they may be the same (eg, multiple cleavage sites cut by a single protease) or different (two or more cleavage sites cut by at least two different proteases). As those skilled in the art will appreciate, constructs containing three or more protease cleavage sites may use one, two, three, etc.; for example some

- 19 045012 designs can use three sites for two different proteases, etc.

The amino acid sequence of the protease cleavage site will depend on the target protease. As is known in the art, there are a number of human proteases that are found in the body and may be associated with disease conditions.

Proteases are known to be secreted by certain diseased cells and tissues, such as tumor or cancer cells, creating a protease-rich microenvironment or protease-rich microenvironment. In some cases, the subject's blood is rich in proteases. In some cases, cells surrounding the tumor release proteases into the tumor microenvironment. Cells surrounding the tumor that secrete proteases include, but are not limited to, tumor stromal cells, myofibroblasts, blood cells, mast cells, B cells, NK cells, regulatory T cells, macrophages, cytotoxic T lymphocytes, dendritic cells, mesenchymal stem cells, polymorphonuclear cells and other cells. In some cases, proteases are present in the subject's blood, such as proteases that target amino acid sequences found in microbial peptides. This feature allows targeted therapeutics, such as antigen-binding proteins, to have additional specificity, since T cells will not be bound by the antigen-binding protein except in the protease-rich microenvironment of the target cells or tissues.

Proteases are proteins that break down proteins, in some cases in a sequence-specific manner. Proteases include, but are not limited to, serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamine proteases, metalloproteases, aspartic peptide lyases, serum proteases, cathepsins (e.g., cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, Cathepsin L, Cathepsin S), kallikreins, hK1, hK10, hK15, KLK7, Granzyme B, plasmin, collagenase, type IV collagenase, stromelysin, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin, actinidain, bromelain, calpain, caspases (eg caspase-3), Mir1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepentesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteases ( MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, meprin, urokinase plasminogen activator (UPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-1 β-converting enzyme, thrombin, FAP ( FAP-α), dipeptidyl peptidase and dipeptidyl peptidase IV (DPP-IV/CD26).

Some suitable proteases and protease cutting sequences are demonstrated in FIG. 5 and 6.

Linkers.

As discussed herein, the various domains of the invention are typically linked to each other using amino acid linkers, which can also impart functionality, including flexibility or inflexibility (eg, steric restriction), as well as in situ protease cutability. . These linkers can be classified in several ways.

The present invention provides domain linkers that are used to connect two or more domains (eg, VH and VL, a tumor antigen-targeting domain (TAB-binding domain) to a VH or VL, half-life extension domain to another component, etc. For example, domain linkers can be non-cutting (NCL), severable (RL), limiting and cutting (ORL), and limiting and non-cutting (ONRL).

1. Uncut linkers.

In some embodiments, the domain linker is not cut. Typically, they can be one of two types: uncut and flexible, which allows the components above and below the linker in the constructs to self-assemble intramolecularly in certain ways; or uncut and confining, in which the two components separated by a linker are not capable of intramolecular self-assembly. It should be noted, however, that in the latter case, although the two domain components that are separated by an uncutable limiting linker do not self-assemble intramolecularly, the other intramolecular components will self-assemble to form pseudo Fv domains.

Uncut but flexible linkers.

In this embodiment, a linker is used to join the domains together to maintain the functionality of the domains, typically through longer flexible domains that are not cut by proteases in situ in the patient. Examples of internal non-cutting linkers suitable for linking domains in polypeptides of this invention include, but are not limited to, (GS)n, (GGS)n, (GGGS)n, (GGSG)n, (GGSGG)n, or (GGGGS) )n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker length may be about 15 amino acids.

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Uncut and limiting linkers.

In some cases, linkers do not contain a cleavage site and are also too short to allow the protein domains separated by the linker to intramolecularly self-assemble and are limiting non-cutting linkers or NLCLs. For example, in Pro186, the active VH and active VL are separated by 8 amino acids (8-mer), which prevents VH and VL from independently assembling into the active antigen-binding domain. In some embodiments, the linker is still flexible; for example, (GGGS)n, where n=2. In other embodiments, although generally less preferred, more stringent linkers may be used, such as those that include proline or bulky amino acids.

2. Cutable linkers.

All prodrug constructs herein contain at least one scissile linker. Thus, in one embodiment, the linker domains are severable (CL), and are sometimes referred to herein as a protease cutting domain (DCP). In this embodiment, the RL contains a protease cleavage site as described herein and depicted in FIG. 5 and fig. 6. In some cases, the RL contains only a protease cutting site. Optionally, depending on the length of the recognition and cutting site, there may be several additional binding amino acids at one or both N- or C-termini of the RL; for example, there may be 1, 2, 3, 4 or 5 amino acids at one or both of the N- and C-termini of the cutting site. Thus, cutable linkers can also be restrictive (eg 8-mer) or flexible.

Of particular interest in the present invention are MMP9 cleavable linkers and meprin cleavable linkers, in particular MMP9 cleavable linkers and meprin cleavable linkers.

Domains according to this invention.

The present invention provides a number of different formats for the prodrug polypeptides of the present invention. The present invention provides limited Fv domains and limited pseudo Fv domains. In addition, the present invention provides multivalent conditionally effective (MCE) proteins that contain two Fv domains but are non-isomerizing constructs. As stated herein, these can be non-isomerizing severable formats or non-isomerizing non-cutting formats, although each construct contains at least one protease cutting domain.

It is important to note that although both of these domains (Fv domain and pseudo Fv domain) are referred to herein as restricted, this means that, as discussed above and demonstrated in FIG. 37, 38 and 39 only one of them needs to be constrained, although usually when both linkers are constrained the protein has better expression.

Those skilled in the art will appreciate that for formats 1, 2, and 4, there are four possibilities for the N- to C-terminal ordering of the limited and pseudo Fv domains of this invention (linkers not shown): aVH-aVL and iVL- iVH, aVH-aVL and iVH-iVL, aVL-aVH and iVL-iVH, aVL-aVH and iVH-iVL. All four were tested and all four have activity, although the first order, aVH-aVL and iVL-iVH, show better expression than the other three. Thus, although the description herein is generally shown in aVH-aVL and iVL-iVH format, all disclosures herein include other orderings for these domains.

It should be noted that generally the N- to C-terminal order for full-length constructs of this invention is based on the aVH-aVL and iVL-iVH orientations.

In addition, it is known in the art that in humans there may be immunogenicity derived from the C-terminal sequences of certain ASDs. Accordingly, in general, and particularly when the C-terminus of constructs ends in odASD (eg, the odASD-HSA domains of many constructs), a histidine tag (His6 or His10) can be used. Many or most of the sequences herein were generated using His6 C-terminal tags for purification reasons, but these sequences can also be used to reduce immunogenicity in humans, as demonstrated in Holland et al., DOI 10.1007/s10875-013-9915-0 and WO2013/024059.

Restricted Fv domains.

This invention provides constrained Fv domains that contain an active VH and an active VL domain that are covalently attached using a constraint linker (which, as defined herein, can be slitable (formats 1 and 3) or uncutable (formats 2 and 4 )). The limiting linker prevents intramolecular association between aVH and aVL in the absence of cleavage. Thus, the restricted Fv domain typically contains a set of six CDRs contained within variable domains, with vhCDR1, vhCDR2 and vhCDR3 VH binding human CD3, and vlCDR1, vCDR2 and vlCDR3 VL binding human CD3, but in a prodrug format (e.g. uncut ) VH and VL are unable to sterically associate to form an active binding domain, preferring instead to associate intramolecularly with the pseudo Fv.

Restricted Fv domains may contain active VH and active VL (aVH and aVL) or inactive VH and VL (iVH and iVL, in which case it is a restricted pseudo Fv domain) or combinations thereof, as

- 21 045012 is described in this document.

As will be appreciated by those skilled in the art, the order of VH and VL in a limited Fv domain can be (N- to C-terminus) either VH-linker-VL or VL-linker-VH.

As discussed herein, for format 1 constructs, the restricted Fv domains may comprise VH and VL linked to the use of a scissile linker, in cases such as those demonstrated in FIG. 5 and fig. 6. In this embodiment, the limited Fv domain has the structure (from N- to C-terminus) vhFR1-vhCDR1-vhFR2-vhCDR2-vhFR3-vhCDR3-vhFR4-ORL-vlFR1-vlCDR1-vlFR2vlCDR2-vlFR3-vlCDR3-vlFR4. Typically, the restricted Fv domain contains active VH and VL domains (e.g., capable of binding CD3 when associated) and thus has the structure (from N- to C-terminus) vhFR1-avhCDR1-vhFR2-avhCDR2-vhFR3- avhCDR3-vhFR4-ORL-vlFR1-avlCDR1-vlFR2avlCDR2-vlFR3-avlCDR3-vlFR4.

As described herein, for format 2 constructs, the restricted Fv domains may contain VH and VL linked by the use of a non-cutting linker. In this embodiment, the limited Fv domain has the structure (from N- to C-terminus) vhFR1-vhCDR1-vhFR2-vhCDR2-vhFR3vhCDR3-vhFR4-ONRL-vlFR1-vlCDR1-vlFR2-vlCDR2-vlFR3-vlCDR3-vlFR4. Typically, the restricted Fv domain contains active VH and VL domains (e.g., capable of binding to CD3 when associated) and thus has the structure (from N- to C-terminus) vhFR1-avhCDR1-vhFR2-avhCDR2-vhFR3avhCDR3 -vhFR4-ONRL-vlFR1-avlCDR1-vlFR2-avlCDR2-vlFR3-avlCDR3-vlFR4.

Of particular use in this invention are limiting non-cutting Fv domains having aVH having SEQ ID NO: 61, aVL having SEQ ID NO: 49, and linker domains having SEQ ID NO: 74.

Limited pseudo Fv domains.

The present invention provides limited pseudo Fv domains containing inactive or pseudo iVH and iVL domains that are covalently linked using a restriction linker (which, as defined herein, can be severable or non-cutable). The limiting linker prevents intramolecular association between iVH and iVL in the absence of cleavage. Thus, the overall restricted pseudo Fv domain includes iVH and iVL with framework regions that allow association (in an unrestricted format) of iVH and iVL, although the resulting pseudo Fv domain does not bind to human protein. iVH domains can assemble with aVL domains, and iVL domains can assemble with aVH domains, although the resulting structures do not bind CD3.

Restricted pseudo Fv domains contain inactive VH and VL (iVH and iVL).

As will be appreciated by those skilled in the art, the order of VH and VL in a limited pseudo Fv domain can be either (N- to C-terminal) VH-linker-VL or VL-linker-VH.

As described herein, limited pseudo Fv domains can contain iVH and iVL linked by the use of a non-cutting linker, as demonstrated in formats 1, 2 and 4, or with cutable linkers, as demonstrated in format 3.

Typically, the restricted Fv domain contains inert VH and VL domains (eg, capable of binding CD3 when associated) and thus has the structure (from N- to C-terminus) vhFR1ivlCDR1-vhFR2-ivlCDR2-vhFR3-ivlCDR3- vhFR4-ONRL -vlFR1-ivhCDR1-vlFR2-ivhCDR2-vlFR3-ivhCDR3vlFR4.

Of particular use in this invention are limited uncut pseudo Fv domains having iVH having SEQ ID NO: 65 or SEQ ID NO: 69, iVL having SEQ ID NO: 53 or SEQ ID NO: 57, and linker domains having SEQ ID NO: 74.

Formats according to this invention.

As discussed herein, the prodrug constructs of this invention can take a number of different formats, including cuttable formats with two AMO binding domains, uncuttable formats with two AMO binding domains (either of which may have the same AMO binding domains or different binding domains ) and uncut formats with a single target domain.

Cuttable formats with dual targeting.

The present invention provides non-isomerizable cut formats such as format 1 in FIG. 1. In this embodiment, the limited Fv domain comprises VH and VL domains, which are linked using restrictive severable linkers, and the limited pseudo Fv domain uses restrictive non-cutting linkers. For ease of discussion, both of these are referred to herein as limiting, but as discussed above and demonstrated in FIG. 37, fig. 38 and fig. 39 when both linkers are restrictive, the protein has better expression.

All constructs in format 1 (as well as other formats) also have a cleavable linker (RL), which is cut by human tumor protease.

This invention relates to prodrug proteins containing from N- to C-terminus (odASDAMO1)-linker domains-restricted Fv domain-linker domains-(odASD-AMO2)-RL-restricted pseudo Fv domain-linker domains-odASD-HSA.

As will be appreciated by those skilled in the art, the order of VH and VL is either limited

- 22 045012

The Fv domain, or the limited pseudo Fv domain can be (from N- to C-terminus) either VH-linker-VL or VL-linker-VH.

Thus, in one embodiment, the prodrug protein comprises, from N- to C-terminus: (odASD-AMO1)-linker domains-aVH-ORL-aVL-linker domains-(odASD-AMO2)-RL-iVL-ONRLiVH-linker domains-odASD -HSA.

Thus, in one embodiment, the prodrug protein comprises, from N- to C-terminus: (odASD-AMO1)-linker domains-aVH-ORL-aVL-linker domains-(odASD-AMO2)-RL-iVH-ORLiVL-linker domains-odASD -HSA.

Thus, in one embodiment, the prodrug protein comprises, from N- to C-terminus: (odASD-AMO1)-linker domains-aVL-ORL-aVH-linker domains-(odASD-AMO2)-RL-iVL-ORLiVH-linker domains-odASD -HSA.

Thus, in one embodiment, the prodrug protein comprises, from N- to C-terminus: (odASD-AMO1)-linker domains-aVL-ORL-aVH-linker domains-(odASD-AMO2)-RL-iVH-ORLiVL-linker domains-odASD -HSA.

In some embodiments, the prodrug construct comprises odASD (AMO1)domain linker-aVH-ORL-aVL-domain linker-odASD (AMO2)-RL-iVL-ONRL-iVH-NRL-odASD ( _1/2 ). In this embodiment, aVH, aVL, iVH and iVL have the sequences shown in FIG. 5.

In some embodiments, the prodrug construct comprises odASD (AMO1)domain linker-aVH-ORL-aVL-domain linker-odASD (AMO2)-RL-iVL-ONRL-iVH-domain linkerASD ( _1/2 ). In this embodiment, aVH, aVL, iVH, iVL have the sequences shown in FIG. 5. In this embodiment, the two targeting domains bind to the same AMO, which may be EGFR, EpCAM, FOLR1 or B7H3, the sequences for which are depicted in FIG. 5.

In some embodiments, the prodrug construct comprises odASD (AMO1)domain linker-aVH-ORL-aVL-domain linker-odASD (AMO2)-RL-iVL-ONRL-iVH-domain linkerASD ( _1/2 ). In this embodiment, aVH, aVL, iVH, iVL have the sequences shown in FIG. 5. In this embodiment, two targeting domains bind to different AMOs.

In some embodiments, the prodrug construct comprises odASD (AMO1)domain linker-aVH-ORL-aVL-domain linker-odASD (AMO2)-RL-iVL-ONRL-iVH-domain linkerASD ( _1/2 ). In this embodiment, aVH, aVL, iVH, iVL have the sequences shown in FIG. 5. In this embodiment, the two targeting domains bind to EGFR and EpCAM, and odASD-AMO and have the sequences shown in FIG. 5.

In some embodiments, the prodrug construct comprises odASD (AMO1)domain linker-aVH-ORL-aVL-domain linker-odASD (AMO2)-RL-iVL-ONRL-iVH-domain linkerASD ( _1/2 ). In this embodiment, aVH, aVL, iVH, iVL have the sequences shown in FIG. 5. In this embodiment, the two targeting domains bind to EGFR and FOLR1, and odASD-AMO and have the sequences shown in FIG. 5.

In some embodiments, the prodrug construct comprises odASD (AMO1)domain linker-aVH-ORL-aVL-domain linker-odASD (AMO2)-RL-iVL-ONRL-iVH-domain linkerASD ( _1/2 ). In this embodiment, aVH, aVL, iVH, iVL have the sequences shown in FIG. 5. In this embodiment, the two targeting domains bind to EGFR and B7H3, and odASD-AMO and have the sequences shown in FIG. 5.

In some embodiments, the prodrug construct comprises odASD (AMO1)domain linker-aVH-ORL-aVL-domain linker-odASD (AMO2)-RL-iVL-ONRL-iVH-domain linkerASD ( _1/2 ). In this embodiment, aVH, aVL, iVH, iVL have the sequences shown in FIG. 5. In this embodiment, the two targeting domains bind to EpCAM and FOLR1 and odASD-AMO and have the sequences shown in FIG. 5.

In some embodiments, the prodrug construct comprises odASD (AMO1)domain linker-aVH-ORL-aVL-domain linker-odASD (AMO2)-RL-iVL-ONRL-iVH-domain linkerASD ( _1/2 ). In this embodiment, aVH, aVL, iVH, iVL have the sequences shown in FIG. 5. In this embodiment, two targeting domains bind to EpCAM and B7H3, and odASD-AMO and have the sequences shown in FIG. 5.

In some embodiments, the prodrug construct comprises odASD (AMO1)domain linker-aVH-ORL-aVL-domain linker-odASD (AMO2)-RL-iVL-ONRL-iVH-domain linkerASD ( _1/2 ). In this embodiment, aVH, aVL, iVH, iVL have the sequences shown in FIG. 5. In this embodiment, the two targeting domains bind to B7H3 and FOLR1, and odASD-AMO and have the sequences shown in FIG. 5.

In some embodiments, the prodrug construct comprises odASD (AMO1)domain linker-aVH-ORL-aVL-domain linker-odASD (AMO2)-RL-iVL-ONRL-iVH-domain linkerASD ( _1/2 ). In this embodiment, aVH, aVL, iVH, iVL have sequences proceeding

- 23 045012 monsterized in Fig. 5. In this embodiment, two targeting domains bind to the same AMO, which may be EGFR, FOLR1, B7H3 or EpCAM, the sequences for which are depicted in FIG. 5, and ORL and RL are selected from a linker that is cut by MMP9 or meprin, and odASD (1/2) has SEQ ID NO: 45.

In format 1, the preferred domain linker is SEQ ID NO: 74 (which also serves as a preferred limited non-cutting linker).

In format 1, the preferred designs are Pro140 and Pro140b.

Uncut formats.

As shown in FIG. 2, the present invention provides non-isomerizing, non-cutting formats. In this embodiment, it is understood that uncut applies only to the limited Fv domain linkage since the prodrug construct has an activating cut site. In this embodiment, the limited Fv domain contains VH and VL domains, which are linked using restrictive non-cutting linkers, and the limited pseudo Fv domain uses restrictive non-cutting linkers.

As will be appreciated by those skilled in the art, the order of VH and VL in either the limited Fv domain or limited pseudo Fv domain can be (from N- to C-terminus) either VH-linker-VL or VL-linker- VH.

This invention relates to prodrug proteins containing from N- to C-terminus odASD (AMO1)-linker domains-restricted Fv domain-linker domains-odASD (AMO2)-cutting linker-restricted-pseudo Fv domain-linker domains-odASD-HSA ,

As will be appreciated by those skilled in the art, the order of VH and VL in either the limited Fv domain or limited pseudo Fv domain can be (from N- to C-terminus) either VH-linker-VL or VL-linker- VH.

Thus, in one embodiment, the prodrug protein comprises, from N- to C-terminus: (odASD-AMO1)-linker domains-aVH-ONRL-aVL-linker domains-(odASD-AMO2)-RL-iVLOONRL-iVH-linker domains-odASD -HSA.

Thus, in one embodiment, the prodrug protein comprises, from N- to C-terminus: (odASD-AMO1)-domain linker-aVH-ONRL-aVL-domain linker-(odASD-AMO2)-RL-iVHONRL-iVL-domain linker-odASD -HSA.

Thus, in one embodiment, the prodrug protein comprises, from N- to C-terminus: (odASD-AMO1)-linker domains-aVL-ONRL-aVH-linker domains-(odASD-AMO2)-RL-iVLOONRL-iVH-linker domains-odASD -HSA.

Thus, in one embodiment, the prodrug protein comprises, from N- to C-terminus: (odASD-AMO1)-linker domains-aVL-ONRL-aVH-linker domains-(odASD-AMO2)-RL-iVHONRL-iVL-linker domains-odASD -HSA.

In some embodiments, the prodrug protein comprises, from N- to C-terminus: (odASD-AMO 1)-domain linker-aVH-ONRL-aVL-domain linker-(odASD-AMO2)-RL-iVL-ONRL-iVH domain linker-odASD -ASP. In this embodiment, aVH, aVL, iVH, iVL have the sequences shown in FIG. 5. In this embodiment, the two targeting domains bind to the same AMO, which may be EGFR, EpCAM, FOLR1 or B7H3, the sequences for which are depicted in FIG. 5.

In some embodiments, the prodrug protein comprises, from N- to C-terminus: (odASD-AMO 1)-domain linker-aVH-ONRL-aVL-domain linker-(odASD-AMO2)-RL-iVL-ONRL-iVH domain linker-odASD -ASP. In this embodiment, aVH, aVL, iVH, iVL have the sequences shown in FIG. 5. In this embodiment, two targeting domains bind to different AMOs.

In some embodiments, the prodrug protein comprises, from N- to C-terminus: (odASD-AMO 1)-domain linker-aVH-ONRL-aVL-domain linker-(odASD-AMO2)-RL-iVL-ONRL-iVH domain linker-odASD -ASP. In this embodiment, aVH, aVL, iVH, iVL have the sequences shown in FIG. 5. In this embodiment, the two targeting domains bind to EGFR and EpCAM, and odASD-AMO and have the sequences shown in FIG. 5.

In some embodiments, the prodrug protein comprises, from N- to C-terminus: (odASD-AMO 1)-domain linker-aVH-ONRL-aVL-domain linker-(odASD-AMO2)-RL-iVL-ONRL-iVH domain linker-odASD -ASP. In this embodiment, aVH, aVL, iVH, iVL have the sequences shown in FIG. 5. In this embodiment, the two targeting domains bind to EGFR and FOLR1, and odASD-AMO and have the sequences shown in FIG. 5.

In some embodiments, the prodrug protein comprises, from N- to C-terminus: (odASD-AMO 1)-domain linker-aVH-ONRL-aVL-domain linker-(odASD-AMO2)-RL-iVL-ONRL-iVH domain linker-odASD -ASP. In this embodiment, aVH, aVL, iVH, iVL have the sequences shown in FIG. 5. In this embodiment, two targeting

- 24 045012 men bind to EGFR and B7H3, and odASD-AMO and have the sequences shown in FIG. 5.

In some embodiments, the prodrug protein comprises, from N- to C-terminus: (odASD-AMO 1)-domain linker-aVH-ONRL-aVL-domain linker-(odASD-AMO2)-RL-iVL-ONRL-iVH domain linker-odASD -ASP. In this embodiment, aVH, aVL, iVH, iVL have the sequences shown in FIG. 5. In this embodiment, two targeting domains bind to EpCAM and FOLR1, and odASD-AMO and have the sequences shown in FIG. 5.

In some embodiments, the prodrug protein comprises, from N- to C-terminus: (odASD-AMO 1)-domain linker-aVH-ONRL-aVL-domain linker-(odASD-AMO2)-RL-iVL-ONRL-iVH domain linker-odASD -ASP. In this embodiment, aVH, aVL, iVH, iVL have the sequences shown in FIG. 5. In this embodiment, two targeting domains bind to EpCAM and B7H3, and odASD-AMO, and have the sequences shown in FIG. 5.

In some embodiments, the prodrug protein comprises, from N- to C-terminus: (odASD-AMO 1)-domain linker-aVH-ONRL-aVL-domain linker-(odASD-AMO2)-RL-iVL-ONRL-iVH domain linker-odASD -ASP. In this embodiment, aVH, aVL, iVH, iVL have the sequences shown in FIG. 5. In this embodiment, the two targeting domains bind to FOLR1 and B7H3, and odASD-AMO, and have the sequences shown in FIG. 5.

In some embodiments, the prodrug protein comprises, from N- to C-terminus: (odASD-AMO 1)-domain linker-aVH-ONRL-aVL-domain linker-(odASD-AMO2)-RL-iVL-ONRL-iVH domain linker-odASD -ASP. In this embodiment, aVH, aVL, iVH, iVL have the sequences shown in FIG. 5. In this embodiment, the two targeting domains bind to the same AMO, which may be EGFR, FOLR1, B7H3 or EpCAM, the sequences for which are depicted in FIG. 5, and ORL and RL are selected from a linker that is cut by MMP9 or meprin, and odASD (1/2) has SEQ ID NO: 45.

In format 2, the preferred domain linker is SEQ ID NO: 74 (which also serves as a preferred limited non-cutting linker).

In format 2, embodiments specifically include, but are not limited to, Pro186, Pro225, Pro226, Pro233, Pro311, Pro312, Pro313, Pro495, Pro246, Pro254, Pro255, Pro256, Pro420, Pro421, Pro432, Pro479, Pro480, Pro187, Pro221, Pro222, Pro223, Pro224, Pro393, Pro394, Pro395, Pro396, Pro429, Pro430 and Pro431.

Designs aimed at one AMO.

As shown in FIG. 4, format 4 designs are also included in the compositions of this invention, which are similar to format 2 designs, but without the second AMO ASD. In this embodiment, it is understood that uncut applies only to the binding of the limited Fv domain, since the prodrug construct has an activating cut site. In this embodiment, the limited Fv domain contains VH and VL domains, which are linked using constraint non-cutting linkers, and the limited pseudo Fv domain uses limiting non-cutting linkers.

As will be appreciated by those skilled in the art, the order of VH and VL in either the limited Fv domain or the limited pseudo Fv domain can be (from N- to C-terminus) either VH-linker-VL or VL-linker- VH.

This invention relates to prodrug proteins containing from N- to C-terminus odASD (AMO)-linker domains-restricted Fv domain-cutting linker-odASD-HSA-restricted pseudo Fv domain. (Note that for all constructs for this format, oASD-HSA typically does not have a His6 tag, although it may be included).

As will be appreciated by those skilled in the art, the order of VH and VL in either the limited Fv domain or limited pseudo Fv domain can be (from N- to C-terminus) either VH-linker-VL or VL-linker- VH.

Thus, in one embodiment, the prodrug protein contains, from N- to C-terminus: (odASD-AMO)-domain linker-aVH-ONRL-aVL-RL-(odASD-HSA)-domain linker-iVL-ONRLiVH,

Thus, in one embodiment, the prodrug protein contains, from N- to C-terminus: (odASD-AMO)-domain linker-aVH-ONRL-aVL-RL-(odASD-HSA)-domain linker-iVH-ONRLiVL,

Thus, in one embodiment, the prodrug protein contains, from N- to C-terminus: (odASD-AMO)-domain linker-aVL-ONRL-aVH-RL-(odASD-HSA)-domain linker-iVH-ONRLiVL,

Thus, in one embodiment, the prodrug protein contains, from N- to C-terminus: (odASD-AMO)-domain linker-aVL-ONRL-aVH-RL-(odASD-HSA)-domain linker-iVL-ONRL-25 045012 iVH,

Thus, in one embodiment, the prodrug protein comprises, from N- to C-terminus: (odASD-AMO)-domain linker-aVH-ONRL-aVL-RL-(odASD-HSA)-domain linker-iVL-ONRLiVH. In this embodiment, aVH, aVL, iVH, iVL have the sequences shown in FIG. 5. In this embodiment, the targeting domain binds to an AMO, which may be EGFR, EpCAM, FOLR1 or B7H3, the sequences for which are depicted in FIG. 5.

In format 4, the preferred domain linker is SEQ ID NO: 74 (which also serves as a preferred limited non-cutting linker).

In format 4, the preferred odASD-HSA is SEQ ID NO: 45.

Two-protein compositions.

In some embodiments, the compositions of this invention contain two different molecules, sometimes called hemi-COBRA™ or hemi constructs, which, in the absence of cutting, intramolecularly bind to form pseudo Fv. In the presence of a protease, the cleavage sites are cut, releasing the inert variable domains, and the protein pair then forms an active antigen-binding domain with CD3, as generally demonstrated in FIG. 3.

What is important in the design of hemi constructs is that the active variable domain and sdASD-AMO remain together after cutting, so that the two cut parts are held together by the tumor antigen receptor on the tumor surface and can then form an active anti-CD3 binding domain.

There are two different general format 3 designs in which each member of the pair has one odASD-AMO (Fig. 3A) and one with two different odASD-AMOs, each with a different AMO (Fig. 3B).

1. Hemi-COBRA™ constructs with single AMO-binding domains (3A format).

In some embodiments, the first hemi-COBRA™ has N-to-C terminus odASD (AMO1)-aVH-RL domain linker-iVL-domain linker-d-ASD (1/2), and the second has odASD (1/ 2) - domain linker-iVH-RL-aVL-domain linker-odASD (AMO2). In this embodiment _, aVH, aVL, iVH, iVL and odASD( ^1/2 ) have the sequences shown in FIG. 5, and odASD-AMOa binds to human EGFR, EpCAM, FOLR1 and/or B7H3 and has the pattern shown in FIG. 5 sequence.

2. Hemi-COBRA™ constructs with double AMO ASD.

In some embodiments, paired prodrug constructs may have two sDASD-AMO binding domains per construct, as demonstrated in FIG. 3B. In these embodiments, the first member of the pair comprises N- to C-terminal odASD-AMO1-domain linker-odASD-AMO2-aVH-RL-iVL-domain linker-odASD (HSA) and the second member comprises N- to C-terminal odASD-AM01 -domain linker-odASD-AMO2-aVL-RL-iVH-domain linker-odASD-HSA.

The two odASD-AMOs for each member of the pair are different, but usually both members (hemi-COBRA™) have the same two odASD-AMOs, for example, both have EGFR and FOLR1 or EGFR and B7H3, etc.

