RU2786444C2 - Fused proteins with albumin-binding domains - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, specifically to fused proteins with an albumin-binding domain (hereinafter – ABD); it can be used for recombinant production of fused protein with ABD. Fused protein with ABD is obtained in a recombinant way, in which ABD is attached to one or several fusing partners, and it contains vhCDR1, vhCDR2, vhCDR3 of a heavy chain of SEQ ID NO: 25 and vlCDR1, vlCDR2, vlCDR3 of a light chain of SEQ ID NO: 29.

EFFECT: invention provides obtainment of fused protein, which shows increased half-life, improved pharmacokinetics in vivo compared to biological preparations without ABD, and increased localization in tumor.

22 cl, 51 dwg, 6 ex

Description

State of the art

Although biological agents are useful in the treatment of many diseases, including various types of cancer, the short half-life of such molecules in the bloodstream represents a major limitation.

Biologics are useful in the treatment of cancer in a variety of ways. Cytokine-based drugs can act directly on cancer cells by influencing the growth and reproduction of such cells. Cytokines can also boost the immune system by stimulating the growth of killer T cells and other cells that attack cancer cells. In addition, cytokines can stimulate cancer cells to send chemicals that attract immune system cells. See, for example, Dranoff, Nature Reviews Cancer 4: 11-22 (2004); and Zhang et al., Proc Natl Acad Sci USA 106 (18): 7513-7518 (2009). Antibodies are desirable as therapeutic agents due to their ability to recognize targets with both specificity and high affinity. Monoclonal antibody drugs, including those that target tumor surface antigens and inhibitory signals that limit T-cell activation, have been a standard component of cancer therapeutics for more than 20 years. See, for example, Weiner, Nat Rev Cancer 15 (6): 361-370 (2015).

The short circulating half-life is a major limitation for many biologics. See, for example, Perdreau et al., European Cytokine Network 21: 297-307 (2010). Such short acting therapeutics require frequent dosing profiles, which may reduce clinical utility, especially in the case of chronic conditions. A long serum half-life is desirable as it would reduce the need for repeated injections of the molecule to achieve a therapeutically relevant serum concentration. Methods for extending the half-life of therapeutic proteins include pegylation, fusion with human serum albumin (HSA), fusion with a human IgG constant fragment (Fc), and fusion with unstructured polypeptides such as XTEN. See, for example, Stohl, BioDrugs 29 (4): 215-239 (2015). Half-life extension technologies create opportunities for new and improved biologics that result in cost savings and the inconvenience of frequent dosing. Thus, there remains a continuing need for new reagents and methods useful for extending the half-lives of protein and peptide therapeutics.

Brief description of the essence of the invention

This document provides compositions that contain an albumin binding domain (ABD). The biologics described herein that contain the subject albumin-binding domains (i.e., albumin-binding domain-based fusion proteins) preferably exhibit increased half-lives and more efficient in vivo pharmacokinetics compared to non-ABD biologics.

In one aspect, this document provides a composition that contains an albumin-binding domain (ABD). The ABD contains: a) a heavy chain variable region that contains vhCDR1, vhCDR2 and vhCDR3 of any of the heavy chain variable regions shown in FIG. 2; and b) a light chain variable region that contains vlCDR1, vlCDR2, and vlCDR3 of any of the light chain variable regions depicted in FIG. 2.

In some embodiments, vhCDR1 contains a vhCDR1 sequence, vhCDR2 contains a vhCDR2 sequence, and vhCDR3 contains a vhCDR3 sequence according to any of the vhCDR1, vhCDR2, and vhCDR3 sequences shown in FIG. 2. In one embodiment, the heavy chain variable region comprises the sequence of any of the heavy chain variable regions shown in FIG. 2. In some embodiments, vlCDR1 contains a vlCDR1 sequence, vlCDR2 contains a vlCDR2 sequence, and vlCDR3 contains a vlCDR3 sequence according to any of the vlCDR1, vhCDR2, and vhCDR3 sequences shown in FIG. 2. In one embodiment, the light chain variable region comprises the sequence of any of the light chain variable regions shown in FIG. 2. In an exemplary embodiment, the albumin binding domain comprises a heavy chain variable region and an A10m3 light chain variable region (FIG. 2D).

In another aspect, this document provides a composition that contains variant IL-15. Variant IL-15 contains one or more amino acid substitutions selected from the group consisting of K86A, K86R, N112A, N112S, N112Q, K86A/N112A, K86R/N112A, K86A/N112S, K86R/N112S, K86A/N112Q, K86R/N112Q K86A/N112A/N79A, K86R/N112A/N79A, K86A/N112A/N79D, K86R/N112A/N79D /N112A/N71Q K86R/N112A/N71Q K86A/N112A/N71D/N79A K86A/N112A/N71D/N79D K86A/N112A/N71Q/N79A K86A/N112A/N71Q/N79D K86R/N112A/N7A9 .

In some embodiments, the variant IL-15 contains the amino acid sequence of any of the variant IL-15 shown in FIG. 3. In certain embodiments, the variant IL-15 further comprises an IL-15 receptor alpha (IL-15Rα) fused to said IL-15.

In one aspect, this document provides an albumin-binding domain (ABD) fusion protein that contains an ABD attached to a fusion partner. The ABD contains a heavy chain variable region that contains vhCDR1, vhCDR2 and vhCDR3 of any of the heavy chain variable regions shown in FIG. 2 and a light chain variable region that contains vlCDR1, vlCDR2, and vlCDR3 of any of the light chain variable regions shown in FIG. 2.

In some embodiments, vhCDR1 contains a vhCDR1 sequence, vhCDR2 contains a vhCDR2 sequence, and vhCDR3 contains a vhCDR3 sequence according to any of the vhCDR1, vhCDR2, and vhCDR3 sequences shown in FIG. 2. In some embodiments, the heavy chain variable region comprises a sequence of any of the heavy chain variable region sequences shown in FIG. 2.

In certain embodiments, vlCDR1 contains the vlCDR1 sequence, vlCDR2 contains the vlCDR2 sequence, and vlCDR3 contains the vlCDR3 sequence according to any of the vlCDR1, vhCDR2, and vhCDR3 sequences shown in FIG. 2. In certain embodiments, the light chain variable region comprises a sequence of any of the light chain variable region sequences shown in FIG. 2.

In an exemplary embodiment, the heavy chain variable region and the light chain variable region comprise a heavy chain variable region and an A10m3 light chain variable region, respectively (FIG. 2D).

In some embodiments, the fusion partner is a cytokine. In certain embodiments, the cytokine is selected from IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, GM-CSF, and IFN-α.

In certain embodiments, the fusion partner is a linking fragment. In some embodiments, the binding fragment is an scFv that contains an scFv heavy chain variable region and an scFv light chain variable region. In some embodiments, the scFv is selected from anti-scFv TGFβ, anti-scFv PD-L1, and anti-scFv TNF. In some embodiments, the scFv is an interleukin anti-scFv. In illustrative embodiments, the scFv is anti-scFv IL-1, anti-scFv IL-6, anti-scFv IL-8, anti-scFv IL-17(A-F), or anti-scFv IL-23.

In some embodiments, the ABD is attached to the specified fusion partner using a linker. In an exemplary embodiment, the linker is (GGGGS) _x where x is an integer from 1 to 10.

In another aspect, this document provides an IL15 albumin binding domain (ABD) fusion protein according to the formula (IL-15)-L-(ABD). The ABD contains a heavy chain variable region that contains vhCDR1, vhCDR2 and vhCDR3 of any of the heavy chain variable regions shown in FIG. 2 and a light chain variable region that contains vlCDR1, vlCDR2, and vlCDR3 of any of the light chain variable regions shown in FIG. 2 and L is a linker.

In some embodiments, vhCDR1 contains a vhCDR1 sequence, vhCDR2 contains a vhCDR2 sequence, and vhCDR3 contains a vhCDR3 sequence according to any of the vhCDR1, vhCDR2, and vhCDR3 sequences shown in FIG. 2. In some embodiments, the heavy chain variable region comprises a sequence of any of the heavy chain variable region sequences shown in FIG. 2.

In certain embodiments, vlCDR1 contains the vlCDR1 sequence, vlCDR2 contains the vlCDR2 sequence, and vlCDR3 contains the vlCDR3 sequence according to any of the vlCDR1, vhCDR2, and vhCDR3 sequences shown in FIG. 2. In certain embodiments, the light chain variable region comprises a sequence of any of the light chain variable region sequences shown in FIG. 2.

In an exemplary embodiment, the heavy chain variable region and the light chain variable region comprise a heavy chain variable region and an A10m3 light chain variable region, respectively (FIG. 2D).

In one embodiment, IL-15 is a variant IL-15 containing one or more amino acid substitutions selected from the group consisting of K86A, K86R, N112A, N112S, N112Q, K86A/N112A, K86R/N112A, K86A/N112S, K86R /N112S, K86A/N112Q, K86R/N112Q, K86A/N112A/N79A, K86R/N112A/N79A, K86A/N112A/N79D, K86R/N112A/N79D, K86A/N112A/N79Q, K86R/N112A/N79A, K186A/N1 /N71D K86R/N112A/N71D /N79D K86R/N112A/N71D/N79A K86R/N112A/N71D/N79D K86R/N112A/N71D/N79Q K86R/N112A/N71Q/N79A , compared to the original IL-15. In some embodiments, the variant IL-15 contains the amino acid sequence of any of the variant IL-15 shown in FIG. 3. In an exemplary embodiment, variant IL-15 contains the amino acid sequence of IL15 K86R/N112A.

In certain embodiments, the IL-15 is wild-type IL-15. In some embodiments, the IL-15 is wild-type IL-15 fused to the IL-15 receptor alpha (IL-15Rα).

In certain embodiments, the linker is selected from any of the linkers shown in FIG. 48. In some embodiments, the linker is (GGGGS) _x where x is an integer from 1 to 10.

In one embodiment, the IL15-ABD fusion protein has an amino acid sequence according to any of the amino acid sequences shown in FIG. 4.

In another aspect, provided herein is an IL12 albumin binding domain (ABD) fusion protein according to the formula (IL-12)-L-(ABD). The ABD contains a heavy chain variable region that contains vhCDR1, vhCDR2 and vhCDR3 of any of the heavy chain variable regions shown in FIG. 2 and a light chain variable region that contains vlCDR1, vlCDR2, and vlCDR3 of any of the light chain variable regions shown in FIG. 2 and L is a linker.

In some embodiments, vhCDR1 contains a vhCDR1 sequence, vhCDR2 contains a vhCDR2 sequence, and vhCDR3 contains a vhCDR3 sequence according to any of the vhCDR1, vhCDR2, and vhCDR3 sequences shown in FIG. 2. In some embodiments, the heavy chain variable region comprises a sequence of any of the heavy chain variable region sequences shown in FIG. 2.

In certain embodiments, vlCDR1 contains the vlCDR1 sequence, vlCDR2 contains the vlCDR2 sequence, and vlCDR3 contains the vlCDR3 sequence according to any of the vlCDR1, vhCDR2, and vhCDR3 sequences shown in FIG. 2. In certain embodiments, the light chain variable region comprises a sequence of any of the light chain variable region sequences shown in FIG. 2.

In an exemplary embodiment, the heavy chain variable region and the light chain variable region comprise a heavy chain variable region and an A10m3 light chain variable region, respectively (FIG. 2D).

In one embodiment, IL-12 is a single chain IL-12 that contains a p35 subunit, a p40 subunit and an IL-12 linker, and the IL-12 linker covalently attaches the p35 subunit to the p40 subunit. In certain embodiments, the linker is selected from any of the linkers shown in FIG. 48. In an exemplary embodiment, the linker is (GGGGS) _x where x is an integer from 1 to 10.

In an exemplary embodiment, the IL12-ABD fusion protein comprises an amino acid sequence according to any of the amino acid sequences of FIG. twenty.

In another aspect, provided herein is an albumin binding domain (ABD) fusion protein having an N-terminal to C-terminal formula selected from: a) (IL-12)-L1-(ABD)-L2-( IL-15); and b) (IL-15)-L1-(ABD)-L2-(IL-12). The ABD contains a heavy chain variable region that contains vhCDR1, vhCDR2 and vhCDR3 of any of the heavy chain variable regions shown in FIG. 2 and a light chain variable region that contains vlCDR1, vlCDR2, and vlCDR3 of any of the light chain variable regions shown in FIG. 2, and L1 and L2 are the first and second linkers, respectively.

In some embodiments, vhCDR1 contains a vhCDR1 sequence, vhCDR2 contains a vhCDR2 sequence, and vhCDR3 contains a vhCDR3 sequence according to any of the vhCDR1, vhCDR2, and vhCDR3 sequences shown in FIG. 2. In some embodiments, the heavy chain variable region comprises a sequence of any of the heavy chain variable region sequences shown in FIG. 2.

In certain embodiments, vlCDR1 contains the vlCDR1 sequence, vlCDR2 contains the vlCDR2 sequence, and vlCDR3 contains the vlCDR3 sequence according to any of the vlCDR1, vhCDR2, and vhCDR3 sequences shown in FIG. 2. In certain embodiments, the light chain variable region comprises a sequence of any of the light chain variable region sequences shown in FIG. 2.

In an exemplary embodiment, the heavy chain variable region and the light chain variable region comprise a heavy chain variable region and an A10m3 light chain variable region, respectively (FIG. 2D).

In some embodiments, the IL-15 contains a wild-type IL-15 polypeptide. In some embodiments, wild-type IL-15 is fused to an IL-15 receptor alpha (IL-15Rα).

In one embodiment, IL-15 is a variant IL-15 that contains one or more amino acid substitutions selected from K86A, K86R, N112A, N112S, N112Q, K86A/N112A, K86R/N112A, K86A/N112S, K86R/N112S, K86A/N112Q K86R/N112Q K86A/N112A/N79A K86R/N112A/N79A K86A/N112A/N79D K86R/N112A/N79D K86A/N112A/N79Q K86R/N112A/N79Q K86A/N112 K86R/N112A/N71D K86A/N112A/N71Q K86R/N112A/N71Q K86A/N112A/N71D/N79A K86A/N112A/N71D/N79D K86A/N112A/N71Q/N79A K86A/N112A/N7D,Q K86R/N112A/N71D/N79A, K86R/N112A/N71D/N79D, K86R/N112A/N71D/N79Q, K86R/N112A/N71Q/N79A, K86R/N112A/N71Q/N79D and K86R/N112A/N71Q/N79Q, compared with original IL-15. In one embodiment, IL15 contains an amino acid sequence according to any of the amino acid sequences shown in FIG. 3.

In certain embodiments, IL-12 is single chain IL-12 that contains a p35 subunit, a p40 subunit, and an IL-12 linker, wherein the IL-12 linker joins the p35 subunit to the p40 subunit.

In some embodiments, each of the first linker and the second linker is independently selected from any of the linkers shown in FIG. 48. In an exemplary embodiment, the linker is (GGGGS) _x where x is an integer from 1 to 10.

In another aspect, this document provides an ABD fusion protein that contains an albumin binding domain (ABD), a cytokine and a linker (L) according to the formula (cytokine)-L-(ABD). The ABD contains a heavy chain variable region that contains vhCDR1, vhCDR2 and vhCDR3 of any of the heavy chain variable regions shown in FIG. 2 and a light chain variable region that contains vlCDR1, vlCDR2, and vlCDR3 of any of the light chain variable regions shown in FIG. 2 and L is a linker. The cytokine is selected from IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, GM-CSF and IFN-α.

In some embodiments, vhCDR1 contains a vhCDR1 sequence, vhCDR2 contains a vhCDR2 sequence, and vhCDR3 contains a vhCDR3 sequence according to any of the vhCDR1, vhCDR2, and vhCDR3 sequences shown in FIG. 2. In some embodiments, the heavy chain variable region comprises a sequence of any of the heavy chain variable region sequences shown in FIG. 2.

In certain embodiments, vlCDR1 contains the vlCDR1 sequence, vlCDR2 contains the vlCDR2 sequence, and vlCDR3 contains the vlCDR3 sequence according to any of the vlCDR1, vhCDR2, and vhCDR3 sequences shown in FIG. 2. In certain embodiments, the light chain variable region comprises a sequence of any of the light chain variable region sequences shown in FIG. 2.

In an exemplary embodiment, the heavy chain variable region and the light chain variable region comprise a heavy chain variable region and an A10m3 light chain variable region, respectively (FIG. 2D).

In some embodiments, the linker is selected from any of the linkers shown in FIG. 48. In an exemplary embodiment, the linker is (GGGGS) _x where x is an integer from 1 to 10.

In another aspect, this document provides a fusion protein with ABD according to the formula: (FP1)-L1-(ABD)-L2-(FP2), where ABD is an albumin-binding domain that includes a variable heavy chain and a variable light chain ; where FP1 and FP2 are the first fusion protein and the second fusion protein, respectively; and where L1 and L2 represent the first and second linker, respectively. The ABD contains a heavy chain variable region that contains vhCDR1, vhCDR2 and vhCDR3 of any of the heavy chain variable regions shown in FIG. 2 and a light chain variable region that contains vlCDR1, vlCDR2, and vlCDR3 of any of the light chain variable regions shown in FIG. 2.

In some embodiments, vhCDR1 contains a vhCDR1 sequence, vhCDR2 contains a vhCDR2 sequence, and vhCDR3 contains a vhCDR3 sequence according to any of the vhCDR1, vhCDR2, and vhCDR3 sequences shown in FIG. 2. In some embodiments, the heavy chain variable region comprises a sequence of any of the heavy chain variable region sequences shown in FIG. 2.

In certain embodiments, vlCDR1 contains the vlCDR1 sequence, vlCDR2 contains the vlCDR2 sequence, and vlCDR3 contains the vlCDR3 sequence according to any of the vlCDR1, vhCDR2, and vhCDR3 sequences shown in FIG. 2. In certain embodiments, the light chain variable region comprises a sequence of any of the light chain variable region sequences shown in FIG. 2.

In an exemplary embodiment, the heavy chain variable region and the light chain variable region comprise a heavy chain variable region and an A10m3 light chain variable region, respectively (FIG. 2D).

In some embodiments, FP1 and FP2 are the first cytokine and the second cytokine, respectively. In an exemplary embodiment, the first cytokine and said second cytokine are selected from IL-2 and IL-12; IL-7 and IL-15; IL-15 and IL-12; IL-18 and GM-CSF; IL-21 and IL-15; GM-CSF and IL-12; GM-CSF and IL-21; and IFN-α and IL-15.

In some embodiments, the first and second fusion partners are selected from anti-scFv PD-L1 and IL-12; anti-scFv PD-L1 and IL-15; anti-scFv PD-L1 and anti-scFv TGFβ; a first anti-scFv PD-L1 and a second scFv PD-L1; anti-scFv TGFβ and IL-12; anti-scFv TGFβ and IL-15; anti-scFv TGFβ and scFv PD-L1; and a first anti-scFv TGFβ and a second anti-scFv TGFβ.

In one embodiment, FP1 and FP2 are the first binding fragment and the second binding fragment, respectively. In certain embodiments, the first binding fragment and the second binding fragment are each scFv. In an exemplary embodiment, the first binding fragment and the second binding fragment are selected from scFv TNF and scFv IL-1; scFv TNF and scFv IL-6; scFv TNF and scFv IL-8; scFv TNF and scFv IL-17 (isoforms A-F); ScFv TNF and scFv IL-23 and first scFv TNF and second scFv TNF.

In some embodiments, each of the first linker and the second linker is independently selected from any of the linkers shown in FIG. 48. In one embodiment, each of the first linker and the second linker is independently (GGGGS) _x , where x is an integer from 1 to 10.

In another aspect, provided herein is an albumin-binding domain (ABD) fusion protein that contains a TGFβ-binding domain and an albumin-binding domain. The albumin binding domain comprises an ABD heavy chain variable region and an ABD light chain variable region having the amino acid sequences of any of the heavy chain variable regions and the light chain variable regions of FIG. 2.

In an exemplary embodiment, the ABD heavy chain variable region and the ABD light chain variable region comprise the amino acid sequence of the heavy chain variable region and the light chain variable region of A10m3.

In one embodiment, the TGF binding domain is a scFv that contains a heavy chain variable region and a 4D9 light chain variable region (FIG. 40B). In some embodiments, the ABD further comprises an IL-12, IL-15, or PD-L1 binding domain or a second TGFβ binding domain.

In another aspect, provided herein is an albumin binding domain (ABD) fusion protein that contains a PD-L1 binding domain and an albumin binding domain. The albumin binding domain comprises an ABD heavy chain variable region and an ABD light chain variable region having the amino acid sequences of any of the heavy chain variable regions and the light chain variable regions of FIG. 2.

In some embodiments, the ABD heavy chain variable region and the ABD light chain variable region comprise the amino acid sequence of the heavy chain variable region and the light chain variable region of A10m3 (FIG. 2D). In certain embodiments, the PD-L1 binding domain is an scFv containing a heavy chain variable region and a 10D12 light chain variable region (FIG. 50).

In some embodiments, the ABD further comprises an IL-12, IL-15, or TGFβ binding domain or a second PD-L1 binding domain.

In another aspect, provided herein is a nucleic acid that encodes any of the albumin-binding domains, variant IL-15, or ABD-based fusion proteins described herein, host cells that contain any such nucleic acids, and methods for making such ABD, variant IL-15, or ABD-based fusion proteins.