The two ODAS-AMOs in some embodiments are selected from those demonstrated in FIG. 5.

Methods for making compositions according to this invention.

The prodrug compositions of this invention are prepared as will generally be understood by those skilled in the art and are described generally below.

The present invention provides nucleic acid compositions that encode prodrug compositions of the present invention. As will be appreciated by those skilled in the art, nucleic acid compositions will depend on the format of the prodrug polypeptide(s). Thus, for example, when a format requires two amino acid sequences, such as format 3 constructs, the two nucleic acid sequences may be included in one or more expression vectors for expression. Likewise, prodrug constructs that are a single polypeptide (formats 1, 2, and 4) require a single nucleic acid in a single expression vector for production.

As is known in the art, nucleic acids encoding the components of this invention can be included in expression vectors, as is known in the art, and depending on the host cells used to obtain the prodrug compositions of this invention. Typically, nucleic acids are functionally linked to any number of regulatory elements (promoters, origins of replication, selectable markers, ribosome binding sites, inducers, etc.). Expression vectors can be extrachromosomal or integrating vectors.

The nucleic acids and/or expression vectors of this invention are then transformed into any number of different types of host cells, as is well known in the art, including mammalian cells, bacteria, yeast, insects and/or fungi, and mammalian cells (for example, CHO cells , cells 293) find use in many embodiments.

- 26 045012

The prodrug compositions of this invention are prepared by culturing host cells containing expression vector(s), as is well known in the art. Once produced, conventional antibody purification steps are performed, including a protein A affinity chromatography step and/or an ion exchange chromatography step.

Formulation and administration of prodrug compositions according to this invention.

The prodrug compositions used in accordance with this invention are prepared for storage by mixing the prodrugs (single proteins in the case of formats 1, 2 and 4 and two proteins in the case of format 3) having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (as generally stated in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]), in the form of lyophilized formulations or aqueous solutions.

The prodrug compositions of this invention are administered to a subject in accordance with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time.

The prodrug compositions of this invention are useful in the treatment of cancer.

Examples

Example 1. Preparation and purification of proconstructs.

Transfections.

Each protein (eg, individual proteins for formats 1, 2, and 4) or pairs of constructs (format 3) were expressed from a separate expression vector (pcdna3.4 derivative). Equal amounts of plasmid DNA encoding the hemi-cobra pair or single-stranded constructs were mixed and transfected into Expi293 cells according to the manufacturer's transfection protocol. Conditioned medium was collected 5 days after transfection by centrifugation (6000 rpm x 25') and filtration (0.2 μm filter). Protein expression was confirmed by SDS-PAGE. The constructs were purified and the final buffer composition was: 25 mM citrate, 75 mM arginine, 75 mM NaCl, 4% sucrose, pH 7. The final preparations were stored at -80°C.

Activation of MMP9.

Recombinant (r) human MMP9 was activated according to the following protocol. Recombinant human MMP-9 (R&D #911-MP-010) at a concentration of 0.44 mg/ml (4.7 µM). p -Aminophenylmercuric acetate (APMA) (Sigma) was prepared at a stock concentration of 100 mM in DMSO. The assay buffer is 50 mM Tris, pH 7.5, 10 mM CaCl ₂ , 150 mM NaCl, 0.05% Brij-35.

Dilute pMMP9 with assay buffer to ~100 µg/ml (25 µl hMMP9 + 75 µl assay buffer).

Add p-aminophenylmercuric acetate (APMA) from the 100 mM stock solution in DMSO to a final concentration of 1 mM (1 µL to 100 µL).

Incubate at 37°C for 24 hours.

Dilute MMP9 to 10 ng/µl (add 900 µl assay buffer to 100 µl activated solution).

The concentration of activated pMMP9 is ~100 nM.

Construct splitting for TDCC analyses.

To cut constructs, 100 μl of a protein sample at a concentration of 1 mg/ml (10.5 μM) in preparation buffer (25 mM citric acid, 75 mM L-arginine, 75 mM NaCl, 4% sucrose) was supplied with CaCl ₂ to 10 mM . Activated pMMP9 was added to a concentration of 20–35 nM. The sample was incubated at room temperature overnight (16-20 hours). Completeness of cutting was confirmed using SDS PAGE (10-20% TG, TG running buffer, 200 V, 1 hour). Samples were typically 98% digestible.

Example 2 T-cell dependent cytotoxicity (TDCC) assays.

Firefly luciferase-transformed HT-29 cells were grown to approximately 80% confluence and separated with Versene (0.48 mM EDTA in PBS-Ca-Mg). Cells were centrifuged and resuspended in TDCC medium (5% heat-inactivated FBS in RPMI 1640 with HEPES, GlutaMax, sodium pyruvate, nonessential amino acids, and β-mercaptoethanol). The purified total human T cell population was thawed, centrifuged, and resuspended in TDCC medium.

A coculture of HT-29_Luc cells and T cells was added to 384-well cell culture plates. Serially diluted COBRA was then added to the coculture and incubated at 37°C for 48 h. Finally, an equal volume of SteadyGlo Luciferase Assay Reagent was added to the plates and incubated for 20 min. The plates were read on a Perkin Elmer Envision with an exposure time of 0.1 s/well. Total luminescence was recorded and data were analyzed on GraphPad Prism 7.

Example 3: Development of a general protocol for an in vivo T cell transfer efficiency model.

These protocols were used in many of the experiments presented in the figures. Tumor cells were implanted subcutaneously (SC) into the right flank of NSG mice (NOD.Cg-Prkdcscid

- 27 045012

Il2rgtm1Wjl/SzJ) (The Jackson Laboratory, Cat. No. 005557) and were allowed to grow until they reached a mature tumor with an average volume of about 200 mm ³ . In parallel, human T cells were cultured in T cell medium (X-VIVO 15 [Lonza, cat. no. 04-418Q], 5% human serum, 1% penicillin/streptomycin, 0.01 mM 2-mercaptoethanol) in G - Rex100M gas permeable flask (Wilson Wolf, cat. no. 81100S) with MACSiBeads from the T-cell activation/expansion kit (Miltenyi, cat. no. 130-091-441) for approximately 10 days and supplemented with recombinant human IL-2 protein. Tumor growth in mice and human T cell activation/expansion were coordinated such that on day 0, study mice were randomized into groups (N=6) based on tumor size; Each was then given an intravenous (IV) injection of 2.5 x 106 cultured human T cells and given a first dose of COBRA or control molecules. Mice were dosed every 3 days for 7 doses (days 0, 3, 6, 9, 12, 15, and 18) and then monitored for an additional 2-3 weeks until tumors reached >2000 mm ³ in volume or the study was terminated . Tumor volume was measured every 3 days.

Example 4. In Vivo Activity with the EGFR/MMP9 pair Hemi-COBRA Pro77 and Pro53.

5x106 LoVo cells or 5x106 HT29 cells were implanted subcutaneously into the right flank of NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) (The Jackson Laboratory, Cat. No. 005557) and allowed to grow until tumors were established. In parallel, human T cells were cultured in T cell medium (XVIVO 15 [Lonza, cat. no. 04-418Q], 5% human serum, 1% penicillin/streptomycin, 0.01 mM 2mercaptoethanol) in a G-Rex100M gas permeable flask (Wilson Wolf, cat. no. 81100S) with MACSiBeads from the T-cell activation/expansion kit (Miltenyi, cat. no. 130-091-441) for 10 days and supplemented with recombinant human IL-2 protein. Tumor growth in mice and human T cell activation/expansion were coordinated such that on day 0, study mice were randomized into groups (N=6) based on tumor size; each was then injected intravenously (IV) with 2.5 x 106 cultured human T cells and given a first dose of COBRA or control molecules. Mice were dosed every 3 days for 7 doses (days 0, 3, 6, 9, 12, 15, and 18) and then monitored until tumor volume reached >2000 mm ³ or the study was stopped. Groups received 0.2 mg/kg (mc) anti-EGFR x CD3-positive control Pro51 bispecific antibody (bsAb), 0.5 mg/kg anti-hen egg lysozyme (HEL) x CD3 bsAb Pro98, 0.5 mc each containing MMP9 cleavable linker, Pro77 and Pro53 anti-EGFR hemi-COBRA pairs, or 0.5 μl of each containing non-cutting linker (NCL), Pro74 and Pro72 anti-EGFR hemi-COBRA pairs. Tumor volume was measured every 3 days.

Example 5. In Vivo Activity with EGFR/MMP9 COBRA Pro140.

5x106 LoVo cells or 5x106 HT29 cells were implanted subcutaneously into the right flank of NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) (The Jackson Laboratory, Cat. No. 005557) and allowed to grow until tumors were developed. In parallel, human T cells were cultured in T cell media (X-VIVO 15 [Lonza, Cat. No. 04-418Q], 5% human serum, 1% penicillin/streptomycin, 0.01 mM 2-mercaptoethanol) in G-Rex100M gas permeable flask (Wilson Wolf, cat. no. 81100S) with MACSiBeads from the T-cell activation/expansion kit (Miltenyi, cat. no. 130-091-441) for 10 days and the addition of recombinant human IL-2 protein. Tumor growth in mice and human T cell activation/expansion were coordinated such that on day 0, study mice were randomized into groups (N=6) based on tumor size; each was then injected intravenously (IV) with 2.5 x 106 cultured human T cells and given a first dose of COBRA or control molecules. Mice were dosed every 3 days for 7 doses (days 0, 3, 6, 9, 12, 15, and 18) and then observed until tumor volume reached >2000 mm ³ or the study was terminated. Groups received 0.2 µL anti-EGFR x CD3 bispecific antibody (bsAb) Pro51, 0.5 µL anti-hen egg lysozyme (HEL) x CD3 bsAb Pro98, or 0.5 µL cleavable linker, MMP9, EGFR COBRA Pro140. Tumor volume was measured every 3 days.

Example 6. In Vivo Activity with EGFR/MMP9 COBRA Pro186.

5x106 HT29 cells were implanted subcutaneously into the right flank of NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) (The Jackson Laboratory, Cat. No. 005557) and allowed to grow until tumor formation. In parallel, human T cells are cultured in T cell medium (X-VIVO 15 [Lonza, Cat. No. 04418Q], 5% human serum, 1% penicillin/streptomycin, 0.01 mM 2-mercaptoethanol) in a G Rex100M gas permeable flask ( Wilson Wolf, cat. no. 81100S) with MACSiBeads from a T-cell activation/expansion kit (Miltenyi, cat. no. 130-091-441) for 10 days and supplemented with recombinant human IL-2 protein. Tumor growth in mice and human T cell activation/expansion were coordinated such that on day 0, study mice were randomized into groups (N=6) based on tumor size; Each was then given an intravenous (IV) injection of 2.5 x 106 cultured human T cells and given a first dose of COBRA or control molecules. Mice were dosed every 3 days for 7 doses (days 0, 3, 6, 9, 12, 15, and 18) and then monitored until tumor volume reached >2000 mm ³ or the study was terminated. Groups received 0.1 mg/kg (mc) bispecific antibody (bsAb) positive control anti-EGFR x CD3 Pro51, 0.3 mc containing non-cutting linker (NCL), control anti-EGFR COBRA Pro214, 0.1 or 0. 3 µA containing MMP9 cleavable linker, anti-EGFR COBRA Pro140, or 0.1 or 0.3 µA containing MMP9 cleavable linker

- 28 045012 linker, anti-EGFR COBRA Pro186. Tumor volume was measured every 3 days. Example. Successful humanization of anti-EGFR sequences. The results are shown below.

Molecule KD (Μ) Cop (1/Ms) Kdis (1/c) Pro22 (parent EGFR) 2.58Ε-09Μ/2.6 nM 2.05Ε+05 5.27Ε-04 Pro90 (hEGFRl) 2.00Ε-09Μ/2.0 nM 2.21Ε+05 4.40Ε-04 Pro48 (EGFR2) 2.89Ε-09Μ/2.9 nM 6.09Ε+05 1.76Ε-03 Pro 137 (hEGFR2) 4.36Ε-09Μ/4.4 nM 5.85Ε+05 2.55Ε-03 Pro51 (hEGFR2) 3.27Ε-09Μ/3.2 nM 6.45Ε+05 2D1E-03 Pro201 (hEGFR2 with 2 sites binding) 2.25Ε-12Μ/2.3πΜ 1.55Ε+06 3.48Ε-06

These results demonstrate that humanization of the EGFR binding domains has been successful and that there is strong avidity for the EGFR target when two binding sites are present on the molecule.

Example. Successful humanization of EpCAM odASD.

The results are shown below.

Clone Human Molecule Binding Affinity (nM) Binding affinity of cynomolgus molecule (nM) cynomolgus/human cross-reactivity VIB-13 2.3 I,6 5 hVIB-13 2.8 12.7 4.5 VIB-23 4.2 46.7 I,1 hVIB-23 4.1 58.8 12.6

These results demonstrate that the humanization of EpCAM binding domains was successful.

Sequence Listing <110> MAVERICK THERAPEUTICS, INC.

<120> LIMITED CONDITIONALLY ACTIVATE BINDING PROTEINS <130> 118459-5005 <150> 62/555,943 <151> 2017-09-08 <150> 62/586,627 <151> 2017-11-15 <150> 6 2/587.318 <151 > 2017-11-16 <160>272 <170> PatentIn version 3.5 <210>1 <211>127 <212> Protein <213> Artificial sequence <220>

<223> Synthetic antigen binding domain -Targeting sdAb - alpha-

EGFR1

- 29 045012 <400> 1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

1. 5 10 15

Gln Ala Gly Gly

Ser Leu Arg Leu Ser Cys 20

Ala Ala Ser Gly Arg Thr 25

Phe Ser Ser Tyr 30

Ala Met Gly Trp Phe Arg 35

Gln Ala Pro Gly Lys Glu 40

Arg Glu Phe Val 45

Val Ala Ile Asn Trp Ser 50

Ser Gly Ser Thr Tyr Tyr

60

Ala Asp Ser Val

Lys Gly Arg Phe Thr Ile 65 70

Ser Arg Asp Asn Ala Lys 75

Asn Thr Met Tyr 80

Leu Gln Met Asn Ser Leu

Lys Pro Glu Asp Thr Ala 90

Val Tyr Tyr Cys 95

Ala Ala Gly Tyr Gln Ile

100

Asn Ser Gly Asn Tyr Asn 105

Phe Lys Asp Tyr

110

Glu Tyr Asp Tyr Trp Gly 115

Gln Gly Thr Gln Val Thr 120

Val Ser Ser

125 <210> 2 <211> 10 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR1 -Targeting sdAb <400>2

Gly Arg Thr Phe Ser Ser Tyr Ala Met Gly . 510 alpha-EGFR1 <210>3 <211>16 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR2 -Targeting sdAb <400>3

Ile Asn Trp Ser Ser Gly Ser Thr Tyr Tyr Ala Asp 1. 5 10 15 alpha-EGFR1

Ser Val Lys Gly

- 30 045012 <210> 4 <211> 18 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR3 - Targeting sdAb - alpha-EGFR1 <400>4

Gly Tyr Gln Ile Asn Ser Gly Asn Tyr Asn Phe Lys Asp Tyr Glu Tyr 1. 5 1015

Asp Tyr <210>5 <211>124 <212> Protein <213> Artificial sequence <220>

<223> Synthetic antigen binding domain -Targeting sdAb

EGFR2 <400>5

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly

1. 5 1015 alphaSer Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr 20 2530

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 4045

Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val 50 5560

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp 65 70 7580

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys

9095

Ala Ala Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp

100 105110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115120 <210>6 <211>10

- 31 045012 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDRl -Targeting sdAb - alpha-EGFR2 <400>6

Gly Arg Thr Ser Arg Ser Tyr Gly Met Gly . 510 <210>7 <211>17 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR2 -Targeting sdAb - alpha-EGFR2 <400>7

Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val Lys 1. 5 1015

Gly < 210>8 < 211>15 < 212> Protein < 213> Artificial sequence <220>

<223> Synthetic sdCDR3 -Targeting sdAb - alpha-EGFR2 <400>8

Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp Tyr

1. 5 1015 <210>9 <211>127 <212> Protein <213> Artificial sequence <220>

<223> Synthetic antigen binding domain - Targeting sdAb - h-alphaEGFR1 <400> 9

Glu Val Gln Leu Val 1.5

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30

- 32 045012

Ala Met Gly Trp Phe Arg 35

Val Ala Ile Asn Trp Ser 50

Lys Gly Arg Phe Thr Ile 65 70

Leu Gln Met Asn Ser Leu

Ala Ala Gly Tyr Gln Ile

100

Glu Tyr Asp Tyr Trp Gly 115

Gln Ala Pro Gly Lys Glu 40

Ser Gly Ser Thr Tyr Tyr

60

Ser Arg Asp Asn Ser Lys 75

Arg Ala Glu Asp Thr Ala 90

Asn Ser Gly Asn Tyr Asn 105

Gln Gly Thr Leu Val Thr 120 <210> 10 <211> 10 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR1 -Targeting sdAb <400>10

Arg Glu Phe Val 45

Ala Asp Ser Val

Asn Thr Leu Tyr 80

Val Tyr Tyr Cys 95

Phe Lys Asp Tyr 110

Val Ser Ser

125

- h-alpha-EGFR1

Gly Arg Thr Phe Ser Ser Tyr Ala Met Gly

1. 510 <210>11 <211>16 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR2 -Targeting sdAb <400>11

Ile Asn Trp Ser Ser Gly Ser Thr Tyr Tyr Ala Asp 1. 5 1015 <210>12 <211>18 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR3 -Targeting sdAb <400> 12

Gly Tyr Gln Ile Asn Ser Gly Asn Tyr Asn Phe Lys

- h-alpha-EGFR1

Ser Val Lys Gly

- h-alpha-EGFR1

Asp Tyr Glu Tyr

- 33 045012

15

Asp Tyr <210> 13 <211> 124 <212> Protein <213> Artificial sequence <220>

<223> Synthetic antigen binding domain - Targeting sdAb - h

EGFR2 <400>13

Gln Val Lys Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly

1. 5 1015 alphaSer Leu Thr Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr 20 2530

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 4045

Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val 50 5560

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 7580

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

9095

Ala Ala Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp

100 105110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115120 <210>14 <211>10 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR1 - Targeting sdAb - h-alpha-EGFR2 <400> 14

Gly Arg Thr Ser Arg Ser Tyr Gly Met Gly

1.5 10

- 34 045012 <210> 15 <211> 17 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR2 - Targeting sdAb - h-alpha-EGFR2 <400> 15

Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val Lys

15

Gly <210> 16 <211> 15 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR3 -Targeting sdAb - h-alpha-EGFR2 <400>16

Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp Tyr

1. 5 1015 <210>17 <211>114 <212> Protein <213> Artificial sequence <220>

<223> Synthetic antigen binding domain - Targeting sdAb

FOLR1 h77-2 <400>17

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1. 5 1015 alphaSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val 2025

Ser Asn Ser 30

Val Met Ala Trp 35

Ala Ile Ile Asn 50

Gly Arg Phe Thr 65

Tyr Arg Gln Thr Pro Gly Asn Glu 40

Ser Ile Gly Ile Thr Asn Tyr Ala 55 60

Ile Ser Arg Asp Asn Ser Lys Asn 70 75

Arg Glu Phe Val 45

Asp Ser Val Lys

Thr Leu Tyr Leu 80

- 35 045012

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Val Cys Asn

9095

Arg Asn Phe Asp Arg Ile Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

100 105110

Ser Ser <210>18 <211>10 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR1 -Targeting sdAb - alpha-FOLR1 h77-2 <400>18

Gly Phe Thr Val Ser Asn Ser Val Met Ala . 510 <210>19 <211>16 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR2 -Targeting sdAb - alpha-FOLR1 h77-2 <400>19

Ile Ile Asn Ser Ile Gly Ile Thr Asn Tyr Ala Asp Ser Val Lys Gly

1. 5 1015 <210>20 <211>6 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR3 - Targeting sdAb - alpha-FOLR1 h77-2 <400>20

Asn Phe Asp Arg Ile Tyr 1. 5 <210>21 <211>113 <212> Protein <213> Artificial sequence <220>

<223> Synthetic antigen binding domain - Targeting sdAb - alpha - 36 045012

FOLR1 h59.3 <400> 21

Gln Val Gln Leu Val 1.5

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

15

Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Asn Thr Phe 20 25

Ser Ile Ser 30

Ala

Met

Gly 35

Trp

Tyr

Arg

Gln

Ala

Pro

Gly

Lys

Gln

Arg

Glu

Trp

Val

Ala

Val

Thr

His

Ser

Asp

Tyr

Ser

Thr

Asn

Tyr

Ala

Asp

Ser

Val

Lys

Gly 65

Arg

Phe

Thr

Ile

Ser 70

Arg

Asp

Asn

Ser

Lys 75

Asn

Thr

Leu

Tyr

Leu 80

Gln

Met

Asn

Ser

Leu

Arg 85

Ala

Glu

Asp

Thr

Ala 90

Val

Tyr

Tyr

Cys

Lys 95

His

Tyr

Gly

Ile

Asp Tyr

Trp

Gly

Gln

Gly Thr

Leu

Val

Thr

Val Ser

100

105

110

Ser < 210> 22 < 211> 10 < 212> Protein < 213> Artificial sequence <220>

<223> Synthetic sdCDR1 -Targeting sdAb - alpha-FOLR1 h59.3 <400>22

Gly Asn Thr Phe Ser Ile Ser Ala Met Gly

1. 510 <210>23 <211>16 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR2 -Targeting sdAb - alpha-FOLR1 h59.3 <400>23

Val Thr His Ser Asp Tyr Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly

15

- 37 045012 <210> 24 <211> 5 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR3 -Targeting sdAb - alpha-FOLR1 h59.3 <400>24

Tyr Gly Ile Asp Tyr 1. 5 <210>25 <211>117 <212> Protein <213> Artificial sequence <220>

<223> Synthetic antigen binding domain -Targeting sdAbs -

FOLR1 h22-4 <400>25

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1. 5 1015 alphaSer Leu Arg Leu Ser Cys

Glu Ala Ser Gly Thr Thr Phe Ser Arg Asp

2530

Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys 3540

Gln Arg Glu Leu Val 45

Ala Ile Ile

Gly Arg Phe 65

Gln Met Asn

Ser Arg Gly Gly Ser Thr Asn Tyr Ala Asp 5560

Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 7075

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 8590

Ser Val Lys

Leu Tyr Leu 80

Tyr Cys Asn 95

Ala Asn Thr Ala Thr Trp

100

Gly Arg Val Phe Trp Gly Gln Gly Thr Leu

105110

Val Thr Val Ser Ser

115 <210>26 <211>10 <212> Protein

- 38 045012 <213> Artificial sequence <220>

<223> Synthetic sdCDRl -Targeting sdAb - alpha-FOLRl h22-4 <400>26

Gly Thr Thr Phe Ser Arg Asp Val Met Gly

1. 510 <210>27 <211>16 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR2 -Targeting sdAb - alpha-FOLR1 h22-4 <400>27

Ile Ile Ser Arg Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly . 5 1015 <210>28 <211>9 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR3 -Targeting sdAb - alpha-FOLR1 h22-4 <400>28

Asn Thr Ala Thr Trp Gly Arg Val Phe 1. 5 <210>29 <211>124 <212> Protein <213> Artificial sequence <220>

< 223 > Synthetic antigen binding domain - Targeting sdAb B7H3 hF7 < 400 > 29

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1. 5 1015 alphaSer Leu Arg Leu Ser Cys Ala Pro Ser Arg Arg Thr Phe His Thr Tyr 20 2530

His Met Gly Trp Phe Arg Gln Ala Pro Gly Lys 3540

Glu Arg Glu Phe Val 45

- 39 045012

Ala Val Ile Asn Trp Ser Gly

55

Gly Ser Thr Val Tyr Ala Asp Ser Val 60

Lys Gly Arg Phe Thr Ile Ser 65 70

Arg Asp Asn Ser Lys Asn Thr Leu Tyr 75 80

Leu Gln Met Asn Ser Leu Arg

Ala Glu Asp Thr Ala Val Tyr Tyr Cys

95

Ala Ala Gly Gly Ala Thr Thr

100

Gln Arg Ala Thr Glu Ala Ser Tyr Asp

105 110

Tyr Trp Gly Gln Gly Thr Leu 115

Val Thr Val Ser Ser

120 < 210> 30 < 211> 10 < 212> Protein < 213> Artificial sequence < 220>

<223> Synthetic sdCDR1 -Targeting sdAb - alpha-B7H3 hF7 <400>30

Arg Arg Thr Phe His Thr Tyr His Met Gly

1. 510 <210>31 <211>17 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR2 -Targeting sdAb - alpha-B7H3 hF7 <400>31

Val Ile Asn Trp Ser Gly Gly Ser Thr Val Tyr Ala Asp Ser Val Lys 1. 5 1015

Gly < 210 > 32 < 211 > 15 < 212 > Protein < 213 > Artificial sequence < 220 >

<223> Synthetic sdCDR3 -Targeting sdAb - alpha-B7H3 hF7 <400>32

Gly Gly Ala Thr Thr Gln Arg Ala Thr Glu Ala Ser Tyr Asp Tyr

- 40 045012

1. 5 <210> 33 <211> 122 <212> Protein <213> Artificial sequence <220>

<223> Synthetic antigen binding domain - Targeting sdAb

B7H3 hF12 <400> 33

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1. 5 10 15 alphaSer Leu Arg Leu Ser Cys

Glu Ala Ser Pro Arg Thr Phe Ser Thr Tyr

thirty

Ser Met Ala Trp Phe Arg Gln Ala Pro Gly Lys 35 40

Glu Arg Ser Phe Val 45

Ala Ala Ile

Asn Trp Ser

Gly Gly Asn Thr Ser Tyr Ala Asp Ser Val

60

Lys Gly Arg 65

Phe Thr Ile

Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 75 80

Leu Gln Met

Asn Ser Leu

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 90 95

Ala Ala Gly

Gly Val Leu

100

Ala His His Asn Tyr Glu Tyr Asp Tyr Trp

105 110

Gly Gln

Gly Thr Leu Val Thr Val Ser Ser

115 120 <210> 34 <211> 10 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR1 -Targeting sdAb - alpha-B7H3 hF12 <400>34

Pro Arg Thr Phe Ser Thr Tyr Ser Met Ala

1. 5 10 <210> 35 <211> 17

- 41 045012 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR2 - Targeting sdAb - alpha B7H3 hF12 <400>35

Ala Ile Asn Trp Ser Gly Gly Asn Thr Ser Tyr Ala Asp Ser Val Lys

1.5 1015

Gly <210>36 <211>13 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR3 -Targeting sdAb - alpha-B7H3 hF12 <400>36

Gly Gly Val Leu Ala His His Asn Tyr Glu Tyr Asp Tyr

1. 510 <210>37 <211>122 <212> Protein <213> Artificial sequence <220>

<223> Synthetic antigen binding domain - Targeting sdAb - alphaEpCAM h13 <400>37

Gln Val Gln Leu Val 1.5

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1015

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Thr Gly Ser 2025

Ile Phe Ser 30

Ile Asn Leu Met Gly Trp 35

Tyr Arg Gln Ala Pro 40

Gly Lys Gln Arg Glu 45

Leu Val Ala Arg Ile Thr 50

Ser Gly Asp Ser Thr 55

Val Tyr Ala Asp Ser 60

Val Lys Gly Arg Phe Thr 65 70

Ile Ser Arg Asp Asn 75

Ser Lys Asn Thr Leu 80

Tyr Leu Gln Met Asn Ser

Leu Arg Pro Glu Asp

Thr Ala Val Tyr Tyr

- 42 045012

Cys Asn Leu Leu Leu Arg Ser Ser Pro Gly Ala Thr Thr Pro Tyr Trp

100

105

110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115120 <210>38 <211>12 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR1 -Targeting sdAb - alpha-EpCAM h13 <400>38

Gly Thr Gly Ser Ile Phe Ser Ile Asn Leu Met Gly . 510 <210>39 <211>16 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR2 - Targeting sdAb - alpha-EpCAM h13 <400> 39

Arg Ile Thr Ser Gly Asp Ser Thr Val Tyr Ala Asp Ser Val Lys Gly

15

15 <210> 40 <211> 12 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR3 -Targeting sdAb - alpha-EpCAM h13 <400>40

Leu Leu Arg Ser Ser Pro Gly Ala Thr Thr Pro Tyr . 510 <210>41 <211>122 <212> Protein <213> Artificial sequence <220>

<223> Synthetic antigen binding domain - Targeting sdAb EpCAM h23 alpha- 43 045012 <400> 41

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

1. 5 10 15

Gln Pro Gly Gly

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Thr Gly 20 25

Ser Ile Phe Ser 30

Ile Asn Leu Met Gly Trp Tyr Arg Gln Ala Pro Gly 35 40

Lys Gln Arg Glu 45

Leu Val Ala Arg Ile Thr Ser Gly Asp Ser Thr Val

55 60

Tyr Ala Asp Ser

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

70 75

Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

90

Lys Asn Thr Leu 80

Ala Val Tyr Tyr 95

Cys Asn Leu Leu Leu Arg Ser Ser Pro Gly Ala Thr

100 105

Thr Pro Tyr Trp

110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 <210> 42 <211> 10 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR1 -Targeting sdAb <400>42 alpha-EpCAM h23

Gly Ser Phe Ser Ala Leu Trp Ala Met Arg

1. 5 <210> 43 <211> 15 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR2 -Targeting sdAb <400>43 alpha-EpCAM h23

Ser Ser Arg Gly Gly Thr Thr Ser Tyr Ala Asp Ser

1. 5 10 15

Val Lys Gly <210> 44

- 44 045012 <211> 7 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR3 -Targeting sdAb - alpha-EpCAM h23 <400>44