In yet another aspect, provided herein is a method for inhibiting or reducing a tumor in a subject in need thereof, the method comprising administering an ABD-based fusion protein to the subject.

Brief description of graphic materials

In FIG. 1A-C show several exemplary albumin-binding domain-based fusion proteins disclosed herein, including fusion proteins in which an albumin-binding domain is fused to a cytokine (e.g., IL-12 or IL-15) and fusion proteins. , in which the albumin-binding domain is attached to 1) two cytokines; 2) two binding fragments (eg scFvs); 3) binding fragment and cytokine.

In FIG. 2A-2Z show sequences of exemplary albumin-binding domains included in certain embodiments of the contemplated albumin-binding domain-based fusion proteins described herein. Included in these figures are the heavy chain variable domain and light chain variable domain sequences, as well as certain vhCDR1-3 and vlCDR1-3 sequences.

In FIG. 3 shows the sequences of exemplary variant IL-15s described herein.

In FIG. 4 shows exemplary embodiments of the IL-15-ABD fusion proteins described herein. The IL-15-ABD fusion proteins shown in FIG. 4, include ABD A10m3.

In FIG. 5 shows the results of studies showing that transcription is not responsible for the low expression of IL-15-A10m3 in HEK293T cells. Verification of IL15-A10m3 mRNA in transfected cells. A) Expression of IL15-A10m3 in HEK293 cannot be detected by Western blot using an anti-His tag antibody (left), or by functional linkage using MSA ELISA (right). M: marker, CK: non-transfected cell culture medium as control, 1-3: medium from 3 independently transfected cell cultures (100, 200 and 250 = 100, 200, 250 μg/ml Zeocin, respectively). 10 μg/ml produced E. coli IL15-A10m3 acted as a positive control. B) mRNA was prepared from 4 independently transfected IL15-A10m3 cells and RT-PCR was performed to quantify the level of mRNA from IL15-A10m3 mRNA compared to the level of housekeeping gene, GAPDH. Lane 1) control using non-transfected cells; 2-5) IL15-A10m3 mRNA transcription appears to be normal in all transfected cells; 6) positive control of GAPDH mRNA.

In FIG. 6 shows the identification of potential IL-15 ubiquitination sites that are putatively protected by the IL-15 receptor alpha (IL-15Rα) that binds to IL-15. (A) K86 in red is a putative ubiquitination site that is adjacent to the IL-15Rα binding sites (marked with asterisks); (B) K86 is a ubiquitination target from UbPred, an online database of ubiquitination sites (www.ubpred.org).

In FIG. 7 shows schematic illustrations of various enhanced stability IL-15-ABD fusion proteins described herein, including IL-15-ABD with IL-15Ra/IL15 "sushi domain" fusion partner (A) and fusion proteins. based on IL-15-ABD, which include variant IL-15 fusion partners having amino acid substitutions at the putative K86 ubiquitination site (B).

In FIG. 8 shows studies evaluating the expression level (FIG. 8A) and the ability of HEK293-produced IL-15-ABD K86R and K86A and IL-15Rα/IL-15-ABD variants to bind to mouse serum albumin (FIG. 8A) and IL-15Rα (Fig. 8B). A) ELISA readings in case of binding of IL15-ABD to mouse serum albumin (MSA) 12 clones IL-1510-A3m3 DT (DT), 12 clones IL-15Rα/IL-15-A10m3 (IL15Ra), 6 mutant clones IL-15 K86A-A10m3 (K86A) and 6 IL-15 mutant clones K86R-A10m3 (K86R). The culture medium from each well of the sample from the 24-well plates was added to the MSA-coated ELISA plates. B) ELISA in the case of IL-15 binding to the IL-15 alpha receptor (IL-15Rα) that was coated on the plate was used to confirm that the K86A (clone A3, yellow asterisk) and K86R (clone R6, green asterisk) had no effect on IL-15 binding activity to IL-15Rα. The intrinsic IL-15Rα in IL-15Rα-IL-15-A10m3 (alpha1 clone, red star) could bind to intrinsic IL15 and thus block its binding to plate-coated IL-15Rα. 10 μg/ml produced E. coli IL-15-A10m3 DT was used as a positive control.

In FIG. 9 shows extended production of clone #6 IL15K86R-A10m3. A) Chromatogram of IL15K86R-A10m3 using a size exclusion column; B) SDS-PAGE analysis of SEC fractions 14 to 42; C) End products (1: IL15K86R-A10m3, 2: IL15Ra-IL15-A10m3) were finally confirmed by SDS-PAGE (left) and Western blotting with anti-His tag antibodies (right).

In FIG. 10 shows the results of in vitro binding assays confirming the ability of IL-15 K86R-A10m3 to bind mouse serum albumin (MSA).

In FIG. 11 shows the results of CTLL2 proliferation assays showing that IL-15 K86R-A10m3 polypeptides produced from HEK293T cells have reduced biological activity compared to wild-type IL-15 and IL-15-A10m3 produced from E. coli.

In FIG. 12 shows the results of studies showing that the reduced biological activity of HEK cell-produced IL-15 K86R-A10m3 is at least partially due to its glycosylation.

In FIG. 13 shows the results of studies showing that the N112A mutation introduced in IL15 K86R-A10m3 can restore IL-15 biological activity comparable to that of deglycosylated IL-15R-A10m3 in CTLL2 proliferation assays (A). (B) Additionally, IL-15R-A10m3 fusion proteins with IL-15 amino acid substitutions N112A, N112Q, and N112S were shown to exhibit increased biological activity inversely proportional to the size of the substituted side chain.

In FIG. 14 shows an experimental design for evaluating the in vivo effects of IL-15 and IL-15-ABD on tumor growth in the B16-F10 mouse melanoma model.

In FIG. 15 is a graph showing the magnitude of B16-F10 tumor growth inhibition in C57BL/6 mice treated with an IL-15-ABD fusion protein 11 days after treatment.

In FIG. 16 shows FACS analyzes depicting the effect of IL-15-ABD treatment on tumor infiltrating lymphocyte populations.

In FIG. 17 and 18 show the generalized result of IL-15-ABD treatment on lymphocyte populations in the spleen and tumors.

In FIG. 19A and B show the results of studies showing the stability of the subject IL-15-ABD in a mouse model (A) and in human serum (B) compared to control IL-15 DT.

In FIG. 20 shows the sequences of exemplary IL-12-ABDs described herein: human mIL-12sc-A10m3 and human IL-12sc-A10m3.

In FIG. 21 and 22 show studies showing that subject IL-12-ABD produced from HEK293T cells is biologically active in in vitro assays and cellular assays.

In FIG. 23 shows an experimental design for evaluating the in vivo effects of IL-12 and IL-12-ABD on tumor growth in the B16-F10 mouse melanoma model. Similar molar concentrations of IL-12 and IL-12 were used at three different concentrations. For example, 3 µg IL-12 is the same molar concentration as 4.5 µg IL-12-ABD, 10 µg IL-12 is the same molar concentration as 15 µg IL-12-ABD, and 20 μg IL-12 is the same molar concentration as 30 μg IL-12-ABD. The molecular weight of IL-12 is 70 kDa and the molecular weight of IL-12-ABD is 107 kDa.

In FIG. 24 shows graphs depicting the growth kinetics of tumors of various groups in IL-12-ABD/IL-12 in vivo assays. In FIG. 24A shows the results of tumor size assessments in the IL-12 and IL-12-ABD groups, FIG. 24B shows IL-12 treatment groups alone, and FIG. 24C shows IL-12-ABD treatment groups separately.

In FIG. 25 shows graphs depicting the kinetics of tumor growth in individual animals in each of the various in vivo IL-12-ABD/IL-12 study groups.

In FIG. 26 shows graphs showing tumor volumes of different groups in IL-12-ABD/IL-12 in vivo assays 10 days after treatment.

In FIG. 27 shows measurements of longitudinal body weight (left) and % body weight (right) in B16-F10 tumor-bearing mice at various time points after treatment with IL-12 or IL-12-ABD.

In FIG. 28 shows Kaplan-Meier pseudo-survival curves for various groups in IL-12-ABD/IL-12 in vivo studies.

In FIG. 29 shows a summary of studies comparing the pharmacodynamic effects of a single dose of IL-12-ABD and IL-12 in B16-F10 tumor-bearing mice after five days. Comparisons of tumor weight, spleen weight, serum IFN-γ, and body weight are shown.

In FIG. 30 shows the result of a study comparing tumor volume in B16-F10 tumor-bearing mice at 10 days treated with IL-12-ABD (1.3 μg), IL-12 (30 μg), or placebo. In this study, IL-12-ABD was administered at a ~30 times lower molar dose than IL-12.

In FIG. 31 further demonstrates the hematopoietic effects of IL-12-ABD and IL-12 in mice from the study shown in FIG. 32, after 3 and 7 days.

In FIG. 32 shows a study showing the effect of single dose combination therapies using IL-12-ABD or IL-12 with anti-PD-1 antibodies on tumor growth in B16-F10 tumor-bearing mice.

In FIG. 33 shows serum results showing an increase in PK for IL12-ABD compared to IL-12 DT.

In FIG. 34 shows the sequences of the exemplary IL-15-ABD-IL-12 of interest described herein: 1) hIL-15 (K86R/N112A)-A10m3-mIL-12sc; 2) mIL-12sc-A10m3-hIL-15 (K86R); and 3) mIL-12sc-A10m3-hIL-15 (K86R/N112A).

In FIG. 35 shows the binding activities of both hIL-15 (K86R)-A10m3-mIL-12sc and mIL-12sc -A10m3-hIL-15 (K86R) to MSA (A), IL-15 alpha receptor and IL-β2 receptor. 12(B) confirmed by ELISA.

In FIG. 36 shows the sequences of additional exemplary IL-15-ABD-IL-12 of interest described herein: 1) hIL-15 (K86R)-A10m3-hIL-12sc; 2) hIL-12sc-A10m3-hIL-15 (K86R); and 3) hIL-12sc-A10m3-hIL-15 (K86R/N112A).

In FIG. 37 and 38 show the results of experiments showing that IL-15-ABD-IL-12 described herein exhibits both IL-12 activity (FIG. 39) and IL-15 activity (FIG. 40).

In FIG. 39 shows the results of a study showing that IL-15-ABD-IL-12 described herein was more active than IL-12-ABD alone.

In FIG. 40A and B show the sequences of two exemplary TGFβ binding domains described herein, 4H7 and 4D9, including the heavy chain variable region, light chain variable region, and scFv form sequences.

In FIG. 41 shows the results of a study showing that the scFv TFFβ described herein are able to block TGFβ1-induced Treg (CD4+Foxp3+) expansion.

In FIG. 42 and 43 demonstrate that TGF-β blockade reverses TGF-β1-induced epithelial-mesenchymal transition (FIG. 44) and migration (FIG. 45). (Fig. 42A). Schematic representation of E-cadherin loss and induced vimentin expression during EMT. (Fig. 44B). Mouse 4T1 cells were cultured in growth media supplemented with TGF-β1 alone or together with 1D11 (panel 3) or anti-scFv TGF-β1 (panel 4), then fixed and stained with anti-E-cadherin (green) and anti- vimentin (purple). The cores were stained with DAPI (blue). Treatment with TGF-β1 induced loss of E-cadherin from cell-to-cell contacts and resulted in increased expression of vimentin. This effect is reversed by the addition of 1D11 or the subject anti-scFv TGF-β1 described herein. In FIG. 43 further demonstrates that the subject TGF-β1 anti-scFvs described herein block TGF-β1 mediated migration of carcinoma cells.

In FIG. 44 is a summary of studies showing that the subject anti-scFv TGF-β1 described herein are able to inhibit TGF-β mediated Smad2 phosphorylation in human (A) and mouse (B) cells. In Case A, the anti-scFv TGF-β1 in question were able to inhibit TGF-β1 mediated Smad2 phosphorylation in human cells in a dose-dependent manner. In case B, the anti-scFv TGF-β1 in question were able to inhibit mouse TGF-β1, -β2 and -β3 mediated phosphorylation of Smad2 in mouse cells.

In FIG. 45A shows the sequences of exemplary TGF-β1-ABD scFv constructs (4D9M-A6m and 4H7-A6m).

In FIG. 45B shows the summary result of the study showing that anti-TGFβ-1-ABD expands anti-scFv TGFβ-1.

In FIG. 46 shows the summary result of the study showing that various anti-scFv TGF-β1-ABD (bivalent in the case of TGF-β1) produced in E. coli or mammalian (HEK) cells are able to bind to mouse serum albumin.

In FIG. 47 shows the summary result of the study showing that TGFβ-1 mediated inhibition of T cell proliferation was reversed (i.e. increased T cell proliferation) with exemplary TGF-β1-ABD scFv constructs (4D9M-A6m and 4H7-A6m) .

In FIG. 48 shows exemplary linkers that can be used with the subject fusion proteins. Such linkers can be used as scFv linkers for the heavy chain variable regions and the light chain variable regions of the ABD. Such linkers can also be used to attach the albumin binding domains described herein to IL-12 and IL-15 fusion partners or to connect components of IL-12 fusion partners (p35 and p40) or IL-12 fusion partners. 15 (IL-15 and IL-15Rα) with each other.

In FIG. 49A-G show the amino acid sequence of exemplary cytokines that can be included in the ABD-based fusion proteins described herein. In FIG. 49A-G also show exemplary cytokine-ABD fusion proteins, with ABD being A10m3. Although such cytokine-ABD fusion proteins are depicted with A10m3 ABD, any ABD, including the ABD described herein, can be included in cytokine-ABD fusion proteins.

In FIG. 50 shows the amino acid sequence of an exemplary anti-PD-L1 binding domain, 10D12, which can be used with the ABD-based fusion proteins described herein. 10D12 binds to hPD-L1 at low pH and cross-reacts with mPD-L1. 10D12 does not bind to hPD-L2 or mPD-L2. In addition, 10D12 blocks PD-1/PD-L1 interaction as well as B71/PD-L1 interaction.

In FIG. 51A and B show several useful combinations of cytokine-ABD-cytokine (A) and binding fragment-ABD-cytokine/binding fragment (B). "A" and "B" in each combination can switch to the opposite orientation. These figures also show the linker-A10m3-linker backbone that can be included in cytokine-ABD-cytokine and ABD-cytokine binding fragment/binding fragment combinations. While the A10m3 ABD is shown, any ABD, including any shown here (eg, FIG. 2), can be used in such designs.

Detailed Description of the Invention

A. Overview

Biologics, including cytokines and antibody-based therapeutics, are useful in the treatment of cancers.

Current and potential cytokine-based therapeutics include those using IL-2, IL-7, IL-12, IL-15, IL-18, IL-21 GM-CSF and IFN-α.

IL-12 is able to mediate immune effector functions in a manner consistent with enhancing the proinflammatory endogenous antitumor immune response. (See, for example, Boggio et al., J Exp Med 188:589-96 (1998); Cavallo et al., Cancer Res 59:414-21 (1999); Yu et al. Int Immunol 8:855-65 (1996) Nastala et al., J Immunol 153:1697:706 (1994) Brunda et al., J Exp Med 178: 1223-30 (1993) IL-12 is known to induce an inflammatory Th1 CD4+ T- cells, and enhances cytotoxicity of CD8+ T cells.Studies have also shown that IL-12 mediated secretion of IFNγ by T cells can reverse T cell anergy and render effector T cells resistant to immunosuppressive regulatory T cells. IL-12 not only activates the adaptive and innate immune systems, but also further modulates the otherwise hostile tumor microenvironment, making IL-12 an ideal candidate for tumor immunotherapy.

IL-15 is able to stimulate T cell proliferation within tumors (see, for example, Miecnik et al., Sci Transl Med 6 (228): 228ra37 (2014)), as well as prolong the survival of effector CD8+ memory T cells, and is critical for development of NK cells. Thus, it is thought that IL-15 may enhance the effects of checkpoint inhibitors and other immunotherapies that use T cells to attack cancer cells. At the same time, the IL-15 monomer has a short half-life of less than 40 minutes in vivo. Modifications to the IL-15 monomer may improve its in vivo pharmacokinetics in the treatment of cancers. These modifications were mainly aimed at improving the trans presentation of IL-15 by the alpha subunit of the IL-15 receptor, IL-15Rα. Such modifications include: 1) pre-association of the fusion of IL-15 and the fusion of its soluble alpha receptor subunit with Fc to form an IL-15:IL-15Rα-Fc complex (see, e.g., Rubinstein et al., Proc Natl Acad Sci USA 103:9166-71 (2006)); 2) expression of hyperagonist protein IL-15-sIL-15Rα-sushi (see, for example, Bessard et al., Molecular Cancer Therapeutics 8: 2736-45 (2009)); and 3) preassociating the human IL-15 mutant IL-15N72D with the IL-15Rα-Fc sushi-Fc fusion complex (see, for example, Zhu et al., Journal of Immunology 183:3598-6007 (2009)).

Monoclonal antibody drugs, including those that target tumor surface antigens and inhibitory signals that limit T-cell activation, have been a standard component of cancer therapeutics for more than 20 years. See, for example, Weiner, Nat Rev Cancer 15 (6): 361-370 (2015).

The short circulating half-life is a major hurdle for many biologics, including cytokine and antibody based drugs. See, for example, Herrington-Symes et al., Advances in Bioscience and Biotechnology 4: 689-698 (2013) and Perdreau et al., European Cytokine Network 21: 297-307 (2010). Such short acting therapeutics require frequent dosing profiles, which may reduce clinical utility, especially in the case of chronic conditions. A long serum half-life is desirable as it would reduce the need for repeated injections of the molecule to achieve a therapeutically relevant serum concentration. Methods for extending the half-life of therapeutic proteins include pegylation, fusion with human serum albumin (HSA), fusion with human IgG constant fragment (Fc), and fusion with unstructured fusion proteins such as XTEN. See, for example, Stohl, BioDrugs 29 (4): 215-239 (2015). Half-life extension technologies create opportunities for improved or new biologics that result in cost savings and the inconvenience of frequent dosing. Thus, there remains a continuing need for new reagents and methods that can lead to extended half-lives of therapeutic agents based on proteins and peptides.

This document provides novel albumin-binding domain (ABD) fusion proteins useful for extending the half-life of biologicals (eg, interleukins and antibodies). Serum albumin has a long half-life in the range of 2-4 weeks due to recycling through the neonatal Fc receptor (FcRn). Albumin is taken up by endothelial cells by macropinocytosis and binds to FcRn in a pH-dependent manner in the acidic environment of the early endosome. Binding of albumin and FcRn helps to deflect albumin molecules from destruction in the lysosomal compartment and redirects albumin molecules to the plasma membrane, where they are released back into the blood plasma due to the neutral pH.

The albumin binding domains (ABDs) described herein do not compete with FcRNs for albumin binding and bind albumin in a pH range that allows ABDs to also undergo endosomal albumin recycling by FcRn when bound to albumin. Thus, biologics that contain the albumin-binding domain (ABD) in question are able to avoid lysosomal degradation by the albumin-FcRn pathway and therefore exhibit longer serum half-lives than non-ABD counterparts.

Moreover, such ABD-containing therapeutics preferentially localize to tumors known to contain high levels of serum albumin. Thus, such ABD-containing therapeutic agents are particularly suitable for the treatment of cancers.

B. Albumin binding domains

In one aspect, this document provides compositions that contain an albumin-binding domain. As used herein, the term "serum albumin" refers to a member of the family of globular proteins produced by the liver that functions primarily as a carrier protein for steroids, fatty acids, and thyroid hormones in the blood. Serum albumin also plays a major role in stabilizing extracellular fluid volume by participating in the maintenance of oncotic pressure in blood plasma, and includes, but is not limited to, human serum albumin (HSA, Genbank accession numbers: NM_000477 and NP_000468) and mouse serum albumin ( MSA, Genbank access numbers: NM_009654 and NP_0033784). The structure of albumin is characterized by several long α-helices and contains eleven different binding domains for hydrophobic compounds. In humans, serum albumin is encoded by the ALB gene.

Fusion proteins that include the albumin-binding domain (ABD) in question can bind serum albumin (SA) to facilitate uptake of the fusion protein into cells by macropinocytosis. In certain embodiments, the ABDs described herein bind over a pH range of about 5.5 to about 7.2. In the early endosome, these SA-bound ABD-based fusion proteins bind to FcRn by SA at an acidic pH (e.g., pH 5.5), which in turn diverts the SA-bound ABD fusion protein away from the cell's lysosomal compartment and redirects to the plasma membrane. At the plasma membrane region, SA dissociates from FcRn due to neutral pH (eg, pH 7.1-7.5) and SA and the ABD fusion protein are released back into the circulation. Because therapeutic agents that contain the ABD in question are capable of binding to albumin in the pH range of about 5.5 to about 7.2, such therapeutic agents also preferentially undergo endosomal albumin recycling by FcRn and thus avoid lysosomal cleavage. Accordingly, therapeutics that contain such ABDs preferably exhibit longer serum half-lives than non-ABD counterparts. Such therapeutic agents are particularly useful in the treatment of cancers known to contain high levels of serum albumin.