Ile Asp Gly His Leu Ala Tyr. 5 <210>45 <211>115 <212> Protein <213> Artificial sequence <220>

<223> Synthetic antigen binding domain - alpha-HSA half-life extension domain <400>45

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn

1.5 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Phe 20 2530

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4045

Ser Ser Ile Ser Gly Ser Gly Arg Asp Thr Leu Tyr Ala Glu Ser Val 50 5560

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 7580

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

9095

Thr Ile Gly Gly Ser Leu Ser Val Ser Ser Gln Gly Thr Leu Val Thr

100 105110

Val Ser Ser

115 <210>46 <211>10 <212> Protein <213> Artificial sequence <220>

- 45 045012 <223> Synthetic sdCDRl alpha-HSA half-life extension domain <400>46

Gly Phe Thr Phe Ser Lys Phe Gly Met Ser

1. 510 <210>47 <211>17 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR2 - alpha-HSA half-life extension <400>47 domain

Ser Ile Ser Gly Ser Gly Arg Asp Thr Leu Tyr Ala Glu Ser Val Lys 1. 5 1015

Gly <210>48 <211>6 <212> Protein <213> Artificial sequence <220>

<223> Synthetic sdCDR3 - alpha-HSA half-life extension <400>48 domain

Gly Gly Ser Leu Ser Val 1. 5 <210>49 <211>109 <212> Protein <213> Artificial sequence <220>

<223> Synthetic antigen binding domain - alpha-CD3 scFv domain alpha-CD3V(L) <400> 49

Gln Thr Val Val 1.5

Thr Gln Glu Pro

Ser Leu Thr Val

Ser Pro Gly Gly

Thr Val Thr Leu

Thr Cys Ala Ser

Ser Thr Gly Ala 25

Val Thr Ser Gly 30

Asn Tyr Pro Asn

Trp Val Gln Gln

Lys Pro Gly Gln

Ala Pro Arg Gly 45

- 46 045012

Leu Ile 50 Gly Gly Thr Lys Phe 55 Leu Val Pro Gly Thr 60 Pro Ala Arg Phe Ser 65 Gly Ser Leu Leu Gly 70 Gly Lys Ala Ala Leu 75 Thr Leu Ser Gly Val 80 Gln Pro Glu Asp Glu Ala 85 Glu Tyr Tyr Cys Thr 90 Leu Trp Tyr Ser Asn 95 Arg Trp Val Phe Gly 100 Gly Gly Thr Lys Leu 105 Thr Val Leu

<210> 50 <211> 14 <212> Protein <213> Artificial sequence <220>

<223> Synthetic aVLCDR1 - alpha CD3 scFV domain - alpha CD3 V(L) <400>50

Ala Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn

1. 510 <210>51 <211>7 <212> Protein <213> Artificial sequence <220>

<223> Synthetic aVLsdCDR2 - alpha CD3 scFV domain - alpha CD3 V(L) <400>51

Gly Thr Lys Phe Leu Val Pro 1. 5 <210>52 <211>9 <212> Protein <213> Artificial sequence <220>

<223> Synthetic aVLsdCDR3 - alpha CD3 scFV domain - alpha CD3 V(L) <400>52

Thr Leu Trp Tyr Ser Asn Arg Trp Val 1. 5 <210>53 <211>110 <212> Protein <213> Artificial sequence

- 47 045012 <220>

<223> Synthetic antigen binding domain - alpha-CD3 scFv domain alpha-CD3V(Li) <400>53

Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1.5 1015

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly 20 2530

Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 4045

Leu Ile Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly Thr Pro Ala Arg 50 5560

Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly 65 70 7580

Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser

9095

Asn Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105110 <210>54 <211>14 <212> Protein <213> Artificial sequence <220>

<223> Synthetic iVLCDR1 - alpha CD3 scFV domain - alpha CD3 V(Li) <400>54

Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn

1. 510 <210>55 <211>8 <212> Protein <213> Artificial sequence <220>

<223> Synthetic iVLsdCDR2 - alpha CD3 scFV domain - alpha CD3 V(Li) <400> 55

Asp Tyr Lys Asp Asp Asp Asp Lys 1. 5 <210> 56

- 48 045012 <211> 9 <212> Protein <213> Artificial sequence <220>

<223> Synthetic iVLsdCDR3 - alpha CD3 scFV domain - alpha CD3 V(Li) <400>56

Val Leu Trp Tyr Ser Asn Arg Trp Val 1. 5 <210>57 <211>109 <212> Protein <213> Artificial sequence <220>

<223> Synthetic antigen binding domain - alpha-CD3 scFv domain alpha-CD3V(Li2) <400>57

Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1.5 1015

Thr Val Thr Leu Thr Cys

Asn Tyr Pro Asn Trp Val 35

Gly Ser Ser Thr Gly Ala Val Thr Ser Gly

2530

Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

4045

Leu Ile Gly Gly Thr Lys Asp Asp

55

Ala Pro Gly Thr

Pro Ala Arg Phe

Ser Gly Ser Leu Leu Gly Gly Lys 65 70

Ala Ala Leu Thr

Leu Ser Gly Val 80

Gln Pro Glu Asp Glu Ala Glu Tyr 85

Tyr Cys Val Leu 90

Trp Tyr Ser Asn 95

Arg Trp Val Phe Gly Gly Gly Thr 100

Lys Leu Thr Val

105

Leu <210> 58 <211> 14 <212> Protein <213> Artificial sequence <220>

<223> Synthetic iVLCDR1 - alpha CD3 scFV domain - alpha CD3 V(Li2) <400> 58

Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn

- 49 045012

1. 5 <210> 59 <211> 7 <212> Protein <213> Artificial sequence <220>

<223> Synthetic 1VLCDR2 - alpha CD3 scFV domain - alpha CD3 V(Li2) <400>59

Gly Thr Lys Asp Asp Ala Pro 1. 5 < 210>60 < 211>9 < 212> Protein < 213> Artificial sequence <220>

<223> Synthetic iVLCDR3 - alpha CD3 scFV domain - alpha CD3 V(Li2) <400>60

Val Leu Trp Tyr Ser Asn Arg Trp Val 1. 5 <210>61 <211>125 <212> Protein <213> Artificial sequence <220>

<223> Synthetic antigen binding domain - alpha-CD3 scFv domain alpha-CD3V(H) <400> 61

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1. 5 10 15

Ser Leu Lys Leu Ser 20

Cys Ala Ala Ser Gly Phe 25

Thr Phe Asn Lys Tyr 30

Ala Ile Asn Trp Val 35

Arg Gln Ala Pro Gly Lys 40

Gly Leu Glu Trp Val 45

Ala Arg Ile Arg Ser 50

Lys Tyr Asn Asn Tyr Ala 55

Thr Tyr Tyr Ala Asp 60

Gln Val Lys Asp Arg 65

Phe Thr Ile Ser Arg Asp 70 75

Asp Ser Lys Asn Thr 80

Ala Tyr Leu Gln Met

Asn Asn Leu Lys Thr Glu

Asp Thr Ala Val Tyr

- 50 045012

Tyr Cys Val Arg His Ala Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp

100

105

110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120125 <210>62 <211>10 <212> Protein <213> Artificial sequence <220>

< 223> Synthetic aVHCDR1 - alpha CD3 scFV domain - alpha CD3 V(H) < 400>62

Gly Phe Thr Phe Asn Lys Tyr Ala Ile Asn

1. 510 <210>63 <211>19 <212> Protein <213> Artificial sequence <220>

<223> Synthetic aVHsdCDR2 - alpha CD3 scFV domain - alpha CD3 V(H) <400>63

Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Gln

1.5 1015

Val Lys Asp <210>64 <211>14 <212> Protein <213> Artificial sequence <220>

< 223> Synthetic aVHsdCDR3 - alpha CD3 scFV domain - alpha CD3 V(H) < 400>64

His Ala Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp Ala Tyr

1. 510 <210>65 <211>126 <212> Protein <213> Artificial sequence <220>

- 51 045012 <223> Synthetic antigen binding domain alpha-CD3V(Hi) <400> 65 alpha-CD3 scFv domain Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

1. 5 10 15

Gln Pro Gly Gly

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr 20 25

Phe Asn Lys Tyr 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly

40

Leu Glu Trp Val 45

Ala

Asp 65

Thr

Arg Ile Arg Ser Lys Tyr Asp 50 55

Ser Val Lys Asp Arg Phe Thr 70

Ala Tyr Leu Gln Met Asn Asn 85

Tyr Lys Asp Asp

Ile Ser Arg Asp 75

Leu Lys Thr Glu 90

Asp Asp Lys Ala

Asp Ser Lys Asn 80

Asp Thr Ala Val 95

Tyr Tyr Cys Val Arg His Gly Asn

100

Phe Gly Asn Ser

105

Tyr Ile Ser Tyr

110

Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

115 120

Ser Ser

125 <210> 66 <211> 10 <212> Protein <213> Artificial sequence <220>

<223> Synthetic iVHsdCDR1 - alpha CD3 scFV <400> 66 domain - alpha CD3 V(Hi)

Gly Phe Thr Phe Asn Lys Tyr Ala Met Asn

1. 5 10 <210> 67 <211> 20 <212> Protein <213> Artificial sequence <220>

<223> Synthetic iVHsdCDR2 - alpha CD3 scFV <400> 67 domain - alpha CD3 V(Hi)

Arg Ile Arg Ser Lys Tyr Asp Tyr Lys Asp Asp Asp

Asp Lys Ala Asp

15

15

- 52 045012

Ser Val Lys Asp <210> 68 <211> 14 <212> Protein <213> Artificial sequence <220>

<223> Synthetic iVHsdCDR3 - alpha CD3 scFV <400> 68 domain - alpha CD3 V(Hi)

His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp

Ala Tyr

1. 5 10 <210> 69 <211> 125 <212> Protein <213> Artificial sequence <220>

<223> Synthetic antigen binding domain alpha-CD3V(Hi2) <400> 69

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

1. 5 10 15 alpha CD3 scFv domain Gln Pro Gly Gly

Ser Leu Lys Leu Ser Cys 20

Ala Ala Ser Gly Phe Thr 25

Phe Asn Lys His 30

Ala Met Asn Trp Val Arg 35

Gln Ala Pro Gly Lys Gly 40

Leu Glu Trp Val 45

Ala Arg Ile Arg Ser Lys 50

Tyr Asn Asn Tyr Ala Thr

60

Ala Tyr Ala Asp

Ser Val Lys Asp Arg Phe 65 70

Thr Ile Ser Arg Asp Asp 75

Ser Lys Asn Thr 80

Ala Tyr Leu Gln Met Asn

Asn Leu Lys Thr Glu Asp 90

Thr Ala Val Tyr 95

Tyr Cys Val Arg His Gly

100

Asn Phe Gly Asn Ser Tyr 105

Ile Ser Tyr Trp 110

Ala Tyr Trp Gly Gln Gly 115

Thr Leu Val Thr Val Ser

120

Ser

125

- 53 045012 <210> 70 <211> 10 <212> Protein <213> Artificial sequence <220>

< 223> Synthetic iVHsdCDR1 - alpha CD3 scFV domain - alpha CD3 V(Hi2) < 400>70

Gly Phe Thr Phe Asn Lys His Ala Met Asn

1. 510 <210>71 <211>19 <212> Protein <213> Artificial sequence <220>

<223> Synthetic iVHsdCDR2 - alpha CD3 scFV domain - alpha CD3 V(Hi2) <400> 71

Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Ala Tyr Ala Asp Ser

15

15

Val Lys Asp <210> 72 <211> 14 <212> Protein <213> Artificial sequence <220>

<223> Synthetic iVHsdCDR3 - alpha CD3 scFV domain - alpha CD3 V(Hi2) <400>72

His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp Ala Tyr . 510 <210>73 <211>15 <212> Protein <213> Artificial sequence <220>

<223> Synthetic linker - alpha-CD3 scFV - Normal, uncut <400>73

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1. 5 1015 <210>74 <211>8 <212> Protein

- 54 045012 <213> Artificial sequence <220>

<223> Synthetic linker - alpha-CD3 scFV - Limited <400>74

Gly Gly Gly Ser Gly Gly Gly Ser 1. 5 < 210>75 < 211>8 < 212> Protein < 213> Artificial sequence <220>

<223>Synthetic cut linker - MMP 2/9 <400>75

Gly Pro Ala Gly Met Lys Gly Leu

1. 5 <210>76 <211>15 <212> Protein <213> Artificial sequence <220>

<223>Synthetic cut linker - MMP 2/9 <400>76

Ser Gly Gly Pro Gly Pro Ala Gly Met Lys Gly Leu Pro Gly Ser

1. 5 1015 <210>77 <211>17 <212> Protein <213> Artificial sequence <220>

< 223 > Synthetic severable linker - MMP 2/9 < 400 > 77

Ser Gly Gly Gly Pro Gly Pro Ala Gly Met Lys Gly Leu Pro Gly Gly

1. 5 10 15

Ser <210>

<211>

<212>

<213>

Protein

Artificial sequence <220>

- 55 045012 <223> Synthetic severable linker - Meprin A/B <400>78

Gly Gly Gly Gly Lys Lys Leu Ala Asp Glu Pro Glu Gly Gly Gly Ser 1. 5 1015 <210>79 <211>8 <212> Protein <213> Artificial sequence <220>

<223> Synthetic cutable linker - Meprin A/B <400>79

Lys Lys Leu Ala Asp Glu Pro Glu 1. 5 <210>80 <211>13 <212> Protein <213> Artificial sequence <220>

<223>Synthetic cutable linker - Meprin A/B (Variant, high performance) <400>80

Gly Gly Gly Lys Phe Leu Ala Asp Glu Pro Glu Gly Gly . 510 <210>81 <211>16 <212> Protein <213> Artificial sequence <220>

<223> Synthetic cutable linker - Cathepsin S,K,L <400>81

Ser Gly Gly Gly Ala Arg Leu Gln Ser Ala Ala Pro Gly Gly Gly Ser 1. 5 1015 <210>82 <211>8 <212> Protein <213> Artificial sequence <220>

< 223> Synthetic severable linker - Cathepsin S,K,L <400> 82

Ala Arg Leu Gln Ser Ala Ala Pro 1.5

- 56 045012 <210> 83 <211> 18 <212> Protein <213> Artificial sequence <220>

<223>Synthetic scissile linker - Meprin/Granzyme B <400>83

Ser Gly Gly Gly Gly Val Tyr Ala Asp Ser Leu Glu Asp Gly Gly Gly 1. 5 1015

Gly Ser < 210>84 < 211>10 < 212> Protein < 213> Artificial sequence <220>

<223>Synthetic scissile linker - Meprin/Granzyme B <400>84

Gly Val Tyr Ala Asp Ser Leu Glu Asp Gly . 510 <210>85 <211>16 <212> Protein <213> Artificial sequence <220>

<223> Synthetic cleavable linker - Matriptase/UAP (MS) <400>85

Gly Gly Gly Ser Leu Ser Gly Arg Ser Asp Asn His Gly Gly Gly Ser 1. 5 1015 < 210>86 < 211>10 < 212> Protein < 213> Artificial sequence < 220>

<223>Synthetic scissile linker - Matriptase/UAP (MS) <400>86

Gly Leu Ser Gly Arg Ser Asp Asn His Gly

1. 5 10 <210> 87

- 57 045012 <211> 17 <212> Protein <213> Artificial sequence <220>

<223>Synthetic scissile linker - Matriptase (MV) <400>87

Ser Gly Gly Gly Ser Phe Thr Arg Gln Ala Arg Val Val Gly Gly Gly 1. 5 1015

Ser <210>88 <211>9 <212> Protein <213> Artificial sequence <220>

<223> Synthetic cleavable linker - Matriptase (MV) <400>88

Ser Phe Thr Arg Gln Ala Arg Val Val 1. 5 <210>89 <211>14 <212> Protein <213> Artificial sequence <220>

<223> Synthetic cleavable linker - Cathepsin S/MMP9/Meprin A <400>89

Ala Arg Leu Gln Ser Ala Ala Pro Ala Gly Leu Lys Gly Ala

1. 510 <210>90 <211>13 <212> Protein <213> Artificial sequence <220>

<223> Synthetic severable linker

MMP9 (Option, highly effective) <400> 90

Gly Gly Pro Gly Pro Ala Gly Met His Gly Leu Pro Gly

1. 5 10 <210> 91 <211> 13 <212> Protein

- 58 045012 <213> Artificial sequence <220>

<223> Synthetic scissile linker - MMP9 (Variant, low efficiency) <400>91

Gly Gly Pro Gly Pro Ala Gly Met Glu Gly Leu Pro Gly

1. 510 <210>92 <211>16 <212> Protein <213> Artificial sequence <220>

<223> Synthetic scissile linker - Thrombin 1 <400>92

Gly Gly Gly Gly Leu Val Pro Arg Gly Ser Leu Gly Gly Gly Gly Ser

1. 5 1015 <210>93 <211>15 <212> Protein <213> Artificial sequence <220>

<223>Synthetic scissile linker - Thrombin 2 <400>93

Ser Ser Gly Gly Gly Met Pro Arg Ser Phe Arg Gly Gly Gly Ser . 5 1015 <210>94 <211>16 <212> Protein <213> Artificial sequence <220>

<223> Synthetic cut linker - Enterokinase/Flag <400>94

Gly Gly Gly Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly Ser 1. 5 1015 < 210>95 < 211>16 < 212> Protein < 213> Artificial sequence <220>

<223> Synthetic cutable linker - KLK7-6 <400> 95

- 59 045012

Ser Gly Gly Gly Gln Asn Pro Tyr Ser Ala

Gly Arg Gly Gly Gly Ser

1. 5 1015 <210>96 <211>16 <212> Protein <213> Artificial sequence <220>

<223>Synthetic cutable linker - KLK7-13 <400>96

Ser Gly Gly Gly Gln Asn Pro Tyr Ser Ala Gly Gly Gly Ser Gly Gly

1. 5 1015 <210>97 <211>16 <212> Protein <213> Artificial sequence <220>

<223> Synthetic cleavable linker - KLK7-11 <400>97

Ser Gly Gly Gly Arg Asn Val Tyr Ser Ala Gly Gly Gly Ser Gly Gly

1. 5 1015 <210>98 <211>16 <212> Protein <213> Artificial sequence <220>

<223> Synthetic cleavable linker - KLK7-10 <400>98

Ser Gly Gly Gly Gln Asn Thr Trp Ser Ala Gly Lys Gly Gly Gly Ser

1. 5 1015 <210>99 <211>16 <212> Protein <213> Artificial sequence <220>

< 223> Synthetic cutable linker - UAP < 400> 99

Gly Gly Gly Ser His Thr Gly Arg Ser Ala Tyr Phe Gly Gly Gly Ser 1. 5 10 15 <210> 100 <211> 8 <212> Protein

- 60 045012 <213> Artificial sequence <220>

<223> Synthetic protease cutting site - MMP7 <400>100

Lys Arg Ala Leu Gly Leu Pro Gly 1. 5 <210>101 <211>24 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - MMP7 <220>

< 221> misc_feature < 222> (1)..(8) < 223> X can be Asp or Glu <220>

< 221> misc_feature < 222> (17)..(24) < 223> X can be Asp or Arg < 400>101

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Pro Leu Ala Leu Trp Arg Ser . 5 1015

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa <210>102 <211>8 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - MMP9 <400>102

Pro Arg Ser Thr Leu Ile Ser Thr 1. 5 < 210>103 < 211>5 < 212> Protein < 213> Artificial sequence <220>

<223> Synthetic protease cutting site - MMP9 <400> 103

- 61 045012

Leu Glu Ala Thr Ala 1. 5 < 210> 104 < 211> 10 < 212> Protein < 213> Artificial sequence <220>

<223>Synthetic protease cutting site - MMP11 <400>104

Gly Gly Ala Ala Asn Leu Val Arg Gly Gly

1. 510 < 210> 105 < 211> 10 < 212> Protein < 213> Artificial sequence <220>

<223> Synthetic protease cutting site - MMP14 <400>105

Ser Gly Arg Ile Gly Phe Leu Arg Thr Ala

1. 510 <210>106 <211>6 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - MMP <400>106

Pro Leu Gly Leu Ala Gly 1. 5 <210>107 <211>6 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - MMP <220>

< 221> misc_feature < 222> (6)..(6) < 223> Xaa can be any naturally occurring amino acid <400> 107

- 62 045012

Pro Leu Gly Leu Ala Xaa 1. 5 <210> 108 <211> 7 <212> Protein <213> Artificial Sequence <220>

<223> Synthetic protease cutting site - MMP <220>

< 221> misc_feature < 222> (5)..(5) <223> X can be Met or Glu <400>108

Pro Leu Gly Cys Xaa Ala Gly 1. 5 <210>109 <211>8 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - MMP <400>109

Glu Ser Pro Ala Tyr Tyr Thr Ala 1. 5 <210>110 <211>6 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - MMP <400>110

Arg Leu Gln Leu Lys Leu

1. 5 <210>111 <211>7 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - MMP <400> 111

Arg Leu Gln Leu Lys Ala Cys

- 63 045012

1. 5 <210> 112 <211> 7 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - MMP2, MMP9, MMP14 <220>

<221> misc_feature <222> (1)..(3)

<223> X can be Cys or Ile or Thr <220> <221> misc_ feature <222> (5) . . (5) <223> X can be His or Orn or Phe

<400> 112

Glu Pro Xaa Gly Xaa Tyr Leu 1. 5 < 210> 113 < 211> 5 < 212> Protein < 213> Artificial sequence <220>

< 223> Synthetic protease cutting site - Urokinase plasminogen activator (UPA) < 400>113

Ser Gly Arg Ser Ala

1. 5 <210>114 <211>4 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - Urokinase plasminogen activator (UPA) <400>114

Asp Ala Phe Lys 1 <210>115 <211>5 <212> Protein <213> Artificial sequence

- 64 045012 <220>

<223> Synthetic plasminogen protease cleavage site (PCA)

Urokinase activator <400> 115

Gly Gly Gly Arg Arg 1. 5 <210> 116 <211> 4 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site <400> 116

Lysosomal enzyme

Gly Phe Leu Gly 1 <210> 117 <211> 4 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site <400> 117

Lysosomal enzyme

Ala Leu Ala Leu 1 <210> 118 <211> 2 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site <400> 118

Lysosomal enzyme

Phe Lys 1 <210> 119 <211> 3 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site <400> 119

Cathepsin B

Asn Leu Leu

- 65 045012 <210> 120 <211> 6 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - Cathepsin D <220>

< 221> misc_feature < 222> (4)..(4) < 223> X can be Glu or Thr < 400>120

Pro Ile Cys Xaa Phe Phe 1. 5 < 210>121 < 211>8 < 212> Protein < 213> Artificial sequence < 220>

<223> Synthetic protease cutting site - Cathepsin K <400>121

Gly Gly Pro Arg Gly Leu Pro Gly 1. 5 < 210>122 < 211>6 < 212> Protein < 213> Artificial sequence <220>

<223>Synthetic protease cutting site - Prostate-specific antigen <400>122

His Ser Ser Lys Leu Gln 1. 5 < 210>123 < 211>7 < 212> Protein < 213> Artificial sequence <220>

<223> Synthetic protease cutting site - Prostate-specific antigen <400> 123

- 66 045012

His Ser Ser Lys Leu Gln Leu 1. 5 <210> 124 <211> 9 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - Prostate-specific antigen <400>124

His Ser Ser Lys Leu Gln Glu Asp Ala 1. 5 <210>125 <211>10 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - Herpes simplex virus protease <400>125

Leu Val Leu Ala Ser Ser Ser Phe Gly Tyr

1. 510 <210>126 <211>10 <212> Protein <213> Artificial sequence <220>

<223>Synthetic protease cutting site - HIV protease <400>126

Gly Val Ser Gln Asn Tyr Pro Ile Val Gly . 510 <210>127 <211>10 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - CMV protease <400> 127

Gly Val Val Gln Ala Ser Cys Arg Leu Ala

1. 5 <210> 128

- 67 045012 <211> 4 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - Thrombin <220>

< 221> misc_feature < 222> (2)..(2) <223> X can be Pro or Ile or Pro <400>128

Phe Xaa Arg Ser <210>129 <211>6 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - Thrombin <400>129

Asp Pro Arg Ser Phe Leu . 5 <210>130 <211>6 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - Thrombin <400>130

Pro Pro Arg Ser Phe Leu 1. 5 < 210>131 < 211>4 < 212> Protein < 213> Artificial sequence <220>

<223> Synthetic protease cutting site - Caspase-3 <400>131

Asp Glu Val Asp <210>132 <211>5

- 68 045012 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - Caspase-3 <400>132

Asp Glu Val Asp Pro 1. 5 < 210>133 < 211>8 < 212> Protein < 213> Artificial sequence <220>

<223> Synthetic protease cutting site - Caspase-3 <400>133

Lys Gly Ser Gly Asp Val Glu Gly 1. 5 < 210>134 < 211>6 < 212> Protein < 213> Artificial sequence <220>

<223>Synthetic protease cutting site - Interleukin-1-beta converting enzyme <400>134

Gly Trp Glu His Asp Gly

1. 5 <210>135 <211>7 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - Enterokinase <400>135

Glu Asp Asp Asp Asp Lys Ala

1. 5 <210>136 <211>9 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - Fap

- 69 045012 <400>136

Lys Gln Glu Gln Asn Pro Gly Ser Thr 1. 5 <210>137 <211>6 <212> Protein <213> Artificial sequence <220>

<223>Synthetic protease cutting site - Kallikrein 2 <400>137

Gly Lys Ala Phe Arg Arg . 5 <210>138 <211>4 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - Plasmin <400>138

Asp Ala Phe Lys 1 <210>139 <211>4 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - Plasmin <400>139

Asp Val Leu Lys 1 <210>140 <211>4 <212> Protein <213> Artificial sequence <220>

<223> Synthetic protease cutting site - Plasmin <400> 140

Asp Ala Phe Lys 1

- 70 045012 <210> 141 <211> 7 <212> Protein <213> Artificial sequence <220>

< 223> Synthetic protease cutting site - Top < 400>141

Ala Leu Leu Leu Ala Leu Leu

1. 5 <210>142 <211>121 <212> Protein <213> Artificial sequence <220>

<223> Synthetic antigen binding domain - alpha-HSA half-life extension domain <400>142

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn

1.5 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Phe 20 2530

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4045

Ser Ser Ile Ser Gly Ser Gly Arg Asp Thr Leu Tyr Ala Glu Ser Val 50 5560

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 7580

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

9095

Thr Ile Gly Gly Ser Leu Ser Val Ser Ser Gln Gly Thr Leu Val Thr

100 105110

Val Ser Ser His His His His His

115120 <210>143 <211>889 <212> Protein <213> Artificial sequence

- 71 045012 <220>

<223>

Synthetic construction

Pro140 Format 1 <400>

143

Gln Val

15

Lys

Leu Glu

Glu Ser

Gly

Gly Gly

Ser Val

Gln

Thr Gly

Gly

Ser

Leu

Arg

Leu Thr

Cys

Ala

Ala

Ser Gly 25

Arg

Thr

Ser

Arg Ser

Tyr

Gly

Met

Gly 35

Trp

Phe

Arg

Gln

Ala

Pro

Gly

Lys

Glu

Arg

Glu

Phe

Val

Ser

Gly

Ile

Ser

Trp

Arg

Gly

Asp

Ser

Thr

Gly

Tyr

Ala

Asp

Ser

Val

Lys 65

Gly

Arg

Phe

Thr

Ile 70

Ser

Arg

Asp

Asn

Ala 75

Lys

Asn

Thr

Val

Asp 80

Leu

Gln

Met

Asn

Ser

Leu 85

Lys

Pro

Glu

Asp

Thr 90

Ala

Ile

Tyr

Tyr

Cys 95

Ala

Ala

Ala

Ala

Gly

Ser

Ala

Trp

Tyr

Gly

Thr

Leu

Tyr

Glu

Tyr

Asp

100

105

110

Tyr

Trp

Gly

Gln

Gly

Thr

Gln

Val

Thr

Val

Ser

Ser

Gly

Gly

Gly

Ser

115

120

125

Gly

Gly

130

Gly

Ser

Glu

Val

Gln

135

Leu

Val

Glu

Ser

Gly

140

Gly

Gly

Leu

Val

Gln

145

Pro

Gly

Gly

Ser

Leu

150

Lys

Leu

Ser

Cys

Ala

155

Ala

Ser

Gly

Phe

Thr

160

Phe

Asn

Lys

Tyr

Ala

Ile 165

Asn

Trp

Val

Arg

Gln 170

Ala

Pro

Gly

Lys

Gly 175

Leu

Glu

Trp

Val

Ala

Arg

Ile

Arg

Ser

Lys

Tyr

Asn

Asn

Tyr

Ala

Thr

180

185

190

Tyr

Tyr

Ala

Asp

Gln

Val

Lys

Asp

Arg

Phe

Thr

Ile

Ser

Arg

Asp

Asp

195

200

205

Ser

Lys

210

Asn Thr

Ala

Tyr

Leu

215

Gln

Met

Asn

Asn

Leu

220

Lys Thr

Glu

Asp

Thr

225

Ala Val Tyr

Tyr

Cys 230

Val Arg

His

Ala

Asn

235

Phe Gly Asn

Ser

Tyr 240

- 72 045012

Ile Ser Tyr Trp Ala Tyr Trp Gly Gln Gly Thr

245 250

Leu Val Thr Val Ser

255

Ser Gly Pro Ala Gly Met

260

Lys Gly Leu Gln Thr Val Val

265

Thr Gln Glu

270

Pro Ser Leu Thr Val

275

Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Ala

280 285

Ser Ser Thr Gly Ala

290

Val Thr Ser Gly Asn

295

Tyr Pro Asn Trp Val Gln 300

Gln Lys Pro Gly Gln 305

Ala Pro Arg Gly Leu 310

Ile Gly Gly Thr Lys Phe

315 320

Leu Val Pro Gly Thr

Pro Ala Arg Phe Ser 25

Gly Ser Leu Leu Gly Gly

335

Lys Ala Ala Leu Thr Leu

340

Ser Gly Val Gln Pro

345

Glu Asp Glu Ala Glu

350

Tyr Tyr Cys Thr Leu Trp Tyr

355

Ser Asn Arg Trp Val Phe Gly Gly Gly

360 365

Thr Lys Leu Thr Val

370

Leu Gly Gly Gly Ser Gly Gly Gly Ser Gln Val

375 380

Lys Leu 385

Arg Leu

Glu Glu Ser Gly Gly Gly Ser 390

Thr Cys Ala Ala Ser Gly Arg 405

Val Gln Thr Gly Gly Ser Leu

395 400

Thr Ser Arg Ser Tyr Gly Met

410 415

Gly Trp Phe Arg Gln Ala Pro 420

Gly Lys Glu Arg Glu Phe Val Ser Gly

425 430

Ile Ser Trp Arg 435 Gly Asp Ser Thr Gly 440 Tyr Ala Asp Ser Val 445 Lys Gly Arg Phe 450 Thr Ile Ser Arg Asp 455 Asn Ala ) Lys Asn Thr 460 Val Asp Leu Gln Met 465 Asn Ser Leu Lys Pro 470 Glu Asp Thr Ala Ile 475 Tyr Tyr Cys Ala Ala 480 Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp Tyr Trp