In some embodiments, the albumin binding domain binds albumin at a site that does not prevent SA from binding to the neonatal Fc receptor (FcRn). As used herein, the term "FcRn" or "neonatal Fc receptor" means a protein that binds the Fc region of an IgG antibody and is encoded at least in part by the FcRn gene. FcRn can be derived from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. As is known in the art, a functional FcRn protein includes two fusion proteins, often referred to as a heavy chain and a light chain. The light chain is a beta-2 microglobulin and the heavy chain is encoded by the FcRn gene. Unless otherwise specified herein, an FcRn or FcRn protein refers to a FcRn heavy chain complex with beta-2-microglobulin.

In some embodiments, the albumin binding domain described or exemplified herein preferably binds specifically to serum albumin (e.g., HSA) at an epitope on the serum albumin molecule that is not involved in the interaction of the serum albumin molecule with FcRn. Binding of the SA-binding moiety to the serum albumin molecule thus preferably does not substantially interfere with, inhibit, prevent, or otherwise reduce the binding of the serum albumin molecule (eg, HSA) to FcRn. Preferably, the albumin binding domain does not compete with the FcRn for binding to the serum albumin molecule. Preferably, the albumin binding domain does not sterically inhibit the binding of serum albumin to FcRn. Preferably, the SA-binding moiety does not change the conformation of the serum albumin molecule such that the albumin cannot interact with the FcRn.

In some embodiments, the albumin binding domain binds SA (e.g., HSA) at a pH of 5.0 ± 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0. In some embodiments, the albumin binding domain binds SA over a pH range of about 5.5 to about 7.2. In some embodiments, the SA binding moiety binds SA at a pH of 5.5.

In some embodiments, the albumin binding domain is a human serum albumin (HSA) binding domain. HSA binding domains include, but are not limited to, albumin binding domains that can bind to an HSA molecule, such as an entire HSA molecule or a fragment of an HSA. In some embodiments, the HSA binding domain also binds mouse serum albumin. In some embodiments, the HSA binding domain also binds cynomolgus albumin. In some embodiments, the HSA binding domain does not bind to bovine serum albumin (BSA).

The albumin binding domains provided herein may comprise a heavy chain variable region alone, or a heavy chain variable region in combination with a light chain variable region. In some embodiments, the albumin-binding domain contains a heavy chain variable region. In some embodiments, the heavy chain variable region comprises vhCDR1, vhCDR2 and vhCDR3 (complementarity determining regions of the heavy chain variable region 1-3). In certain embodiments, the antigen binding domain also contains a light chain variable region. In some embodiments, the light chain variable region comprises vlCDR1, vlCDR2, and vlCDR3 (complementarity determining regions of the light chain variable region 1-3).

In some embodiments, the albumin binding domain comprises a heavy chain variable region that comprises vhCDR1, vhCDR2, and vhCDR3 of any of the heavy chain variable regions shown in FIG. 2. In some embodiments, the albumin binding domain comprises vhCDR1, vhCDR2, and vhCDR3 of the A10m3 heavy chain variable region, as shown in FIG. 2D. In some embodiments, the albumin binding domain comprises vhCDR1, vhCDR2, and vhCDR3 A10m3, as shown in FIG. 2D.

In some embodiments, the albumin binding domain also contains a light chain variable region. In an exemplary embodiment, the albumin binding domain comprises a light chain variable region that contains vlCDR1, vlCDR2 and vlCDR3 of any of the light chain variable regions as shown in FIG. 2. In some embodiments, the albumin binding domain comprises vlCDR1, vlCDR2, and vlCDR3 of the A10m3 light chain variable region, as shown in FIG. 2D. In some embodiments, the albumin binding domain comprises vlCDR1, vlCDR2, and vlCDR3 A10m3, as shown in FIG. 2D.

In some embodiments, the albumin binding domain (eg, HSA binding domain) is an antibody or antibody fragment. In some embodiments, the albumin binding domain (eg, HSA binding domain) is a scFv.

In some embodiments, in which the ABD contains both a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region are joined to each other by a linker (eg, an scFv linker). In some embodiments, the linker is attached to the heavy chain variable region at its C-terminus and to the light chain variable region at its N-terminus. Suitable linkers are described herein and in FIG. 48. In some embodiments, the linker is a (Gly4Ser) _x linker, where x is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the linker is a (Gly4Ser) ₅ linker.

In some embodiments, the albumin binding domain also contains a heavy chain variable region that contains vhCDR1, vhCDR2, and vhCDR3 A10m3, and a light chain variable region that contains vlCDR1, vlCDR2, and vlCDR3 A10m3 (FIG. 2D). In one embodiment, the albumin binding domain comprises a heavy chain variable region sequence and an ABD A10m3 variable light chain sequence shown in FIG. 2D.

C. Interleukin-15 variants

In another aspect, this document provides compositions that contain variant IL-15 with increased in vivo stability and/or biological activity compared to wild-type IL-15.

As used herein, the terms "interleukin 15", "IL-15", and "IL15" refer to an interleukin that binds and signals through a complex consisting of the alpha chain of the IL-15 specific receptor, the beta chain of the IL receptor -2/IL-15 (CD122) and common gamma chain (gamma-C, CD132) (Genbank accession numbers: NM_00000585 and NP_000576 (human); and NM_001254747 and NP_001241676 (mouse)).

IL-15 has been shown to stimulate T cell proliferation within tumors (see, for example, Miecnik et al., Sci Transl Med 6 (228):228ra37 (2014)). IL-15 is also capable of prolonging the survival of effector CD8+ memory T cells and is critical for NK cell development. It is thought that IL-15 may enhance the effects of checkpoint inhibitors and other immunotherapies that T cells use to attack cancer cells. Thus, without being bound by any particular mode of action, the IL-15s described herein are believed to be useful in the treatment of cancers.

At the same time, the IL-15 monomer has a short half-life of less than 40 minutes in vivo. Modifications to the IL-15 monomer may lead to an improvement in its in vivo pharmacokinetics in the treatment of cancers. These modifications were mainly aimed at improving the trans presentation of IL-15 by the alpha subunit of the IL-15 receptor, IL-15Rα. Such modifications include: 1) pre-association of the fusion of IL-15 and the fusion of its soluble alpha receptor subunit with Fc to form an IL-15:IL-15Rα-Fc complex (see, e.g., Rubinstein et al., Proc Natl Acad Sci USA 103:9166–71 (2006)); 2) expression of hyperagonist protein IL-15-sIL-15Rα-sushi (see, for example, Bessard et al., Molecular Cancer Therapeutics 8: 2736-45 (2009)); and 3) pre-association of the human IL-15 mutant IL-15N72D with the IL-15Rα-Fc sushi-Fc fusion complex (see, e.g., Zhu et al., Journal of Immunology 183:3598-6007 (2009)).

In some embodiments, the implementation of IL-15 is a variant of the original IL-15 with increased stability compared to wild-type IL-15. In specific embodiments, the variant IL-15 is a wild-type human IL-15 variant. In an exemplary embodiment, the variant IL-15 contains an amino acid substitution at position K86 of the original IL-15 shown in FIG. 3. Described herein, K86 is a putative ubiquitin-dependent cleavage site (see Example 2) when used with certain cell types (eg, HEK293 T cells). Thus, without being bound by any particular mode of action, the removal of the K86 ubiquitination site by amino acid substitution is believed to increase the stability of IL-15 (see Examples 2 and 3).

In certain embodiments, IL-15 is a variant IL-15 having an amino acid substitution at position N112. The amino acid position N112 is the main site of biological activity of IL-15, as it is decisive for the proper interaction of the IL-15/IL-15 gamma receptor, especially when IL-15 is attached to the ABD. Thus, without being bound by any particular mode of action, it is believed that mutations at the N112 position may lead to an increase in one or more of the functions of IL-15, including, but not limited to, promoting T cell proliferation in tumor microenvironments, increasing survival of CD8+ T cells and activation of NK cell development.

Certain amino acid substitutions that result in increased stability and/or biological activity of IL-15 in vivo include, but are not limited to: K86A, K86R, N112A, N112S, N112Q, K86A/N112A, K86R/N112A, K86A/N112S, K86R/N112S K86A/N112Q K86R/N112Q K86A/N112A/N79A K86R/N112A/N79A K86A/N112A/N79D K86R/N112A/N79D K86A/N112A/N79Q K86R/N112A/N79 N112A/N71D K86R/N112A/N71D K86A/N112A/N71Q K86R/N112A/N71Q K86A/N112A/N71D/N79A K86A/N112A/N71D/N79D K86A/N112A/N71Q/N79A/N11A K86R/N112A/N71D/N79A, K86R/N112A/N71D/N79D, K86R/N112A/N71D/N79Q, K86R/N112A/N71Q/N79A, K86R/N112A/N71Q/N79D and K86R/N112A N79Q. Exemplary variant IL-15s that contain one or more of these amino acid substitutions are shown in FIG. 3. In an exemplary embodiment, variant IL-15 contains the amino acid substitutions K86A and N112A.

In one embodiment, the IL-15 described herein (wild type IL-15 and variant IL-15) is fused to alpha IL-15R. Such IL-15, presented in trans with its receptor, has been shown to have a long half-life and higher activity compared to native Il-15 alone. See, for example, Wu, J Mol Genet Med 7, 85 (2013).

D. IL-12

In another aspect, this document provides compositions that contain IL-12. The terms "interleukin 12", "IL-12", and "IL12" as used herein all refer to interleukin, which is a heterodimeric cytokine encoded by the IL-12A and IL-12B genes (Genbank accession numbers: NM_000882 (IL-12A ) and NM_002187 (IL-12B)). IL-12 consists of a bundle of four alpha helices and is involved in the differentiation of native T cells into TH1 cells. IL-12 binds to the IL-12 receptor, which is a heterodimeric receptor formed by IL-12R-β1 and IL-12R-β2. IL-12 is known as a T cell stimulating factor that can stimulate T cell growth and function. In particular, IL-12 can stimulate the production of interferon gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) from T cells and natural killer (NK) cells and lead to a decrease in IL-4 mediated suppression of IFN- γ. IL-12 may further mediate an increase in the cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes. In addition, IL-12 may also have anti-angiogenic activity by increasing the production of interferon gamma, which in turn leads to an increase in the production of a chemokine-10 inducible protein (IP-10 or CXCL10). IP-10 then mediates this anti-angiogenic effect. Without being bound by any particular mode of action, it is believed that IL-12, due to its ability to induce immune responses and its anti-angiogenic activity, can be used to treat cancers.

In some embodiments, the IL-12 is mouse IL-12. In other embodiments, the IL-12 is human IL-12.

In some embodiments, IL-12 is a single chain IL-12 polypeptide comprising an IL-12 p35 subunit fused to an IL-12 p40 subunit. Such single chain IL-12 polypeptides preferably retain one or more of the biological activities of wild-type IL-12. In some embodiments, a single chain IL-12 polypeptide described herein is of the formula, N-terminus to C-terminus, (p40)-(L)-(p35), where "p40" is the p40 subunit of IL-12 , "p35" is the p35 subunit of IL-12, and L is a linker. In other embodiments, single chain IL-12 corresponds to the formula, N-terminus to C-terminus, (p35)-(L)-(p40). Any suitable linker can be used in a single chain IL-12 polypeptide, including linkers as described herein and disclosed in FIG. 49C. Suitable linkers may include, for example, linkers having the amino acid sequence (GGGGS) _x , where x is an integer from 1 to 10. Other suitable linkers include, for example, the amino acid sequence GGGGGGS. Exemplary IL-12 single chain linkers that can be used with the subject IL-12 single chain polypeptides are also described in Lieschke et al., Nature Biotechnology 15:35-40 (1997), which is incorporated herein by reference in its entirety and especially in connection with describing IL-12 polypeptide linkers.

In one exemplary embodiment, the single chain IL-12 polypeptide is a single chain human IL-12 polypeptide (i.e., it contains the p35 and p40 subunit of human IL-12). In certain embodiments, the single chain IL-12 polypeptide is a single chain murine IL-12 polypeptide. Exemplary mouse and human single chain IL-12 are shown in FIG. 20 (shown as fused peptide to ABD) and 49C.

E. ABD-based fusion proteins

In one aspect, this document provides ABD-based compositions that comprise an albumin-binding domain attached to one or more fusion partners (eg, a first fusion partner, a second fusion partner, etc.) via a linker. As described herein, ABD-based fusion proteins can undergo FcRn-mediated endosomal recycling and thus preferably exhibit an increased half-life compared to counterparts that do not contain such ABDs.

ABDs suitable for such ABD-based fusion proteins include, but are not limited to, those described herein. The amino acid sequences of such ABDs, including vhCDR1-3, vlCDR1-3, heavy chain variable region and light chain variable region sequences, are disclosed, for example, in FIG. 2. In some embodiments, the ABD fusion protein comprises a heavy chain variable region that comprises vhCDR1, vhCDR2, and vhCDR3 of any of the ABD heavy chain variable regions of FIG. 2 and a light chain variable region that contains vlCDR1, vlCDR2, and vCDR3 of any of the ABD light chain variable regions of FIG. 2. In some embodiments, the ABD comprises a heavy chain variable region having the vhCDR1, vhCDR2, and vhCDR3 ABDs disclosed in FIG. 2 and a light chain variable region having the vhCDR1, vhCDR2 and vhCDR3 ABD disclosed in FIG. 2. In some embodiments, the ABD fusion protein comprises the heavy chain variable region and the light chain variable region of the ABD disclosed in FIG. 2.

In one exemplary embodiment, the ABD fusion protein comprises a heavy chain variable region that contains vhCDR1, vhCDR2, and vhCDR3 of the A10m3 heavy chain variable region, and a light chain variable region that contains vlCDR1, vlCDR2, and vCDR3 of the A10m3 light chain variable region (Figure 2D). ). In some embodiments, the ABD comprises a heavy chain variable region having vhCDR1, vhCDR2 and vhCDR3 A10m3 and a light chain variable region having vhCDR1, vhCDR2 and vhCDR3 A10m3 (FIG. 2D). In some embodiments, the ABD fusion protein comprises a heavy chain variable region and an A10m3 light chain variable region (FIG. 2D).

The ABD-based fusion proteins described herein contain a fusion partner. In some embodiments, the merger partner includes two merger partners (a first merger partner (FP1) and a second merger partner (FP2). In embodiments that include two merger partners, the merger partners can be attached to ABD in multiple orientations In some embodiments, the ABD fusion protein is of the formula, N-terminus to C-terminus: FP1-ABD-FP2, FP1-PF2-ABD, or ABD-FP1-FP2, where FP1 is the first partner merger, and FP2 is the second merger partner.

Any suitable fusion partner for which it is desirable to extend the half-life of the fusion partner can be included in the subject ABD-based fusion proteins. Fusion partners may include, for example, cytokines (eg, interferons and interleukins), growth factors, polypeptides, proteins, and hormones (eg, growth hormones, parathyroid hormone).

In certain embodiments, the fusion partner is an antibody-based binding fragment that contains a heavy chain variable region and a light chain variable region. Such binding fragments can bind to any target of interest, including, for example, tumor-specific targets or cytokines. In an exemplary embodiment, the fusion partner is a single chain variable fragment (scFv). Antibody-based fusion partners also include, but are not limited to, ds-scFv, single domain antibodies (sdAb), diabodies, dsFv, ds-scFv, Fab, and full length antibodies. Antibody-based fusion partners also include multispecific (eg, bispecific) antibodies and fragments.

The contemplated ABD-based fusion proteins described herein use linkers between components (ABD and fusion partners) and within components. For example, scFv fusion partners use standard peptide linkers, typically based on glycine and serine, to attach heavy and light chain variable regions to form scFvs. In addition, standard peptide linkers are used to attach ABD to fusion partners (eg, cytokine fusion partners). In addition, linkers are used to attach components of certain fragments, such as the p35 and p40 subunits of IL-12 and IL-15 to IL-15Rα. In some embodiments, the linker is a (Gly4Ser) _x linker, where x is 1, 2, 3, 4, 6, 7, or 8. In specific embodiments, the ABD is connected to fusion partners via (Gly4Ser) ₅ linkers.

As shown in this document, there are a number of suitable linkers that can be used, including traditional peptide bonds obtained using recombinant techniques. The linker peptide may advantageously contain the following amino acid residues: Gly, Ser, Ala or Thr. The linker peptide must have a length that is sufficient to link the two molecules so that they take the correct conformation relative to each other, in order to ensure that they retain the necessary activity. In one embodiment, the linker is about 1 to 50 amino acids in length, preferably about 1 to 30 amino acids in length. In one embodiment, linkers from 1 to 20 amino acids in length can be used, with linkers of about 5 to about 10 amino acids in length finding use in some embodiments. Suitable linkers include glycine-serine polymers, including, for example, (GS)n, (GSGGS)n, (GGGGS)n and (GGGS)n, where n is an integer, from at least one (and typically 3 to 4), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Alternatively, various non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, may find use as linkers, i. may find use as linkers.

Other linker sequences may contain any sequence of any length of the CL/CH1 domain, but not all residues of the CL/CH1 domain; for example, the first 5-12 amino acid residues of the CL/CH1 domains. Linkers can be derived from the light chain of immunoglobulins, such as Cκ or Cλ. Linkers can be derived from immunoglobulin heavy chains of any isotype, including, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cσ, Cε, and Cμ. Linker sequences can also be derived from other proteins, such as Ig-like proteins (eg, TCR, FcR, KIR), hinge-derived sequences, and other naturally occurring sequences from other proteins.

Often the linker is inherently immune to the extracellular environment. As used herein, the term "substantially immune to the extracellular environment" in the context of a linker means that no more than about 20%, 15%, 10%, 5%, 3%, or no more than about 1% of the linkers in a sample of the conjugate compound the antibody-drug is cleaved when the antibody-drug conjugate compound is present in the extracellular environment (eg, blood plasma).

Whether the linker is substantially immune to the extracellular environment can be determined, for example, by incubating the antibody drug conjugate compound with blood plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying determining the amount of free drug present in blood plasma.

In other, non-mutually exclusive, embodiments, the implementation of the linker promotes cellular internalization. In some embodiments, the linker stimulates cellular internalization when conjugated to a therapeutic agent (ie, in the environment of a linker-therapeutic agent fragment of an antibody-drug conjugate compound, as described herein). In yet another embodiment, the linker promotes cellular internalization when conjugated to both the auristatin compound and the ABD-based fusion proteins of the invention.

Various exemplary linkers that can be used with the compositions and methods of this invention are described in WO 2004-010957, US Publication No. 2006/0074008, US Publication No. 20050238649, and US Publication No. 2006/0024317 (each of which is incorporated herein by reference in its entirety and for all purposes).

Exemplary linkers that can be used with the subject ABDs as domain linkers, scFv linkers, and attachment components of certain fusion partners are further illustrated in FIG. 48.

In some embodiments, the linker is a "domain linker" used to link any two domains as defined herein together (eg, a cytokine fusion partner (eg, interleukin) and ABD). While any suitable linker may be used, many embodiments use a glycine-serine polymer, including, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least one (and typically 3 to 4 to 5), as well as any peptide sequence that allows recombinant attachment of two domains with sufficient length and flexibility so that each domain retained its biological function.

In some embodiments, the fusion partner is a scFv that contains a heavy chain variable region and a light chain variable region. In such embodiments, the ABD heavy chain variable region is attached to the light chain variable region using an scFv linker.

Exemplary ABD-based fusion proteins are further described below.

1. Cytokine-ABD fusion proteins

In some embodiments, the ABD fusion protein contains a cytokine fusion partner, i. refers to fusion proteins based on the cytokine-albumin-binding domain (cytokine-ABD) (Fig. 1). In some embodiments, the cytokine-ABD fusion protein comprises IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-27, GM-CSF, or IFN-α. Contemplated cytokine-ABD fusion proteins are useful for immunomodulatory effects in a subject when such immunomodulatory effects are desired (eg, treatment of cancer or an autoimmune disease). In addition, the subject cytokine ABDs exhibit a longer half-life and improved pharmacokinetic properties compared to interleukin-based therapeutics alone.

Any ABD can be used with the cytokine-ABD fusion proteins described herein. In some embodiments, the implementation of the ABD contains the variable region of the heavy chain of ABD. In certain embodiments, the ABD contains an ABD light chain variable region. In exemplary embodiments, the ABD is an scFv that contains a heavy chain variable region linked to a light chain variable region via a linker (eg, any of the linkers disclosed herein and in FIG. 48).

In certain embodiments, the heavy chain variable region comprises vhCDR1-3 any of the ABD heavy chain variable regions described herein, including those ABD heavy chain variable regions shown in FIG. 2. In certain embodiments, the ABD heavy chain variable region comprises vhCDR1-3 of the A10m3 heavy chain variable region (FIG. 2D). In some embodiments, the ABD heavy chain variable region contains vhCDR1-3 A10m3, as shown in FIG. 2D. In illustrative embodiments, the ABD heavy chain variable region has the amino acid sequence of the A10m3 heavy chain variable region.

In certain embodiments, the ABD contains an ABD light chain variable region. In some embodiments, the ABD light chain variable region comprises vlCDR1-3 any of the ABD light chain variable regions described herein, including those ABD light chain variable regions shown in FIG. 2. In certain embodiments, the ABD light chain variable region comprises vlCDR1-3 of the A10m3 light chain variable region (FIG. 2D). In some embodiments, the ABD light chain variable region contains vlCDR1-3 A10m3, as shown in FIG. 2D. In exemplary embodiments, the ABD light chain variable region has the A10m3 light chain variable region sequence.

The amino acid sequences of exemplary cytokines that can be used in contemplated cytokine-ABD fusions, as well as exemplary cytokine-ABD fusion proteins, where ABD is A10m3, are shown in FIG. 49A-G.

In some embodiments, IL-ABD corresponds to the formula, N-terminus to C-terminus, cytokine-L-ABD or ABD-L-cytokine, where L is a linker that attaches the cytokine to ABD (eg, a peptide linker). In some embodiments, the ABD cytokine comprises a heavy chain variable region, and the cytokine is fused to the N-terminus of the ABD heavy chain variable region. In some embodiments, the cytokine is fused to the C-terminus of the ABD heavy chain variable region. In an exemplary embodiment, the interleukin-ABD contains an ABD that also contains a light chain variable region (eg, scFv ABD). In some embodiments, the cytokine is fused to the C-terminus of the light chain variable region. In a certain embodiment, the N-terminus of the cytokine is attached to the ABD. In other embodiments, the C-terminus of the cytokine is fused to the ABD.

In certain embodiments, the cytokine is a cytokine selected from IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-27, GM-CSF, and IFN-α. Cytokine molecules include, for example, full-length cytokines and fragments of cytokines, for example, the part that is important for the function of a particular cytokine (eg, the part of an interleukin that binds to its receptor).

In some embodiments, the implementation of the cytokine-ABD contains an IL-2 molecule or a fragment thereof. As used herein, the terms "interleukin 2", "IL-2", and "IL2" refer to a member of the cytokine family having four alpha-helical bundles and signaling via the IL-2 receptor (Genbank accession numbers: NM_000586 and NP_000577 (human) and NM_008366 and NP_032392 (mouse)). IL-2 plays critical roles in immune system function, tolerance and immunity, mainly as a result of its direct effects on T cells. In the thymus, IL-2 prevents autoimmune disease by promoting the differentiation of certain immature T cells into regulatory T cells that destroy other T cells that are primed to attack normal healthy cells in the body. IL-2 has been used in the treatment of cancers (malignant melanoma, renal cell carcinoma) in high doses with intermittent dosing and has been widely used in continuous dosing. Exemplary IL-2-ABDs are shown in FIG. 49A.

In some embodiments, the cytokine-ABD fusion protein comprises an IL-7 molecule or a fragment thereof. As used herein, the terms "interleukin 7", "IL-7" and "IL7" (Genbank accession numbers: NM_000880 and NP_000871 (human); and NM_008371 and NP_032397 (mouse)) refer to a member of the cytokine family, which is hematopoietic growth factor secreted by stromal cells in the bone marrow and thymus and binds to the IL-7 receptor. Interleukin-7 (IL-7) is a non-hematopoietic cell-derived cytokine with a central role in the adaptive immune system. It promotes the development of lymphocytes in the thymus, maintains homeostasis due to the survival of naive T-cells and memory T-cells in the periphery. In addition, it plays an important role in lymph node (LN) organogenesis and in maintaining activated T cells recruited to secondary lymphoid organs (SLOs). IL-7 is an ideal solution for the recovery of immunocompromised patients with cancer as a result of the expansion of peripheral T cells. In animal models, IL-7 has been shown to prolong the survival of tumor-bearing hosts. Exemplary IL-7-ABDs are shown in FIG. 49b.

In certain embodiments, the cytokine-ABD contains an IL-12 molecule or a fragment thereof. In some embodiments, IL-12 is the single chain IL-12 described herein and shown in FIG. 20 (shown as a fusion peptide to ABD) and 49C.

In certain embodiments, the cytokine-ABD contains an IL-15 molecule or a fragment thereof. In some embodiments, the IL-15-ABD contains variant IL-15 as described herein and in FIG. 3 and 4. In some embodiments, IL-15 is wild-type IL-15 as shown in FIG. 3 (eg, "original IL-15"). In some embodiments, wild-type IL-15 is fused to an IL-15 receptor alpha (IL-15Rα).

In certain embodiments, the cytokine-ABD contains an IL-21 molecule or a fragment thereof. As used herein, the terms "interleukin 21", "IL-21" and "IL21" (Genbank accession numbers NM_001207006 and NP_001193935 (human) and NM_0001291041 and NP_001277970 (mouse)) refer to a member of the cytokine family that binds to the receptor IL-21 and has strong regulatory effects on cells of the immune system, including natural killer (NK) cells and cytotoxic cells, and binds to the IL-21 receptor, which can destroy virus-infected or cancer cells. Thus, without being bound by any particular mode of action, the subject cytokine ABD having IL-21 is believed to be useful in the treatment of various types of cancer. Exemplary IL-21-ABDs are shown in FIG. 49E.

Other suitable cytokines that may be included in the contemplated cytokine-ABD include, but are not limited to, IL-27, IFN-α, and GM-CSF. In certain embodiments, the interleukin-ABD polypeptide also comprises an interferon-alpha, interferon-beta, or GM-CSF molecule. Such molecules are suitable for delivery to tumors and reducing toxicity.

In some embodiments, the implementation of the cytokine-ABD contains two cytokines attached to the ABD, selected from the following: IL-2 and IL-12; IL-7 and IL-15; IL-15 and IL-12; IL-18 and GMC SF; IL-21 and IL-15; GMC-SF and IL-12; GMC-SF and IL-21; and IFN-α and IL-15. In FIG. 1 shows several exemplary orientations in which two cytokines can be attached to an ABD. Any ABD can be used in such a fusion protein, including those containing any of the ABD heavy chain variable domains and light chain variable domains shown in FIG. 2. In some embodiments, ABD is A10m3.

Any linker can be used to attach each cytokine to the ABD, including any linker shown in FIG. 48. An exemplary linker-A10m3-linker backbone sequence for use in such fusion proteins is shown in FIG. 50. Exemplary linkers that can be used include (GGGGS) _x linkers, where X is 1 to 10. In certain embodiments, (GGGGS) ₅ is used to attach a cytokine to an ABD.

In some embodiments, the implementation of the cytokine-ABD corresponds to the formula selected from the following (from the N-terminus to the C-terminus): cytokine 1-L1-ABD-L2-cytokine 2; cytokine 1-L1-cytokine 2-L2-ABD; and ABD-L1-cytokine 1-L2-cytokine 2, where L1 and L2 are linkers that bind cytokines and ABD components, and cytokine 1 and cytokine 2 are selected from the following pairs of cytokines: IL-2 and IL-12; IL-7 and IL-15; IL-15 and IL-12; IL-18 and GM-CSF; IL-21 and IL-15; GM-CSF and IL-12; GM-CSF and IL-21; and IFN-α and IL-15. In some embodiments, ABD is A10m3 (FIG. 2D).

In such cytokine-ABDs, any cytokine may be "cytokine 1" and the other cytokine may be "cytokine 2". For example, in one embodiment of the IL-2 and IL-12 cytokine pair, IL-2 is "cytokine 1" and IL-12 is "cytokine 2". In another embodiment, pairs of cytokines IL-2 and IL-12. IL-12 is "cytokine 1" and IL-2 is "cytokine 2".

In some embodiments, the implementation of the cytokine-ABD contains two identical interleukins. In other embodiments, the interleukin-ABD polypeptide contains two different interleukins (eg, IL-12 and IL-15) described above. ABD-based fusion proteins that include certain combinations of cytokines are disclosed in FIG. 51. Exemplary cytokine-ABD fusion proteins that include a combination of various cytokines are further detailed below.

A. IL-12 and IL-15

In certain embodiments, the cytokine-ABD fusion protein comprises IL-12 and IL-15. Exemplary IL-12 and IL-15 sequences that may be included in such embodiments are shown in FIG. 3 and 49C. Cytokine ABDs that include both IL-12 fusion partner and IL-15 fusion partner are believed to exhibit the antitumor effects of both interleukins. The combination of IL-12 and IL-15 has been shown to induce increased antitumor activity compared to either cytokine alone. This increase in antitumor activity correlated with reactivation of each cytokine receptor through synergistic induction of IFN-γ. In addition, IL-12, in combination with IL-15, has been shown to stimulate antitumor activity in peritoneal macrophages via nitric oxide synthesis. Without being bound by any particular mode of action, it is believed that polypeptides having both an IL-12 fusion partner and an IL-15 fusion partner are able to rapidly activate an innate response (IL-12) as well as effectively stimulate T- cells and support CD8+ memory T cells (IL-15). Animal studies show that sequential delivery of cells expressing IL-12 and IL-15 also resulted in cure in mice under therapeutic conditions of advanced tumors. Depletion of CD8+ cells resulted in the loss of protection of said therapy, suggesting clonal expansion of tumor CTL. See, for example, Croce et al., Clin Cancer Res 11(2 Pt 1):735-42 (2005).

In some embodiments, the implementation of the cytokine-ABD corresponds to the formula, from the N-terminus to the C-terminus:

a) (IL-12)-L1-(ABD)-L2-(IL-15); or

b) (IL-15)-L1-(ABD)-L2-(IL-12).

Any suitable ABD may be used, including, for example, an ABD having an ABD heavy chain variable region that contains vhCDR1-3 of any of the ABD heavy chain variable regions shown in FIG. 2. In an exemplary embodiment, the heavy chain variable region comprises vhCDR1-3 of the A10m3 heavy chain variable region (FIG. 2D). In certain embodiments, the ABD comprises an ABD light chain variable region that contains vlCDR1-3 of the A10m3 light chain variable region. In an exemplary embodiment, the ABD is an A10m3 scFv.

Any suitable IL-12 and IL-15 may be used. In some embodiments, IL-15 is a variant IL-15 described herein (see, for example, FIG. 3). IL-15 can also be wild-type IL-15 or wild-type IL-15 that is attached to IL-15R. In one embodiment, IL-15 is a variant IL-15 selected from those shown in FIG. 3. In an exemplary embodiment, Il-15 is a variant IL-15 having the amino acid substitutions K86R and N112A.

IL-12s that can be used include those IL-12s that have a p35 and p40 domain. In some embodiments, IL-12 is single chain IL-12 as described herein (see, for example, FIG. 20 as shown as part of an IL-12-ABD fusion polypeptide and FIG. 49C).

In such embodiments, L1 and L2 are the first and second linker, respectively, L1 and L2 can be any linker that is suitable for joining the IL-15 and IL-12 domains to the ABD domain (for example, the linkers shown in Fig. 48 ). Illustrative linkers that can be used include (GGGGS) _x linkers, where X is 1 to 10. In certain embodiments, each of L1 and L2 is (GGGGS) ₅ .

The sequences of exemplary ABD polypeptides that include IL-12 and IL-15 are shown in FIG. 34 and 36.

B. IL-2 and IL-12

In certain embodiments, the implementation of the cytokine-ABD contains IL-2 and IL-12.

IL-2 and IL-12 mutually activate each other's receptors and use separate signaling pathways to induce different but complementary biological effects. Both IL-2 and IL-12 can stimulate mitogen- or CD3-activated T cells to proliferate and produce IFN-γ. In addition, studies show that delivery of both IL-2 and IL-12 genes to B16 melanoma-bearing mice caused a significant reduction in tumor burden and an increase in overall survival (see, for example, Dietrich et al., Arch Surg 387(34 ):177-82 (2002)). Thus, cytokine-ABD fusion proteins having both IL-2 and IL-12 are considered to be useful in reducing tumors and treating cancers.

C. IL-2 and IL-15

In certain embodiments, the cytokine-ABD fusion protein comprises IL-2 and IL-15. Both IL-2 and IL-15 are able to stimulate the proliferation of NK cells and activated T cells, as well as support the expansion of effector T cells. Cytokine-ABD fusion proteins that contain both IL-2 and IL-15 are believed to be useful in reducing tumors and treating cancers.

D. IL-7 and IL-12

In certain embodiments, the cytokine-ABD fusion protein comprises an IL-7 fusion partner and an IL-12 fusion partner.

Interleukin-7 (IL-7) is a non-hematopoietic cell-derived cytokine with a central role in the adaptive immune system. It promotes the development of lymphocytes in the thymus, maintains homeostasis due to the survival of naive T-cells and memory T-cells in the periphery. In addition, it plays an important role in lymph node (LN) organogenesis and in maintaining activated T cells recruited to secondary lymphoid organs (SLOs). The immune activity of cancer patients is suppressed and is characterized by fewer T cells, less effector immune cell infiltration, higher levels of depleted effector cells, and higher levels of immunosuppressive cytokines such as transforming growth factor β (TGF-β). IL-7 is an ideal solution for the recovery of immunocompromised patients with cancer as a result of the expansion of peripheral T cells. In animal models, IL-7 has been shown to prolong the survival of tumor-bearing hosts. See Gao et al., Int. J. Mol. sci. 16:10267-10280 (2015).

IL-12 acts directly on CD8+ T cells to enhance their IL-7 mediated proliferation. Cytokine-ABD fusion proteins that contain an IL-7 and IL-12 binding domain are believed to preferentially promote CD8+ T cell proliferation and enhance cytolytic activity against tumors.

IL-7 and IL-15

In certain embodiments, the cytokine-ABD fusion protein comprises IL-7 and IL-15. As mentioned above, interleukins 7 and 15 are considered to be potent pro-inflammatory cytokines that are capable of reducing tumor progression. Cytokine-ABD fusion proteins that contain both IL-7 and IL-15 are believed to be useful in reducing tumors and treating cancers.

E. IL-12 and IL-21

In certain embodiments, the implementation of the cytokine-ABD contains IL-12 and IL-21.

As mentioned above, IL-12 is able to stimulate the proliferation of NK cells and activated T cells and support the expansion of effector T cells. IL-21 is a regulator of NK and T cell function that links the innate and adaptive immune systems. IL-21 promotes maturation of NK cells from bone marrow progenitors, activates human peripheral NK cells, promotes NK expansion and maturation, and enhances CD8+ T cell-mediated effector functions.

Cytokine-ABD fusion proteins that contain both IL-12 and IL-21 are believed to be useful in reducing tumors and treating cancers.

F. IL-12 and IL-18

In certain embodiments, the implementation of the cytokine-ABD contains IL-12 and IL-18.

IL-18 is known to induce IFN-γ production, promote Th1 cell development and NK activation. IL-12 is known to induce IL-18 receptor activation in an IFN-γ dependent manner. Administration of mouse mammary carcinoma cells SCK co-expressing IL-18 and IL-12 to mice reduced tumor burden and inhibited angiogenesis (see, e.g., Coughlin et al., J Clin Invest 101(6):1441-1452 (1998) ). Administration of IL-18 in combination with IL-12 to tumor-bearing mice synergistically induced prolongation of serum IFN-γ levels, while tumor-bearing mice treated with IL-18 or IL-12 alone induced a minimal level of IFN-γ in serum, which declined rapidly (see, for example, Subleski et al., Cancer Res 66 (22):11005-1012 (2006)). Exemplary IL-18-ABDs are shown in FIG. 37.

Cytokine-ABD fusion proteins that contain both an IL-12 fusion partner and an IL-21 fusion partner are believed to be useful in reducing tumors and treating cancers.

G. GM-CSF and IL-12

In certain embodiments, the implementation of the cytokine-ABD contains GM-CSF and IL-18. GM-CSF regulates the differentiation and proliferation of hematopoietic progenitor cells. GM-CSF also leads to an increase in the ability of APC to process and present antigen, which in turn leads to the activation of cytotoxic T cells, increased production of IFN-γ and, ultimately, tumor regression. Both GM-CSF and IL-12 are capable of inducing significant antitumor responses in several different preclinical tumor models, including a liver tumor model and a lung tumor model. (See, for example, Kilinc et al., J. Immunol. 177(10):6962-973 (2006)).

Cytokine-ABD fusion proteins that contain both a GM-CSF fusion partner and an IL-12 fusion partner are believed to be useful in reducing tumors and treating cancers.

H. IFN-alpha and IL-12

The joint nature of these two cytokines goes beyond mere similar biological effects. For example, IL-12, which is well known to induce IFN-γ production, may result in the production of additional soluble factors that enhance IFN-α signaling. It is known that interferons, including interferon alpha, cause apoptosis in malignant cells. See, for example, Thyrell, L. et al., Oncogene 21, 1251-262 (2002).

Cytokine-ABD fusion proteins that contain both IFN alpha and IL-12 are believed to be useful in reducing tumors and treating cancers.

Additional cytokine-cytokine combinations that can be included in contemplated cytokine-ABD fusion proteins are shown in FIG. 51A.

2. Fusion Protein Binding Fragment-ABD

In some embodiments, the ABD-based fusion proteins contain a binding moiety (eg, scFv), i.e. fusion protein BM-ABD. In some embodiments, the BM-ABD contains a single binding fragment. In some embodiments, the BM-ABD contains two binding fragments (eg, scFv). In other embodiments, the BM-ABD contains a cytokine and a binding fragment (FIG. 1B). In FIG. 1 shows several exemplary orientations in which binding fragment or binding fragment-cytokine/binding fragment-binding fragment combinations can be attached to an ABD.

Binding fragments that are suitable for practical implementation with the subject BM-ABD binding fragments are based on the heavy chain variable domain and the light chain variable domain of antibodies. In some embodiments, the implementation of the binding fragment contains a heavy chain variable domain and a light chain variable domain. In some embodiments, the binding fragment is a single chain variable fragment (scFv).

Any ABD can be used in such a fusion protein, including those containing any of the ABD heavy chain variable domains and light chain variable domains shown in FIG. 2. In certain embodiments, the ABD contains the heavy and light chain variable domains of A10m3. In some embodiments, the ABD is scFv A10m3.

Any linker can be used to attach the cytokine and/or binding fragment to the ABD, including any linker shown in FIG. 48. In an exemplary embodiment, the linker is (GGGGS) ₅ . An exemplary linker-A10m3-linker backbone sequence for use in such fusion proteins is shown in FIG. 48.

Exemplary combinations of two binding fragments (eg, scFv) or a cytokine and a binding fragment, which may be such ABD-based fusion proteins, are shown in FIG. 51b.

In some embodiments, the BM-ABD contains an anti-TGFβ binding domain (eg, anti-scFv TGFβ). Exemplary anti-scFv TGFβ sequences are shown in FIG. 45. In some embodiments, the anti-scFv TGFβ comprises a heavy chain variable domain and a light chain variable domain of anti-scFv TGFβ 4G9. In some embodiments, the ABD fusion protein comprises an anti-scFv TGFβ and a cytokine or additional binding fragment, wherein the cytokine or additional binding fragment is a second anti-scFv TGFβ-, IL-15-, IL-12-, or anti-PD- L1 binding domain (10D12). See fig. 51b.

In some embodiments, the BM-ABD contains an anti-PD-L1 binding domain (eg, an anti-PD-L1 scFv). In some embodiments, the anti-PD-L1 scFv comprises a heavy chain variable domain and an anti-PD-L1 10D12 scFv 4D9 light chain variable domain. In some embodiments, the ABD fusion protein comprises an anti-PD-L1 scFv and a cytokine or additional binding fragment, wherein the cytokine or additional binding fragment is a second anti-PD-L1 scFv, IL-15-, IL-12-, or anti -TGFβ binding domain (4D9). See fig. 51b.

A. TGF-β-binding fragments

In certain embodiments, the BM-ABD provided herein contains a TGF-β binding fragment. As used herein, the terms "TGF-β", "TGFβ", "TGFb", and "transforming growth factor beta" all refer to a member of the cytokine family that is involved in the control of proliferation, cell differentiation, and other functions in most cells and exists for at least three isoforms: TGFβ1 (Genbank accession numbers NM_000660 and NP_000651 (human) and NM_011577 and NP_035707 (mouse)), TGFβ2 (Genbank accession numbers NM_001135599 and NP_001129071 (human)) and NM_009363 and NP_33 (accession number) and TGFβ3 in Genbank: NM_003239). Members of the TGFβ family have a 20-30 amino acid N-terminal signal peptide that is required for secretion from cells, a pro-region, and a 112-113 amino acid C-terminal region that becomes the mature TGFβ molecule upon its release as a pro-region in the result of proteolytic cleavage. In some embodiments, the mature TGFβ protein can dimerize to form an active 25 kDa molecule with many conserved structural motifs, including nine cysteine residues, eight of which form disulfide bonds in the TGFβ molecule to form a cysteine knot structure. The ninth conserved cysteine forms a bond with the ninth cysteine of another TGFβ to form a dimer.

Without being bound by any particular mode of action, it is believed that the contemplated BM-ABD, which binds TGFβ, can be used to treat subjects with cancer (eg, advanced cancer). In certain embodiments, the BM-ABD contains a TGFβ1 binding fragment. In certain embodiments, the BM-ABD contains a TGFβ2 binding fragment. In certain embodiments, the BM-ABD contains a TGFβ3 binding fragment. In some embodiments, the multivalent binding polypeptide comprises a binding moiety that can bind TGFβ1, TGFβ2, and/or TGFβ3, or any combination thereof (e.g., binds TGFβ1 and TGFβ2; binds TGFβ2 and TGFβ3; binds TGFβ1 and TGFβ3; or binds TGFβ1, TGFβ2, and TGFβ3 ). In some embodiments, the TGFβ-binding fragment binds TGFβ1, TGFβ2 and TGFβ3. In some embodiments, the TGFβ binding fragment comprises the heavy chain variable domain and the light chain variable domain of the TGFβ binding fragment of FIG. 40. In one embodiment, the TGFβ binding fragment comprises the heavy chain variable domain and the light chain variable domain of the TGFβ binding fragment 4D9 (FIG. 40B). In a specific embodiment, the TGFβ binding fragment is scFv 4D9. 4D9 has been shown to prevent expansion of regulatory CD4 ⁺ FoxP3 ⁺ T cells, prevent Smad activation (eg, Smad2 phosphorylation), and also prevent cellular epithelial-mesenchymal transition and/or migration of cancer cells.