- 73 045012

485

490

495

Gly

Ala

Pro

Ser 545

Gln

Asp

Gly

Glu

Gly 625

Val

Leu

Met

Arg

Ser 705

Ala

Gln

Gly

Gly Thr Gln

500

Met Lys

515

Ser

530

Leu

Thr

Ser

Lys

Asp

Lys

Thr

Gly

Gly

Val

Ala

Pro

Gly

Gln

Asp

Ala

Tyr

610

Thr

Gln

Lys

Asn

Lys

Val

Leu

Ser

Val

550

Ala 565

Gly

Thr

580

Ala

Leu

Thr

595

Tyr

Cys

Val

Leu

Lys

Leu

Leu

Trp

Ile

690

Leu

Thr

Val

630

Thr

Pro

Val

Glu

Ser 645

Ser

Cys

660

Val

Arg

Ala

Gln

675

Arg

Ser

Lys

Tyr

Val Lys

Tyr Leu

Asp

Arg

Phe

710

Gln

Met

Asn 725

Val Ser Ser

505

Gly Ser

520

Pro

535

Gly

Gly

Ser

Gly

Gly Pro Gly

510

Pro

Thr

Pro

Pro

Leu

Ser

Gly

Gln

Thr

Val

Val Thr

525

Gln

Glu

Thr

Asn

Arg

Ala

Ser

Trp

615

Leu

Gly

Ala

Ala

Gly

Leu

Arg

Val

Tyr

555

Ile 570

Phe

585

Gly

600

Tyr

Gly

Gly

Ser

Pro

Asp

695

Ser

Val

Gln

Ser

Gly

Gly

Gly

Asn

Arg

Gly

Leu

Ser

635

Val

650

Phe

Thr

665

Gly

Lys

Gly

680

Tyr

Lys

Asp

Asp

Thr Ile

Asn Leu

Ser

Lys

Arg

Asp

715

Thr

Glu 730

Thr

540

Pro

Gly

Gly

Pro

Leu

Thr

Cys

Gly

Asn

Asp

Ser

Glu

Trp

620

Gly

Gln

Phe

Leu

Trp

Tyr

Leu

Val

Lys

Gln

560

Asp 575

Leu

Gly

590

Asp

Glu

Ala

605

Val

Phe

Gly

Gly

Gly

Gly

Ser

Glu

640

Pro

Gly

Gly

Ser 655

Asn

Lys

Tyr

670

Glu

Trp

685

Ala

Val

Ala

Asp

700

Asp

Lys

Ala

Asp

Asp Ser

Asp Thr

Lys

Asn

Thr

720

Ala

Val

Tyr 735

- 74 045012

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser

740745

Tyr Ile Ser Tyr Trp

750

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

755 760765

Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

770 775780

Leu Val Gln Pro 785

Phe Thr Phe Ser

Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

790 795800

Lys Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly

805 810815

Lys Gly Leu Glu Trp Val

820

Ser Ser Ile Ser Gly Ser Gly Arg Asp Thr

825830

Leu

Ala

Tyr Ala Glu Ser Val 835

Lys Thr Thr Leu Tyr 850

Lys Gly Arg Phe Thr 840

Leu Gln Met Asn Ser 855

Ile Ser Arg Asp Asn 845

Leu Arg Pro Glu Asp 860

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Val

865 870875

Ser Ser

880

Gln Gly Thr Leu Val Thr Val Ser Ser

885 <210>144 <211>890 <212> Protein <213> Artificial sequence <220>

< 223> Synthetic construction - Pro140b Format 1 < 400> 144

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly

1. 5 10 15

Ser Leu Arg Leu Thr Cys Ala Ala 20

Ser Gly Arg Thr Ser Arg Ser Tyr

thirty

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys 35 40

Glu Arg Glu Phe Val

Ser Gly Ile Ser Trp Arg Gly Asp Ser

Thr Gly Tyr Ala Asp Ser Val

- 75 045012

Lys 65

Leu

Ala

Tyr

Gly

Gln 145

Phe

Leu

Tyr

Ser

Thr 225

Ile

Ser

Pro

Ser

Gly

Gln

Ala

Trp

Arg

Met

Ala

Gly

Gly

130

Pro

Asn

Glu

Tyr

Phe

Asn

Ala

Thr Ile

Ser Leu

Gly

Ser

100

Gln

Gly

Thr

115

Gly

Gly

Lys

Trp

Ala

Lys

210

Ala

Ser

Lys

Ser

Ser

Lys

Ala

Gln

Ser

Glu

Val

Gly

Tyr

Val

Ser

Leu

150

Ala

Ile 165

Ala

Arg

180

Asp

Gln

Val

195

Asn

Val

Tyr

Lys

Leu

Ser

290

Thr

Tyr

Trp

Leu

Ala

Tyr

Ala

Tyr

Cys 230

Tyr 245

Ala

Asp

260

Thr Val

275

Thr Gly Ala

Ser

Val

Arg

Asp

Asn Ala

Lys

Asn

Thr

Val Asp

Pro

Trp

Val

Gln

135

Lys

Asn

Ile

Lys

Glu

Tyr

Asp Thr 90

Ala

Ile

Tyr

Tyr Cys 95

Gly

Thr

Leu

Tyr

Glu

Tyr

Asp

105

110

Thr

120

Leu

Leu

Trp

Arg

Asp

Leu

215

Val

Trp

Glu

Pro

Val

Ser

Ser

Gly

Gly

Gly

Ser

125

Val

Glu

Ser

Gly

140

Gly

Gly

Leu

Val

Ser

Cys

Ala

155

Ala

Ser

Gly

Phe

Thr

160

Val

Arg

Gln 170

Ala

Pro

Gly

Lys

Gly 175

Ser

Lys

Tyr

Asn

Asn

Tyr

Ala

Thr

185

190

Arg

Phe

Thr

Ile

Ser

Arg

Asp

Asp

200

Gln

Met

Asn

Asn

Leu

220

205

Lys

Thr

Glu

Asp

Arg

Gly

Pro

Gly

Thr

295

His

Gln

Glu

Ala

Asn

235

Phe

Gly

Asn

Ser

Tyr 240

Gly

Thr 250

Leu

Val

Thr

Val

Ser 255

Gln

265

Gly

280

Ser Gly

Thr

Asn

Thr

Val

Val

Thr

Gln

270

Glu

Val

Tyr

Thr

Leu

Pro

300

Thr Cys

Ala

285

Asn Trp Val

Gln

- 76 045012

Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Lys Phe

305 310 315320

Leu Val Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly

325 330335

Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu

340 345350

Tyr Tyr Cys Thr Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly Gly

355 360365

Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Gln Val

370 375380

Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly Ser Leu

385 390 395400

Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr Gly Met

405 410415

Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Gly

420 425430

Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val Lys Gly

435 440445

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp Leu Gln

450 455460

Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala

465 470 475480

Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp Tyr Trp

485 490495

Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Ser Lys Lys

500 505510

Leu Ala Asp Glu Pro Glu Gly Gly Gly Ser Gln Thr Val Val Thr Gln

515 520525

Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys

530 535540

Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val

545 550 555560

- 77 045012

Gln Gln Lys Pro Gly Gln Ala Pro Arg 565

Asp Asp Asp Asp Lys Gly Thr Pro Ala

580 5

Gly Gly Lys Ala Ala Leu Thr Leu Ser

595 600

Ala Glu Tyr Tyr Cys Val Leu Trp Tyr

610 615

Gly Gly Thr Lys Leu Thr Val Leu Gly 625 630

Glu Val Gln Leu Val Glu Ser Gly Gly 645

Ser Leu Lys Leu Ser Cys Ala Ala Ser

660 6

Ala Met Asn Trp Val Arg Gln Ala Pro

675 680

Ala Arg Ile Arg Ser Lys Tyr Asp Tyr

690 695

Asp Ser Val Lys Asp Arg Phe Thr Ile 705 710

Thr Ala Tyr Leu Gln Met Asn Asn Leu 725

Tyr Tyr Cys Val Arg His Gly Asn Phe

740 7

Trp Ala Tyr Trp Gly Gln Gly Thr Leu

755 760

Gly Gly Ser Gly Gly Gly Ser Glu Val

770 775

Gly Leu Val Gln Pro Gly Asn Ser Leu 785 790

Gly Phe Thr Phe Ser Lys Phe Gly Met 805

Leu Ile Gly Asp Tyr Lys

570 575

Phe Ser Gly Ser Leu Leu

590

Val Gln Pro Glu Asp Glu

605

Asn Arg Trp Val Phe Gly 620

Gly Ser Gly Gly Gly Ser

635 640

Leu Val Gln Pro Gly Gly

650 655

Phe Thr Phe Asn Lys Tyr

670

Lys Gly Leu Glu Trp Val

685

Asp Asp Asp Asp Lys Ala 700

Arg Asp Asp Ser Lys Asn

715 720

Thr Glu Asp Thr Ala Val

730 735

Asn Ser Tyr Ile Ser Tyr

750

Thr Val Ser Ser Gly Gly

765

Leu Val Glu Ser Gly Gly 780

Leu Ser Cys Ala Ala Ser

795 800

Trp Val Arg Gln Ala Pro

810 815

- 78 045012

Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser

820 825

Gly Ser Gly Arg Asp

830

Thr Leu Tyr Ala

835

Asn Ala Lys Thr

850

Asp Thr Ala Val 865

Glu Ser Val Lys Gly Arg 840

Thr Leu Tyr Leu Gln Met 855

Tyr Tyr Cys Thr Ile Gly 870

Phe Thr Ile Ser Arg Asp 845

Asn Ser Leu Arg Pro Glu 860

Gly Ser Leu Ser Val Ser

875 880

Ser Gln Gly Thr Leu Val Thr Val Ser Ser

885890 < 210 > 145 < 211 > 889 < 212 > Protein < 213 > Artificial sequence < 220 >

<223> Synthetic construction - Pro186 Format 2 <400>145

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly

1.5 1015

Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr 20 2530

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 4045

Ser Gly 50 Ile Ser Trp Arg Gly 55 Asp Ser Thr Gly Tyr 60 Ala Asp Ser Val Lys 65 Gly Arg Phe Thr Ile 70 Ser Arg Asp Asn Ala 75 Lys Asn Thr Val Asp 80 Leu Gln Met Asn Ser Leu 85 Lys Pro Glu Asp Thr 90 Ala Ile Tyr Tyr Cys 95 Ala Ala Ala Ala Gly 100 Ser Ala Trp Tyr Gly 105 Thr Leu Tyr Glu Tyr 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Ser

115 120 125

- 79 045012

Gly

Gln 145

Phe

Leu

Tyr

Ser

Thr 225

Ile

Ser

Pro

Ser

Gln 305

Leu

Lys

Tyr

Thr

Gly Gly 130

Pro

Gly

Ser

Gly

Glu

Ser

Asn

Lys

Tyr

Ala

Glu

Tyr

Trp

Val

Ala

Lys

210

Ala

Ser

Gly

Ser

Val

Leu

150

Ile 165

Ala

Arg

180

Asp

Gln

Val

195

Asn

Val

Tyr

Gly

Leu

Ser

290

Lys

Val

Ala

Tyr

Thr

Tyr

Trp

Gly

Ala

Tyr

Ala

Tyr

Cys 230

Tyr 245

Ser

260

Thr

275

Thr

Gly

Gly

Val

Ala

Ser

Val

Pro

Pro

Ala

Cys

Lys

370

Gly

Gln

Ala

310

Gly

Thr

Pro 325

Leu

Thr

Leu

340

Thr Leu

Trp

355

Leu Thr Val

Leu

Gln Leu

135

Lys

Leu

Val

Glu

Ser

Gly Gly 140

Gly

Leu

Val

Ser

Cys

Ala

155

Ala

Ser

Gly

Phe

Thr

160

Asn

Trp

Val

Arg

Gln 170

Ala

Pro

Gly

Lys

Gly 175

Ile

Lys

Arg

Asp

Leu

215

Val

Trp

Gly

Pro

Ser

Lys

Tyr

Asn

Asn

Tyr

Ala

Thr

185

190

Arg

Phe

Thr

Ile

Ser

Arg

Asp

Asp

200

Gln

Met

Asn

Asn

Leu

220

205

Lys

Thr

Glu

Asp

Arg

Gly

Gly

Gly

Thr

295

Pro

Ala

Ser

Tyr

His

Gln

Ser

Ala

Asn

235

Phe

Gly

Asn

Ser

Tyr 240

Gly

Thr

250

Leu

Val

Thr

Val

Ser 255

Gln

265

Gly

280

Ser

Gly

Thr

Val

Val

Thr

Gln

270

Glu

Thr

Val

Thr

Leu

Thr

285

Cys

Ala

Arg

Arg

Gly

Ser

Gly

375

Gly

Phe

Val

Asn

Leu

Ser

Tyr

Ile

315

Gly 330

Gln

345

Asn

Arg

Pro

Trp

360

Gly Gly

Ser

Gly

Pro

300

Asn

Trp

Val

Gln

Gly

Gly

Thr

Lys

Phe

320

Ser

Glu

Val

Leu

Leu

Gly

Gly 335

Asp

Phe

Gly

380

Glu

Ala

Glu

350

Gly Gly

Gly

365

Gly Ser Gln

Val

- 80 045012

Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly Ser Leu

385 390 395400

Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr Gly Met

405 410415

Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Gly

420 425430

Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val Lys Gly

435 440445

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp Leu Gln

450 455460

Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala

465 470 475480

Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp Tyr Trp

485 490495

Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ser Gly Gly Pro Gly Pro

500 505510

Ala Gly Met Lys Gly Leu Pro Gly Ser Gln Thr Val Val Thr Gln Glu

515 520525

Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly

530 535540

Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln

545 550 555560

Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Asp Tyr Lys Asp

565 570575

Asp Asp Asp Lys Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly

580 585590

Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala

595 600605

Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly

610 615620

Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Glu

- 81 045012

625

Val

Leu

Met

Arg

Ser 705

Ala

Tyr

Ala

Gly

Leu 785

Phe

Lys

Leu

Ala

Thr 865

630

635

640

Gln Leu Val Glu Ser Gly Gly Gly Leu Val

645 650

Lys Leu Ser Cys Ala

660

Ala Ser Gly Phe Thr 6 65

Gln Pro Gly Gly Ser

655

Phe Asn Lys Tyr Ala

670

Asn Trp Val Arg Gln

675

Ala Pro Gly Lys Gly

680

Leu Glu Trp Val Ala

685

Ile Arg Ser Lys Tyr 690

Val Lys Asp Arg Phe

710

Tyr Leu Gln Met Asn

725

Cys Val Arg His Gly

740

Tyr Trp Gly Gln Gly

755

Ser Gly Gly Gly Ser 770

Val Gln Pro Gly Asn

790

Thr Phe Ser Lys Phe

805

Gly Leu Glu Trp Val

820

Tyr Ala Glu Ser Val

835

Lys Thr Thr Leu Tyr 850

Asp Tyr Lys Asp Asp 695

Asp Asp Lys Ala Asp 700

Thr Ile Ser Arg Asp

715

Asp Ser Lys Asn Thr

720

Asn Leu Lys Thr Glu

730

Asn Phe Gly Asn Ser

745

Thr Leu Val Thr Val

760

Glu Val Gln Leu Val

775

Ser Leu Arg Leu Ser

795

Gly Met Ser Trp Val

810

Ser Ser Ile Ser Gly

825

Lys Gly Arg Phe Thr 840

Leu Gln Met Asn Ser 855

Ala Val Tyr Tyr Cys Thr Ile 870

Asp Thr Ala Val Tyr

735

Tyr Ile Ser Tyr Trp

750

Ser Ser Gly Gly Gly

765

Glu Ser Gly Gly Gly 780

Cys Ala Ala Ser Gly

800

Arg Gln Ala Pro Gly

815

Ser Gly Arg Asp Thr

830

Ile Ser Arg Asp Asn

845

Leu Arg Pro Glu Asp 860

Gly Gly Ser Leu Ser Val Ser Ser

875 880

- 82 045012

Gln Gly Thr Leu Val Thr Val Ser Ser

885 <210>

<211>

<212>

<213>

146

890

Protein

Artificial sequence <220>

<223>

Synthetic construction

Pro187 Format 2 <400>

146

Gln Val

15

Lys

Leu Glu

Glu Ser

Gly

Gly Gly

Ser Val

Gln

Thr Gly

Gly

Ser

Leu

Arg

Leu Thr

Cys

Ala

Ala

Ser Gly 25

Arg

Thr

Ser

Arg Ser 30

Tyr

Gly

Met

Gly 35

Trp

Phe

Arg

Gln

Ala

Pro

Gly

Lys

Glu

Arg

Glu

Phe

Val

Ser

Gly

Ile

Ser

Trp

Arg

Gly

Asp

Ser

Thr

Gly

Tyr

Ala

Asp

Ser

Val

Lys 65

Gly

Arg

Phe

Thr

Ile 70

Ser

Arg

Asp

Asn

Ala 75

Lys

Asn

Thr

Val

Asp 80

Leu

Gln

Met

Asn

Ser

Leu 85

Lys

Pro

Glu

Asp

Thr

Ala

Ile

Tyr

Tyr

Cys 95

Ala

Ala

Ala

Ala

Gly

Ser

Ala

Trp

Tyr

Gly

Thr

Leu

Tyr

Glu

Tyr

Asp

100

105

110

Tyr

Trp

Gly

Gln

Gly

Thr

Gln

Val

Thr

Val

Ser

Ser

Gly

Gly

Gly

Ser

115

120

125

Gly

Gly

130

Gly

Ser

Glu

Val

Gln

135

Leu

Val

Glu

Ser

Gly

140

Gly

Gly

Leu

Val

Gln

145

Pro

Gly

Gly

Ser

Leu

150

Lys

Leu

Ser

Cys

Ala

155

Ala

Ser

Gly

Phe

Thr

160

Phe

Asn

Lys

Tyr

Ala

Ile 165

Asn

Trp

Val

Arg

Gln 170

Ala

Pro

Gly

Lys

Gly 175

Leu

Glu

Trp

Val

Ala Arg

Ile

Arg

Ser

Lys Tyr

Asn

Asn

Tyr

Ala Thr

180

185

190

Tyr

Tyr

Ala

Asp Gln Val

Lys

Asp

Arg Phe Thr

Ile

Ser

Arg Asp Asp

- 83 045012

195

200

205

Ser

Thr 225

Ile

Ser

Pro

Ser

Gln 305

Leu

Lys

Tyr

Thr

Lys 385

Arg

Gly

Ile

Lys Asn

210

Ala

Val

Ser

Gly

Ser

Thr

Tyr

Tyr

Trp

Gly

Gly

Leu

Ser

290

Lys

Val

Ala

Tyr

Ala

Tyr

Leu Gln

215

Met

Asn

Asn

Leu Lys Thr Glu Asp 220

Tyr

Cys 230

Val

Arg

His

Ala

Asn

235

Phe Gly Asn Ser Tyr

240

Ala

Tyr 245

Trp

Gly

Gln

Gly

Thr 250

Leu Val Thr Val Ser

255

Ser

260

Thr

275

Thr

Gly

Gly

Gly

Gly

Ser

Gln

265

Thr

Val Val Thr Gln Glu

270

Val

Ala

Ser

Pro

Gly

Gly

280

Thr

Val

Thr Leu Thr Cys Ala

285

Pro

Pro

Ala

Cys

Lys

370

Leu

Leu

Trp

Ser

Gly

Gln

Gly

Thr

Leu

Val

Ala

310

Pro 325

Thr

Leu

340

Thr

Leu

Trp

355

Leu

Glu

Thr

Phe

Trp

Thr

Glu

Cys

Arg

Val

Leu

Ser

Gly 390

Ala

Ala 405

Gln Ala

420

Arg Gly Asp

435

Thr

295

Pro

Ala

Ser

Tyr

Ser

Gly

Asn

Tyr

Pro Asn Trp Val Gln 300

Arg

Arg

Gly

Ser

Gly

375

Gly

Ser

Pro

Ser

Gly

Leu

Ile

315

Gly Gly Thr Lys Phe

320

Phe

Val

Ser

Gly 330

Gln

345

Asn

Arg

Pro

Trp

360

Gly

Gly

Gly

Gly

Thr

Gly

Ser

Gly

Ser

Val

Gln

395

Arg

Thr

Ser 410

Lys

Glu Arg

425

Gly Tyr Ala

440

Ser Leu Leu Gly Gly

335

Glu Asp Glu Ala Glu

350

Val Phe Gly Gly Gly

365

Gly Gly Ser Gln Val 380

Thr Gly Gly Ser Leu

400

Arg Ser Tyr Gly Met

415

Glu Phe Val Ser Gly

430

Asp Ser Val Lys Gly 445

- 84 045012

Arg

Met 465

Ala

Gly

Leu

Glu

Gly 545

Gln

Asp

Gly

Ala

Gly 625

Glu

Ser

Ala

Ala

Phe Thr Ile Ser Arg 450

Asn Ser Leu Lys Pro

470

Ala Gly Ser Ala Trp

485

Gln Gly Thr Gln Val

500

Ala Asp Glu Pro Glu

515

Pro Ser Leu Thr Val 530

Ser Ser Thr Gly Ala

550

Gln Lys Pro Gly Gln

565

Asp Asp Asp Lys Gly

580

Gly Lys Ala Ala Leu

595

Glu Tyr Tyr Cys Val 610

Gly Thr Lys Leu Thr

630

Val Gln Leu Val Glu

645

Leu Lys Leu Ser Cys

660

Met Asn Trp Val Arg

675

Arg Ile Arg Ser Lys 690

Asp Asn Ala Lys Asn 455

Glu Asp Thr Ala Ile

475

Tyr Gly Thr Leu Tyr

490

Thr Val Ser Ser Gly

505

Gly Gly Gly Ser Gln

520

Ser Pro Gly Gly Thr 535

Val Thr Ser Gly Asn

555

Ala Pro Arg Gly Leu

570

Thr Pro Ala Arg Phe

585

Thr Leu Ser Gly Val 600

Leu Trp Tyr Ser Asn 615

Val Leu Gly Gly Gly

635

Ser Gly Gly Gly Leu

650

Ala Ala Ser Gly Phe 6 65

Gln Ala Pro Gly Lys

680

Tyr Asp Tyr Lys Asp 695

Thr Val Asp Leu Gln 460

Tyr Tyr Cys Ala Ala

480

Glu Tyr Asp Tyr Trp

495

Gly Gly Ser Lys Lys

510

Thr Val Val Thr Gln

525

Val Thr Leu Thr Cys 540

Tyr Pro Asn Trp Val

560

Ile Gly Asp Tyr Lys

575

Ser Gly Ser Leu Leu

590

Gln Pro Glu Asp Glu

605

Arg Trp Val Phe Gly 620

Ser Gly Gly Gly Ser

640

Val Gln Pro Gly Gly

655

Thr Phe Asn Lys Tyr

670

Gly Leu Glu Trp Val

685

Asp Asp Asp Lys Ala 700

- 85 045012

Asp Ser Val Lys Asp Arg Phe Thr

705 710

Ile Ser Arg Asp Asp Ser Lys Asn

715 720

Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val

725 730735

Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr

740 745750

Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

755 760765

Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

770 775780

Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser

785 790 795800

Gly Phe Thr Phe Ser Lys Phe Gly Met Ser Trp Val Arg Gln Ala Pro

805 810815

Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Arg Asp

820 825830

Thr Leu Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

835 840845

Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu

850 855860

Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Val Ser

865 870 875880

Ser Gln Gly Thr Leu Val Thr Val Ser Ser

885890 <210>147 <211>895 <212> Protein <213> Artificial sequence <220>

<223> Synthetic construction - Pro225 (FL aB7H3 hF7 MMP9 linker) Format 2 <400> 147

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1. 5 10 15

- 86 045012

Ser

His

Ala

Lys 65

Leu

Ala

Tyr

Gly

Gln 145

Phe

Leu

Tyr

Ser

Thr 225

Ile

Ser

Leu

Met

Val

Gly

Gln

Ala

Trp

Arg Leu Ser

Gly Trp 35

Phe

Ile

Asn

Trp

Cys

Arg

Ser

Ala

Gln

Gly

Arg

Met

Gly

Gly

Gly

130

Pro

Asn

Glu

Tyr

Phe

Thr

Ile 70

Ser

Asn

Gly

Ser

Leu 85

Arg

Ala

Thr

Thr

100

Gln

Gly

Thr

Leu

115

Gly

Gly

Lys

Trp

Ala

Lys

210

Ala

Ser

Gly

Ser

Glu

Val

Gly

Tyr

Val

Ser

Leu

150

Ala

Ile 165

Ala

Arg

180

Asp

Gln

Val

195

Asn

Val

Tyr

Gly

Thr

Tyr

Trp

Gly

Ala

Tyr

Ala

Tyr

Cys 230

Tyr 245

Ser Gly

260

Pro Ser Arg

Ala Pro

Gly

Gly

Ser

Thr

Arg

Asp

Asn

Ala

Glu

Asp

Gln

Arg

Val

Gln

135

Lys

Asn

Ile

Lys

Arg

Lys

Val

Ser 75

Thr 90

Ala

Thr

105

Thr

120

Leu

Leu

Trp

Arg

Asp

Leu

215

Val

Trp

Gly

Val

Ser

Thr

Glu

Tyr

Lys

Ala

Glu

Ser

Val

Glu

Ser

Ser

Cys

Ala

155

Val

Arg

Gln 170

Ser

Lys

Tyr

185

Arg

Phe

Thr

200

Gln

Met

Asn

Asn

Arg

Gly

Gly

His

Gln

Ser

Ala

Asn

235

Gly

Thr 250

Gln Thr

265

Phe His Thr

Arg Glu

Ala

Asp

Tyr

Phe

Ser

Asn

Val

Ala

Gly

Gly

140

Ala

Ala

Asn

Ile

Thr

Tyr

Ser

Leu

Tyr

Val

Val

Tyr 80

Cys 95

Tyr

110

Gly

Gly

Asp

Ser

125

Gly

Gly

Leu

Val

Ser

Gly

Phe

Thr

160

Pro

Gly

Lys

Gly 175

Asn

Tyr

Ala

Thr

Ser

Leu

220

Phe

Leu

Val

190

Arg

Asp

Asp

205

Lys

Gly

Val

Val

Thr

Glu

Asp

Asn

Thr

Thr

Ser

Tyr 240

Val

Ser 255

Gln Glu

270

- 87 045012

Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val

275280

Thr Leu Thr Cys Ala

285

Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln

290 295300

Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Lys Phe

305 310 315320

Leu Val Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly

325 330335

Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu

340 345350

Tyr Tyr Cys Thr Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly Gly

355 360365

Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Gln Val

370 375380

Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

385 390 395400

Arg Leu Ser Cys Ala Pro Ser Arg Arg Thr Phe His Thr Tyr His Met

405 410415

Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Val

420 425430

Ile Asn Trp Ser Gly Gly Ser Thr Val Tyr Ala Asp Ser Val Lys Gly

435 440445

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

450 455460

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala

465 470 475480

Gly Gly Ala Thr Thr Gln Arg Ala Thr Glu Ala Ser Tyr Asp Tyr Trp

485 490495

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ser Gly Gly Pro Gly Pro

500 505510

Ala Gly Met Lys Gly Leu Pro Gly Ser Gln Thr Val Val Thr Gln Glu

- 88 045012

515

520

525

Pro

Ser Leu

530

Thr

Val

Ser

Pro Gly 535

Gly

Thr

Val

Thr Leu

540

Thr

Cys

Gly

Ser

545

Ser

Thr

Gly

Ala

Val

550

Thr

Ser

Gly

Asn

Tyr

555

Pro

Asn

Trp

Val

Gln

560

Gln

Lys

Pro

Gly

Gln

Ala 565

Pro

Arg

Gly

Leu

Ile 570

Gly

Asp

Tyr

Lys

Asp 575

Asp

Asp

Asp

Lys

Gly

Thr

Pro

Ala

Arg

Phe

Ser

Gly

Ser

Leu

Leu

Gly

580

585

590

Gly

Lys

Ala

Ala

Leu

Thr

Leu

Ser

Gly

Val

Gln

Pro

Glu

Asp

Glu

Ala

595

600

605

Glu

Tyr

610

Tyr

Cys

Val

Leu

Trp

615

Tyr

Ser

Asn

Arg

Trp

620

Val

Phe

Gly

Gly

Gly 625

Thr

Lys

Leu

Thr

Val

630

Leu

Gly

Gly

Gly

Ser

635

Gly

Gly

Gly

Ser

Glu

640

Val

Gln

Leu

Val

Glu

Ser 645

Gly

Gly

Gly

Leu

Val

650

Gln

Pro

Gly

Gly

Ser 655

Leu

Lys

Leu

Ser

Cys

Ala

Ala

Ser

Gly

Phe

Thr

Phe

Asn

Lys

Tyr

Ala

660

665

670

Met

Asn

Trp

Val

Arg

Gln

Ala

Pro

Gly

Lys

Gly

Leu

Glu

Trp

Val

Ala

675

680

685

Arg

Ile

690

Arg

Ser

Lys

Tyr

Asp

695

Tyr

Lys

Asp

Asp

Asp

700

Asp

Lys

Ala

Asp

Ser

705

Val

Lys

Asp

Arg

Phe

710

Thr

Ile

Ser

Arg

Asp

715

Asp

Ser

Lys

Asn

Thr

720

Ala

Tyr

Leu

Gln

Met

Asn 725

Asn

Leu

Lys

Thr

Glu 730

Asp

Thr

Ala

Val

Tyr 735

Tyr

Cys

Val

Arg

His Gly

Asn

Phe

Gly

Asn Ser

Tyr

Ile

Ser

Tyr Trp

Ala

Tyr

Trp

740

745

750

Gly Gln Gly

Thr

Leu

Val Thr Val

Ser

Ser

Gly Gly Gly

755

760

765

- 89 045012

Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

770 775780

Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

785 790 795800

Phe Thr Phe Ser Lys Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly

805 810815

Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Arg Asp Thr

820 825830

Leu Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

835 840845

Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

850 855860

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Val Ser Ser

865 870 875880

Gln Gly Thr Leu Val Thr Val Ser Ser His His His His His

885 890895 <210>148 <211>891 <212> Protein <213> Artificial sequence <220>

<223> Synthetic construction - Pro226 (FL aB7H3 hF12 MMP9 linker) Format 2 <400>148