In some embodiments, the TGFβ-binding fragment-ABD fusion protein is further fused to another binding fragment or cytokine. In certain embodiments, the other binding fragment is a PD-L1 binding fragment or another TGFβ binding fragment. In some embodiments, this cytokine is IL-15 or IL-12 (see FIG. 51B).

B. PD-L1 binding fragments

In certain embodiments, the BM-ABD provided herein comprises a programmed cell death protein 1 (PD-L1) ligand 1 binding fragment. All the terms "programmed cell death protein ligand 1", "programmed death protein ligand 1", "PDL1", and "PD-L1" as used herein (Genbank accession numbers NM_001267706 and NP_001254635 (human) and NM_021893 and NP_068693 (mouse) ) refers to a member of the 40 kDa type 1 transmembrane protein family that binds to the PD1 receptor found on activated T cells, B cells, and myeloid cells to modulate activation or inhibition. Upregulation of PD-L1 helps cancers elude the immune system. See, for example, Hamanishi et al., Proc Natl Acad Sci USA 104(9): 3360-5 (2007). Thus, it is believed that the considered BM-ABDs, which contain a PD-L1 binding fragment, may be useful in the treatment of cancers.

In one embodiment, the PD-L1 binding fragment contains the heavy chain variable domain and the light chain variable domain of the PD-L1 binding fragment 10D12 (FIG. 50). In a specific embodiment, the PD-L1 binding fragment is scFv 10D12 (FIG. 50). 10D12 binds to hPD-L1 at low pH and cross-reacts with mPD-L1. 10D12 does not bind to hPD-L2 or mPD-L2. In addition, 10D12 blocks PD-1/PD-L1 interaction as well as B71/PD-L1 interaction.

In some embodiments, the PD-L1-binding fragment-ABD fusion protein is further fused to another binding fragment or cytokine. In certain embodiments, the other binding fragment is a TGFβ binding fragment or another PD-L1 binding fragment. In some embodiments, this cytokine is IL-15 or IL-12 (see FIG. 51B).

C. TNF and other binding fragments

In one embodiment, the ABD fusion protein provided herein comprises a tumor necrosis factor (TNF) binding fragment. Without being bound by any particular mode of action, such ABD-based fusion proteins are believed to be useful as anti-inflammatory and/or antitumor agents. In some embodiments, the TNF-binding fragment is scFv. In specific embodiments, the TNF-binding fragment-ABD fusion protein is further attached to another fusion partner, which is a binding fragment or an inhibitory peptide. In certain embodiments, the second binding fragment is a second TNF binding fragment, IL-1 binding fragment, IL-6 binding fragment, IL-8 binding fragment, IL-17- (isoforms A-F) binding fragment, or IL-IL -23-linking fragment.

In another embodiment, the ABD fusion protein contains a binding fragment selected from IL-1-, IL-6-, IL-8-, IL-17- (A-F) and IL-23-binding fragment.

In some embodiments, such TNF- and interleukin-binding fragments-ABDs are useful in the treatment of diseases such as rheumatoid arthritis, Crohn's disease, psoriatic arthritis, ankylosing spondylitis, ulcerative colitis, chronic plaque psoriasis, and TNF-based diseases.

F. Diagnostic pathways

In another aspect, this document provides methods for imaging and/or detecting tumors. In some aspects, the methods include contacting a tumor cell, tumor cell culture, tumor vasculature cell, tumor vasculature culture, tumor tissue, and other tissues and cells with an ABD-based fusion protein of the present invention that is labeled.

The ABD fusion protein also finds use in in vitro or in vivo imaging of tumors or autoimmune diseases associated with the antigen binding partners of the ABD fusion protein herein. In some embodiments, the fusion protein described herein is used for both diagnosis and treatment, or only for diagnosis. In some embodiments, the ABD fusion protein in question is labeled.

Diagnosis can be performed either in vivo, by administering a diagnostic protein that allows imaging of the whole body as described below, or in vitro on samples taken from the patient. "Sample" in this context includes any number of objects, including, but not limited to, biological fluids (including, but not limited to, blood, urine, serum, lymph, saliva, anal and vaginal secretions, sweat and semen), as well as tissue samples, such as biopsies of the relevant tissues.

By "tagged" herein is meant that the ABD fusion protein disclosed herein has one or more elements, isotopes, or chemical compounds attached to enable detection during a screening or diagnostic procedure. Typically, labels fall into several classes: a) immune labels, which may be an epitope included as a fusion partner that is recognized by an antibody, b) isotopic labels, which may be radioactive or heavy isotopes, c) labels based on small molecules, which may include fluorescent and colorimetric dyes, or molecules such as biotin, which allow other labeling methods, and d) labels, such as particles (including vials for ultrasonic labeling) or paramagnetic labels, which allow imaging of the organism Labels can be incorporated into proteins at any position (eg, using one or more of the linkers described herein) and can be incorporated in vitro or in vivo during protein expression, as is known in the art. Specific labels include optical dyes including, but not limited to, chromophores, luminophores, and fluorophores, the latter being specific in many cases. Fluorophores can be either "small molecule" fluores or protein fluores.

By "fluorescent label" is meant any molecule that can be detected by its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl coumarins, pyrene, malacite green, stilbene, lucifer yellow, Cascade BlueJ, Texas Red, IAEDANS, EDANS, BODIPY FL , LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon Green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633 , Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow, and R-Phycoerythrin (PE) (Molecular Probes, Eugene, OR), FITC, Rhodamine, and Texas Red (Pierce, Rockford, IL), Cy5, Cy5.5 , Cy7 (Amersham Life Science, Pittsburgh, PA). Suitable optical dyes, including fluorophores, are described in the Molecular Probes Handbook by Richard P. Haugland, incorporated by reference in its entirety.

Suitable protein fluorescent labels also include, but are not limited to, green fluorescent protein, including GFP of Renilla, Ptilosarcus or Aequorea species (Chalfie et al., Science 263:802-805 (1994)), EGFP (Clontech Laboratories, Inc., Genbank accession number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), luciferase (Ichiki et al., J. Immunol. 150:5408-5417 (1993 ); WO99/49019, US Pat. All of the above references in this paragraph are expressly incorporated herein by reference.

G. Preparation of albumin-binding domains and fusion proteins

As will be appreciated by those skilled in the art, standard protocols are used to obtain the ABDs in question. General methods for molecular biology, expression, purification and screening of antibodies are described in Antibody Engineering, edited by Kontermann & Dubel, Springer, Heidelberg, 2001; and Hayhurst & Georgiou, Curr Opin Chem Biol 5:683-689 (2001); Maynard & Georgiou, Annu Rev Biomed Eng 2:339-76 (2000).

In one embodiment disclosed herein, nucleic acids are generated that encode a fusion protein to an ABD and which can then be cloned into host cells, expressed and analyzed as needed. In this way, nucleic acids, and in particular DNA, can be obtained which encode each protein sequence. These practical implementations are performed using well known procedures. For example, various methods that may find use in generating ABD-based fusion proteins, analogous to producing antibodies, are disclosed herein, described in Molecular Cloning - A Laboratory Manual, 3rd Ed. (Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001) and Current Protocols in Molecular Biology (John Wiley & Sons), both incorporated by reference in their entirety. There are many techniques that can be used to efficiently obtain DNA encoding the ABD disclosed in this document. Such methods include, but are not limited to, gene assembly methods, PCR-based methods and methods using PCR variations, ligase-based chain reaction methods, pooled oligonucleotide methods, such as those used in synthetic shuffling. , reduced-fidelity amplification methods, and methods using randomly mutated oligonucleotides, classical site-directed mutagenesis methods, cassette mutagenesis, and other gene amplification and synthesis methods. As is known in the art, there are many commercially available kits and methods for gene assembly, mutagenesis, vector subcloning, and the like, and such commercial products find use in generating nucleic acids that encode ABD-based fusion proteins.

The ABDs disclosed herein can be produced by culturing a host cell transformed with a nucleic acid, such as an expression vector containing a nucleic acid encoding ABD-based fusion proteins, under appropriate conditions so as to induce or cause expression of the protein. Conditions suitable for expression will vary depending on the choice of expression vector and host cell and will be readily ascertained by one of ordinary skill in the art using standard experimentation. A wide range of suitable host cells can be used, including, but not limited to, mammalian, bacterial, insect, yeast, and plant cells. For example, various cell lines that may find use in the preparation of the ABD-based fusion proteins disclosed herein are described in the ^ATCC® Cell Line Catalog available from the American Type Culture Collection.

In one embodiment, ABDs are expressed in mammalian expression systems, including systems in which expression constructs are introduced into mammalian cells using a virus, such as a retrovirus or adenovirus. Any mammalian cell can be used, for example, human, mouse, rat, hamster, and primate cells. Suitable cells also include known research cells, including but not limited to Jurkat T cells, NIH3T3, CHO, BHK, COS, HEK293, PER C.6, HeLa, Sp2/0, NS0 cells, and their options. In an alternative embodiment, the library proteins are expressed in bacterial cells. Bacterial expression systems are well known in the art and include Escherichia coli (E. coli), Bacillus subtilis, Streptococcus cremoris, and Streptococcus lividans. In alternative embodiments, ABD-based fusion proteins are produced in insect cells (eg, Sf21/Sf9, Trichoplusia ni Bti-Tn5b1-4) or yeast cells (eg, S. cerevisiae, Pichia, etc.). In an alternative embodiment, the ABD-based polypeptides are expressed in vitro using cell-free translation systems. In vitro translation systems derived from both prokaryotic (eg, E. coli) and eukaryotic (eg, wheat germ, rabbit reticulocytes) cells are available and can be selected based on expression levels and functional properties of the protein of interest. For example, as will be appreciated by those skilled in the art, in vitro translation is required for some display technologies, such as ribosome display. In addition, ABD-based fusion proteins can be obtained using chemical synthesis methods. Also, transgenic expression systems can be either animal (eg, cow, sheep or goat milk, developing chicken embryos, whole insect larvae, etc.) or vegetable (eg, corn, tobacco, duckweed, etc.).

Nucleic acids that encode the ABD-based fusion proteins disclosed herein can be included in an expression vector for protein expression. A variety of expression vectors can be used to express the protein. Expression vectors may contain self-replicating extrachromosomal vectors or vectors that integrate into the host's genome. Expression vectors are designed to be compatible with the host cell type. Thus, expression vectors that find use in making the antibodies disclosed herein include, but are not limited to, vectors that allow for protein expression in mammalian cells, bacteria, insect cells, yeasts, and in vitro systems. As is known in the art, a variety of expression vectors are available, commercially or otherwise, which may find use in expressing the antibodies disclosed herein.

The disclosed ABD-based fusion proteins may be encoded by multiple nucleic acid molecules. For example, the heavy and light chain variable regions can be introduced into the host cell independently of each other. Although they are present in individual nucleic acids, their expression results in the formation of a single polypeptide.

Expression vectors typically contain a protein operably linked to control or regulatory sequences, selectable markers, any fusion partners, and/or additional elements. By "operably linked" as used herein is meant that a nucleic acid is in a functional relationship with another nucleic acid sequence. Typically, these expression vectors contain a transcriptional and translational regulation nucleic acid operably linked to a nucleic acid encoding multivalent ABD-based fusion proteins and are typically suitable for the host cell used to express the protein. Typically, transcription and translation regulation sequences may comprise promoter sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences. As is also known in the art, expression vectors typically contain a selectable gene or marker that allows selection of transformed host cells containing the expression vector. Selectable genes are well known in the art and will vary depending on the host cell used.

In one embodiment, the ABD is purified or isolated after expression. ABD and ABD-based fusion proteins can be isolated or purified by various methods known to those skilled in the art. Purification may be particularly useful for separating the heterodimeric heavy chain molecules from the homodimeric heavy chain molecules described herein. Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic, affinity, size exclusion or gel filtration, and reverse phase, carried out at atmospheric pressure or at high pressure using systems such as FPLC and HPLC. Purification methods also include electrophoretic techniques, isoelectric focusing techniques, immunological, precipitation, dialytic, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques in combination with protein concentration are also suitable. Fusion, Ni+2 affinity chromatography is used if a His tag is used, or immobilized anti-flag antibody if a flag tag is used. For general guidance on suitable purification techniques, see, for example, incorporated by reference in its entirety Protein Purification: Principles and Practice, 3rd Ed., Scopes, Springer-Verlag, NY, 1994, incorporated by reference in its entirety. The degree of purification required will vary depending on the screening or antibody application. In some cases, cleaning is not required.

H. Therapeutic routes for using albumin-binding domains and ABD-based fusion proteins

The contemplated ABDs and ABD-based fusion proteins find use in a variety of therapeutic applications, as described herein.

In one aspect, provided herein is a method for inhibiting tumor growth in a subject in need thereof using the contemplated albumin binding domain (ABD) fusion proteins described herein. Suitable ABD-based fusion proteins include, but are not limited to, those disclosed in FIG. 4, 20, 34, 36, 40, 45, 50-51.

In some embodiments, the ABD fusion protein comprises IL-12 or IL-15 (eg, IL-12-ABD or IL-15-ABD fusion protein). The IL-15-ABD fusion proteins described herein are capable of inhibiting tumor growth in a dose-dependent manner. This IL-15 mediated inhibition of tumor growth is accompanied by an increase in tumor infiltrating lymphocytes, including cytotoxic T lymphocytes (CTL) and activated natural killer (NK) cells. In certain embodiments, the ABD fusion protein comprises an IL-12 molecule. In some embodiments, the ABD fusion protein comprises IL-15. In still other embodiments, the implementation of the cytokine-ABD contains IL-12 and IL-15. In some embodiments, the implementation of IL-15-ABD contains variant IL-15, described in this document (see, for example, Fig. 3).

This document also provides a method of treating a subject having cancer by administering an albumin binding domain (ABD) fusion protein to the subject. In some embodiments, the ABD fusion protein comprises IL-12 or IL-15 (eg, IL-12-ABD or IL-15-ABD fusion protein). IL-12 and IL-15 are cytokines for immunomodulating the tumor microenvironment due to their ability to proliferate and prolong the survival of CD8+ T cells. Other suitable ABD-based fusion proteins include, but are not limited to, those disclosed in FIG. 4, 20, 34, 36, 40, 45, 50-51.

Examples of cancers to be treated herein include, but are not limited to, carcinoma, blastoma, sarcoma, certain types of leukemia, or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., squamous cell carcinoma), lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell lung cancer, peritoneal cancer, hepatocellular cancer, gastric cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer , salivary gland carcinoma, kidney cancer, prostate cancer, vaginal cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, skin cancer/melanoma, and head and neck cancer and metastases associated with any of the primary tumors .

In another aspect, provided herein is a method for increasing the proliferation and/or survival of a CD8+ T cell. In some embodiments, the method includes contacting the cell with an albumin-binding domain fusion protein that contains IL-12 and/or IL-15 (eg, an IL-12-ABD or IL-15-ABD fusion protein). In certain embodiments, the ABD fusion protein comprises an IL-12 molecule. In some embodiments, the ABD fusion protein comprises an IL-15 molecule. In still other embodiments, the ABD fusion protein comprises IL-12 and IL-15.

I. Pharmaceutical formulations, administration and dosage

In another aspect, provided herein is a therapeutic composition comprising any contemplated albumin-binding domain (ABD) polypeptide and a carrier. The contemplated therapeutic compositions used in the practice of the above methods may be formulated into pharmaceutical compositions containing a carrier suitable for the desired mode of delivery. Suitable carriers include any substance which, in combination with the therapeutic composition, retains the antitumor function of the therapeutic composition and does not generally react with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like. (See, in general, Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

1. Compositions for in vivo administration

Albumin binding domain (ABD) fusion protein formulations used in accordance with the present invention are prepared for storage by mixing an ABD fusion protein having the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) fusion proteins; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA or DPTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG) of various molecular weights.

The composition herein may also contain more than one active compound as needed for the particular indication being treated, preferably with complementary activities that do not adversely affect each other. For example, it may be desirable to impart other specific features to ABD-based fusion proteins. Alternatively or in addition, the composition may contain a cytotoxic agent, a cytokine, a growth inhibitor and/or a small molecule antagonist. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

Active ingredients can also be entrapped into microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, into microcapsules based on hydroxymethylcellulose or gelatin and microcapsules based on poly-(methyl methacylate), respectively, in colloidal delivery systems. drugs (eg, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Formulations to be used for in vivo administration must be sterile or nearly sterile. This is easily achieved by filtration using sterile filter membranes.

Sustained release formulations can be obtained. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate , non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid and glycolic acid allow molecules to be released for over 100 days, certain hydrogels release proteins for shorter periods of time.

When encapsulated albumin-binding domain-based fusion proteins remain in the body for a long time, they can denature or aggregate upon exposure to moisture at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Rational strategies can be developed for stabilization, depending on the mechanism used. For example, if the mechanism of aggregation is found to be through the formation of intermolecular S-S bonds through thiodisulfide exchange, stabilization can be achieved by modification of sulfhydryl residues, lyophilization from acidic solutions, moisture control, use of appropriate additives, and development of specific polymer matrix compositions.

2. Methods of administration

The subject fusion proteins and albumin-binding domain therapeutic agents are administered to the subject according to known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, using intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intradermal, intraarticular , intrasynovial, intrathecal, oral, local or inhalation routes. Intravenous or subcutaneous administration of the antibody is preferred.

3. Methods of treatment

In the methods provided herein, therapy is used to provide a positive therapeutic response to a disease or pathological condition. By "positive therapeutic response" is meant normalization of the disease or condition and/or normalization of symptoms associated with the disease or condition. For example, a positive therapeutic response may refer to one or more of the following normalization of the disease: (1) reduction in the number of tumor cells; (2) increased death of tumor cells; (3) inhibition of tumor cell survival; (5) inhibiting (ie slowing down to some extent, preferably stopping) tumor growth; (6) increase patient survival; and (7) some relief from one or more symptoms associated with the disease or condition.

Positive therapeutic responses for any given disease or condition can be determined using standardized response criteria specific to that disease or condition. Tumor response can be assessed for changes in tumor morphology (i.e., total tumor burden, tumor size, etc.) using screening techniques such as magnetic resonance imaging (MRI), radiography, computed tomography (CT), scanning skeletal bone, endoscopy, and tumor biopsy, including bone marrow aspiration (BMA) and circulating tumor cell counts.

In addition to these positive therapeutic responses, the subject undergoing therapy may experience a positive effect in the form of normalization of the symptoms associated with the disease.

Thus, for example, in the case of B-cell tumors, the subject may experience a reduction in so-called B-symptoms, ie. nocturnal hot flashes, fever, weight loss and/or urticaria. In the case of pre-malignant pathological conditions, therapy with a multivalent therapeutic agent can block and/or prolong the time to the development of an associated malignant condition, for example, the development of multiple myeloma in subjects suffering from unexplained monoclonal gammopathy (MGUS).

Normalization of the disease can be characterized as a complete response. By "complete response" is meant the absence of clinically detectable disease with normalization of any previously abnormal radiographic findings, bone marrow and cerebrospinal fluid (CSF), or abnormal monoclonal protein in the case of myeloma.

Such a response may be maintained for at least 4-8 weeks, or sometimes 6-8 weeks, following treatment with the methods in question. Alternatively, normalization of the disease may be classified as a partial response. By "partial response" is meant at least approximately 50% reduction in all measured tumor burden (i.e., the number of malignant cells present in the subject, or the measured mass of tumor masses, or the amount of abnormal monoclonal protein) in the absence of new lesions that may persist for 4-8 weeks or 6-8 weeks.

Treatment includes a "therapeutically effective amount" of drugs used. "Therapeutically effective amount" refers to an amount effective at dosages and for periods of time necessary to achieve the desired therapeutic result.

The therapeutically effective amount may vary depending on such factors as the disease state, the age, sex and body weight of the individual, and the ability of the drugs to elicit the desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of an antibody or antibody portion are offset by therapeutically beneficial effects.

A "therapeutically effective amount" in the case of tumor therapy can also be measured by its ability to stabilize the progression of the disease. The ability of a compound to inhibit cancer can be evaluated in an animal model predictive of efficacy in human tumors.

Alternatively, this property of the composition can be assessed by testing the ability of the compound to inhibit cell growth or induce apoptosis using in vitro assays known to those skilled in the art. A therapeutically effective amount of a therapeutic compound may result in a reduction in tumor size or otherwise relieve symptoms in a subject. One of skill in the art will be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration chosen.