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1. 5 1015

Ser Leu Arg Leu Ser Cys

Glu Ala Ser Pro Arg Thr Phe Ser Thr Tyr

2530

Ser Met Ala Trp Phe Arg 35

Ala Ala Ile Asn Trp Ser 50

Lys Gly Arg Phe Thr Ile 65 70

Gln Ala Pro Gly Lys 40

Gly Gly Asn Thr Ser 55

Ser Arg Asp Asn Ser 75

Glu Arg Ser Phe Val 45

Tyr Ala Asp Ser Val 60

Lys Asn Thr Leu Tyr 80

- 90 045012

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

90

Ala Val Tyr Tyr Cys 95

Ala Ala Gly Gly Val Leu Ala His His Asn Tyr

100 105

Glu Tyr Asp Tyr Trp

110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly

115 120

Gly Gly Ser Gly Gly

125

Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

130 135

Gly Leu Val Gln Pro 140

Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser

145 150 155

Gly Phe Thr Phe Asn

160

Lys Tyr Ala Ile Asn Trp Val Arg Gln Ala Pro

165 170

Gly Lys Gly Leu Glu

175

Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn

180 185

Tyr Ala Thr Tyr Tyr

190

Ala Asp Gln Val Lys Asp Arg Phe Thr Ile Ser

195 200

Arg Asp Asp Ser Lys

205

Asn Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys

210 215

Thr Glu Asp Thr Ala 220

Val Tyr Tyr Cys Val Arg His Ala Asn Phe Gly

225 230 235

Asn Ser Tyr Ile Ser

240

Tyr Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val

245 250

Thr Val Ser Ser Gly

255

Gly Gly Ser Gly Gly 260 Gly Ser Gln Thr Val 265 Val Leu Thr Val Ser 275 Pro Gly Gly Thr Val 280 Thr Leu Thr Gly 290 Ala Val Thr Ser Gly 295 Asn Tyr Pro Asn Pro 305 Gly Gln Ala Pro Arg 310 Gly Leu Ile Gly Gly 315 Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu

325 330

Thr Gln Glu Pro Ser

270

Thr Cys Ala Ser Ser

285

Trp Val Gln Gln Lys 300

Thr Lys Phe Leu Val

320

Leu Gly Gly Lys Ala

335

- 91 045012

Ala Leu Thr Leu Ser Gly Val Gln Pro

340 34

Cys Thr Leu Trp Tyr Ser Asn Arg Trp

355 360

Leu Thr Val Leu Gly Gly Gly Ser Gly

370 375

Gln Glu Ser Gly Gly Gly Leu Val Gln

385 390

Ser Cys Glu Ala Ser Pro Arg Thr Phe

405

Phe Arg Gln Ala Pro Gly Lys Glu Arg

420 42

Trp Ser Gly Gly Asn Thr Ser Tyr Ala

435 440

Thr Ile Ser Arg Asp Asn Ser Lys Asn

450 455

Ser Leu Arg Ala Glu Asp Thr Ala Val

465 470

Val Leu Ala His His Asn Tyr Glu Tyr

485

Leu Val Thr Val Ser Ser Ser Gly Gly

500 50

Gly Leu Pro Gly Ser Gln Thr Val Val

515 520

Val Ser Pro Gly Gly Thr Val Thr Leu

530 535

Ala Val Thr Ser Gly Asn Tyr Pro Asn 545 550

Gln Ala Pro Arg Gly Leu Ile Gly Asp

565

Gly Thr Pro Ala Arg Phe Ser Gly Ser

580 58

Asp Glu Ala Glu Tyr Tyr

350

Phe Gly Gly Gly Thr Lys 365

Gly Ser Gln Val Gln Leu 380

Gly Gly Ser Leu Arg Leu

395 400

Thr Tyr Ser Met Ala Trp

410 415

Phe Val Ala Ala Ile Asn

430

Ser Val Lys Gly Arg Phe

445

Leu Tyr Leu Gln Met Asn 460

Tyr Cys Ala Ala Gly Gly

475 480

Tyr Trp Gly Gln Gly Thr

490 495

Gly Pro Ala Gly Met Lys

510

Gln Glu Pro Ser Leu Thr

525

Cys Gly Ser Ser Thr Gly 540

Val Gln Gln Lys Pro Gly

555 560

Lys Asp Asp Asp Asp Lys

570 575

Leu Gly Gly Lys Ala Ala

590

- 92 045012

Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu

595 600

Ala Glu Tyr Tyr Cys

605

Val Leu Trp Tyr

610

Ser Asn Arg Trp Val

615

Phe Gly Gly Gly Thr Lys Leu 620

Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val

625 630 635 640

Glu Ser Gly Gly Gly Leu 645 Val Gln Pro Gly Gly 650 Ser Leu Lys Leu Ser 655 Cys Ala Ala Ser Gly 660 Phe Thr Phe Asn Lys 6 65 Tyr Ala Met Asn Trp 670 Val Arg Gln Ala Pro 675 Gly Lys Gly Leu Glu 680 Trp Val Ala Arg Ile 685 Arg Ser Lys Tyr 690 Asp 1 Tyr Lys Asp Asp 695 Asp ) Asp Lys Ala Asp 700 Ser Val Lys Asp Arg 705 Phe Thr Ile Ser Arg 710 Asp Asp Ser Lys Asn 715 Thr Ala Tyr Leu Gln 720 Met Asn Asn Leu Lys Thr 725 Glu Asp Thr Ala Val 730 Tyr Tyr Cys Val Arg 735 His Gly Asn Phe Gly 740 Asn Ser Tyr Ile Ser 745 Tyr Trp Ala Tyr Trp 750 Gly Gln Gly Thr Leu 755 Val Thr Val Ser Ser 760 Gly Gly Gly Gly Ser 765 Gly Gly Gly Ser 770 Glu 1 Val Gln Leu Val 775 Glu ) Ser Gly Gly Gly 780 Leu Val Gln Pro Gly 785 Asn Ser Leu Arg Leu 790 Ser Cys Ala Ala Ser 795 Gly Phe Thr Phe Ser 800 Lys Phe Gly Met Ser Trp 805 Val Arg Gln Ala Pro 810 Gly Lys Gly Leu Glu 815 Trp Val Ser Ser Ile 820 Ser Gly Ser Gly Arg 825 Asp Thr Leu Tyr Ala 830 Glu

Ser Val Lys Gly Arg Phe Thr Ile

Ser Arg Asp Asn Ala Lys Thr Thr

- 93 045012

835

840

845

Leu Tyr Leu Gln Met Asn 850

Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr

855 860

Tyr Cys Thr 865

Ile Gly Gly Ser Leu Ser Val 870

Ser Ser Gln Gly Thr Leu

875 880

Val Thr Val Ser Ser His His His His His

885

890 <210> 149 <211> 895 <212> Protein <213> Artificial sequence <220>

<223> Synthetic construction - Pro233 (humanized Pro186) 2

Format <400> 149

Gln Val Lys Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1. 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala 20

Ser Gly Arg Thr Ser Arg Ser Tyr

thirty

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys 35 40

Glu Arg Glu Phe Val 45

Ser Gly Ile Ser 50

Lys Gly Arg Phe 65

Leu Gln Met Asn

Trp Arg Gly Asp Ser 55

Thr Ile Ser Arg Asp 70

Ser Leu Arg Ala Glu 85

Thr Gly Tyr Ala Asp 60

Asn Ala Lys Asn Ser 75

Asp Thr Ala Leu Tyr 90

Ser Val

Leu Tyr 80

Tyr Cys 95

Ala Ala Ala Ala Gly

100

Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp

105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 115 120

Gly Gly Gly Ser Glu Val Gln Leu Val Glu 130 135

Ser Ser Gly Gly Gly Ser

125

Ser Gly Gly Gly Leu Val

140

Gln Pro Gly Gly Ser Leu Lys Leu

Ser Cys Ala Ala Ser Gly Phe Thr

- 94 045012

145

Phe

Leu

Tyr

Ser

Thr 225

Ile

Ser

Pro

Ser

Gln 305

Leu

Lys

Tyr

Thr

Lys 385

Asn

Glu

Tyr

150

155

160

Lys

Trp

Ala

Lys

210

Ala

Ser

Gly

Ser

Tyr Ala Ile

165

Val Ala

180

Arg

Asp

Gln

Val

195

Asn

Val

Tyr

Gly

Leu

Ser

290

Lys

Val

Ala

Tyr

Thr

Tyr

Trp

Gly

Asn

Trp

Val Arg Gln

170

Ala

Pro

Gly Lys Gly

175

Ile

Lys

Arg

Asp

Ala

Tyr

Leu

215

Tyr

Cys 230

Val

Ala

Tyr 245

Trp

Ser

260

Thr

275

Thr

Gly

Gly

Gly

Val

Ala

Ser

Pro

Pro

Pro

Ala

Cys

Lys

370

Gly

Gln

Gly

Thr

Leu

Val

Ala

310

Pro 325

Thr

Leu

340

Thr

Leu

Trp

355

Leu Thr

Val

Leu

Leu Val Glu

Ser

Gly 390

Ser Lys

185

Arg

Phe

200

Gln

Met

Asn

Tyr

Thr

Asn

Asn

Asn

Tyr Ala

190

Thr

Ile

Ser

Arg

Asp

Asp

205

Arg

Gly

Gly

Gly

Thr

295

Pro

Ala

Ser

Tyr

His

Gln

Ser

Ala

Asn

235

Gly

Thr 250

Gln

265

Gly

280

Ser

Gly

Thr

Thr

Val

Asn

Tyr

Arg

Arg

Gly

Ser

Gly

375

Gly

Leu

Ile

315

Phe

Val

Ser

Gly 330

Gln

345

Asn

Arg

Pro

Trp

360

Gly

Gly

Ser

Gly

Gly Gly

Val

Val

Arg 395

Leu

220

Phe

Leu

Val

Thr

Lys

Thr

Glu

Asp

Gly

Asn

Ser

Tyr 240

Val

Val

Leu

Pro

300

Gly

Ser

Glu

Val

Thr

Thr

Val

Ser 255

Gln

270

Thr

285

Asn

Trp

Cys

Val

Glu

Ala

Gln

Gly

Thr

Lys

Phe

320

Leu

Leu

Gly

Gly 335

Asp

Phe

Gly

380

Glu

Ala

Glu

350

Gly

Gly

Gly

365

Gly Ser

Gln

Val

Pro Gly Gly

Ser

Leu

400

- 95 045012

Thr Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser

405 410

Arg Ser Tyr Gly Met

415

Gly Trp Phe Arg Gln Ala Pro 420

Gly Lys Glu Arg Glu Phe Val Ser Gly

425 430

Ile Ser Trp Arg Gly Asp

435

Ser Thr Gly Tyr Ala Asp Ser Val Lys Gly

440 445

Arg Phe Thr

450

Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln

455 460

Met

465

Asn

Ser

Leu

Arg

Ala

470

Glu

Asp

Thr

Ala

Leu

475

Tyr

Tyr

Cys

Ala

Ala

480

Ala

Ala

Gly

Ser

Ala

Trp 485

Tyr

Gly

Thr

Leu

Tyr 490

Glu

Tyr

Asp

Tyr

Trp 495

Gly

Gln

Gly

Thr

Leu

Val

Thr

Val

Ser

Ser

Ser

Gly

Gly

Pro

Gly

Pro

500

505

510

Ala

Gly

Met

Lys

Gly

Leu

Pro

Gly

Ser

Gln

Thr

Val

Val

Thr

Gln

Glu

515

520

525

Pro Ser Leu Thr Val

530

Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly

535 540

Ser Ser Thr 545

Gln Lys Pro

Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn

550 555

Gly Gln Ala Pro Arg Gly Leu Ile Gly Asp

565 570

Trp Val Gln

560

Tyr Lys Asp

575

Asp Asp Asp Lys Gly Thr Pro Ala Arg Phe

580 585

Ser Gly Ser Leu Leu Gly

590

Gly Lys Ala Ala Leu Thr Leu 595

Ser Gly Val Gln Pro Glu Asp Glu Ala

600 605

Glu Tyr Tyr Cys Val Leu 610

Gly Thr Lys Leu Thr Val

625 630

Val Gln Leu Val Glu Ser

645

Trp Tyr Ser Asn Arg 615

Leu Gly Gly Gly Ser

635

Gly Gly Gly Leu Val 650

Trp Val Phe Gly Gly 620

Gly Gly Gly Ser Glu

640

Gln Pro Gly Gly Ser 655

- 96 045012

Leu

Lys

Leu Ser Cys

660

Ala

Ala

Ser Gly Phe 6 65

Thr

Phe

Asn Lys Tyr

670

Ala

Met

Asn

Trp Val

675

Arg

Gln

Ala

Pro Gly

680

Lys

Gly

Leu

Glu Trp

685

Val

Ala

Arg

Ile

690

Arg

Ser

Lys

Tyr

Asp

695

Tyr

Lys

Asp

Asp

Asp

700

Asp

Lys

Ala

Asp

Ser

705

Val

Lys

Asp

Arg

Phe

710

Thr

Ile

Ser

Arg

Asp

715

Asp

Ser

Lys

Asn

Thr

720

Ala

Tyr

Leu

Gln

Met

Asn

725

Asn

Leu

Lys

Thr

Glu 730

Asp

Thr

Ala

Val

Tyr 735

Tyr

Cys

Val

Arg

His

Gly

Asn

Phe

Gly

Asn

Ser

Tyr

Ile

Ser

Tyr

Trp

Ala

Tyr

Trp

740

745

750

Gly

Gln

Gly

Thr

Leu

Val

Thr

Val

Ser

Ser

Gly

Gly

Gly

755

760

765

Gly

Ser

770

Gly

Gly

Gly

Ser

Glu

775

Val

Gln

Leu

Val

Glu

780

Ser

Gly

Gly

Gly

Leu

785

Val

Gln

Pro

Gly

Asn

790

Ser

Leu

Arg

Leu

Ser

795

Cys

Ala

Ala

Ser

Gly 800

Phe

Thr

Phe

Ser

Lys

Phe 805

Gly

Met

Ser

Trp

Val

810

Arg

Gln

Ala

Pro

Gly 815

Lys

Gly

Leu

Glu

Trp

Val

Ser

Ser

Ile

Ser

Gly

Ser

Gly

Arg

Asp

Thr

Leu

Tyr

Ala

820

825

830

Glu

Ser

Val

Lys

Gly

Arg

Phe

Thr

Ile

Ser

Arg

Asp

Asn

835

840

845

Ala

Lys

850

Thr

Thr

Leu

Tyr

Leu

855

Gln

Met

Asn

Ser

Leu

860

Arg

Pro

Glu

Asp

Thr

865

Ala Val

Tyr

Tyr

Cys 870

Thr Ile

Gly

Gly

Ser

875

Leu Ser

Val

Ser

Ser

880

Gln

Gly Thr

Leu

Val

Thr 885

Val Ser

Ser

His

His 890

His His

His

His

895 <210>

<211>

150

875

- 97 045012 <212> Protein <213> Artificial sequence <220>

<223> Synthetic construction - Pro311 (FL aFOLRl h77.2 MMP9 linker) Format 2 <400> 150

Gln Val Gln Leu Val Glu Ser Gly

1.5 10

Gly Gly Leu Val Gln Pro Gly Gly 15

Ser Leu Arg Leu Ser Cys Ala Ala 20

Ser Gly Phe Thr Val Ser Asn Ser

thirty

Val Met Ala Trp Tyr Arg Gln Thr

40

Pro Gly Asn Glu Arg Glu Phe Val

Ala Ile Ile Asn Ser Ile Gly Ile

55

Thr Asn Tyr Ala Asp Ser Val Lys 60

Gly Arg Phe Thr Ile Ser Arg Asp 65 70

Asn Ser Lys Asn Thr Leu Tyr Leu

80

Gln Met Asn Ser Leu Arg Ala Glu 85

Asp Thr Ala Val Tyr Val Cys Asn

95

Arg Asn Phe Asp Arg Ile Tyr Trp

100

Gly Gln Gly Thr Leu Val Thr Val

105 110

Ser Ser Gly Gly Gly Ser Gly Gly

115 1

Gly Ser Glu Val Gln Leu Val Glu 0 125

Ser Gly Gly Gly Leu Val Gln Pro

130 135

Gly Gly Ser Leu Lys Leu Ser Cys

140

Ala Ala Ser Gly Phe Thr Phe Asn

145 150

Lys Tyr Ala Ile Asn Trp Val Arg

155 160

Gln Ala Pro Gly Lys Gly Leu Glu

165

Trp Val Ala Arg Ile Arg Ser Lys

170 175

Tyr Asn Asn Tyr Ala Thr Tyr Tyr 180

Ala Asp Gln Val Lys Asp Arg Phe

185 190

Thr Ile Ser Arg Asp Asp Ser Lys

195 2

Asn Thr Ala Tyr Leu Gln Met Asn 0 205

Asn Leu Lys Thr Glu Asp Thr Ala

Val Tyr Tyr Cys Val Arg His Ala

- 98 045012

210

215

220

Asn 225

Thr

Thr

Val

Tyr

Ile 305

Gly

Pro

Trp

Gly

Gln 385

Val

Arg

Asp

Thr

Phe

Gly

Asn

Ser Tyr

230

Ile

Ser

Tyr

Trp Ala

235

Tyr

Trp

Gly

Gln Gly

240

Leu

Val

Thr

Val

Val

Leu

Pro

290

Gly

Ser

Glu

Val

Thr

Thr

Val Ser

245

Gln

260

Thr

275

Asn

Gly

Leu

Asp

Phe

Gly

370

Pro

Ser

Glu

Ser

Cys

Glu

Ala

Ser

Gly

Gly

Gly Ser

250

Gly

Gly

Gly

Ser Gln

255

Pro

Ser

Leu

Thr

265

Ser

Ser

Thr

280

Gly

Val

Ala

Ser

Pro

Gly

Gly

270

Thr

Val

Thr

Ser

285

Gly

Asn

Trp

Thr

Leu

Glu

Val

Gln

Gln

295

Lys

Pro

Gly

Gln

Ala

300

Pro

Arg

Gly

Leu

Lys

Gly

Phe

310

Leu

Val

Pro

Gly

Thr

315

Pro

Ala

Arg

Phe

Ser

320

Gly 325

Ala

Glu

340

Gly

Gly

Gly

355

Gly

Gly

Asn

Phe

Val

Leu

450

Ser

Gln

Val

Lys

Tyr

Thr

Gly

Ser

Leu

390

Ser

Val

Met 405

Val

Ala

420

Lys Gly

435

Tyr Leu Gln

Ile

Arg

Met

Ala

Ala

Leu

Thr 330

Leu

Ser

Gly

Val

Gln 335

Tyr

Lys

Gln

375

Arg

Ala

Ile

Phe

Cys

Thr

Leu

Trp

Tyr

Ser

Asn

Arg

345

350

Leu

Thr

Val

Leu

Gly

Gly

Gly

Ser

360

365

Leu

Leu

Trp

Asn

Thr

Asn

455

Val

Glu

Ser

Gly

380

Gly

Gly

Leu

Val

Ser

Cys

Ala

395

Ala

Ser

Gly

Phe

Thr

400

Tyr

Ser

Arg

Gln 410

Thr

Pro

Gly

Asn

Glu 415

Ile

425

Ile

440

Ser Leu

Ser

Arg

Gly

Ile

Thr

Asn

Tyr

430

Arg

Ala

Asp

Asn

Glu

460

Ala

Ser Lys

Asn

445

Asp Thr Ala

Val

- 99 045012

Tyr Val Cys Asn Arg Asn Phe Asp Arg Ile Tyr Trp Gly Gln Gly Thr

465 470 475480

Leu Val Thr Val Ser Ser Ser Gly Gly Pro Gly Pro Ala Gly Met Lys

485 490495

Gly Leu Pro Gly Ser Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr

500 505510

Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly

515 520525

Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly

530 535540

Gln Ala Pro Arg Gly Leu Ile Gly Asp Tyr Lys Asp Asp Asp Asp Lys

545 550 555560

Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala

565 570575

Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys

580 585590

Val Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly Gly Thr Lys Leu

595 600605

Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val

610 615620

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser

625 630 635640

Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr Ala Met Asn Trp Val

645 650655

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser

660 665670

Lys Tyr Asp Tyr Lys Asp Asp Asp Asp Lys Ala Asp Ser Val Lys Asp

675 680685

Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln

690 695700

Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg

705 710 715720

- 100 045012

His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp Ala Tyr Trp Gly

725 730 735

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

740 745 750

Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

755 760 765

Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

770 775 780

Lys Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

785 790 795 800

Trp Val Ser Ser Ile Ser Gly Ser Gly Arg Asp Thr Leu Tyr Ala Glu

805 810 815

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr

820 825 830

Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr

835 840 845

Tyr Cys Thr Ile Gly Gly Ser Leu Ser Val Ser Ser Gln Gly Thr Leu

850 855 860

Val Thr Val Ser Ser His His His His His

865 870 875 <210> 151 <211> 873 <212> Protein <213> Artificial sequence <220>

<223> Synthetic construction - Pro312 (FL aFOLR1 h59.3 MMP9 linker) <400> 151

Gln Val Gln Leu Val 1.5

Glu Ser Gly Gly Gly Leu Val

15

Gln Pro Gly Gly

Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Asn Thr Phe 20 25

Ser Ile Ser 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys 35 40

Gln Arg Glu Trp Val 45

- 101 045012

Ala Val Thr His Ser Asp Tyr Ser Thr Asn Tyr Ala Asp Ser Val Lys 50 5560

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 7580

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Lys

9095

His Tyr Gly Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105110

Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser

115 120125

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala

130 135140

Ala Ser Gly Phe Thr Phe Asn Lys Tyr Ala Ile Asn Trp Val Arg Gln

145 150 155160

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr

165 170175

Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Gln Val Lys Asp Arg Phe Thr

180 185190

Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn

195 200205

Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Ala Asn

210 215220

Phe Gly Asn Ser Tyr Ile Ser Tyr Trp Ala Tyr Trp Gly Gln Gly Thr

225 230 235240

Leu Val Thr Val Ser Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln Thr

245 250255

Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val

260 265270

Thr Leu Thr Cys Ala Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr

275 280285

Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile

290 295300

- 102 045012

Gly 305

Ser

Glu

Val

Gly

Pro 385

Ser

Glu

Ser

Leu

Tyr 465

Thr

Pro

Pro

Thr

Pro 545

Gly Thr Lys Phe Leu Val Pro Gly Thr Pro Ala Arg Phe Ser Gly

310 315 320

Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro

325 330 335

Asp Glu Ala Glu Tyr Tyr Cys Thr Leu Trp Tyr Ser Asn Arg Trp

340 345 350

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly

355 360 365

Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

370 375 380

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Asn Thr Phe

390 395 400

Ile Ser Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg

405 410 415

Trp Val Ala Val Thr His Ser Asp Tyr Ser Thr Asn Tyr Ala Asp

420 425 430

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

435 440 445

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

450 455 460

Cys Lys His Tyr Gly Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val

470 475 480

Val Ser Ser Ser Gly Gly Pro Gly Pro Ala Gly Met Lys Gly Leu

485 490 495

Gly Ser Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser

500 505 510

Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val

515 520 525

Ser Gly Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala

530 535 540

Arg Gly Leu Ile Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly Thr

550 555 560

- 103 045012

Pro Ala Arg Phe Ser Gly Ser Leu

565

Leu Gly Gly Lys Ala Ala Leu Thr

570 575

Leu Ser Gly Val Gln Pro

580

Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu

585 590

Trp Tyr Ser Asn Arg Trp Val 595

Phe Gly Gly Gly Thr Lys Leu Thr Val

600 605

Leu Gly Gly Gly Ser Gly Gly Gly Ser Glu Val

610 615

Gln Leu Val Glu Ser

620

Gly Gly Gly Leu Val Gln Pro

625 630

Ala Ser Gly Phe Thr Phe Asn

645

Gly Gly Ser Leu Lys Leu Ser Cys Ala 635 640

Lys Tyr Ala Met Asn Trp Val Arg Gln 650 655

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg

660 665

Ile Arg Ser Lys Tyr

670

Asp Tyr Lys Asp Asp Asp Asp Lys Ala Asp

675 680

Ser Val Lys Asp Arg Phe 685

Thr Ile Ser Arg Asp Asp 690

Asn Leu Lys Thr Glu Asp

705 710

Asn Phe Gly Asn Ser Tyr

725

Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn 695700

Thr Ala Val Tyr Tyr Cys Val Arg His Gly

715720

Ile Ser Tyr Trp Ala Tyr Trp Gly Gln Gly 730735

Thr Leu Val Thr Val

740

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser

745 750

Glu Val Gln Leu Val Glu Ser Gly Gly

755 760

Ser Leu Arg Leu Ser Cys Ala Ala Ser

770 775

Gly Met Ser Trp Val Arg Gln Ala Pro

785 790

Gly Leu Val Gln Pro Gly Asn 765

Gly Phe Thr Phe Ser Lys Phe 780

Gly Lys Gly Leu Glu Trp Val 795 800

Ser Ser Ile Ser Gly Ser Gly Arg Asp

Thr Leu Tyr Ala Glu Ser Val

- 104 045012

805 810 815

Lys Gly Arg Phe Thr

820

Ile Ser Arg Asp Asn

825

Ala Lys Thr Thr Leu Tyr

830

Leu Gln Met Asn Ser

835

Leu Arg Pro Glu Asp

840

Thr Ala Val Tyr Tyr Cys 845

Thr Ile Gly Gly Ser 850

Leu Ser Val Ser Ser

855

Gln Gly Thr Leu Val Thr 860

Val Ser Ser His His 865

His His His His 870 <210> 152 <211> 881 <212> Protein <213> Artificial sequence <220>

<223> Synthetic construction - Pro313 (FL aFOLR1 h22.4 MMP9 linker) Format 2 <400>152

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1. 5 1015

Ser Leu Arg Leu Ser Cys

Glu Ala Ser Gly Thr Thr Phe Ser Arg Asp

2530

Val Met Gly Trp

Tyr Arg Gln Ala Pro 40

Gly Lys Gln Arg Glu Leu Val 45

Ala Ile Ile Ser 50

Arg Gly Gly Ser Thr 55

Gly Arg Phe Thr 65

Ile Ser Arg Asp Asn 70

Gln Met Asn Ser

Leu Arg Ala Glu Asp 85

Asn Tyr Ala Asp Ser Val Lys 60

Ser Lys Asn Thr Leu Tyr Leu 75 80

Thr Ala Val Tyr Tyr Cys Asn 90 95

Ala Asn

Thr Ala Thr Trp Gly Arg Val Phe Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Ser Gly Gly