Examples

Example 1 Screening and Identification of Human Serum Albumin Binding Domains

Solid phase panning and solution panning methods were performed to identify phage scFv albumin-binding domains (ABDs). Albumin-binding domains, which were selected from the primary screening of human serum albumin binding domains, were subsequently screened for cross-reactivity with mouse serum albumin using standard ELISA techniques. Primary candidate albumin-binding domains generated using the screening methods were sequenced and then analyzed for concentration-dependent target binding, pH stability, FcRn binding interference, and binding kinetics. In particular, candidate ABDs were selected for their ability to bind human serum albumin (kD ~ 20-60 nM), mouse serum albumin (kD ~ 10-30 nM) and cynomolgus serum albumin (kD ~ 20-60 nM) at low pH (pH 5.5) and neutral pH (pH 7.7). Candidate ABDs were analyzed to ensure that they did not compete with FcRn binding to serum albumin. As described herein, ABDs that bind at this pH and do not compete for FcRn binding are able to undergo FcRn-mediated endosomal recycling. Thus, biologics (eg, cytokines and antibody-based biologics) that contain such ABDs are also capable of such FcRn-mediated recycling and thus exhibit a longer half-life compared to counterparts that do not contain such ABDs.

Five albumin-binding clones were selected based on these criteria: A9, A10, A6, 2B4, 2H10. Of these five A10 clones were selected based on high expression levels and the best activity profile. A10 was then mutated to exclude regions that could be expected to elicit immunogenicity. Of these variants, A10 A10m3 was selected as an example due to its high affinity for serum albumin.

The sequences of exemplary human serum albumin binding domains are shown in FIG. 2, including A10m3 (FIG. 2D).

Example 2 Variant IL-15 and IL-15-ABD

Expression of IL15-A10m3 protein by HEK293T cells is extremely low, which cannot be explained by transcription

IL-15-ABD constructs (IL-15-A10m3) were generated in HEK293 cells in three independently transfected cell cultures and evaluated by Western blot using either an anti-His tag antibody (Fig. 5A, left) or functional binding of ELISA to mouse serum albumin (Fig. 5A, right). As shown in FIG. 5A, IL-15-A10m3 expression could not be assessed by either of these methods. To assess whether IL-15-A10m3 expression was deficient in HEK293 cells due to low levels of transcription, mRNA was obtained from four independently transfected IL-15-A10m3 cells and RT-PCR was performed to quantify the level of IL15-A10m3 mRNA (Fig. 5B, lanes 2-5) compared to mRNA for the housekeeping gene, GAPDH (FIG. 5B, lane 6). As shown in FIG. 5B, IL15-A10m3 mRNA was detected in significant amounts in transfected cells relative to this control GAPDH, suggesting that the low levels of expression of IL-15-A10m3 produced in HEK293 cells are not transcriptional, but most likely related to translation or post-translational process.

Identification of a putative ubiquitination site in IL15 that is adjacent to the IL15 alpha receptor binding site

Studies have shown that the IL15 protein is expressed in cells but is highly unstable with a short half-life. Co-expression of IL15Rα with IL15 in the same cell significantly increased the amount of cell surface IL15R α as well as IL15. Further studies confirmed that IL15Rα acts as an IL15 chaperone and binds IL-15 intracellularly to protect and stabilize IL-15 prior to secretion. These results suggest that translation cannot explain the low IL15 production. Most likely, post-translational modification (PTM) may play a role in intracellular instability of IL15, and intracellular instability of IL15 can be overcome by blocking IL15Rα specific but as yet unknown post-translational modification. Ubiquitination is a well-described mechanism that allows cells to mark intracellular proteins for cleavage.

Given that IL15 is a very potent pro-inflammatory cytokine whose expression is tightly controlled by cells, it is possible that cells use ubiquitination to actively control IL15 protein levels. Potential ubiquitination sites on IL-15 were identified and were putatively protected when bound to IL-15Rα (FIG. 6). In particular, the amino acid K86 is a putative ubiquitination site that is adjacent to the IL-15/IL-15Rα binding site (Figure 6A), suggesting that IL15Rα binding to IL15 blocks the availability of a ubiquitin ligase (eg, E3) to K86 in protein IL15. K86 was further verified as a ubiquitination site using UbPred, an online ubiquitination site database (www.ubpred.org), FIG. 6b.

K86 Mutation in IL15 Restores IL15-A10m3 Protein Expression in HEK293T Cells

To assess whether ubiquitination at K86 affects the intracellular stability of IL-15, IL-15 variants containing amino acid substitutions at K86, including K86A and K86R, were generated. The sequences of several of these IL-15 variants are shown in FIG. 3. Without being bound by any particular mode of action, it is believed that amino acid substitutions at these specific ubiquitination sites result in ubiquitination-resistant IL-15 that exhibits increased stability compared to wild-type IL-15. These IL-15 variants were attached to ABD (A10m3) to further extend the half-life (FIG. 4). In FIG. 7 provides schematic illustrations of such IL-15-ABD fusion proteins, including IL-15-ABD with the "sushi domain" (A) IL-15Rα/IL15, and IL-15-ABD fusion proteins that contain IL-15 variants. , having amino acid substitutions at the site of K86 ubiquitination (B).

IL-15-ABD variants K86R and K86A and IL-15Rα/IL15-ABD produced in HEK293 cells were evaluated for their ability to bind mouse serum albumin (MSA) and IL-15Rα. As shown in FIG. 8A, HEK293-produced IL-15-ABD variants K86R and K86A (12 clones) and IL-15Rα/IL15-ABD (12 clones) show higher expression compared to wild-type HEK-293-produced IL-15-ABD (12 clones). ). Such constructs were also capable of binding to MSA. Moreover, as shown in FIG. 8B, the K86R (clone R6, green asterisk) and K86A (clone A3, yellow asterisk) substitutions did not affect the ability of the variants to bind to IL-15Rα. Interestingly, IL15R α fused IL15-A10m3 could not show any binding to IL15R, α, indicating that the internal sushi domain of IL15R, α bound to IL15 intramolecularly and thus blocked binding to the external IL15R, α applied to the tablet. This is consistent with our and other findings that IL15 binding to the sushi domain of IL15R α increases IL15 expression (FIG. 7B).

One particular HEK293 cell-produced IL-15-ABD variant containing the K86R amino acid substitution (IL-15 K86R-A10m3) was scaled up and the IL-15 K86R-A10m3 construct was evaluated for in vitro serum albumin binding. As shown in FIG. 9, IL-15-ABD having mutation K86 (IL-15 K86R-A10m3) and IL-15Rα/IL15-ABD (IL-15Rα/IL15-A10m3) were able to be produced in HEK 293 cells in increased amounts, which was confirmed SDS-PAGE (Fig. 9C, left) and Western blot with anti-His tag antibody (right). In addition, as shown in FIG. 10, K86R-A10m3 shows binding to mouse serum albumin (MSA).

The biological activity of IL15 K86R-A10m3 produced from HEK293T cells is impaired, which can be eliminated by deglycosylation.

The biological activity of various IL-15-ABDs was studied using CTLL2 proliferation assays. Investigated IL-15-ABDs include wild-type IL-15-A10m3 and three different IL-15 K86R-A10m3 produced in HEK293T cells. As controls, commercially available IL-15 and IL-15-ABD of our own production were used, both prepared using E. coli. As shown in FIG. 11, IL-15 K86R-A10m3 produced from HEK293T showed a significantly reduced ability to stimulate CTLL2 proliferation compared to controls produced in E. coli, including commercial wild-type (R&D) IL-15 and IL-15-A10m3 produced by native way. Considering the difference in CTLL2 proliferation assay between IL15 K86R-A10m3 from HEK293T and IL15-A10m3 from E. coli, it was hypothesized that because IL15-A10m3 produced from E. coli does not undergo classical N-glycosylation as occurs in cells mammals, it is possible that glycosylation of IL15 K86R-A10m3 in HEK293 T cells may disrupt the interaction of IL15 K86R-A10m3 with its receptors.

To assess whether the reduced bioavailability of HEK cell-produced IL-15 K86R-A10m3 is due to glycosylation, IL-15 K86R-A10m3 was deglycosylated using PNGase and CTLL2 proliferation assays to assess the bioavailability of deglycosylated IL15 K86R-A10m3. After treatment with IL-15 K86R-A10m3 PNGase mixture under native conditions, the glycan was completely removed and visualized by SDS-PAGE followed by glycan staining (Fig. 12A, left) and Coomassie blue staining (Fig. 12A, right). 1) mixture of IL-15R-A10m3 + 5 µl of PNGase; 2) a mixture of protein + 10 µl of PNGase; 3) there is no enzyme control. As shown in FIG. 12B, IL-15 deglycosylation of K86R-A10m3 (blue) almost completely restored activity in CTLL2 proliferation assays compared to untreated samples (purple, yellow). IL-15 DT from R&D systems (red) and home-produced E. coli IL-15-A10m3 (black) were used as positive controls.

N112 IL-15 K86R-A10m3 is critical for its biological activity to activate CTLL2 proliferation

The N112 amino acid position of IL-15 K86R-A10m3 is a major site for the biological activity of IL-15, especially in the context of IL-15-ABD, as it is critical for the proper establishment of an interaction between IL-15 and the IL15 gamma receptor. IL-15 variant K86R was further mutated in N112A to determine if mutations at this site could restore the biological activity of IL-15, similar to deglycosylated IL-15. In particular, IL15 K86R-A10m3 was mutated to further include the N112Q, N112A, or N112S IL-15 amino acid substitution, and CTTLL2 proliferation assays were performed to test for biological activity. As shown in FIG. 13A, introduction of the N112A amino acid substitution in IL-15 K86R-A10m3 (blue) restored its biological activity comparable to that of deglycosylated IL-15 K86R-A10m3 (green) in CTLL2 proliferation assays, while the N112Q mutation (red) ) had no effect on biological activity relative to the original IL-15R-A10m3 without deglycosylation (yellow). IL-15 DT (black) from R&D systems served as a positive control.

Mutations with different side chains in N112 were investigated to further demonstrate the effect of size on biological activity. As shown in FIG. 13B, N112Q (large size, red), N112S (medium size, green) and N112A (small size, blue) showed increased biological activity, inversely proportional to the size of the side chains. Moreover, the putative hydrogen bond formed by IL-15 N112 and IL15 gamma receptor Y103 did not appear to be important for this activity, since N112A cannot form such a bond. IL-15 DT from R&D systems (black) and IL-15-A10m3 E. coli own production (purple) were used as positive controls; original IL15 K86R-A10m3 without deglycosylation was used as a negative control (yellow).

Example 3 In vivo activity of variant IL-15 and IL-15-ABD

The ability of IL-15 and IL-15-ABD to inhibit tumor growth was evaluated using the B16-F10 mouse melanoma model. As shown in FIG. 14, mice were treated with IL-15, placebo PBS, or various doses of IL-15-ABD by intravenous injection at four different time points, 48 hours apart. As shown in FIG. 16, IL-15-ABD inhibits tumor growth in a dose-dependent manner.

FACS analyzes were performed to further evaluate the profile of tumor infiltrating lymphocyte populations in IL-15-ABD treated mice based on these studies. As shown in FIG. 16, tumors in mice treated with IL-15-ABD showed an increase in NK cell populations. These data, combined with observations of increased tumor accumulation and persistence in mice treated with IL-15-ABD as described above, suggest that ABD enhances the pro-inflammatory effect of IL-15 in tumors. The effects of IL-15-ABD treatment on lymphocyte populations in the spleen and tumors are shown in FIG. 17 and 18. As shown in FIG. 17 and 18, FACS analysis of lymphocyte populations shows a 3-6 fold increase in tumor infiltrating CTL and NK cell populations in IL-15 ABD treated mouse tumors. No significant differences were observed in the spleen. In general, the results of these studies show the immunomodulatory ability of IL-15-ABD in relation to the tumor in vivo.

To assess the ability of ABD-based fusion proteins to increase the half-life of IL-15, C57B mice were intravenously injected with 5 μg of IL-15-ABD or IL-15 alone and then serum concentrations of IL-15-ABD and IL-15 were measured. As shown in FIG. 20A, IL-15-ABD showed a higher PK compared to IL-15 DT. T ½β IL-15 = 0.6 hours, similar to those reported in the public domain (~ 0.5 hours). The results of the study show that ABD increases the T½β of IL-15 to ~7.0 hours, which is ~10 times greater. IL-15-ABD was also analyzed for stability in human serum using cell assay. As shown in FIG. 19B, IL-15-ABD was more stable in human serum compared to commercial control IL-15 without ABD.

Example 4 IL-12-ABD

Mouse-ABD single chain IL-12 constructs were generated in HEK293T cells and purified by size exclusion chromatography. IL-12-A10m3 produced from HEK293T cells was fully active in both in vitro and cellular assays. As shown in FIG. 21A, IL12-A10m3 is able to bind to mouse serum albumin with an equilibrium dissociation constant (KD) of 2.1 nM. IL12-A10m3 produced from HEK293T was also able to stimulate human PBMC proliferation comparable to native mouse IL12 and commercially available mouse IL-12 (R&D) (FIG. 21B). In addition, IL12-A10m3 produced from HEK293T stimulated interferon-gamma secretion from human PBMCs comparable to that of domestically produced mouse IL-12 and commercially available mouse IL-12 (R&D) (Fig. 22).

Treatment with IL-12-ABD results in tumor volume reduction in vivo.

The ability of IL-12 and IL-12-ABD to inhibit tumor growth was evaluated using the B16-F10 mouse melanoma model. As shown in FIG. 23, mice were treated with either IL-12-ABD or IL-12 at three similar doses by intravenous injection on day 7 post-tumor inoculation (day 0) when tumor volume reached 100 mm ³ . Tumor growth was monitored every 2 days after treatment for 10 days. Placebo PBS acted as a control. As shown in FIG. 24-26 and 28, both IL-12 and IL-12-ABD were able to reduce tumor growth in a dose-dependent manner. Moreover, IL-12-ABD was able to reduce tumor volume more effectively than IL-12 alone at similar concentrations (see, for example, Figure 26 at 10 days and Figure 28, median days for 50 % of tumors reached 2000 mm ³ ). Longitudinal body weight measurements of mice from these studies show minimal changes in body weight across all IL-12-ABD treatment groups (FIG. 27). The absence of significant weight changes observed indicates the absence of IL-12-ABD toxicity in the treatment groups during the 12 day follow-up course of treatment.

Further characterization of the pharmacodynamic effects of a single dose of IL-12-ABD (4.5 μg IL-12-ABD, same molar dose as control 3 μg IL-12) in mice bearing B16-F10 tumors after 5 days demonstrated, that IL-12-ABD exhibited a similar higher tumor growth suppression compared to a similar molar dose of control IL-12. IL-12-ABD-treated mice also showed a corresponding increase in immune activation, as evidenced by an increase in spleen weight and IFN-γ, with no effect on mouse body weight compared to controls (FIG. 29).

In FIG. 30 further shows the result of a study comparing tumor volume in B16-F10 tumor-bearing mice at 10 days treated with IL-12-ABD (1.3 μg), IL-12 (30 μg), or placebo. Although IL-12 (1 μg) and IL-12-ABD (1.3 μg) are molar equivalent and have the same in vitro biological activity, IL-12-ABD is ~30 times more potent than IL -12, in vivo (compare results at day 10 in Fig. 30, 1.3 μg IL-12-ABD > 30 μg IL-12). In FIG. 31 further demonstrates the hematopoietic effects of IL-12-ABD and IL-12 in mice from the study shown in FIG. 30, after 3 and 7 days. As shown in FIG. 31, mice treated with IL-12-ABD on day 3 showed a transient decrease in WBC, neutrophils and lymphocytes compared to mice treated with IL-12 and placebo control. At the same time, these cell populations returned to normal on day 7. In addition, IFN-γ levels in mice treated with IL-12-ABD were higher on days 3 and 7 compared to mice treated with IL-12 and controls. .

Evaluation of the antitumor effects of IL-12-ABD or IL-12 in combination with an anti-PD-1 antibody in vivo.

The effect of single dose combination therapies using IL-12-ABD or IL-12 with anti-PD-1 antibodies was evaluated in mice bearing B16-F10 tumors after 8 days (FIG. 32).

Animals (aged 7-10 weeks) were divided into 8 groups (8 animals per group) 10 days after inoculation of tumor cells B16-F10 (2×10 ⁴ cells/mouse). Animals were assigned based on tumor volume. At the time of distribution, the average tumor volume per group was 100 mm ³ . On day 0 (when tumors reached 100 mm ³ ), each group received a single intravenous dose of either PBS (placebo) or IL12-ABD (1.5 μg, 5 μg, 15 μg) or IL15-ABD-IL12 (1.7 mcg, 6 17, 17)

The groups were examined for body weight, tumor volume and pseudo-survival. Body weights were measured prior to tumor inoculation and during tumor measurements. The size of the tumor was measured every 2 days in two dimensions using a caliper and the volume was expressed in mm ³ using the formula: V = 0.5 xaxb ² where a and b are the long and short diameters of the tumor. The study was conducted in the form of pseudo-survival; each mouse was euthanized when its tumor reached 2000 mm ³ or when it was determined that it was dying.

As shown in FIG. 33, IL-12-ABD was more effective than treatment with either anti-PD-1 or a molar equivalent dose of recombinant IL-12. Moreover, IL-12-ABD was as effective as the combination of recombinant IL-12 and anti-PD-1 treatment. Interestingly, the addition of anti-PD-1 Ab to recombinant IL-12 increased the efficacy of either treatment alone, while anti-PD-1 treatment provided no additional benefit for IL-12-ABD.

To evaluate the ability of ABD-based fusion proteins to increase the half-life of IL-12, C57B mice were intravenously injected with 5 μg of IL-12-ABD or IL-12 alone and serum concentrations of IL-12-ABD and IL-12 were assessed. As shown in FIG. 33, IL-12-ABD showed a higher PK than IL-12 DT. T ½β IL-12 = 2.5 hours, similar to those reported in the public domain (~ 3.5 hours). The results of the study show that ABD extended the T ½β of IL-12 to 9.5 hours, which was an increase of ~ 4 times.

Example 5 Bispecific IL-15-ABD-IL-12

Constructs IL-15-ABD-IL-12, hIL15 (K86R/N112A)-A10m3-mIL-12sc and mIL-12sc-A10m3-hIL15 (K86R/N112A) were generated in HEK293T cells and purified by size exclusion chromatography. The sequences of these constructs are shown in FIG. 34. The ability of hIL15 (K86R/N112A)-A10m3-mIL-12sc and mIL-12sc-A10m3-hIL15 (K86R/N112A) to bind MSA, IL12 beta 2 receptor and IL-15 alpha receptor was assessed by ELISA. As shown in FIG. 35, both IL-15-ABD-IL-12 constructs were able to bind to MSA in a dose dependent manner in cell culture medium. In addition, both bispecific constructs were able to bind to the IL-12 beta 2 receptor and IL-15 alpha receptor in a dose-dependent manner in cell culture medium. As shown in FIG. 35, IL-12/IL-15-ABD having an N-terminal to C-terminal orientation, IL-15-ABD-IL-12, showed higher antigen binding compared to IL-12-ABD-IL- fifteen. Additional bispecific compounds that contain IL-15 and IL-12 are disclosed in FIG. 36.

Assessing IL-15-ABD-IL-12 for IL-12 and IL-15 activity

IL-15-ABD-IL-12 was further evaluated for IL-12 and IL-15 activity (FIGS. 37 and 38).

To assess IL-12 activity, PBMC lymphocytes were blasted with PHA-P for four days and rhIL-2 for the third day. The lymphoblasts were then treated with either IL-15-ABD-IL-12 or control IL-12 for two days and IL-12 activity was assessed based on lymphoblast proliferation and IFN-γ secretion (FIG. 37A). IL-15 activity was assessed using the CTLL-2 cytotoxic T-lymphocyte proliferation assay (FIG. 38A).

As shown in FIG. 37, IL-15-ABD-IL-12 exhibited IL-12 activity as assessed by lymphoblast proliferation (FIG. 37B) and IFN-γ secretion (FIG. 37C). In addition, IL-15-ABD-IL-12 showed IL-15 in the CTLL-2 proliferation assay (FIG. 38B). Thus, the considered IL-15-ABD-IL-12 exhibited the biological activity of both IL-12 and IL-15.

Antitumor Effects of IL-15-ABD-IL-12 in B16-F10 Mouse Melanoma Model

Without being bound by any particular mode of action, IL-15/IL-12 ABDs are believed to provide synergistic biological activity. Specifically, IL-12 increases IL-15 alpha receptor activity, NK/T cell IFN-γ and TH1 immunity, while decreasing Treg cell activity. IL-15 increases the activity of the beta 1 receptor of IL-12 and NK cells, while reducing the loss of CD8 memory cells.

The antitumor effects and pseudo-survival of IL-12-ABD versus IL15-ABD-IL12 were evaluated using the B16-F10 mouse melanoma model (FIG. 39).

Animals (aged 7-10 weeks) were divided into 8 groups (8 animals per group) 10 days after inoculation of tumor cells B16-F10 (2×10 ⁴ cells/mouse). Animals were assigned based on tumor volume. At the time of distribution, the average tumor volume per group was 100 mm ³ . On day 0 (when tumors reached 100 mm ³ ), each group received a single intravenous dose of either PBS (placebo) or molar equivalent doses of IL12-ABD (1.5 μg, 5 μg, 15 μg) or IL15-ABD-IL12 ( 1.7 µg, 6 µg, 17 µg).

As shown in FIG. 39, IL-15-ABD-IL-12 was more effective in antitumor activity than IL-12-ABD in the B16-F10 mouse model at the same molar concentration. Other similar in vivo studies show that free IL-12 (5 µg) in combination with IL-15 (1 µg) is less than 50% more effective than IL-15-ABD-IL-12 (6 µg) (data not shown).

Example 6 Anti-TGFβ-ABD

After biopanning and screening using surface plasmon resonance technology, anti-hTGFβ1 binding domains were identified: 1A10, 1F11, 2H6, 4B9, 4C10, 4D9, 4G3, 4G6, 4H4, 4H7 and 6H11. These clones showed cross-reactivity with hTGFβ2, 3 and mTGFβ1 and potential inhibition of hTGFβ1 binding to its receptor II. Clones were then selected for scFv purification and further characterization.

ScFv were screened for cross-reactivity with mouse and human TGF-1 using standard ELISA techniques. ELISA binding shows that 2H6, 4G3, 4H7, 4B9, 4D9 and 6H11 have high cross-reactivity with both mTGFβ-1 and hTGFβ-1.

Binding and blocking by ELISA was performed to determine whether anti-scFv TGFβ-1 is able to bind TGFβ-1 and block its interaction with TGFβR-II. All of 2H6, 4G3, 4H7, 4B9 and 4D9 show high efficiency in blocking and inhibiting the interaction of mTGFβ and mTGFβR-II.

Some of these anti-scFv TGFβ-1 have been investigated for their ability to interfere with the biological activity of TGFβ-1. The sequences of anti-scFv TGFβ 14H7 and 4D9 are shown in FIG. 40A and B.

Blockade of TGFβ1-induced expansion of CD4 ⁺ Foxp3 ⁺ regulatory T cells

Regulatory T cells (Treg) are able to influence the homeostasis of the immune system. Such Tregs are required to maintain autotolerance, since defects can lead to severe autoimmune diseases. In cancer, tumor cells are able to secrete cytokines that affect the homeostasis of the immune system. In particular, tumor cells can secrete TGFβ, which can then influence the amount of circulating Tregs. It has been previously demonstrated that exposure to TGFβ1 leads to the expansion of CD4 ⁺ Foxp3 ⁺ Treg subsets from CD4 ⁺ Foxp3 ^- T cells. These induced Tregs can then contribute to the induction of an anergic T cell response by inhibiting the activation of tumor antigen-specific cytotoxic CD8 ⁺ T cells.

As shown in FIG. 41, recombinant TGFβ1 is able to stimulate the expansion of CD4 ⁺ Foxp3 ⁺ Treg from a mixed population of T cells isolated from healthy human PBMCs. At the same time, blockade of TGFβ by all of the anti-TGFβ 1D11 antibody or scFv TGF-β1 2H6, 4H7 and 4D9 significantly inhibits TGFβ-induced expansion of CD4 ⁺ Foxp3 ⁺ Treg also in a dose-dependent manner. Thus, such TGFβ1scFv are useful in reducing TReg expansion in cancers.

Blockade of the TGFβ1-induced epithelial-mesenchymal transition (EMT)

It is known that exposure to TGFβ induces the epithelial-mesenchymal transition. During this process, epithelial cells transdifferentiate from an organized, polarized, and tightly bound epithelial layer of cells with a pebble-like morphology to disorganized and motile cells that appear mesenchymal in morphology. During EMT, the invasive capacity of the cells is activated and thus the oncogenic capacity of the cells is enhanced. E-cadherin is a widely used epithelial cell marker and is localized at adhesion junctions between epithelial cells. Loss of E-cadherin is a strong EMT marker that indicates the process of transdifferentiation. (Fig. 42A). In addition, vimentin is associated with highly mobile cells. Thus, induction of vimentin expression in cells is also indicative of increased motility and increased localized invasion in vivo.

As shown in FIG. 42 and 43, blockade of TGF-β with anti-TGF-β 1D11 antibody or scFv TGF-β1 reverses TGF-β1-induced epithelial-mesenchymal transition (FIG. 42) and migration (FIG. 43). Mouse 4T1 cells were cultured in growth medium supplemented with TGF-β1 (panel 2); TGF-β1 and 1D11 (panel 3); or TGF-β1 and anti-scFv TGF-β1 (panel 4), then fixed and stained with anti-E-cadgeriun (green) and anti-vimentin (purple). The nuclei were counterstained with DAPI (blue). Treatment with TGF-β1 induced loss of E-cadherin from cell-to-cell contacts and resulted in increased expression of vimentin. This effect is reversed by the addition of 1D11 or the subject anti-scFv TGF-β1 described herein (FIG. 42, panels 3 and 4). In addition, the anti-scFv TGF-β1 described herein is able to block TGF-β1 mediated migration of carcinoma cells (FIG. 43).

Neutralization of TGFβ1-induced Smad activation

The TGFβ superfamily consists of pleiotropic cytokines that regulate various biological processes, including proliferation, differentiation, migration, cell survival, angiogenesis, wound healing, and immune surveillance. In humans, the predominant isoform is TGFβ1, which is expressed in various tissue types. TGFβ inhibits the proliferation of most normal epithelial cells. In addition, in the early stages of cancers of epithelial origin, TGFβ acts as an inhibitor of cell growth. Thus, during the onset of cancer, TGFβ acts as a tumor suppressor. At the same time, at later stages of cancer progression, tumor cells become resistant to the effects of the inhibitory growth of TGFβ, and TGFβ acts as a tumor promoter. Indeed, TGFβ1 has been shown to be overexpressed in various tumors. Activation of the TGFβ pathway occurs through binding of the TGFβ ligand to the type II TGFβ receptor (TβRII), which then induces association and oligomerization between TβRII and TβRI. When this oligomer is formed, Smad2 and Smad3 are recruited and phosphorylated by TβRI. Phosphorylated Smad2 or Smad3 then binds to Smad4 in the cytoplasm, and this complex translocates to the nucleus, where it interacts with the promoter region and activates the transcription of target genes. Thus, activation of the TGFβ pathway is measurable by rapid phosphorylation of Smad2 upon addition of TGFβ to serum-deprived cells. At the same time, effective blockade of TGFβ will inhibit Smad2 phosphorylation. Herein, the absence of Smad2 phosphorylation can be used as a measure of the effective blockade of TGFβ by the anti-scFv TGFβ constructs in question.

Using serum-depleted human (FIG. 44A) or mouse (FIG. 44B) cells, it was determined that the addition of recombinant human TGFβ1 (FIG. 44A) or mouse TGF-β1, -β2 and -β3 (FIG. 44B) induced Smad2 phosphorylation . This phosphorylation is reduced in a dose-dependent manner when TGFβ is pre-incubated with control anti-TGFβ 1D11 antibody or TGF-β1 2H6, 4H7 and 4D9 scFv constructs. These data suggest that the 2H6, 4H7, and 4D9 scFv constructs are able to sequester TGFβ1 and inhibit its interaction with TβRII/TβRI, thereby inhibiting the TGFβ activation cascade during advanced cancer.

Anti-TGFβ-1-ABD 4D9

The sequences of exemplary TGF-β1 scGv-ABD constructs (4D9M-A6m and 4H7-A6m) are shown in FIG. 45A. As shown in FIG. 45B, anti-TGFβ-1-ABD extended T½β of anti-scFv TGFβ-1 from 106 minutes to 10.6 hours.

Anti-scFv TGF-β1-ABD (bivalent for TGF-β1) produced in E. coli and HEK cells was evaluated for binding to mouse serum albumin (FIG. 47). Three different orientations of the constructs were evaluated: two anti-scFv TGF-β1 attached to the N-terminus of ABD ("Bi N Term"); two anti-scFv TGF-β1 attached to the C-terminus of ABD ("Bi C Term"); or one anti-TGF-β1 scFv attached to each of the N-terminus and C-terminus of ABD ("Bi Mid"). As shown in FIG. 46, all constructs exhibited binding to mouse serum albumin. With respect to constructs produced in E. coli, Bi Mid showed more efficient binding to MSA than N Term orientation. With respect to constructs produced in HEK cells, the Bi N Term orientation exhibited more efficient binding to MSA than the Bi Mid or Bi C term orientation.

As shown in FIG. 47, TGFβ-1 mediated inhibition of T cell proliferation was reversed (ie, increased T cell proliferation) with these constructs (FIG. 46B). In addition, 4D9M-ABD has been shown to block the binding of human TGFβ-1 and human TGFβ-3 to cognate receptors (data not shown).

All references cited are expressly incorporated herein by reference in their entirety.

While the specific embodiments of the invention have been described above for illustrative purposes, those skilled in the art will appreciate that numerous variations on the details may be made without deviating from the invention described in the appended claims.

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SEQUENCE LISTING

<110> Sonnet Bio Therapeutics

<120> Albumin Binding Domain Fusion Proteins

<130> 116076-5007-WO

<140> PCT/US2018/000085

<141> 2018-02-20

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Thr Ala Thr Ile Thr Cys Gly Gly Asn Asn Ile Gly Thr Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Ala Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Val Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Arg Ser Asp His

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 22

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 22

Asn Ile Gly Thr Lys Ser

fifteen

<210> 23

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 23

Ala Asp Ser

one

<210> 24

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 24

Gln Val Trp Asp Ser Arg Ser Asp His Leu Trp Val

1 5 10

<210> 25

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 25

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Asp Asp Ala

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Val Lys Gly Leu Tyr Ser Asn Pro Arg Gly Gly Ala Phe Asp Ile Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 26

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 26

Gly Ile Thr Phe Asp Asp Ala Val

fifteen

<210> 27

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 27

Ile Ser Ser Asn Ser Gly Tyr Ile

fifteen

<210> 28

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 28

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

1 5 10 15

<210> 29

<211> 110

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 29

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

1 5 10 15

Thr Ala Thr Ile Thr Cys Gly Gly Asn Asn Ile Gly Thr Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Ala Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Val Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Arg Ser Asp His

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 30

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 30

Asn Ile Gly Thr Lys Ser

fifteen

<210> 31

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 31

Ala Asp Ser

one

<210> 32

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 32

Gln Val Trp Asp Ser Arg Ser Asp His Leu Trp Val

1 5 10

<210> 33

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 33

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Asn Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Ala Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

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

65 70 75 80

Leu Val Trp Thr Ser Leu Lys Ala Ser Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Gln Arg Trp Gly Ser Ser Ser Phe Asp Leu Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 34

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 34

Gly Tyr Ser Phe Asn Ser Tyr Trp

fifteen

<210> 35

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 35

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 36

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 36

Ala Arg Gln Arg Trp Gly Ser Ser Ser Phe Asp Leu

1 5 10

<210> 37

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 37

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

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

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

35 40 45

Ile Tyr Glu Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

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

85 90 95

Ser Ala Gly Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly

100 105 110

<210> 38

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 38

Ser Ser Asn Ile Gly Asn Asn Tyr

fifteen

<210> 39

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 39

Glu Asn Asn

one

<210> 40

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 40

Gly Thr Trp Asp Ser Ser Leu Ser Ala Gly Val

1 5 10

<210> 41

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 41

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Asn Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

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

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Ala Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gln Arg Trp Gly Ser Ser Ser Phe Asp Ala Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 42

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 42

Gly Tyr Ser Phe Asn Ser Tyr Trp

fifteen

<210> 43

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 43

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 44

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 44

Ala Arg Gln Arg Trp Gly Ser Ser Ser Phe Asp Ala

1 5 10

<210> 45

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 45

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

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

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

35 40 45

Ile Tyr Glu Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

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

85 90 95

Ser Ala Gly Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly

100 105 110

<210> 46

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 46

Ser Ser Asn Ile Gly Asn Asn Tyr

fifteen

<210> 47

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 47

Glu Asn Asn

one

<210> 48

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 48

Gly Thr Trp Asp Ser Ser Leu Ser Ala Gly Val

1 5 10

<210> 49

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 49

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Glu Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Leu His Gly Val Val Ser Arg Gly Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 50

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 50

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 51

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 51

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 52

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 52

Ala Arg Leu His Gly Val Val Ser Arg Gly Asp Tyr

1 5 10

<210> 53

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 53

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

1 5 10 15

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

20 25 30

Asn Leu Val Ser Trp Tyr Gln Gln Tyr Pro Gly Lys Ala Pro Lys Leu

35 40 45

Ile Ile Tyr Glu Val Thr Lys Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

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

85 90 95

Asn Ala Val Ala Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly

100 105 110

<210> 54

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 54

Ser Ser Leu Ala Gly Ser Tyr Asn Leu

fifteen

<210> 55

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 55

Glu Val Thr

one

<210> 56

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 56

Ser Ser Tyr Ala Gly Arg Asn Ala Val Ala Val

1 5 10

<210> 57

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 57

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Glu Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Ala Tyr Tyr Cys

85 90 95

Ala Arg Leu His Gly Val Val Ser Arg Gly Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 58

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 58

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 59

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 59

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 60

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 60

Ala Arg Leu His Gly Val Val Ser Arg Gly Asp Tyr

1 5 10

<210> 61

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 61

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

1 5 10 15

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

20 25 30

Asn Leu Val Ser Trp Tyr Gln Gln Tyr Pro Gly Lys Ala Pro Lys Leu

35 40 45

Ile Ile Tyr Glu Val Thr Lys Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

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

85 90 95

Asn Ala Val Ala Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly

100 105 110

<210> 62

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 62

Ser Ser Leu Ala Gly Ser Tyr Asn Leu

fifteen

<210> 63

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 63

Glu Val Thr

one

<210> 64

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 64

Ser Ser Tyr Ala Gly Arg Asn Ala Val Ala Val

1 5 10

<210> 65

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 65

Glu Val Gln Leu Val Glu Thr Gly Gly Gly Val Val Arg Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Lys Ser Ser Gly Trp Tyr Pro Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 66

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 66

Gly Phe Thr Phe Ser Ser Tyr Ala

fifteen

<210> 67

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 67

Ile Ser Gly Ser Gly Gly Ser Thr

fifteen

<210> 68

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 68

Ala Lys Ser Ser Gly Trp Tyr Pro Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 69

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 69

Ser Tyr Glu Leu Met Gln Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Ser Gly Asn Asn Leu Gly Asp Lys Tyr Thr

20 25 30

Ser Trp Tyr Gln His Lys Pro Gly Gln Ser Pro Val Met Val Ile Tyr

35 40 45

Gln Asp Thr Lys Arg Pro Ser Gly Ile Ala Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Leu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Thr Gly Thr Ala Val

85 90 95

Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly

100 105

<210> 70

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 70

Asn Leu Gly Asp Lys Tyr

fifteen

<210> 71

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 71

Gln AspThr

one

<210> 72

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 72

Gln Ala Trp Asp Thr Gly Thr Ala Val

fifteen

<210> 73

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 73

Glu Val Gln Leu Val Glu Thr Gly Gly Gly Val Val Arg Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Lys Ser Ser Gly Trp Tyr Pro Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 74

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 74

Gly Phe Thr Phe Ser Ser Tyr Ala

fifteen

<210> 75

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 75

Ile Ser Gly Ser Gly Gly Ser Thr

fifteen

<210> 76

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 76

Ala Lys Ser Ser Gly Trp Tyr Pro Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 77

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 77

Ser Tyr Glu Leu Met Gln Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Ser Gly Asn Asn Leu Gly Asp Lys Tyr Thr

20 25 30

Ser Trp Tyr Gln His Lys Pro Gly Gln Ser Pro Val Met Val Ile Tyr

35 40 45

Gln Asp Thr Lys Arg Pro Ser Gly Ile Ala Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Leu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Thr Gly Thr Ala Val

85 90 95

Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly

100 105

<210> 78

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 78

Asn Leu Gly Asp Lys Tyr

fifteen

<210> 79

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 79

Gln AspThr

one

<210> 80

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 80

Gln Ala Trp Asp Thr Gly Thr Ala Val

fifteen

<210> 81

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 81

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Gln Ala Phe Gly Tyr Asn Phe Asn Asn Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

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

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Arg Ser Thr Val Thr Lys Ser Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210> 82

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 82

Gly Tyr Asn Phe Asn Asn Tyr Trp

fifteen

<210> 83

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 83

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 84

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 84

Ala Arg Arg Ser Thr Val Thr Lys Ser Phe Asp Ile

1 5 10

<210> 85

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 85

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

1 5 10 15

Thr Ala Thr Leu Thr Cys Thr Gly Asn Ser Asn Asn Val Gly Lys Gln

20 25 30

Gly Ala Ala Trp Leu Gln Gln His Gln Gly His Pro Pro Lys Leu Leu

35 40 45

Ser Tyr Ser Ser Ile Asn Arg Pro Val Gly Ile Ser Glu Arg Phe Ser

50 55 60

Ala Ser Arg Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Leu Gln

65 70 75 80

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

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 86

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 86

Ser Asn Asn Val Gly Lys Gln Gly

fifteen

<210> 87

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 87

Ser Ser Ile

one

<210> 88

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 88

Ser Ala Trp Asp Ser Ser Leu Ser Ala Trp Val

1 5 10

<210> 89

<211> 130

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 89

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser

20 25 30

Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

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

100 105 110

Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val

115 120 125

Ser Ser

130

<210> 90

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 90

Gly Gly Ser Ile Ser Ser Ser Ser Tyr Tyr

1 5 10

<210> 91

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 91

Ile Tyr Tyr Ser Gly Ser Thr

fifteen

<210> 92

<211> 22

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 92

Ala Arg Arg Ser Ala Trp Ser Ser Gly Trp Ser Thr Leu Arg Tyr Tyr

1 5 10 15

Tyr Tyr Gly Met Asp Val

twenty

<210> 93

<211> 109

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 93

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ala Thr Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Asn Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Glu Arg Ser Asn Trp Ser Arg

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 94

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 94

Gln Ser Ile Ala Thr Tyr

fifteen

<210> 95

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 95

Asp Ala Ser

one

<210> 96

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 96

Gln Glu Arg Ser Asn Trp Ser Arg Leu Thr

1 5 10

<210> 97

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 97

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Val Pro Gly Arg Gly Leu Glu Trp Leu

35 40 45

Ala Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Glu Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Leu His Gly Val Val Ser Arg Gly Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 98

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 98

Gly Tyr Ser Phe Thr Asn Tyr Trp

fifteen

<210> 99

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 99

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 100

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 100

Ala Arg Leu His Gly Val Val Ser Arg Gly Asp Tyr

1 5 10

<210> 101

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 101

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

1 5 10 15

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

20 25 30

Asn Leu Val Ser Trp Tyr Gln Gln Tyr Pro Gly Lys Ala Pro Lys Leu

35 40 45

Ile Ile Tyr Glu Val Thr Lys Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

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

85 90 95

Asn Ala Val Ala Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly

100 105 110

<210> 102

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 102

Ser Ser Leu Ala Gly Ser Tyr Asn Leu

fifteen

<210> 103

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 103

Glu Val Thr

one

<210> 104

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 104

Ser Ser Tyr Ala Gly Arg Asn Ala Val Ala Val

1 5 10

<210> 105

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 105

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Glu Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Glu Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Leu His Gly Val Val Ser Arg Gly Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 106

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 106

Gly Tyr Ser Phe Thr Asn Tyr Trp

fifteen

<210> 107

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 107

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 108

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 108

Ala Arg Leu His Gly Val Val Ser Arg Gly Asp Tyr

1 5 10

<210> 109

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 109

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

1 5 10 15

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

20 25 30

Asn Leu Val Ser Trp Tyr Gln Gln Tyr Pro Gly Lys Ala Pro Lys Leu

35 40 45

Ile Ile Tyr Glu Val Thr Lys Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

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

85 90 95

Asn Ala Val Ala Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly

100 105 110

<210> 110

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 110

Ser Ser Leu Ala Gly Ser Tyr Asn Leu

fifteen

<210> 111

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 111

Glu Val Thr

one

<210> 112

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 112

Ser Ser Tyr Ala Gly Arg Asn Ala Val Ala Val

1 5 10

<210> 113

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 113

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Glu Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Glu Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Leu His Gly Val Val Ser Arg Gly Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 114

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 114

Gly Tyr Ser Phe Thr Asn Tyr Trp

fifteen

<210> 115

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 115

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 116

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 116

Ala Arg Leu His Gly Val Val Ser Arg Gly Asp Tyr

1 5 10

<210> 117

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 117

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

1 5 10 15

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

20 25 30

Asn Leu Val Ser Trp Tyr Gln Gln Tyr Pro Gly Lys Ala Pro Lys Leu

35 40 45

Ile Ile Tyr Glu Val Thr Lys Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

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

85 90 95

Asn Ala Val Ala Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly

100 105 110

<210> 118

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 118

Ser Ser Leu Ala Gly Ser Tyr Asn Leu

fifteen

<210> 119

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 119

Glu Val Thr

one

<210> 120

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 120

Ser Ser Tyr Ala Gly Arg Asn Ala Val Ala Val

1 5 10

<210> 121

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 121

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr Leu Phe

65 70 75 80

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

85 90 95

Ala Arg His His Gly Arg Leu Tyr Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 122

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 122

Gly Phe Thr Phe Ser Asn Tyr Ala

fifteen

<210> 123

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 123

Ile Ser Gly Ser Ser Asn Ser Ile

fifteen

<210> 124

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 124

Ala Arg His His Gly Arg Leu Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 125

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 125

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

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Glu Lys Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Met Val Ile Tyr