115 120

Gly Ser Glu Val Gln

125

- 105 045012

Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

130 135

Gly Gly Ser Leu Lys 140

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn

145 150 155

Lys Tyr Ala Ile Asn

160

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

165 170

Trp Val Ala Arg Ile

175

Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr

180 185

Ala Asp Gln Val Lys

190

Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys

195 200

Asn Thr Ala Tyr Leu

205

Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala

210 215

Val Tyr Tyr Cys Val 220

Arg His Ala Asn Phe Gly Asn Ser Tyr Ile Ser

225 230 235

Tyr Trp Ala Tyr Trp

240

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly

245 250

Gly Gly Ser Gly Gly

255

Gly Ser Gln Thr Val Val Thr Gln Glu Pro Ser

260 265

Leu Thr Val Ser Pro

270

Gly Gly Thr Val Thr Leu Thr Cys Ala Ser Ser

275 280

Thr Gly Ala Val Thr

285

Ser Gly Asn Tyr Pro Asn Trp Val Gln Gln Lys

290 295

Pro Gly Gln Ala Pro 300

Arg Gly Leu Ile Gly Gly Thr Lys Phe Leu Val

305 310 315

Pro Gly Thr Pro Ala

320

Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala

325 330

Ala Leu Thr Leu Ser

335

Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr

340 345

Cys Thr Leu Trp Tyr

350

Ser Asn Arg Trp Val Phe Gly Gly Gly Thr Lys

355 360

Leu Thr Val Leu Gly

365

Gly Gly Ser Gly Gly Gly Ser Gln Val Gln Leu

370 375

Val Glu Ser Gly Gly 380

- 106 045012

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Glu Ala Ser

385 390 395400

Gly Thr Thr Phe Ser Arg Asp Val Met Gly Trp Tyr Arg Gln Ala Pro

405 410415

Gly Lys Gln Arg Glu Leu Val Ala Ile Ile Ser Arg Gly Gly Ser Thr

420 425430

Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

435 440445

Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

450 455460

Thr Ala Val Tyr Tyr Cys Asn Ala Asn Thr Ala Thr Trp Gly Arg Val

465 470 475480

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ser Gly Gly Pro

485 490495

Gly Pro Ala Gly Met Lys Gly Leu Pro Gly Ser Gln Thr Val Val Thr

500 505510

Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr

515 520525

Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp

530 535540

Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Asp Tyr

545 550 555560

Lys Asp Asp Asp Asp Lys Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu

565 570575

Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp

580 585590

Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn Arg Trp Val Phe

595 600605

Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly

610 615620

Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

625 630 635640

- 107 045012

Gly

Tyr

Val

Ala

Asn 705

Val

Tyr

Gly

Gly

Ser 785

Pro

Asp

Asp

Glu

Ser 865

His

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys

645 650 655

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

660 665 670

Ala Arg Ile Arg Ser Lys Tyr Asp Tyr Lys Asp Asp Asp Asp Lys

675 680 685

Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys

690 695 700

Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala

710 715 720

Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser

725 730 735

Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly

740 745 750

Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

755 760 765

Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala

770 775 780

Gly Phe Thr Phe Ser Lys Phe Gly Met Ser Trp Val Arg Gln Ala

790 795 800

Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Arg

805 810 815

Thr Leu Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

820 825 830

Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro

835 840 845

Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Val

850 855 860

Ser Gln Gly Thr Leu Val Thr Val Ser Ser His His His His

870 875 880

- 108 045012 <210> 153 <211> 898 <212> Protein <213> Artificial sequence <220>

<223> Synthetic design - Pro246 (FL haEGFR1/haEGFR2 heterologous

COBRA with MMP9 linker) < 400 > 153

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1. 5 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 2530

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 4045

Val Ala Ile Asn Trp Ser Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 5560

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 7580

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

9095

Ala Ala Gly Tyr Gln Ile Asn Ser Gly Asn Tyr Asn Phe Lys Asp Tyr

100 105110

Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly

115 120125

Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

130 135140

Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser

145 150 155160

Gly Phe Thr Phe Asn Lys Tyr Ala Ile Asn Trp Val Arg Gln Ala Pro

165 170175

Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn

180 185190

- 109 045012

Tyr

Arg

Thr 225

Asn

Thr

Thr

Thr

Trp 305

Thr

Leu

Glu

Gly

Ser 385

Gly

Tyr

Val

Ala Thr Tyr Tyr Ala Asp Gln Val Lys Asp Arg Phe Thr Ile Ser

195 200 205

Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys

210 215 220

Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Ala Asn Phe Gly

230 235 240

Ser Tyr Ile Ser Tyr Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val

245 250 255

Val Ser Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln Thr Val Val

260 265 270

Gln Glu Pro 275 Ser Leu Thr Val 2( Ser 80 Pro Gly Gly Thr Val 285 Thr Leu Cys 290 Ala Ser Ser Thr Gly 295 Ala ) Val Thr Ser Gly 300 Asn Tyr Pro Asn Val Gln Gln Lys Pro 310 Gly Gln Ala Pro Arg 315 Gly Leu Ile Gly Gly 320 Lys Phe Leu Val Pro 325 Gly Thr Pro Ala Arg 330 Phe Ser Gly Ser Leu 335 Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Glin Pro Glu Asp

340 345 350

Ala Glu Tyr Tyr Cys Thr Leu Trp Tyr Ser Asn Arg Trp Val Phe

355 360 365

Gly 370 Gly Thr Lys Leu Thr 375 Val Leu Gly Gly Gly 380 Ser Gly Gly Gly Gln Val Lys Leu Val 390 Glu Ser Gly Gly Gly 395 Val Val Arg Pro Gly 400 Ser Leu Thr Leu Ser 405 Cys Ala Ala Ser Gly 410 Arg Thr Ser Arg Ser 415 Gly Met Gly Trp 420 Phe Arg Gln Ala Pro 425 Gly Lys Glu Arg Glu 430 Phe Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser

435 440 445

- 110 045012

Val

Tyr 465

Cys

Asp

Pro

Thr

Thr 545

Trp

Tyr

Leu

Asp

Phe 625

Gly

Gly

Lys

Trp

Lys Gly Arg Phe Thr 450

Leu Gln Met Asn Ser

470

Ala Ala Ala Ala Gly

485

Tyr Trp Gly Gln Gly

500

Gly Pro Ala Gly Met

515

Gln Glu Pro Ser Leu 530

Cys Gly Ser Ser Thr

550

Val Gln Gln Lys Pro

565

Lys Asp Asp Asp Asp

580

Leu Gly Gly Lys Ala

595

Glu Ala Glu Tyr Tyr 610

Gly Gly Gly Thr Lys

630

Ser Glu Val Gln Leu

645

Gly Ser Leu Lys Leu

660

Tyr Ala Met Asn Trp

675

Val Ala Arg Ile Arg 690

Ile Ser Arg Asp Asn 455

Leu Arg Ala Glu Asp

475

Ser Ala Trp Tyr Gly

490

Thr Leu Val Thr Val

505

Lys Gly Leu Pro Gly

520

Thr Val Ser Pro Gly 535

Gly Ala Val Thr Ser

555

Gly Gln Ala Pro Arg

570

Lys Gly Thr Pro Ala

585

Ala Leu Thr Leu Ser

600

Cys Val Leu Trp Tyr 615

Leu Thr Val Leu Gly

635

Val Glu Ser Gly Gly

650

Ser Cys Ala Ala Ser 6 65

Val Arg Gln Ala Pro

680

Ser Lys Tyr Asp Tyr 695

Ala Lys Asn Ser Leu 460

Thr Ala Leu Tyr Tyr

480

Thr Leu Tyr Glu Tyr

495

Ser Ser Ser Gly Gly

510

Ser Gln Thr Val Val

525

Gly Thr Val Thr Leu 540

Gly Asn Tyr Pro Asn

560

Gly Leu Ile Gly Asp

575

Arg Phe Ser Gly Ser

590

Gly Val Gln Pro Glu

605

Ser Asn Arg Trp Val 620

Gly Gly Ser Gly Gly

640

Gly Leu Val Gln Pro

655

Gly Phe Thr Phe Asn

670

Gly Lys Gly Leu Glu

685

Lys Asp Asp Asp Asp 700

- 111 045012

Lys Ala Asp Ser Val Lys Asp Arg Phe Thr Ile

705 710715

Ser Arg Asp Asp Ser

720

Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr

725 730735

Ala Val Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile

740 745750

Ser Tyr Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

755 760765

Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser

770 775780

Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala

785 790 795800

Ala Ser Gly Phe Thr Phe Ser Lys Phe Gly Met Ser Trp Val Arg Gln

805 810815

Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly

820 825830

Arg Asp Thr Leu Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser

835 840845

Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg

850 855860

Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser

865 870 875880

Val Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser His His His

885 890895

His His <210>154 <211>893 <212> Protein <213> Artificial sequence <220>

<223> Synthetic design - Pro256 (FL haEpCAM VIB13/haEGFR1 heterologous COBRA MMP9 linker)

- 112 045012 <400> 154

Gln Val Gln Leu Val Glu Ser Gly Gly

1.5 10

Ser Leu Thr Leu Ser Cys Ala Ala Ser

2

Ile Asn Leu Met Gly Trp Tyr Arg Gln 35 40

Leu Val Ala Arg Ile Thr Ser Gly Asp

55

Val Lys Gly Arg Phe Thr Ile Ser Arg 65 70

Tyr Leu Gln Met Asn Ser Leu Arg Pro 85

Cys Asn Leu Leu Leu Arg Ser Ser Pro

100 1

Gly Gln Gly Thr Leu Val Thr Val Ser

115 120

Gly Ser Glu Val Gln Leu Val Glu Ser

130 135

Gly Gly Ser Leu Lys Leu Ser Cys Ala 145 150

Lys Tyr Ala Ile Asn Trp Val Arg Gln

165

Trp Val Ala Arg Ile Arg Ser Lys Tyr

180 1

Ala Asp Gln Val Lys Asp Arg Phe Thr

195 200

Asn Thr Ala Tyr Leu Gln Met Asn Asn

210 215

Val Tyr Tyr Cys Val Arg His Ala Asn 225 230

Tyr Trp Ala Tyr Trp Gly Gln Gly Thr

Leu Val Gln Pro Gly Gly 15

Thr Gly Ser Ile Phe Ser 30

Pro Gly Lys Gln Arg Glu 45

Thr Val Tyr Ala Asp Ser 60

Asn Ser Lys Asn Thr Leu 75 80

Asp Thr Ala Val Tyr Tyr

95

Ala Thr Thr Pro Tyr Trp

110

Gly Gly Gly Ser Gly Gly

125

Gly Gly Leu Val Gln Pro 140

Ser Gly Phe Thr Phe Asn

155 160

Pro Gly Lys Gly Leu Glu

170 175

Asn Tyr Ala Thr Tyr Tyr

190

Ser Arg Asp Asp Ser Lys

205

Lys Thr Glu Asp Thr Ala 220

Gly Asn Ser Tyr Ile Ser

235 240

Val Thr Val Ser Ser Gly

- 113 045012

245

250

255

Gly

Leu

Thr

Pro 305

Pro

Ala

Cys

Leu

Val 385

Ser

Phe

Trp

Thr

Ser 465

Gly

Gly

Thr

Ser Gly Gly

260

Val Ser

275

Gly

290

Ala

Val

Gly

Gln

Ala

Gly

Thr

Pro

Leu

Thr

Leu

Thr

Leu

Thr

370

Glu

Cys

Arg

Arg

Gly

Ser

Gln Thr Val

265

Val

Thr Gln Glu Pro Ser

270

Pro

Gly

Gly

Thr Val

280

Thr

Leu

Thr Cys Ala Ser Ser

285

Thr

Pro

Ala

Ser

Arg 310

Arg 325

Ser

340

Trp

Tyr

Gly

Ser

355

Val

Leu

Gly

Gly

Ser

Gly

Gly

Gly 390

Ala

Ala

Ser

Gly 405

Gln

Gly

Ile

450

Ala

Pro

Gly

420

Asp

435

Ser

Arg

Ser

Thr

Asp

Asn

Leu Arg

Ser Ala

Ala

Glu

Asp 470

Trp

Tyr

Gly 485

Gly

295

Asn

Tyr

Pro

Asn

Trp Val Gln Gln Lys 300

Gly

Leu

Ile

Gly

Gly 315

Thr Lys Phe Leu Val

320

Phe

Ser

Gly

Ser

Leu 330

Leu Gly Gly Lys Ala

335

Val

Asn

Gln

Pro

Glu

345

Arg

Trp

360

Asp

Glu Ala Glu Tyr Tyr

350

Val

Phe

Gly Gly Gly Thr Lys

365

Gly

375

Val

Arg

Lys

Gly

Ser

Gly

Gly

Gly

Ser Gln Val Lys Leu 380

Val

Arg

Pro

Gly 395

Gly Ser Leu Thr Leu

400

Thr

Glu

Tyr

Ala

455

Ser

Arg

Ser 410

Tyr Gly Met Gly Trp

415

Arg

Glu

425

Ala

Asp

Phe

Ser

440

Lys

Asn

Ser

Leu

Thr Ala

Thr Leu

Leu

Tyr

Tyr

475

Tyr

Glu

Tyr 490

Val Ser Gly Ile Ser

430

Val Lys Gly Arg Phe

445

Tyr Leu Gln Met Asn 460

Cys Ala Ala Ala Ala

480

Asp Tyr Trp Gly Gln

495

- 114 045012

Gly Thr Leu Val Thr Val Ser Ser Ser

500 5

Met Lys Gly Leu Pro Gly Ser Gln Thr

515 520

Leu Thr Val Ser Pro Gly Gly Thr Val

530 535

Thr Gly Ala Val Thr Ser Gly Asn Tyr 545 550

Pro Gly Gln Ala Pro Arg Gly Leu Ile 565

Asp Lys Gly Thr Pro Ala Arg Phe Ser

580 5

Ala Ala Leu Thr Leu Ser Gly Val Gln

595 600

Tyr Cys Val Leu Trp Tyr Ser Asn Arg

610 615

Lys Leu Thr Val Leu Gly Gly Gly Ser 625 630

Leu Val Glu Ser Gly Gly Gly Leu Val 645

Leu Ser Cys Ala Ala Ser Gly Phe Thr

660 6

Trp Val Arg Gln Ala Pro Gly Lys Gly

675 680

Arg Ser Lys Tyr Asp Tyr Lys Asp Asp

690 695

Lys 705 Asp Arg Phe Thr Ile 710 Ser Arg Asp Leu Gln Met Asn Asn Leu 725 Lys Thr Glu Val Arg His Gly Asn Phe Gly Asn Ser

740 7

Gly Pro Gly Pro Ala Gly

510

Val Thr Gln Glu Pro Ser

525

Leu Thr Cys Gly Ser Ser 540

Asn Trp Val Gln Gln Lys

555 560

Asp Tyr Lys Asp Asp Asp

570 575

Ser Leu Leu Gly Gly Lys

590

Glu Asp Glu Ala Glu Tyr 605

Val Phe Gly Gly Gly Thr 620

Gly Gly Ser Glu Val Gln

635 640

Pro Gly Gly Ser Leu Lys

650 655

Asn Lys Tyr Ala Met Asn

670

Glu Trp Val Ala Arg Ile

685

Asp Lys Ala Asp Ser Val 700

Ser Lys Asn Thr Ala Tyr

715 720

Thr Ala Val Tyr Tyr Cys

730 735

Ile Ser Tyr Trp Ala Tyr

750

- 115 045012

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

755 760765

Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

770 775780

Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

785 790 795800

Phe Ser Lys Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

805 810815

Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Arg Asp Thr Leu Tyr

820 825830

Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

835 840845

Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala

850 855860

Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Val Ser Ser Gln Gly

865 870 875880

Thr Leu Val Thr Val Ser Ser His His His His His

885890 < 210 > 155 < 211 > 885 < 212 > Protein < 213 > Artificial sequence < 220 >

<223> Synthetic construct - Pro420 (FL aFOLR1 h77.2/haEGFR1 heterologous COBRA MMP9 linker) <400>155

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1. 5 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val 2025

Ser Asn Ser 30

Val Met Ala Trp Tyr Arg 35

Ala Ile Ile Asn Ser Ile 50

Gln Thr Pro Gly Asn 40

Gly Ile Thr Asn Tyr 55

Glu Arg Glu Phe Val 45

Ala Asp Ser Val Lys 60

- 116 045012

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys 65 70 75

Asn Thr Leu Tyr Leu 80

Gln

Met

Asn

Ser

Leu Arg

Ala

Glu

Asp

Thr Ala

Val

Tyr

Val

Cys Asn 95

Arg

Asn

Phe

Asp

Arg

Ile

Tyr

Trp

Gly

Gln

Gly

Thr

Leu

Val

Thr

Val

100

105

110

Ser

Ser

Gly

Gly

Gly

Ser

Gly

Gly

Gly

Ser

Glu

Val

Gln

Leu

Val

Glu

115

120

125

Ser Gly Gly Gly Leu Val 130

Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys

135 140

Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr Ala Ile Asn Trp Val Arg

145 150 155 160

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys

165 170 175

Tyr Asn Asn

Tyr Ala Thr Tyr Tyr Ala Asp Gln Val

180 185

Lys Asp Arg Phe

190

Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn

195 200 205

Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Ala

210 215 220

Asn Phe Gly Asn Ser Tyr

225 230

Ile Ser Tyr Trp Ala Tyr Trp Gly Gln Gly

235 240

Thr Leu Val Thr Val Ser

245

Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln

250 255

Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val

260 265

Ser Pro Gly Gly Thr

270

Val Thr Leu Thr Cys Ala

275

Tyr Pro Asn Trp Val Gln 290

Ser Ser Thr Gly Ala 280

Gln Lys Pro Gly Gln 295

Val Thr Ser Gly Asn 285

Ala Pro Arg Gly Leu 300

Ile Gly Gly Thr Lys Phe Leu Val

305 310

Pro Gly Thr Pro Ala Arg Phe Ser

315 320

- 117 045012

Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln

325 330335

Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Thr Leu Trp Tyr Ser Asn Arg

340 345350

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Ser

355 360365

Gly Gly Gly Ser Gln Val Lys Leu Val Glu Ser Gly Gly Gly Val Val

370 375380

Arg Pro Gly Gly Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Arg Thr

385 390 395400

Ser Arg Ser Tyr Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu

405 410415

Arg Glu Phe Val Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr

420 425430

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

435 440445

Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

450 455460

Leu Tyr Tyr Cys Ala Ala Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu

465 470 475480

Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

485 490495

Ser Gly Gly Pro Gly Pro Ala Gly Met Lys Gly Leu Pro Gly Ser Gln

500 505510

Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr

515 520525

Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Asn

530 535540

Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu

545 550 555560

Ile Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly Thr Pro Ala Arg Phe

- 118 045012

565

570

575

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

580 585590

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn

595 600605

Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly

610 615620

Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

625 630 635640

Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe

645 650655

Thr Phe Asn Lys Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys

660 665670

Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asp Tyr Lys Asp

675 680685

Asp Asp Asp Lys Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg

690 695700

Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr

705 710 715720

Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn

725 730735

Ser Tyr Ile Ser Tyr Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

740 745750

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu

755 760765

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu

770 775780

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Phe Gly Met Ser Trp

785 790 795800

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser

805 810815

- 119 045012

Gly Ser Gly Arg Asp Thr Leu Tyr Ala Glu Ser

820825

Val Lys Gly Arg Phe

830

Thr Ile Ser Arg Asp Asn Ala Lys Thr 835840

Ser Leu Arg Pro Glu Asp Thr Ala Val 850855

Ser Leu Ser Val Ser Ser Gln Gly Thr

865870

Thr Leu Tyr Leu Gln Met Asn 845

Tyr Tyr Cys Thr Ile Gly Gly 860

Leu Val Thr Val Ser Ser His 875 880

His His His His

885 <210>156 <211>885 <212> Protein <213> Artificial sequence <220>

<223> Synthetic construct - Pro421 (FL haEGFR1/aFOLR1 h77.2 heterologous COBRA MMP9 linker) <400> 156

Gln Val Lys Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1. 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala 20

Ser Gly Arg Thr Ser Arg Ser Tyr

thirty

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys 35 40

Glu Arg Glu Phe Val 45

Ser Gly Ile Ser 50

Lys Gly Arg Phe 65

Leu Gln Met Asn

Trp Arg Gly Asp Ser 55

Thr Ile Ser Arg Asp 70

Ser Leu Arg Ala Glu 85

Thr Gly Tyr Ala Asp 60

Asn Ala Lys Asn Ser 75

Asp Thr Ala Leu Tyr 90

Ser Val

Leu Tyr 80

Tyr Cys 95

Ala Ala Ala Ala Gly Ser Ala

100

Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp

105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

115 120

Ser Gly Gly Gly Ser

125

- 120 045012

Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

130 135140

Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr

145 150 155160

Phe Asn Lys Tyr Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly

165 170175

Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr

180 185190

Tyr Tyr Ala Asp Gln Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp

195 200205

Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp

210 215220

Thr Ala Val Tyr Tyr Cys Val Arg His Ala Asn Phe Gly Asn Ser Tyr

225 230 235240

Ile Ser Tyr Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

245 250255

Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln Thr Val Val Thr Gln Glu

260 265270

Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Ala

275 280285

Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln

290 295300

Gln 305 Lys Pro Gly Gln Ala 310 Pro Arg Gly Leu Ile 315 Gly Gly Thr Lys Phe 320 Leu Val Pro Gly Thr Pro 325 Ala Arg Phe Ser Gly 330 Ser Leu Leu Gly Gly 335 Lys Ala Ala Leu Thr 340 Leu Ser Gly Val Gln 345 Pro Glu Asp Glu Ala 350 Glu Tyr Tyr Cys Thr 355 Leu Trp Tyr Ser Asn 360 Arg Trp Val Phe Gly 365 Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Gln Val

370 375 380

- 121 045012

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

385 390 395400

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asn Ser Val Met

405 410415

Ala Trp Tyr Arg Gln Thr Pro Gly Asn Glu Arg Glu Phe Val Ala Ile

420 425430

Ile Asn Ser Ile Gly Ile Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg

435 440445

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met

450 455460

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Val Cys Asn Arg Asn

465 470 475480

Phe Asp Arg Ile Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

485 490495

Ser Gly Gly Pro Gly Pro Ala Gly Met Lys Gly Leu Pro Gly Ser Gln

500 505510

Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr

515 520525

Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Asn

530 535540

Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu

545 550 555560

Ile Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly Thr Pro Ala Arg Phe

565 570575

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

580 585590

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn

595 600605

Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly

610 615620

Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

625 630 635640

- 122 045012

Val

Thr

Gly

Asp

Asp 705

Glu

Ser

Val

Val

Ser 785

Val

Gly

Thr

Ser

Ser 865

His

Gln Pro Gly Gly Ser Leu Lys

645

Phe Asn Lys Tyr Ala

660

Leu Glu Trp Val Ala

675

Asp Asp Lys Ala Asp 690

Asp Ser Lys Asn Thr

710

Asp Thr Ala Val Tyr

725

Tyr Ile Ser Tyr Trp

740

Ser Ser Gly Gly Gly

755

Glu Ser Gly Gly Gly 770

Cys Ala Ala Ser Gly

790

Arg Gln Ala Pro Gly

805

Ser Gly Arg Asp Thr

820

Ile Ser Arg Asp Asn

835

Leu Arg Pro Glu Asp 850

Leu Ser Val Ser Ser

870

His His His

Leu Ser Cys Ala Ala Ser Gly Phe

650 655

Met Asn Trp Val Arg 6 65

Arg Ile Arg Ser Lys

680

Ser Val Lys Asp Arg 695

Ala Tyr Leu Gln Met

715

Tyr Cys Val Arg His

730

Ala Tyr Trp Gly Gln

745

Gly Ser Gly Gly Gly

760

Leu Val Gln Pro Gly 775

Phe Thr Phe Ser Lys

795

Lys Gly Leu Glu Trp

810

Leu Tyr Ala Glu Ser

825

Ala Lys Thr Thr Leu 840

Thr Ala Val Tyr Tyr 855

Gln Gly Thr Leu Val

875

Gln Ala Pro Gly Lys

670

Tyr Asp Tyr Lys Asp

685

Phe Thr Ile Ser Arg 700

Asn Asn Leu Lys Thr

720

Gly Asn Phe Gly Asn

735

Gly Thr Leu Val Thr

750

Ser Glu Val Gln Leu

765

Asn Ser Leu Arg Leu 780

Phe Gly Met Ser Trp

800

Val Ser Ser Ile Ser

815

Val Lys Gly Arg Phe

830

Tyr Leu Gln Met Asn

845

Cys Thr Ile Gly Gly 860

Thr Val Ser Ser His

880

- 123 045012

885 <210>

<211>

<212>

<213>

157

896

Protein

Artificial sequence <220>

<223>

Synthetic construction

Pro393 (Pro186 S9 linker) <400>

157

Gln Val

15

Lys

Leu Glu

Glu Ser

Gly

Gly Gly

Ser Val

Gln

Thr Gly

Gly

Ser

Leu

Arg

Leu Thr

Cys

Ala

Ala

Ser Gly 25

Arg

Thr

Ser

Arg Ser 30

Tyr

Gly

Met

Gly 35

Trp

Phe

Arg

Gln

Ala

Pro

Gly

Lys

Glu

Arg

Glu

Phe

Val

Ser

Gly

Ile

Ser

Trp

Arg

Gly

Asp

Ser

Thr

Gly

Tyr

Ala

Asp

Ser

Val

Lys 65

Gly

Arg

Phe

Thr

Ile 70

Ser

Arg

Asp

Asn

Ala 75

Lys

Asn

Thr

Val

Asp 80

Leu

Gln

Met

Asn

Ser

Leu 85

Lys

Pro

Glu

Asp

Thr 90

Ala

Ile

Tyr

Tyr

Cys 95

Ala

Ala

Ala

Ala

Gly

Ser

Ala

Trp

Tyr

Gly

Thr

Leu

Tyr

Glu

Tyr

Asp

100

105

110

Tyr

Trp

Gly

Gln

Gly

Thr

Gln

Val

Thr

Val

Ser

Ser

Gly

Gly

Gly

Ser

115

120

125

Gly

Gly

130

Gly

Ser

Glu

Val

Gln

135

Leu

Val

Glu

Ser

Gly

140

Gly

Gly

Leu

Val

Gln

145

Pro

Gly

Gly

Ser

Leu

150

Lys

Leu

Ser

Cys

Ala

155

Ala

Ser

Gly

Phe

Thr

160

Phe

Asn

Lys

Tyr

Ala

Ile 165

Asn

Trp

Val

Arg

Gln 170

Ala

Pro

Gly

Lys

Gly 175

Leu

Glu

Trp

Val

Ala Arg

Ile

Arg

Ser

Lys Tyr

Asn

Asn

Tyr

Ala Thr

Tyr

Tyr

Ala

180

185

190

Asp Gln Val

Lys

Asp

Arg Phe Thr

Ile

Ser

Arg Asp Asp

195

200

205

- 124 045012

Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn

210 215

Leu Lys Thr Glu Asp 220

Thr

225

Ala

Val

Tyr

Tyr

Cys 230

Val

Arg

His

Ala

Asn

235

Phe

Gly

Asn

Ser

Tyr 240

Ile

Ser

Tyr

Trp

Ala

Tyr 245

Trp

Gly

Gln

Gly

Thr 250

Leu

Val

Thr

Val

Ser 255

Ser

Gly

Gly

Gly

Ser

Gly

Gly

Gly

Ser

Gln

Thr

Val

Val

Thr

Gln

Glu

260

265

270

Pro

Ser

Leu

Thr

Val

Ser

Pro

Gly

Gly

Thr

Val

Thr

Leu

Thr

Cys

Ala

275

280

285

Ser Ser Thr Gly Ala Val 290

Thr Ser

295

Gly Asn Tyr Pro Asn Trp Val Gln 300

Gln Lys 305

Leu Val

Pro

Pro

Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr

310 315

Lys Phe

320

Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu

325 330

Gly Gly

335

Lys Ala Ala Leu Thr Leu

340

Ser Gly Val Gln Pro Glu Asp Glu Ala Glu

345 350

Tyr Tyr Cys Thr Leu Trp Tyr

355

Ser Asn Arg Trp Val Phe Gly Gly Gly

360 365

Thr Lys Leu Thr Val 370

Leu Gly Gly Gly Ser Gly Gly Gly Ser Gln Val

375 380

Lys 385 Leu Glu Glu Ser Gly 390 Gly Gly Ser Val Gln 395 Thr Gly Gly Ser Leu 400 Arg Leu Thr Cys Ala Ala 405 Ser Gly Arg Thr Ser 410 Arg Ser Tyr Gly Met 415 Gly Trp Phe Arg Gln 420 Ala Pro Gly Lys Glu 425 Arg Glu Phe Val Ser 430 Gly Ile Ser Trp Arg 435 Gly Asp Ser Thr Gly 440 Tyr Ala Asp Ser Val 445 Lys Gly

Arg Phe Thr Ile Ser Arg Asp Asn Ala

Lys Asn Thr Val Asp Leu Gln

- 125 045012

450

455

460

Met 465

Ala

Gly

Ala

Glu

Gly 545

Gln

Asp

Gly

Ala

Gly 625

Glu

Ser

Ala

Ala

Asn

Ala

Gln

Ala

Ser

Gly

Gly

Pro

Pro

530

Ser

Gln

Asp

Gly

Leu

Ser

Thr

Lys Pro

470

Ala Trp

485

Gln

500

Ala

Gly

Val

Leu

515

Ser

Ser

Lys

Asp

Lys

Glu

610

Gly

Val

Leu

Met

Glu

Tyr

Thr

Lys

Leu

Thr

Val

Thr

Gly

Ala

550

Pro

Gly

Gln 565

Asp

Lys

Gly

580

Ala

Ala

Leu

595

Tyr

Thr

Gln

Lys

Asn

Arg

690

Tyr

Cys

Val

Lys

Leu

Thr

630

Leu

Leu

Val

Glu 645

Ser

Cys

660

Trp Val

Arg

675

Ile Arg Ser

Lys

Asp

Gly

Val

Gly

Ser

535

Val

Ala

Thr

Thr

Thr

Thr

Ser

Ala Ile

475

Leu Tyr

490

Ser

505

Ala

Gly

Gly

Gln

520

Pro

Thr

Pro

Pro

Leu

Leu

615

Val

Ser

Ala

Gln

Gly

Gly

Thr

Ser

Gly

Asn

555

Tyr

Glu

Ala

Thr

Tyr

Tyr

Arg

Val

Val

540

Tyr

Cys

Asp

Leu

Arg

Ala

Gly

Leu 570

Arg

Phe

Ile

Ser

585

Ser

Gly

Val

Gln

600

Trp

Leu

Gly

Ala

Ala

Tyr

695

Tyr

Gly

Gly

Ser

Ser

Gly

Gly

Asn

Gly 635

Leu 650

Gly

Phe

665

Pro

Gly

Lys

680

Asp Tyr

Lys

Asp

Ala Ala

480

Tyr Trp

495

Gln

510

Val

Thr

Ser

Gln

525

Thr

Leu

Thr

Cys

Pro

Gly

Gly

Pro

Arg

620

Ser

Val

Thr

Gly

Asn

Asp

Ser

Trp

Tyr

Val

560

Lys 575

Leu

Leu

590

Glu

605

Trp

Gly

Gln

Phe

Leu

Asp

700

Asp

Glu

Val

Phe

Gly

Gly

Gly

Ser

640

Pro

Gly

Gly 655

Asn

Lys

Tyr

670

Glu Trp

Val

685

Asp Asp Lys

Ala

- 126 045012

Asp Ser Val Lys Asp Arg Phe Thr

705 710

Ile Ser Arg Asp Asp

715

Ser Lys Asn

720

Thr Ala Tyr Leu Gln Met Asn Asn

725

Leu Lys Thr Glu Asp

730

Thr Ala Val

735

Tyr Tyr Cys Val Arg His Gly Asn 740

Phe Gly Asn Ser Tyr 745

Ile Ser Tyr 750

Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

755 760 765

Gly Gly Ser

770

Gly Gly Gly Ser Glu Val

775

Gln Leu Val Glu Ser Gly Gly 780

Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser

785 790 795 800

Gly Phe Thr Phe Ser Lys Phe Gly Met Ser Trp Val Arg Gln Ala Pro

805 810 815

Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Arg Asp

820 825 830

Thr Leu Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

835 840 845

Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu

850 855 860

Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Val Ser

865 870 875 880

Ser Gln Gly Thr Leu Val Thr Val Ser Ser His His His His His

885 890 895 <210> 158 <211> 896 <212> Protein <213> Artificial sequence <220>

< 223> Synthetic construction - Pro394 (Pro186 ST14(MV) linker) < 400> 158

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly 1. 5 10 15

Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr

- 127 045012

Gly

Ser

Lys 65

Leu

Ala

Tyr

Gly

Gln 145

Phe

Leu

Tyr

Ser

Thr 225

Ile

Ser

Met

Gly

Gly

Gln

Ala

Trp

Gly Trp 35

Phe

Arg

Gln

Ala Pro

Gly

Lys

Glu

Arg Glu

Phe

Val

Ile

Ser

Trp

Arg

Gly

Asp

Ser

Thr

Gly

Tyr

Ala

Asp

Ser

Val

Arg

Met

Ala

Gly

Gly

130

Pro

Asn

Glu

Tyr

Phe

Thr

Ile 70

Ser

Arg

Asp

Asn

Ala 75

Lys

Asn

Thr

Val

Asp 80

Asn

Ala

Ser

Leu 85

Lys

Pro

Glu

Asp

Thr 90

Ala

Ile

Tyr

Tyr

Cys 95

Gly

Ser

Ala

Trp

Tyr

Gly

Thr

Leu

Tyr

Glu

Tyr

Asp

100

105

110

Gln

Gly

Thr

Gln

Val

Thr

Val

Ser

Ser

Gly

Gly

Gly

Ser

115

120

125

Gly

Gly

Lys

Trp

Ala

Lys

210

Ala

Ser

Gly

Ser

Glu

Val

Gln

135

Leu

Val

Glu

Ser

Gly

140

Gly

Gly

Leu

Val

Gly

Tyr

Val

Ser

Leu

150

Lys

Leu

Ser

Cys

Ala

155

Ala

Ser

Gly

Phe

Thr

160

Ala

Ile 165

Asn

Trp

Val

Arg

Gln 170

Ala

Pro

Gly

Lys

Gly 175

Ala

Arg

Ile

Arg

Ser

Lys

Tyr

Asn

Asn

Tyr

Ala

Thr

180

185

190

Asp

Gln

Val

Lys

Asp

Arg

Phe

Thr

Ile

Ser

Arg

Asp

Asp

195

200

205

Asn

Val

Tyr

Gly

Thr

Tyr

Trp

Gly

Ala

Tyr

Leu

215

Gln

Met

Asn

Asn

Leu

220

Lys

Thr

Glu

Asp

Tyr

Cys 230

Val

Arg

His

Ala

Asn

235

Phe

Gly

Asn

Ser

Tyr 240

Ala

Tyr 245

Trp

Gly

Gln

Gly

Thr 250

Leu

Val

Thr

Val

Ser 255

Ser Gly

Gly

Gly

Ser

Gln Thr

Val

Val

Thr

Gln Glu

260

265

270

- 128 045012

Pro

Ser Leu Thr

275

Val

Ser

Pro Gly Gly

280

Thr

Val

Thr Leu Thr

285

Cys

Ala

Ser

Ser Thr

290

Gly

Ala

Val

Thr Ser

295

Gly

Asn

Tyr

Pro Asn

300

Trp

Val

Gln

Gln

305

Lys

Pro

Gly

Gln

Ala

310

Pro

Arg

Gly

Leu

Ile

315

Gly

Gly

Thr

Lys

Phe

320

Leu

Val

Pro

Gly

Thr

Pro 325

Ala

Arg

Phe

Ser

Gly 330

Ser

Leu

Leu

Gly

Gly 335

Lys

Ala

Ala

Leu

Thr

Leu

Ser

Gly

Val

Gln

Pro

Glu

Asp

Glu

Ala

Glu

340

345

350

Tyr

Tyr

Cys

Thr

Leu

Trp

Tyr

Ser

Asn

Arg

Trp

Val

Phe

Gly

Gly

Gly

355

360

365

Thr

Lys

370

Leu

Thr

Val

Leu

Gly

375

Gly

Gly

Ser

Gly

Gly

380

Gly

Ser

Gln

Val

Lys

385

Leu

Glu

Glu

Ser

Gly 390

Gly

Gly

Ser

Val

Gln

395

Thr

Gly

Gly

Ser

Leu

400

Arg

Leu

Thr

Cys

Ala

Ala 405

Ser

Gly

Arg

Thr

Ser 410

Arg

Ser

Tyr

Gly

Met 415

Gly

Trp

Phe

Arg

Gln

Ala

Pro

Gly

Lys

Glu

Arg

Glu

Phe

Val

Ser

Gly

420

425

430

Ile

Ser

Trp

Arg

Gly

Asp

Ser

Thr

Gly

Tyr

Ala

Asp

Ser

Val

Lys

Gly

435

440

445

Arg

Phe

450

Thr

Ile

Ser

Arg

Asp

455

Asn

Ala

Lys

Asn

Thr

460

Val

Asp

Leu

Gln

Met

465

Asn

Ser

Leu

Lys

Pro

470

Glu

Asp

Thr

Ala

Ile

475

Tyr

Tyr

Cys

Ala

Ala

480

Ala

Ala

Gly

Ser

Ala

Trp 485

Tyr

Gly

Thr

Leu

Tyr 490

Glu

Tyr

Asp

Tyr

Trp 495

Gly

Gln

Gly

Thr

Gln Val

Thr

Val

Ser

Ser Gly

Gly

Gly

Ser

Phe Thr

Arg

Gln

Ala

500

505

510

Arg Val Val

Gly

Gly

Gly Ser Gln

Thr

Val

Val Thr Gln

515

520

525

- 129 045012

Glu Pro Ser Leu Thr Val Ser Pro Gly

530 535

Gly Ser Ser Thr Gly Ala Val Thr Ser 545 550

Gln Gln Lys Pro Gly Gln Ala Pro Arg 565

Asp Asp Asp Asp Lys Gly Thr Pro Ala

580 5

Gly Gly Lys Ala Ala Leu Thr Leu Ser

595 600

Ala Glu Tyr Tyr Cys Val Leu Trp Tyr

610 615

Gly Gly Thr Lys Leu Thr Val Leu Gly 625 630

Glu Val Gln Leu Val Glu Ser Gly Gly 645

Ser Leu Lys Leu Ser Cys Ala Ala Ser

660 6

Ala Met Asn Trp Val Arg Gln Ala Pro

675 680

Ala Arg Ile Arg Ser Lys Tyr Asp Tyr

690 695

Asp Ser Val Lys Asp Arg Phe Thr Ile 705 710

Thr Ala Tyr Leu Gln Met Asn Asn Leu 725

Tyr Tyr Cys Val Arg His Gly Asn Phe

740 7

Trp Ala Tyr Trp Gly Gln Gly Thr Leu

755 760

Gly Gly Ser Gly Gly Gly Ser Glu Val

770 775

Thr Val Thr Leu Thr Cys 540

Asn Tyr Pro Asn Trp Val

555 560

Leu Ile Gly Asp Tyr Lys

570 575

Phe Ser Gly Ser Leu Leu

590

Val Gln Pro Glu Asp Glu

605

Asn Arg Trp Val Phe Gly 620

Gly Ser Gly Gly Gly Ser

635 640

Leu Val Gln Pro Gly Gly

650 655

Phe Thr Phe Asn Lys Tyr

670

Lys Gly Leu Glu Trp Val

685

Asp Asp Asp Asp Lys Ala 700

Arg Asp Asp Ser Lys Asn

715 720

Thr Glu Asp Thr Ala Val

730 735

Asn Ser Tyr Ile Ser Tyr

750

Thr Val Ser Ser Gly Gly

765

Leu Val Glu Ser Gly Gly 780

- 130 045012

Gly 785

Leu

Val

Gln

Pro Gly

790

Asn

Ser

Leu

Arg Leu

795

Ser

Cys

Ala

Ala Ser

800

Gly

Phe

Thr

Phe

Ser Lys

805

Phe

Gly

Met

Ser Trp

810

Val

Arg

Gln

Ala Pro

815

Gly

Lys

Gly

Leu

Glu

Trp

Val

Ser

Ser

Ile

Ser

Gly

Ser

Gly

Arg

Asp

Thr

Leu

Tyr

820

825

830

Ala

Glu

Ser

Val

Lys

Gly

Arg

Phe

Thr

Ile

Ser

Arg

Asp

835

840

845

Asn

Ala

850

Lys

Thr

Thr

Leu

Tyr

855

Leu

Gln

Met

Asn

Ser

860

Leu

Arg

Pro

Glu

Asp 865

Thr Ala

Val

Tyr

Tyr 870

Cys Thr

Ile

Gly

Gly 875

Ser Leu

Ser

Val

Ser

880

Ser

Gln Gly

Thr

Leu

Val

885

Thr Val

Ser

Ser

His 890

His His

His

His

His 895 <210>

<211>

<212>

<213>

159

896

Protein

Artificial sequence <220>

<223>

Synthetic construction

Pro395 (Pro186 CathS linker) <400>

159

Gln Val

15

Lys

Leu Glu

Glu Ser

Gly

Gly Gly

Ser Val

Gln

Thr

Gly

Gly

Ser

Leu

Arg

Leu Thr

Cys

Ala

Ala

Ser Gly 25

Arg

Thr

Ser

Arg

Ser

Tyr

Gly

Met

Gly 35

Trp

Phe

Arg

Gln

Ala

Pro

Gly

Lys

Glu

Arg

Glu

Phe

Val

Ser

Gly

Ile

Ser

Trp

Arg

Gly

Asp

Ser

Thr

Gly

Tyr

Ala

Asp

Ser

Val

Lys 65

Gly

Arg

Phe

Thr

Ile 70

Ser

Arg

Asp

Asn

Ala 75

Lys

Asn

Thr

Val

Asp 80

Leu

Gln

Met

Asn

Ser

Leu 85

Lys

Pro

Glu

Asp

Thr 90

Ala

Ile

Tyr

Tyr

Cys 95

- 131 045012

Ala

Tyr

Gly

Gln 145

Phe

Leu

Tyr

Ser

Thr 225

Ile

Ser

Pro

Ser

Gln 305

Leu

Lys

Ala

Trp

Ala Ala Gly

100

Gly Gln

115

Gly

130

Pro

Asn

Glu

Tyr

Ser

Ala

Trp Tyr Gly

105

Thr

Leu Tyr Glu Tyr Asp

110

Gly

Gly

Lys

Trp

Ala

Lys

210

Ala

Ser

Gly

Ser

Ser

Gly

Tyr

Val

Gly

Thr

Gln

Val Thr

120

Val

Ser

Ser Gly Gly Gly Ser

125

Glu

Ser

Ala

Val

Leu

150

Ile 165

Ala

Arg

180

Asp

Gln

Val

195

Asn

Val

Tyr

Gly

Leu

Ser

290

Lys

Val

Ala

Thr

Tyr

Trp

Gly

Ala

Tyr

Tyr

Cys 230

Ala

Tyr 245

Ser

260

Thr

275

Thr

Gly

Gly

Val

Ala

Ser

Val

Pro

Pro

Ala

Gly

Gln

Ala

310

Gly

Thr

Pro 325

Leu

Thr Leu

340

Gln

135

Leu

Val

Glu

Ser

Gly Gly Gly Leu Val 140

Lys

Leu

Ser

Cys

Ala

155

Ala Ser Gly Phe Thr

160

Asn

Trp

Val

Arg

Gln 170

Ala Pro Gly Lys Gly

175

Ile

Lys

Arg

Asp

Leu

215

Val

Trp

Gly

Pro

Ser

Lys

Tyr

185

Arg

Phe

Thr

200

Gln

Met

Asn

Asn

Arg

Gly

Gly

Gly

Thr

295

Pro

Ala

Ser

His

Gln

Ser

Ala

Asn

235

Gly

Thr 250

Gln

265

Gly

280

Ser

Gly

Thr

Asn

Thr

Val

Tyr

Arg

Arg

Gly

Gly

Leu

Ile

315

Phe

Val

Ser

Gly 330

Glin Pro

345

Asn Asn Tyr Ala Thr

190

Ile Ser Arg Asp Asp

205

Leu Lys Thr Glu Asp 220

Phe Gly Asn Ser Tyr

240

Leu Val Thr Val Ser

255

Val Val Thr Gln Glu

270

Thr Leu Thr Cys Ala

285

Pro Asn Trp Val Gln 300

Gly Gly Thr Lys Phe

320

Ser Leu Leu Gly Gly

335

Glu Asp Glu Ala Glu

350

- 132 045012

Tyr Tyr Cys Thr Leu Trp Tyr Ser Asn

355 360

Thr Lys Leu Thr Val Leu Gly Gly Gly

370 375

Lys Leu Glu Glu Ser Gly Gly Gly Ser

385 390

Arg Leu Thr Cys Ala Ala Ser Gly Arg

405

Gly Trp Phe Arg Gln Ala Pro Gly Lys

420 42

Ile Ser Trp Arg Gly Asp Ser Thr Gly

435 440

Arg Phe Thr Ile Ser Arg Asp Asn Ala

450 455

Met Asn Ser Leu Lys Pro Glu Asp Thr 465 470

Ala Ala Gly Ser Ala Trp Tyr Gly Thr

485

Gly Gln Gly Thr Gln Val Thr Val Ser

500 50

Leu Gln Ser Ala Ala Pro Gly Gly Gly

515 520

Glu Pro Ser Leu Thr Val Ser Pro Gly

530 535

Gly Ser Ser Thr Gly Ala Val Thr Ser

545 550

Gln Gln Lys Pro Gly Gln Ala Pro Arg

565

Asp Asp Asp Asp Lys Gly Thr Pro Ala

580 58

Gly Gly Lys Ala Ala Leu Thr Leu Ser

Trp Val Phe Gly Gly Gly

365

Gly Gly Gly Ser Gln Val 380

Gln Thr Gly Gly Ser Leu

395 400

Ser Arg Ser Tyr Gly Met

410 415

Arg Glu Phe Val Ser Gly

430

Ala Asp Ser Val Lys Gly 445

Asn Thr Val Asp Leu Gln 460

Ile Tyr Tyr Cys Ala Ala

475 480

Tyr Glu Tyr Asp Tyr Trp

490 495

Gly Gly Gly Ser Ala Arg

510

Gln Thr Val Val Thr Gln

525

Thr Val Thr Leu Thr Cys 540

Asn Tyr Pro Asn Trp Val

555 560

Leu Ile Gly Asp Tyr Lys

570 575

Phe Ser Gly Ser Leu Leu

590

Val Gln Pro Glu Asp Glu

- 133 045012

595

600

605

Ala Glu Tyr Tyr Cys Val 610

Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly

615 620

Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser

625 630 635 640

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

645 650 655

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr

660 665 670

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

675 680 685

Ala Arg Ile Arg Ser Lys Tyr Asp Tyr Lys Asp Asp Asp Lys Ala

690 695 700

Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn

705 710 715 720

Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val

725 730 735

Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr

740 745 750

Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

755 760 765

Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

770 775 780

Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser

785 790 795 800

Gly Phe Thr Phe Ser Lys Phe Gly Met Ser Trp Val Arg Gln Ala Pro

805 810 815

Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Arg Asp

820 825 830

Thr Leu Tyr Ala Glu Ser Val Lys Gly Arg Phe

835 840

Thr Ile Ser Arg Asp

845

- 134 045012

Asn

Ala Lys

850

Thr

Thr

Leu

Tyr Leu

855

Gln

Met

Asn

Ser Leu

860

Arg

Pro

Glu

Asp

865

Thr Ala

Val

Tyr

Tyr 870

Cys Thr

Ile

Gly

Gly 875

Ser Leu

Ser

Val

Ser

880

Ser

Gln Gly

Thr

Leu

Val

885

Thr Val

Ser

Ser

His 890

His His

His

His

His 895 <210>

<211>

<212>

<213>

160

897

Protein

Artificial sequence <220>

<223>

Synthetic construction

Pro396 (Pro186 MMP9v linker) <400>

160

Gln Val

15

Lys

Leu Glu

Glu Ser

Gly

Gly Gly

Ser Val

Gln

Thr

Gly

Gly

Ser

Leu

Arg

Leu Thr

Cys

Ala

Ala

Ser Gly 25

Arg

Thr

Ser

Arg

Ser

Tyr

Gly

Met

Gly 35

Trp

Phe

Arg

Gln

Ala

Pro

Gly

Lys

Glu

Arg

Glu

Phe

Val

Ser

Gly

Ile

Ser

Trp

Arg

Gly

Asp

Ser

Thr

Gly

Tyr

Ala

Asp

Ser

Val

Lys 65

Gly

Arg

Phe

Thr

Ile 70

Ser

Arg

Asp

Asn

Ala 75

Lys

Asn

Thr

Val

Asp 80

Leu

Gln

Met

Asn

Ser

Leu 85

Lys

Pro

Glu

Asp

Thr 90

Ala

Ile

Tyr

Tyr

Cys 95

Ala

Ala

Ala

Ala

Gly

Ser

Ala

Trp

Tyr

Gly

Thr

Leu

Tyr

Glu

Tyr

Asp

Tyr

Trp

Gly

100

105

110

Gln

Gly

Thr

Gln

Val

Thr

Val

Ser

Ser

Gly

Gly

Gly

Ser

115

120

125

Gly

Gly

130

Gly

Ser

Glu

Val

Gln

135

Leu

Val

Glu

Ser

Gly

140

Gly

Gly

Leu

Val

Gln

145

Pro Gly

Gly

Ser

Leu

150

Lys Leu

Ser

Cys

Ala

155

Ala Ser

Gly

Phe

Thr

160

Phe

Asn Lys

Tyr

Ala

Ile

Asn Trp

Val

Arg

Gln

Ala Pro

Gly

Lys

Gly

- 135 045012

165

170

175

Leu

Tyr

Ser

Thr 225

Ile

Ser

Pro

Ser

Gln 305

Leu

Lys

Tyr

Thr

Lys 385

Arg

Glu Trp Val Ala Arg

Tyr

180

Ala Asp

195

Lys

210

Asn

Thr

Gln

Val

Ala

Val

Tyr

Ser

Tyr

Trp

Gly

Gly

Gly

Ser

Leu

Ser

290

Lys

Val

Ala

Tyr

Ala

Tyr

Ala

Tyr

Cys 230

Tyr 245

Ser

260

Thr

275

Thr

Gly

Gly

Val

Ala

Ser

Val

Pro

Pro

Ala

Cys

Lys

370

Gly

Gln

Ala

310

Gly

Thr

Pro 325

Leu

Thr

Leu

340

Thr

Leu

Trp

355

Leu

Thr

Val

Leu

Leu Glu

Leu Thr

Glu

Cys

Ser

Gly 390

Ala

Ala 405

Ile Arg Ser Lys

185

Lys

Asp Arg

200

Leu

215

Val

Trp

Gly

Pro

Gln

Met

Phe

Asn

Arg

Gly

Gly

Gly

Thr

295

Pro

Ala

Ser

Tyr

His

Gln

Ser

Ala

Gly

Tyr Asn Asn Tyr Ala Thr

190

Thr

Ile

Ser Arg

205

Asp

Asp

Asn

Asn

235

Thr

250

Gln

265

Gly

280

Ser

Gly

Thr

Thr

Val

Arg

Arg

Gly

Ser

Gly

375

Gly

Phe

Val

Asn

Leu

Ser

Tyr

Ile

315

Gly 330

Gln

345

Asn

Arg

Pro

Trp

360

Gly

Gly

Ser

Gly

Gly Gly

Ser Gly

Ser

Val

Gln

395

Arg

Thr

Ser 410

Leu

220

Phe

Leu

Val

Thr

Lys

Gly

Val

Val

Leu

Pro

300

Gly

Ser

Glu

Val

Thr

Glu

Asp

Asn

Thr

Thr

Ser

Tyr 240

Val

Ser 255

Gln

270

Thr

285

Asn

Trp

Cys

Val

Glu

Ala

Gln

Gly

Thr

Lys

Phe

320

Leu

Leu

Gly

Gly 335

Asp

Phe

Gly

380

Glu

Ala

Glu

350

Gly

Gly

Gly

365

Gly

Ser

Gln

Val

Thr Gly

Arg Ser

Gly

Tyr

Ser

Gly

Leu

400

Met 415

- 136 045012

Gly Trp Phe Arg Gln Ala Pro Gly 420

Lys Glu Arg Glu Phe Val Ser Gly

425 430

Ile Ser Trp Arg Gly Asp Ser Thr

435 44

Gly Tyr Ala Asp Ser Val Lys Gly 0 445

Arg Phe Thr Ile Ser Arg Asp Asn

450 455

Ala Lys Asn Thr Val Asp Leu Gln 460

Met Asn Ser Leu Lys Pro Glu Asp

465 470

Thr Ala Ile Tyr Tyr Cys Ala Ala

475 480

Ala Ala Gly Ser Ala Trp Tyr Gly

485

Thr Leu Tyr Glu Tyr Asp Tyr Trp

490 495

Gly Gln Gly Thr Gln Val Thr Val 500

Ser Ser Gly Ser Gly Gly Pro Gly

505 510

Pro Ala Gly Met His Gly Leu Pro

515 5

Gly Gly Ser Gln Thr Val Val Thr 0 525

Gln Glu Pro Ser Leu Thr Val Ser

530 535

Pro Gly Gly Thr Val Thr Leu Thr 540

Cys Gly Ser Ser Thr Gly Ala Val

545 550

Thr Ser Gly Asn Tyr Pro Asn Trp

555 560

Val Gln Gln Lys Pro Gly Gln Ala

565

Pro Arg Gly Leu Ile Gly Asp Tyr

570 575

Lys Asp Asp Asp Asp Lys Gly Thr 580

Pro Ala Arg Phe Ser Gly Ser Leu

585 590

Leu Gly Gly Lys Ala Ala Leu Thr

595 6

Leu Ser Gly Val Gln Pro Glu Asp 0 605

Glu Ala Glu Tyr Tyr Cys Val Leu

610 615

Trp Tyr Ser Asn Arg Trp Val Phe 620

Gly Gly Gly Thr Lys Leu Thr Val

625 630

Leu Gly Gly Gly Ser Gly Gly Gly

635 640

Ser Glu Val Gln Leu Val Glu Ser

645

Gly Gly Gly Leu Val Gln Pro Gly

650 655

Gly Ser Leu Lys Leu Ser Cys Ala 660

Ala Ser Gly Phe Thr Phe Asn Lys

665 670

- 137 045012

Tyr

Val

Ala 705

Asn

Val

Tyr

Gly

Gly 785

Ser

Pro

Asp

Asp

Glu 865

Ser

His

Ala Met Asn Trp Val Arg Gln Ala Pro Gly

675 680

Ala Arg Ile Arg Ser 690

Asp Ser Val Lys Asp

710

Thr Ala Tyr Leu Gln

725

Tyr Tyr Cys Val Arg

740

Trp Ala Tyr Trp Gly

755

Gly Gly Ser Gly Gly 770

Gly Leu Val Gln Pro

790

Gly Phe Thr Phe Ser

805

Gly Lys Gly Leu Glu

820

Thr Leu Tyr Ala Glu

835

Asn Ala Lys Thr Thr 850

Asp Thr Ala Val Tyr

870

Ser Gln Gly Thr Leu

885 <210> 161 <211> 896

Lys Gly Leu Glu Trp

685

Lys Tyr Asp Tyr Lys 695

Arg Phe Thr Ile Ser

715

Met Asn Asn Leu Lys

730

His Gly Asn Phe Gly 745

Gln Gly Thr Leu Val

760

Gly Ser Glu Val Gln 775

Gly Asn Ser Leu Arg

795

Lys Phe Gly Met Ser

810

Trp Val Ser Ser Ile

825

Ser Val Lys Gly Arg 840

Leu Tyr Leu Gln Met 855

Tyr Cys Thr Ile Gly

875

Val Thr Val Ser Ser

890

Asp Asp Asp Asp Lys 700

Arg Asp Asp Ser Lys

720

Thr Glu Asp Thr Ala

735

Asn Ser Tyr Ile Ser

750

Thr Val Ser Ser Gly

765

Leu Val Glu Ser Gly 780

Leu Ser Cys Ala Ala

800

Trp Val Arg Gln Ala

815

Ser Gly Ser Gly Arg

830

Phe Thr Ile Ser Arg

845

Asn Ser Leu Arg Pro 860

Gly Ser Leu Ser Val

880

His His His His

895

- 138 045012 <212> Protein <213> Artificial sequence <220>

<223> Synthetic construction - Pro429 (Pro186 Meprin/GranzymeB linker) <400>161

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly

1.5 1015

Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr 20 2530

Gly

Met

Gly 35

Trp

Phe

Arg

Gln

Ala

Pro

Gly

Lys

Glu

Arg

Glu

Phe

Val

Ser

Gly

Ile

Ser

Trp

Arg

Gly

Asp

Ser

Thr

Gly

Tyr

Ala

Asp

Ser

Val

Lys 65

Gly

Arg

Phe

Thr

Ile 70

Ser

Arg

Asp

Asn

Ala 75

Lys

Asn

Thr

Val

Asp 80

Leu

Gln

Met

Asn

Ser

Leu 85

Lys

Pro

Glu

Asp

Thr 90

Ala

Ile

Tyr

Tyr

Cys 95

Ala

Ala

Ala

Ala

Gly

Ser

Ala

Trp

Tyr

Gly

Thr

Leu

Tyr

Glu

Tyr

Asp

100

105

110

Tyr

Trp

Gly

Gln

Gly

Thr

Gln

Val

Thr

Val

Ser

Ser

Gly

Gly

Gly

Ser

115

120

125

Gly

Gly

130

Gly

Ser

Glu

Val

Gln

135

Leu

Val

Glu

Ser

Gly

140

Gly

Gly

Leu

Val

Gln

145

Pro

Gly

Gly

Ser

Leu

150

Lys

Leu

Ser

Cys

Ala

155

Ala

Ser

Gly

Phe

Thr

160

Phe

Asn

Lys

Tyr

Ala

Ile 165

Asn

Trp

Val

Arg

Gln 170

Ala

Pro

Gly

Lys

Gly 175

Leu

Glu

Trp

Val

Ala

Arg

Ile

Arg

Ser

Lys

Tyr

Asn

Asn

Tyr

Ala

Thr

Tyr

Tyr

Ala

180

185

190

Asp Gln

Val

Lys

Asp

Arg

Phe

Thr

Ile

Ser

Arg Asp

Asp

195

200

205

Ser

Lys

210

Asn Thr Ala

Tyr

Leu

215

Gln Met

Asn

Asn

Leu

220

Lys Thr Glu

Asp

- 139 045012

Thr 225

Ile

Ser

Pro

Ser

Gln 305

Leu

Lys

Tyr

Thr

Lys 385

Arg

Gly

Ile

Arg

Met 465

Ala

Val

Tyr

Tyr Cys

230

Val

Arg

His

Ala Asn

235

Phe Gly Asn Ser Tyr

240

Ser

Tyr

Trp

Ala Tyr

245

Trp

Gly

Gln

Gly Thr

250

Leu Val Thr Val Ser

255

Gly

Gly

Gly

Ser

260

Ser

Leu

Thr

275

Val

Gly

Gly

Gly

Ser

Gln

265

Ser

Pro

Gly

Gly

280

Thr

Thr

Val

Ser

290

Thr

Gly

Ala

Val

Thr

295

Ser

Gly

Asn

Tyr

Lys

Pro

Gly

Gln

Ala

310

Pro

Arg

Gly

Leu

Ile

315

Val

Ala

Tyr

Pro

Gly

Thr

Pro 325

Ala

Arg

Phe

Ser

Gly 330

Ala

Cys

Lys

370

Leu

Leu

Trp

Ser

Leu

Thr

Leu

Ser

Gly

Val

Gln

Pro

340

345

Thr

Leu

Trp

Tyr

Ser

Asn

Arg

Trp

355

360

Leu

Glu

Thr

Phe

Trp

Phe

450

Thr

Glu

Cys

Arg

Val

Leu

Gly

375

Gly

Gly

Ser

Gly

Ser

Gly 390

Gly

Gly

Ser

Val

Gln

395

Ala

Ala 405

Gln

420

Arg

Gly

Ala

Asp

435

Thr Ile

Ser

Arg

Ser

Gly

Arg

Thr

Ser 410

Pro

Gly

Lys

Glu

425

Ser

Thr

Gly

Tyr

Arg

Ala

440

Asn Ser Leu

Lys

Pro

470

Asp

455

Asn

Ala

Lys

Asn

Glu Asp

Thr

Ala

Ile

475

Val Val Thr Gln Glu

270

Thr Leu Thr Cys Ala

285

Pro Asn Trp Val Gln 300

Gly Gly Thr Lys Phe

320

Ser Leu Leu Gly Gly

335

Glu Asp Glu Ala Glu

350

Val Phe Gly Gly Gly

365

Gly Gly Ser Gln Val 380

Thr Gly Gly Ser Leu

400

Arg Ser Tyr Gly Met

415

Glu Phe Val Ser Gly

430

Asp Ser Val Lys Gly 445

Thr Val Asp Leu Gln 460

Tyr Tyr Cys Ala Ala

480

- 140 045012

Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr

485 490

Glu Tyr Asp Tyr Trp

495

Gly Gln Gly Thr Gln Val Thr Val 500

Ser Ser Ser Gly Gly Gly Val Tyr

505 510

Ala Asp Ser Leu Glu Asp Gly Gly Gly Ser Gln Thr Val Val Thr Gln

515 520 525

Glu Pro Ser Leu Thr Val

530

Ser Pro Gly Gly Thr Val Thr Leu Thr Cys

535 540

Gly Ser 545

Gln Gln

Ser Thr

Lys Pro

Gly Ala Val Thr Ser Gly Asn

550 555

Gly Gln Ala Pro Arg Gly Leu

565 570

Tyr Pro Asn Trp Val

560

Ile Gly Asp Tyr Lys

575

Asp Asp Asp Asp Lys

580

Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu

585 590

Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

595 600

Gln Pro Glu Asp Glu

605

Ala Glu Tyr Tyr Cys Val 610

Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly

615 620

Gly Gly Thr 625

Glu Val Gln

Lys Leu Thr Val 630

Leu Val Glu Ser 645

Leu Gly Gly Gly Ser Gly Gly Gly Ser 635 640

Gly Gly Gly Leu Val Gln Pro Gly Gly 650 655

Ser Leu Lys

Leu Ser Cys Ala Ala 660

Ser Gly Phe Thr Phe Asn Lys Tyr

665 670

Ala Met Asn Trp 675 Val Arg Gln Ala Pro 680 Gly Lys Gly Leu Glu 685 Trp Val Ala Arg 690 Ile Arg Ser Lys Tyr 695 Asp Tyr) Lys Asp Asp 700 Asp Asp 1 Lys Ala Asp 705 Ser Val Lys Asp Arg 710 Phe Thr Ile Ser Arg 715 Asp Asp Ser Lys Asn 720 Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val

- 141 045012

725

730

735

Tyr

Tyr

Cys Val Arg

740

His

Gly

Asn Phe Gly

745

Asn

Ser

Tyr Ile Ser

750

Tyr

Trp

Ala

Tyr Trp

755

Gly

Gln

Gly

Thr Leu

760

Val

Thr

Val

Ser Ser

765

Gly

Gly

Gly

Gly

770

Ser

Gly

Gly

Gly

Ser

775

Glu

Val

Gln

Leu

Val

780

Glu

Ser

Gly

Gly

Gly 785

Leu

Val

Gln

Pro

Gly 790

Asn

Ser

Leu

Arg

Leu

795

Ser

Cys

Ala

Ala

Ser

800

Gly

Phe

Thr

Phe

Ser

Lys 805

Phe

Gly

Met

Ser

Trp 810

Val

Arg

Gln

Ala

Pro 815

Gly

Lys

Gly

Leu

Glu

Trp

Val

Ser

Ser

Ile

Ser

Gly

Ser

Gly

Arg

Asp

Thr

Leu

Tyr

820

825

830

Ala

Glu

Ser

Val

Lys

Gly

Arg

Phe

Thr

Ile

Ser

Arg

Asp

835

840

845

Asn

Ala

850

Lys

Thr

Thr

Leu

Tyr

855

Leu

Gln

Met

Asn

Ser

860

Leu

Arg

Pro

Glu

Asp 865

Thr Ala

Val

Tyr

Tyr 870

Cys Thr

Ile

Gly

Gly 875

Ser Leu

Ser

Val

Ser

880

Ser

Gln Gly

Thr

Leu

Val

885

Thr Val

Ser

Ser

His 890

His His

His

His

His 895 <210>

<211>

<212>

<213>

162

895

Protein

Artificial sequence <220>

<223>

Synthetic construction

Pro430 (Pro186 MMP9-2 linker) <400>

162

Gln Val

15

Lys

Leu Glu

Glu Ser

Gly

Gly Gly

Ser Val

Gln

Thr Gly

Gly

Ser Leu

Arg

Leu Thr

Cys Ala

Ala

Ser Gly 25

Arg Thr

Ser

Arg Ser 30

Tyr

Gly Met

Gly 35

Trp Phe

Arg Gln

Ala

Pro Gly

Lys Glu

Arg

Glu Phe

Val

- 142 045012

Ser

Lys 65

Leu

Ala

Tyr

Gly

Gln 145

Phe

Leu

Tyr

Ser

Thr 225

Ile

Ser

Pro

Ser

Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val

55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95

Ala Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Ser

115 120 125

Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

130 135 140

Pro Gly Gly Ser Leu 150 Lys Leu Ser Cys Ala 155 Ala Ser Gly Phe Thr 160 Asn Lys Tyr Ala Ile 165 Asn Trp Val Arg Gln 170 Ala Pro Gly Lys Gly 175 Glu Trp Val Ala 180 Arg Ile Arg Ser Lys 185 Tyr Asn Asn Tyr Ala 190 Thr Tyr Ala Asp 195 Gln Val Lys Asp Arg 200 Phe Thr Ile Ser Arg 205 Asp Asp Lys 210 Asn 1 Thr Ala Tyr Leu 215 Gln Met Asn Asn Leu 220 Lys 1 Thr Glu Asp Ala Val Tyr Tyr Cys 230 Val Arg His Ala Asn 235 Phe Gly Asn Ser Tyr 240 Ser Tyr Trp Ala Tyr 245 Trp Gly Gln Gly Thr 250 Leu Val Thr Val Ser 255 Gly Gly Gly Ser 260 Gly Gly Gly Ser Gln 265 Thr Val Val Thr Gln 270 Glu Ser Leu Thr 275 Val Ser Pro Gly Gly 280 Thr Val Thr Leu Thr 285 Cys Ala Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln

- 143 045012

290

295

300

Gln 305

Leu

Lys

Tyr

Thr

Lys 385

Arg

Gly

Ile

Arg

Met 465

Ala

Gly

Ala

Pro

Lys

Pro

Gly

Gln Ala

310

Pro

Arg

Gly

Leu Ile

315

Gly

Gly

Thr

Lys Phe

320

Val

Ala

Tyr

Pro

Gly

Thr Pro

325

Ala

Arg

Phe

Ser Gly

330

Ser

Leu

Leu

Gly Gly

335

Ala

Cys

Lys

370

Leu

Leu

Trp

Ser

Leu

Thr

Leu

Ser

Gly

Val

Gln

Pro

Glu

Asp

Glu

Ala

Glu

340

345

350

Thr

Leu

Trp

Tyr

Ser

Asn

Arg

Trp

Val

Phe

Gly

Gly

Gly

355

360

365

Leu

Glu

Thr

Phe

Trp

Phe

450

Asn

Ala

Gln

Gly

Thr

Glu

Cys

Arg

Val

Leu

Gly

375

Gly

Gly

Ser

Gly

Gly

380

Gly

Ser

Gln

Val

Ser

Gly 390

Gly

Gly

Ser

Val

Gln

395

Thr

Gly

Gly

Ser

Leu

400

Ala

Ala 405

Gln

420

Arg

Gly

Ala

Asp

435

Thr

Ser

Gly

Gly

Leu

Ser

530

Ile

Ser

Arg

Ser

Gly

Arg

Thr

Ser 410

Arg

Ser

Tyr

Gly

Met 415

Pro

Gly

Lys

Glu

425

Ser

Thr

Gly

Tyr

Arg

Ala

Glu

Phe

Val

Ser

430

Gly

Asp

Ser

Val

Lys

Gly

440

445

Leu

Lys

Pro

470

Ser

Thr

Ala

Trp 485

Gln

500

Lys Gly

515

Leu Thr Val

Val

Ala

Ser

Asp

455

Asn

Ala

Lys

Asn

Thr

460

Val

Asp

Leu

Gln

Glu

Tyr

Thr

Pro

Asp

Thr

Ala

Ile

475

Tyr

Tyr

Cys

Ala

Ala

480

Gly

Thr

Leu

Tyr 490

Glu

Tyr

Asp

Tyr

Trp 495

Val

Gly

Pro

535

Ser

Ser

505

Ser

520

Gly Gly

Ser

Gly

Gly

Pro

Gly

510

Gln

Thr

Thr

Val

Pro

Val

Val

Thr

540

Thr Gln

Glu

525

Leu Thr Cys

Gly

- 144 045012

Ser 545

Gln

Asp

Gly

Glu

Gly 625

Val

Leu

Met

Arg

Ser 705

Ala

Tyr

Ala

Gly

Leu 785

Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln

550 555560

Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Asp Tyr Lys Asp

565 570575

Asp Asp Lys Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly

580 585590

Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala

595 600605

Tyr Tyr Cys Val Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly

610 615620

Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Glu

630 635640

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

645 650655

Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr Ala

660 665670

Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

675 680685

Ile Arg Ser Lys Tyr Asp Tyr Lys Asp Asp Asp Asp Lys Ala Asp

690 695700

Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

710 715720

Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr

725 730735

Cys Val Arg His 740 Gly Asn Phe Gly Asn 745 Ser Tyr Ile Ser Tyr 750 Trp Tyr Trp Gly 755 Gln Gly Thr Leu Val 760 Thr Val Ser Ser Gly 765 Gly Gly Ser 770 Gly Gly Gly Ser Glu 775 Val Gln Leu Val Glu 780 Ser Gly Gly Gly Val Gln Pro Gly Asn 790 Ser Leu Arg Leu Ser 795 Cys Ala Ala Ser Gly 800

- 145 045012

Phe Thr Phe Ser Lys Phe Gly Met

805

Ser Trp Val Arg Gln Ala Pro Gly

810 815

Lys Gly Leu Glu Trp Val

820

Ser Ser Ile Ser Gly Ser Gly Arg Asp Thr

825 830

Leu

Ala

Tyr Ala Glu Ser Val 835

Lys Thr Thr Leu Tyr 850

Lys Gly Arg Phe Thr 840

Leu Gln Met Asn Ser 855

Ile Ser Arg Asp Asn 845

Leu Arg Pro Glu Asp 860

Thr Ala Val 865

Gln Gly Thr

Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Val

870 875

Ser Ser

880

Leu Val Thr Val Ser Ser His His His His His

885

890

895 <210> 163 <211> 896 <212> Protein <213> Artificial sequence <220>

<223> Synthetic construction - Pro431 (Pro186 ST14(MS) linker) <400>163

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly 1. 5 1015

Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr 20 2530

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 4045

Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val 50 5560

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp 65 70 7580

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys

9095

Ala Ala Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp

100 105110

- 146 045012

Tyr

Gly

Gln 145

Phe

Leu

Tyr

Ser

Thr 225

Ile

Ser

Pro

Ser

Gln 305

Leu

Lys

Tyr

Trp Gly Gln

115

Gly Gly 130

Pro

Gly

Ser

Gly

Gly

Glu

Ser

Thr

Val

Leu

150

Asn

Lys

Tyr

Ala

Ile 165

Glu

Trp

Val

Ala

Arg

Tyr

Ala

Lys

210

Ala

Ser

Gly

Ser

180

Asp

Gln

Val

195

Asn

Val

Tyr

Gly

Leu

Ser

290

Lys

Val

Ala

Tyr

Thr

Tyr

Trp

Gly

Ala

Tyr

Ala

Tyr

Cys 230

Tyr 245

Ser

260

Thr

275

Thr

Gly

Gly

Val

Ala

Ser

Val

Pro

Pro

Ala

Cys

Gly

Gln

Ala

310

Gly

Thr

Pro 325

Leu

Thr Leu

340

Thr Leu Trp

355

Gln Val Thr

120

Gln Leu

135

Val

Lys

Leu

Ser

Asn

Trp

Val

Ile

Lys

Arg

Asp

Leu

215

Val

Trp

Gly

Pro

Ser

Val

Glu

Cys

Arg

Ser

Ser

Ala

155

Gln 170

Lys

Tyr

185

Arg

Phe

Thr

200

Gln

Met

Asn

Asn

Arg

Gly

Gly

Gly

Thr

295

Pro

Ala

Ser

Tyr

His

Gln

Ser

Ala

Asn

235

Gly

Thr 250

Gln

265

Gly

280

Ser

Gly

Thr

Asn

Thr

Val

Tyr

Arg

Arg

Gly

Ser

Gly

Leu

Ile

315

Phe

Val

Ser

Gly 330

Glin Pro

345

Asn Arg Trp

360

Ser Gly Gly

125

Gly Gly 140

Ala

Ala

Asn

Ile

Ser

Pro

Asn

Ser

Leu

220

Phe

Leu

Val

Thr

Gly

Gly

Gly

Tyr

Gly

Leu

Phe

Lys

Ser

Val

Thr

160

Gly 175

Ala

Thr

190

Arg

Asp

Asp

205

Lys

Gly

Val

Val

Leu

Pro

300

Gly

Ser

Glu

Val

Thr

Glu

Asp

Asn

Thr

Thr

Ser

Tyr 240

Val

Ser 255

Gln

270

Thr

285

Asn

Trp

Cys

Val

Glu

Ala

Gln

Gly

Thr

Lys

Phe

320

Leu

Leu

Gly

Gly 335

Asp

Phe

Glu

Ala Glu

350

Gly Gly Gly

365

- 147 045012

Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Gln Val

370 375380

Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly Ser Leu

385 390 395400

Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr Gly Met

405 410415

Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Gly

420 425430

Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val Lys Gly

435 440445

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp Leu Gln

450 455460

Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala

465 470 475480

Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp Tyr Trp

485 490495

Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Ser Leu Ser

500 505510

Gly Arg Ser Asp Asn His Gly Gly Gly Ser Gln Thr Val Val Thr Gln

515 520525

Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys

530 535540

Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val

545 550 555560

Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Asp Tyr Lys

565 570575

Asp Asp Asp Asp Lys Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu

580 585590

Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu

595 600605

Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly

610 615620

- 148 045012

Gly 625

Glu

Ser

Ala

Ala

Asp 705

Thr

Tyr

Trp

Gly

Gly 785

Gly

Gly

Thr

Asn

Asp

Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser

630 635640

Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

645 650655

Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr

660 665670

Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

675 680685

Arg Ile Arg Ser Lys Tyr Asp Tyr Lys Asp Asp Asp Asp Lys Ala

690 695700

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn

710 715720

Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val

725 730735

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr

740 745750

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

755 760765

Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

770 775780

Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser

790 795800

Phe Thr Phe Ser Lys Phe Gly Met Ser Trp Val Arg Gln Ala Pro

805 810815

Lys Gly Leu Glu 820 Trp Val Ser Ser Ile 825 Ser Gly Ser Gly Arg 830 Asp Leu Tyr Ala 835 Glu Ser Val Lys Gly 840 Arg Phe Thr Ile Ser 845 Arg Asp Ala 850 Lys Thr Thr Leu Tyr 855 Leu Gln Met Asn Ser 860 Leu Arg Pro Glu Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Val Ser

- 149 045012

865 870 875880

Ser Gln Gly Thr Leu Val Thr Val Ser Ser His His His His His

885 890895 <210>164 <211>763 <212> Protein <213> Artificial sequence <220>

<223> Synthetic construct - Pro258 (Pro186 with a single aEGFR and central aHSA domain) <400> 164 domain

Gln Val Lys Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1. 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala

Ser Gly Arg Thr Ser Arg Ser Tyr

thirty

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys 35 40

Glu Arg Glu Phe Val

Ser Gly Ile Ser 50

Lys Gly Arg Phe 65

Leu Gln Met Asn

Trp Arg Gly Asp Ser 55

Thr Ile Ser Arg Asp 70

Ser Leu Arg Ala Glu 85

Thr Gly Tyr Ala Asp 60

Asn Ala Lys Asn Ser 75

Asp Thr Ala Leu Tyr 90

Ser Val

Leu Tyr 80

Tyr Cys 95

Ala Ala Ala Ala Gly

100

Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp

105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Ser

115 120 125

Gly Gly 130 Gly Ser Glu Val Gln 135 Leu Val Glu Ser Gly 140 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr 145 Phe Asn Lys Tyr Ala 150 Ile Asn Trp Val Arg 155 Gln Ala Pro Gly Lys 160 Gly Leu Glu Trp Val Ala 165 Arg Ile Arg Ser Lys 170 Tyr Asn Asn Tyr Ala 175 Thr

- 150 045012

180

185

190

Tyr

Ser

Thr 225

Ile

Ser

Pro

Ser

Gln 305

Leu

Lys

Tyr

Thr

Gly 385

Val

Thr

Tyr Ala Asp

195

Lys Asn

210

Ala

Val

Ser

Gly

Ser

Gln

Val

Lys Asp Arg

200

Phe

Thr

Ile Ser Arg

205

Asp

Asp

Thr

Tyr

Tyr

Trp

Gly

Gly

Leu

Ser

290

Lys

Val

Ala

Tyr

Ala

Tyr

Leu Gln

215

Met

Asn

Asn

Leu Lys 220

Thr

Glu

Asp

Tyr

Cys 230

Val

Arg

His

Ala

Asn

235

Phe

Gly

Asn

Ser

Tyr 240

Ala

Tyr 245

Trp

Gly

Gln

Gly

Thr 250

Leu

Val

Thr

Val

Ser 255

Ser

260

Thr

275

Thr

Gly

Val

Ala

Gly

Gly

Gly

Ser

Gln

265

Ser

Pro

Gly

Gly

280

Thr

Thr

Val

Val

Thr

Gln

270

Val

Thr

Leu

Thr

285

Cys

Glu

Ala

Pro

Pro

Ala

Cys

Lys

370

Leu

Gln

Phe

Gly

Gln

Gly

Thr

Leu

Val

Ala

310

Pro 325

Thr

Leu

340

Thr

Leu

Trp

355

Leu

Thr

Val

Leu

Pro

Pro

Ser

Gly

Gly

Lys

Ser

Glu

390

Asn

Ser 405

Phe Gly

420

Thr

295

Pro

Ala

Ser

Tyr

Ser

Gly

Asn

Tyr

Pro

300

Asn

Trp

Val

Gln

Arg

Arg

Gly

Ser

Ser

375

Val

Leu

Met

Gly

Leu

Ile

315

Gly

Gly

Thr

Lys

Phe

320

Phe

Val

Ser

Gly 330

Gln

345

Asn

Arg

Pro

Trp

Ser

Glu

Val

360

Gly

Gln

Arg

Ser

Gly

Leu

Leu

Trp

Leu

Leu

Gly

Gly 335

Asp

Phe

Glu

Ala

Glu

350

Gly

Gly

Gly

365

Pro

Gly

Pro

380

Ala

Gly

Met

Lys

Val

Glu

395

Ser

Gly

Gly

Gly

Leu

400

Ser

Cys 410

Ala

Ala

Ser

Gly

Phe 415

Val Arg

Gln

Ala

Pro

Gly Lys

425

430

- 151 045012

Gly Leu Glu Trp Val Ser Ser Ile Ser

435 440

Tyr Ala Glu Ser Val Lys Gly Arg Phe

450 455

Lys Thr Thr Leu Tyr Leu Gln Met Asn 465 470

Ala Val Tyr Tyr Cys Thr Ile Gly Gly 485

Gly Thr Leu Val Thr Val Ser Ser Gly

500 5

Ser Gln Thr Val Val Thr Gln Glu Pro

515 520

Gly Thr Val Thr Leu Thr Cys Gly Ser

530 535

Gly Asn Tyr Pro Asn Trp Val Gln Gln 545 550

Gly Leu Ile Gly Asp Tyr Lys Asp Asp 565

Arg Phe Ser Gly Ser Leu Leu Gly Gly

580 5

Gly Val Gln Pro Glu Asp Glu Ala Glu

595 600

Ser Asn 610 Arg Trp Val Phe Gly 615 Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val 625 Gly Leu Val Gln Pro 630 Gly Gly Ser Leu Gly Phe Thr Phe Asn 645 Lys Tyr Ala Met Gly Lys Gly 660 Leu Glu Trp Val Ala 6 Arg

675 680

Ser Gly Arg Asp Thr Leu

445

Ile Ser Arg Asp Asn Ala 460

Leu Arg Pro Glu Asp Thr

475 480

Leu Ser Val Ser Ser Gln

490 495

Gly Gly Ser Gly Gly Gly

510

Leu Thr Val Ser Pro Gly

525

Thr Gly Ala Val Thr Ser 540

Pro Gly Gln Ala Pro Arg

555 560

Asp Lys Gly Thr Pro Ala

570 575

Ala Ala Leu Thr Leu Ser

590

Tyr Cys Val Leu Trp Tyr 605

Lys Leu Thr Val Leu Gly 620

Leu Val Glu Ser Gly Gly

635 640

Leu Ser Cys Ala Ala Ser

650 655

Trp Val Arg Gln Ala Pro

670

Arg Ser Lys Tyr Asp Tyr 685

- 152 045012

Lys Asp Asp Asp Asp Lys Ala Asp

690 695

Ser Val Lys Asp Arg Phe Thr Ile 700

Ser Arg Asp Asp Ser Lys Asn

705 710

Lys Thr Glu Asp Thr Ala Val

725

Gly Asn Ser Tyr Ile Ser Tyr

740

Thr Ala Tyr Leu Gln Met Asn Asn Leu

715720

Tyr Tyr Cys Val Arg His Gly Asn Phe

730735

Trp Ala Tyr Trp Gly Gln Gly Thr Leu

745750

Val Thr Val Ser Ser His His His His His

755760 <210>165 <211>896 <212> Protein <213> Artificial sequence <220>

<223> Synthetic construction - Pro221 - Cutable option <400> 165

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly

1. 5 10 15 linker

Ser Leu Arg Leu Thr Cys Ala Ala

Ser Gly Arg Thr Ser Arg Ser Tyr

thirty

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys 35 40

Glu Arg Glu Phe Val 45

Ser Gly Ile Ser 50

Lys Gly Arg Phe 65

Leu Gln Met Asn

Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val 5560

Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp 70 7580

Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 9095

Ala Ala Ala Ala Gly Ser Ala

100

Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp

105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser

115 120

Ser Gly Gly Gly Ser

125

- 153 045012

Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

130 135140

Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr

145 150 155160

Phe Asn Lys Tyr Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly

165 170175

Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr

180 185190

Tyr Tyr Ala Asp Gln Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp

195 200205

Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp

210 215220

Thr Ala Val Tyr Tyr Cys Val Arg His Ala Asn Phe Gly Asn Ser Tyr

225 230 235240

Ile Ser Tyr Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

245 250255

Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln Thr Val Val Thr Gln Glu

260 265270

Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Ala

275 280285

Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln

290 295300

Gln 305 Lys Pro Gly Gln Ala 310 Pro Arg Gly Leu Ile 315 Gly Gly Thr Lys Phe 320 Leu Val Pro Gly Thr Pro 325 Ala Arg Phe Ser Gly 330 Ser Leu Leu Gly Gly 335 Lys Ala Ala Leu Thr 340 Leu Ser Gly Val Gln 345 Pro Glu Asp Glu Ala 350 Glu Tyr Tyr Cys Thr 355 Leu Trp Tyr Ser Asn 360 Arg Trp Val Phe Gly 365 Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Gln Val

370 375 380

- 154 045012

Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly Ser Leu

385 390 395400

Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr Gly Met

405 410415

Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Gly

420 425430

Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val Lys Gly

435 440445

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp Leu Gln

450 455460

Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala

465 470 475480

Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp Tyr Trp

485 490495

Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ser Gly Gly Gly Gln Asn

500 505510

Pro Tyr Ser Ala Gly Arg Gly Gly Gly Ser Gln Thr Val Val Thr Gln

515 520525

Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys

530 535540

Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val

545 550 555560

Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Asp Tyr Lys

565 570575

Asp Asp Asp Asp Lys Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu

580 585590

Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu

595 600605

Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly

610 615620

Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser

625 630 635640

- 155 045012

Glu Val Gln Leu Val Glu Ser Gly

645

Gly Gly Leu Val Gln Pro Gly Gly

650 655

Ser Leu Lys

Leu Ser Cys Ala Ala 660

Ser Gly Phe Thr Phe Asn Lys Tyr

665 670

Ala

Met

Asn

Trp

Val

Arg

Gln

Ala

Pro

Gly

Lys

Gly

Leu

Glu

Trp

Val

675

680

685

Ala

Arg

690

Ile

Arg

Ser

Lys

Tyr

695

Asp

Tyr

Lys

Asp

Asp

700

Asp

Asp

Lys

Ala

Asp 705

Ser

Val

Lys

Asp

Arg 710

Phe

Thr

Ile

Ser

Arg 715

Asp

Asp

Ser

Lys

Asn

720

Thr

Ala

Tyr

Leu

Gln

Met

725

Asn

Asn

Leu

Lys

Thr 730

Glu

Asp

Thr

Ala

Val

735

Tyr

Tyr

Cys

Val

Arg

His

Gly

Asn

Phe

Gly

Asn

Ser

Tyr

Ile

Ser

Tyr

740

745

750

Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

755 760 765

Gly Gly Ser Gly Gly Gly Ser Glu Val

770 775

Gln Leu Val Glu Ser Gly Gly 780

Gly Leu Val Gln Pro Gly Asn

785 790

Ser Leu Arg Leu Ser Cys Ala Ala Ser

795 800

Gly Phe Thr Phe Ser Lys Phe

805

Gly Met Ser Trp Val Arg Gln Ala Pro

810 815

Gly Lys Gly Leu Glu Trp Val

820

Ser Ser Ile Ser Gly Ser Gly Arg Asp

825 830

Thr Leu Tyr Ala Glu

835

Ser Val Lys Gly Arg Phe Thr 840

Ile Ser Arg Asp 845

Asn Ala 850 Lys Thr Thr Leu Tyr 855 Leu Gln Met Asn Ser 860 Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Val Ser 865 870 875 880 Ser Gln Gly Thr Leu Val Thr Val Ser Ser His His His His His His

- 156 045012

885

890

895 <210>

<211>

<212>

<213>

166

896

Protein

Artificial sequence <220>

<223>

Synthetic construction

Pro222

Option with cut linker <400>

166

Gln Val

15

Lys

Leu Glu

Glu Ser

Gly

Gly Gly

Ser Val

Gln

Thr Gly

Gly

Ser

Leu

Arg

Leu Thr

Cys

Ala

Ala

Ser Gly 25

Arg

Thr

Ser

Arg Ser 30

Tyr

Gly

Met

Gly 35

Trp

Phe

Arg

Gln

Ala

Pro

Gly

Lys

Glu

Arg

Glu

Phe

Val

Ser

Gly

Ile

Ser

Trp

Arg

Gly

Asp

Ser

Thr

Gly

Tyr

Ala

Asp

Ser

Val

Lys 65

Gly

Arg

Phe

Thr

Ile 70

Ser

Arg

Asp

Asn

Ala 75

Lys

Asn

Thr

Val

Asp 80

Leu

Gln

Met

Asn

Ser

Leu 85

Lys

Pro

Glu

Asp

Thr 90

Ala

Ile

Tyr

Tyr

Cys 95

Ala

Ala

Ala

Ala

Gly

Ser

Ala

Trp

Tyr

Gly

Thr

Leu

Tyr

Glu

Tyr

Asp

100

105

110

Tyr

Trp

Gly

Gln

Gly

Thr

Gln

Val

Thr

Val

Ser

Ser

Gly

Gly

Gly

Ser

115

120

125

Gly

Gly

130

Gly

Ser

Glu

Val

Gln

135

Leu

Val

Glu

Ser

Gly

140

Gly

Gly

Leu

Val

Gln

145

Pro

Gly

Gly

Ser

Leu

150

Lys

Leu

Ser

Cys

Ala

155

Ala

Ser

Gly

Phe

Thr

160

Phe

Asn

Lys

Tyr

Ala

Ile 165

Asn

Trp

Val

Arg

Gln 170

Ala

Pro

Gly

Lys

Gly 175

Leu

Glu

Trp

Val

Ala Arg

Ile

Arg

Ser

Lys Tyr

Asn

Asn

Tyr

Ala Thr

Tyr

Tyr

Ala

180

185

190

Asp Gln Val

Lys

Asp

Arg Phe Thr

Ile

Ser

Arg Asp Asp

195

200

205

- 157 045012

Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp

210 215220

Thr Ala Val Tyr Tyr Cys Val Arg His Ala Asn Phe Gly Asn Ser Tyr

225 230 235240

Ile Ser Tyr Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

245 250255

Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln Thr Val Val Thr Gln Glu

260 265270

Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Ala

275 280285

Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln

290 295300

Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Lys Phe

305 310 315320

Leu Val Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly

325 330335

Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu

340 345350

Tyr Tyr Cys Thr Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly Gly

355 360365

Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Gln Val

370 375380

Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly Ser Leu

Claims (6)

1. A protein containing the amino acid sequence SEQ ID NO: 145. 2. Nucleic acid encoding a protein according to claim 1. 3. An expression vector containing the nucleic acid according to claim 2. 4. A host cell containing the expression vector according to claim 3. 5. A method for producing a protein, comprising culturing a host cell according to claim 4 under conditions under which said protein is expressed, and isolating said protein. 6. The use of protein according to claim 1, characterized in that it is aimed at obtaining a drug for the treatment of cancer.