35 40 45

Gln Asp Arg Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Thr Thr Thr Pro Gly

85 90 95

Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly

100 105

<210> 126

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 126

Lys Leu Gly Glu Lys Tyr

fifteen

<210> 127

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 127

Gln Asp Arg

one

<210> 128

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 128

Gln Ala Trp Asp Thr Thr Thr Pro Gly

fifteen

<210> 129

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 129

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Asp Asp Tyr

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

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

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe

65 70 75 80

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

85 90 95

Ala Arg His His Gly Arg Leu Tyr Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 130

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 130

Gly Ile Thr Phe Asp Asp Tyr Val

fifteen

<210> 131

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 131

Ile Ser Ser Asn Ser Gly Tyr Ile

fifteen

<210> 132

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 132

Ala Arg His His Gly Arg Leu Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 133

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 133

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

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Glu Lys Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Met Val Ile Tyr

35 40 45

Gln Asp Arg Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Thr Thr Thr Pro Gly

85 90 95

Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly

100 105

<210> 134

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 134

Lys Leu Gly Glu Lys Tyr

fifteen

<210> 135

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 135

Gln Asp Arg

one

<210> 136

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 136

Gln Ala Trp Asp Thr Thr Thr Pro Gly

fifteen

<210> 137

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 137

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Glu Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Leu His Gly Val Val Ser Arg Gly Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 138

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 138

Gly Tyr Ser Phe Ser Ser Tyr Trp

fifteen

<210> 139

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 139

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 140

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 140

Ala Arg Leu His Gly Val Val Ser Arg Gly Asp Tyr

1 5 10

<210> 141

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 141

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

1 5 10 15

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

20 25 30

Asn Leu Val Ser Trp Tyr Gln Gln Tyr Pro Gly Lys Ala Pro Lys Leu

35 40 45

Ile Ile Tyr Glu Val Thr Lys Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

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

85 90 95

Asn Ala Val Ala Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly

100 105 110

<210> 142

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 142

Ser Ser Leu Ala Gly Ser Tyr Asn Leu

fifteen

<210> 143

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 143

Glu Val Thr

one

<210> 144

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 144

Ser Ser Tyr Ala Gly Arg Asn Ala Val Ala Val

1 5 10

<210> 145

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 145

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Asn Phe Asn Ser Tyr

20 25 30

Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Gly Pro Pro Phe

50 55 60

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

65 70 75 80

Leu Gln Trp Ser Ser Leu Arg Pro Ser Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Leu Val Gly Glu Gly Arg Ser Ile Gln Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 146

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 146

Gly Tyr Asn Phe Asn Ser Tyr Trp

fifteen

<210> 147

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 147

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 148

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 148

Ala Arg Leu Val Gly Glu Gly Arg Ser Ile Gln Tyr

1 5 10

<210> 149

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 149

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

1 5 10 15

Ser Val Thr Leu Thr Cys Thr Leu Thr Ser Gly Tyr Arg Asn Tyr Lys

20 25 30

Val Asp Trp Tyr Gln Gln Arg Pro Gly Lys Gly Pro Arg Phe Val Met

35 40 45

Arg Val Gly Thr Gly Gly Ile Val Gly Ser Lys Gly Asp Gly Ile Pro

50 55 60

Asp Arg Phe Ser Ala Leu Gly Ser Gly Leu Asn Arg Tyr Leu Thr Ile

65 70 75 80

Lys Asp Ile Gln Glu Glu Asp Glu Ser Asp Tyr Tyr Cys Gly Ala Asp

85 90 95

Tyr Gly Ser Gly Ser Asn Phe Leu Val Val Phe Gly Gly Gly Thr Lys

100 105 110

Leu Thr Val Leu Gly

115

<210> 150

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 150

Ser Gly Tyr Arg Asn Tyr Lys

fifteen

<210> 151

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 151

Val Gly Thr Gly Gly Ile Val Gly

fifteen

<210> 152

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 152

Gly Ala Asp Tyr Gly Ser Gly Ser Asn Phe Leu Val Val

1 5 10

<210> 153

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 153

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Asp Phe Thr Arg Tyr

20 25 30

Trp Ile Val Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Tyr Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Gly Pro Pro Phe

50 55 60

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

65 70 75 80

Leu Gln Trp Ser Ser Leu Arg Pro Ser Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Leu Val Gly Glu Gly Arg Ser Ile Gln Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 154

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 154

Gly Tyr Asp Phe Thr Arg Tyr Trp

fifteen

<210> 155

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 155

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 156

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 156

Ala Arg Leu Val Gly Glu Gly Arg Ser Ile Gln Tyr

1 5 10

<210> 157

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 157

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

1 5 10 15

Ser Val Thr Leu Thr Cys Thr Leu Thr Ser Gly Tyr Arg Asn Tyr Lys

20 25 30

Val Asp Trp Tyr Gln Gln Arg Pro Gly Lys Gly Pro Arg Phe Val Met

35 40 45

Arg Val Gly Thr Gly Gly Ile Val Gly Ser Lys Gly Asp Gly Ile Pro

50 55 60

Asp Arg Phe Ser Ala Leu Gly Ser Gly Leu Asn Arg Tyr Leu Thr Ile

65 70 75 80

Lys Asp Ile Gln Glu Glu Asp Glu Ser Asp Tyr Tyr Cys Gly Ala Asp

85 90 95

Tyr Gly Ser Gly Ser Asn Phe Leu Val Val Phe Gly Gly Gly Thr Lys

100 105 110

Val Thr Val Leu Gly

115

<210> 158

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 158

Ser Gly Tyr Arg Asn Tyr Lys

fifteen

<210> 159

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 159

Val Gly Thr Gly Gly Ile Val Gly

fifteen

<210> 160

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 160

Gly Ala Asp Tyr Gly Ser Gly Ser Asn Phe Leu Val Val

1 5 10

<210> 161

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 161

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Arg Ser

20 25 30

Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Glu Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Leu His Gly Val Val Ser Arg Gly Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 162

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 162

Gly Tyr Ser Phe Thr Arg Ser Trp

fifteen

<210> 163

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 163

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 164

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 164

Ala Arg Leu His Gly Val Val Ser Arg Gly Asp Tyr

1 5 10

<210> 165

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 165

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

1 5 10 15

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

20 25 30

Asn Leu Val Ser Trp Tyr Gln Gln Tyr Pro Gly Lys Ala Pro Lys Leu

35 40 45

Ile Ile Tyr Glu Val Thr Lys Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

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

85 90 95

Asn Ala Val Ala Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly

100 105 110

<210> 166

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 166

Ser Ser Leu Ala Gly Ser Tyr Asn Leu

fifteen

<210> 167

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 167

Glu Val Thr

one

<210> 168

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 168

Ser Ser Tyr Ala Gly Arg Asn Ala Val Ala Val

1 5 10

<210> 169

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 169

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

1 5 10 15

Ser Pro Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Glu Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Glu Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Leu His Gly Val Val Ser Arg Gly Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 170

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 170

Gly Tyr Ser Phe Thr Asn Tyr Trp

fifteen

<210> 171

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 171

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 172

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 172

Ala Arg Leu His Gly Val Val Ser Arg Gly Asp Tyr

1 5 10

<210> 173

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 173

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

1 5 10 15

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

20 25 30

Asn Leu Val Ser Trp Tyr Gln Gln Tyr Pro Gly Lys Ala Pro Lys Leu

35 40 45

Ile Ile Tyr Glu Val Thr Lys Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

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

85 90 95

Asn Ala Val Ala Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly

100 105 110

<210> 174

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 174

Ser Ser Leu Ala Gly Ser Tyr Asn Leu

fifteen

<210> 175

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 175

Glu Val Thr

one

<210> 176

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 176

Ser Ser Tyr Ala Gly Arg Asn Ala Val Ala Val

1 5 10

<210> 177

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 177

Glu Val Gln Leu Val Glu Thr Gly Gly Gly Val Val Arg Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Lys Ser Ser Gly Trp Tyr Pro Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 178

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 178

Gly Phe Thr Phe Asp Asp Tyr Ala

fifteen

<210> 179

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 179

Ile Ser Gly Ser Gly Gly Ser Thr

fifteen

<210> 180

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 180

Ala Lys Ser Ser Gly Trp Tyr Pro Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 181

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 181

Ser Tyr Glu Leu Met Gln Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Ser Gly Asn Asn Leu Gly Asp Lys Tyr Thr

20 25 30

Ser Trp Tyr Gln His Lys Pro Gly Gln Ser Pro Val Met Val Ile Tyr

35 40 45

Gln Asp Thr Lys Arg Pro Ser Gly Ile Ala Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Leu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Thr Gly Thr Ala Val

85 90 95

Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly

100 105

<210> 182

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 182

Asn Leu Gly Asp Lys Tyr

fifteen

<210> 183

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 183

Gln AspThr

one

<210> 184

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 184

Gln Ala Trp Asp Thr Gly Thr Ala Val

fifteen

<210> 185

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 185

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

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

35 40 45

Ser Gly Ile Ser Gly Ser Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr Leu Phe

65 70 75 80

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

85 90 95

Ala Arg His His Gly Arg Leu Tyr Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 186

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 186

Gly Phe Thr Phe Ser Asn Tyr Ala

fifteen

<210> 187

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 187

Ile Ser Gly Ser Ser Gly Ser Ile

fifteen

<210> 188

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 188

Ala Arg His His Gly Arg Leu Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 189

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 189

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

1 5 10 15

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

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Thr Val

85 90 95

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105

<210> 190

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 190

Ser Leu Arg Ser Tyr Tyr

fifteen

<210> 191

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 191

Gly Lys Asn

one

<210> 192

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 192

Asn Ser Arg Asp Ser Ser Gly Thr Val Val

1 5 10

<210> 193

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 193

Glu Val Gln Leu Val Glu Thr Gly Gly Gly Val Val Arg Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Lys Ser Ser Gly Trp Tyr Pro Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 194

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 194

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 195

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 195

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 196

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 196

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

1 5 10 15

<210> 197

<211> 110

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 197

Ser Tyr Glu Leu Met Gln Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Ser Gly Asn Asn Leu Gly Asp Lys Tyr Thr

20 25 30

Ser Trp Tyr Gln His Lys Pro Gly Gln Ser Pro Val Met Val Ile Tyr

35 40 45

Gln Asp Thr Lys Arg Pro Ser Gly Ile Ala Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Leu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Thr Gly Thr Ala Val

85 90 95

Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly Leu Gly Gly

100 105 110

<210> 198

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 198

Asn Leu Gly Asp Lys Tyr

fifteen

<210> 199

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 199

Gln AspThr

one

<210> 200

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 200

Gln Ala Trp Asp Thr Gly Thr Ala Val

fifteen

<210> 201

<211> 253

<212> PRT

<213> Artificial Sequence

<220>

<223> A10m3 amino acid sequence

<400> 201

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Asp Asp Ala

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Val Lys Gly Leu Tyr Ser Asn Pro Arg Gly Gly Ala Phe Asp Ile Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val His Ser Ser

130 135 140

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

145 150 155 160

Ala Thr Ile Thr Cys Gly Gly Asn Asn Ile Gly Thr Lys Ser Val His

165 170 175

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Ala

180 185 190

Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Val Ser Gly Ser Asn

195 200 205

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

210 215 220

Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Arg Ser Asp His Leu

225 230 235 240

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

245 250

<210> 202

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy chain

<400> 202

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Asp Asp Ala

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Val Lys Gly Leu Tyr Ser Asn Pro Arg Gly Gly Ala Phe Asp Ile Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr

115 120 125

<210> 203

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light chain

<400> 203

Val His Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala

1 5 10 15

Pro Gly Gln Thr Ala Thr Ile Thr Cys Gly Gly Asn Asn Ile Gly Thr

20 25 30

Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu

35 40 45

Val Val Tyr Ala Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Val

50 55 60

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

65 70 75 80

Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Arg

85 90 95

Ser Asp His Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

gly

<210> 204

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Parental IL15

<400> 204

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 205

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> IL15 K86A

<400> 205

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Ala Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 206

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> IL15 K86R

<400> 206

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Arg Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 207

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> IL15 N112A

<400> 207

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Ala

100 105 110

Thr Ser

<210> 208

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> IL15 N112S

<400> 208

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Ser

100 105 110

Thr Ser

<210> 209

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> IL15 N112Q

<400> 209

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Gln

100 105 110

Thr Ser

<210> 210

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> IL15 K86R/N112A

<400> 210

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Arg Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Ala

100 105 110

Thr Ser

<210> 211

<211> 392

<212> PRT

<213> Artificial Sequence

<220>

<223> Parental IL15-A10m3

<400> 211

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

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

130 135 140

Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Arg Ser Leu Arg Leu Ser

145 150 155 160

Cys Ala Ala Ser Gly Ile Thr Phe Asp Asp Ala Val Met His Trp Val

165 170 175

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Ser Ser

180 185 190

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

195 200 205

Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Arg

210 215 220

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Lys Gly Leu Tyr

225 230 235 240

Ser Asn Pro Arg Gly Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met

245 250 255

Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly

260 265 270

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

275 280 285

Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Thr Cys

290 295 300

Gly Gly Asn Asn Ile Gly Thr Lys Ser Val His Trp Tyr Gln Gln Lys

305 310 315 320

Pro Gly Gln Ala Pro Val Leu Val Val Tyr Ala Asp Ser Asp Arg Pro

325 330 335

Ser Gly Ile Pro Glu Arg Val Ser Gly Ser Asn Ser Gly Asn Thr Ala

340 345 350

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

355 360 365

Cys Gln Val Trp Asp Ser Arg Ser Asp His Leu Trp Val Phe Gly Gly

370 375 380

Gly Thr Lys Leu Thr Val Leu Gly

385 390

<210> 212

<211> 392

<212> PRT

<213> Artificial Sequence

<220>

<223> IL15 K86A -A10m3 K86A

<400> 212

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Ala Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

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

130 135 140

Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Arg Ser Leu Arg Leu Ser

145 150 155 160

Cys Ala Ala Ser Gly Ile Thr Phe Asp Asp Ala Val Met His Trp Val

165 170 175

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Ser Ser

180 185 190

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

195 200 205

Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Arg

210 215 220

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Lys Gly Leu Tyr

225 230 235 240

Ser Asn Pro Arg Gly Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met

245 250 255

Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly

260 265 270

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

275 280 285

Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Thr Cys

290 295 300

Gly Gly Asn Asn Ile Gly Thr Lys Ser Val His Trp Tyr Gln Gln Lys

305 310 315 320

Pro Gly Gln Ala Pro Val Leu Val Val Tyr Ala Asp Ser Asp Arg Pro

325 330 335

Ser Gly Ile Pro Glu Arg Val Ser Gly Ser Asn Ser Gly Asn Thr Ala

340 345 350

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

355 360 365

Cys Gln Val Trp Asp Ser Arg Ser Asp His Leu Trp Val Phe Gly Gly

370 375 380

Gly Thr Lys Leu Thr Val Leu Gly

385 390

<210> 213

<211> 392

<212> PRT

<213> Artificial Sequence

<220>

<223> IL15 K86R-A10m3

<400> 213

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Arg Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

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

130 135 140

Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Arg Ser Leu Arg Leu Ser

145 150 155 160

Cys Ala Ala Ser Gly Ile Thr Phe Asp Asp Ala Val Met His Trp Val

165 170 175

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Ser Ser

180 185 190

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

195 200 205

Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Arg

210 215 220

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Lys Gly Leu Tyr

225 230 235 240

Ser Asn Pro Arg Gly Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met

245 250 255

Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly

260 265 270

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

275 280 285

Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Thr Cys

290 295 300

Gly Gly Asn Asn Ile Gly Thr Lys Ser Val His Trp Tyr Gln Gln Lys

305 310 315 320

Pro Gly Gln Ala Pro Val Leu Val Val Tyr Ala Asp Ser Asp Arg Pro

325 330 335

Ser Gly Ile Pro Glu Arg Val Ser Gly Ser Asn Ser Gly Asn Thr Ala

340 345 350

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

355 360 365

Cys Gln Val Trp Asp Ser Arg Ser Asp His Leu Trp Val Phe Gly Gly

370 375 380

Gly Thr Lys Leu Thr Val Leu Gly

385 390

<210> 214

<211> 392

<212> PRT

<213> Artificial Sequence

<220>

<223> IL15R K86R/ N112A-A10m3

<400> 214

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Arg Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Ala

100 105 110

Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

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

130 135 140

Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Arg Ser Leu Arg Leu Ser

145 150 155 160

Cys Ala Ala Ser Gly Ile Thr Phe Asp Asp Ala Val Met His Trp Val

165 170 175

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Ser Ser

180 185 190

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

195 200 205

Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Arg

210 215 220

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Lys Gly Leu Tyr

225 230 235 240

Ser Asn Pro Arg Gly Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met

245 250 255

Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly

260 265 270

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

275 280 285

Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Thr Cys

290 295 300

Gly Gly Asn Asn Ile Gly Thr Lys Ser Val His Trp Tyr Gln Gln Lys

305 310 315 320

Pro Gly Gln Ala Pro Val Leu Val Val Tyr Ala Asp Ser Asp Arg Pro

325 330 335

Ser Gly Ile Pro Glu Arg Val Ser Gly Ser Asn Ser Gly Asn Thr Ala

340 345 350

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

355 360 365

Cys Gln Val Trp Asp Ser Arg Ser Asp His Leu Trp Val Phe Gly Gly

370 375 380

Gly Thr Lys Leu Thr Val Leu Gly

385 390

<210> 215

<211> 38

<212> PRT

<213> Artificial Sequence

<220>

<223> IL-15 ubiquitination sites

<400> 215

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr

35

<210> 216

<211> 38

<212> PRT

<213> Artificial Sequence

<220>

<223> IL-15 ubiquintination sites

<400> 216

Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser

1 5 10 15

Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala

20 25 30

Asn Asn Ser Leu Ser Ser

35

<210> 217

<211> 38

<212> PRT

<213> Artificial Sequence

<220>

<223> IL-15 ubiquitination sites

<400> 217

Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu

1 5 10 15

Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln

20 25 30

Met Phe Ile Asn Thr Ser

35

<210> 218

<211> 799

<212> PRT

<213> Artificial Sequence

<220>

<223> mIL12sc-A10m3

<400> 218

Met Trp Glu Leu Glu Lys Asp Val Tyr Val Val Glu Val Asp Trp Thr

1 5 10 15

Pro Asp Ala Pro Gly Glu Thr Val Asn Leu Thr Cys Asp Thr Pro Glu

20 25 30

Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln Arg His Gly Val Ile Gly

35 40 45

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

50 55 60

Gln Tyr Thr Cys His Lys Gly Gly Glu Thr Leu Ser His Ser His Leu

65 70 75 80

Leu Leu His Lys Lys Glu Asn Gly Ile Trp Ser Thr Glu Ile Leu Lys

85 90 95

Asn Phe Lys Asn Lys Thr Phe Leu Lys Cys Glu Ala Pro Asn Tyr Ser

100 105 110

Gly Arg Phe Thr Cys Ser Trp Leu Val Gln Arg Asn Met Asp Leu Lys

115 120 125

Phe Asn Ile Lys Ser Ser Ser Ser Ser Pro Asp Ser Arg Ala Val Thr

130 135 140

Cys Gly Met Ala Ser Leu Ser Ala Glu Lys Val Thr Leu Asp Gln Arg

145 150 155 160

Asp Tyr Glu Lys Tyr Ser Val Ser Cys Gln Glu Asp Val Thr Cys Pro

165 170 175

Thr Ala Glu Glu Thr Leu Pro Ile Glu Leu Ala Leu Glu Ala Arg Gln

180 185 190

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

195 200 205

Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Met Lys Pro Leu Lys Asn

210 215 220

Ser Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Ser Trp Ser Thr Pro

225 230 235 240

His Ser Tyr Phe Ser Leu Lys Phe Phe Val Arg Ile Gln Arg Lys Lys

245 250 255

Glu Lys Met Lys Glu Thr Glu Glu Gly Cys Asn Gln Lys Gly Ala Phe

260 265 270

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

275 280 285

Cys Val Gln Ala Gln Asp Arg Tyr Tyr Asn Ser Ser Cys Ser Lys Trp

290 295 300

Ala Cys Val Pro Cys Arg Val Arg Ser Gly Gly Gly Gly Ser Gly Gly

305 310 315 320

Gly Gly Ser Gly Gly Gly Gly Ser Arg Val Ile Pro Val Ser Gly Pro

325 330 335

Ala Arg Cys Leu Ser Gln Ser Arg Asn Leu Leu Lys Thr Thr Asp Asp

340 345 350

Met Val Lys Thr Ala Arg Glu Lys Leu Lys His Tyr Ser Cys Thr Ala

355 360 365

Glu Asp Ile Asp His Glu Asp Ile Thr Arg Asp Gln Thr Ser Thr Leu

370 375 380

Lys Thr Cys Leu Pro Leu Glu Leu His Lys Asn Glu Ser Cys Leu Ala

385 390 395 400

Thr Arg Glu Thr Ser Ser Thr Thr Arg Gly Ser Cys Leu Pro Pro Gln

405 410 415

Lys Thr Ser Leu Met Met Thr Leu Cys Leu Gly Ser Ile Tyr Glu Asp

420 425 430

Leu Lys Met Tyr Gln Thr Glu Phe Gln Ala Ile Asn Ala Ala Leu Gln

435 440 445

Asn His Asn His Gln Gln Ile Ile Leu Asp Lys Gly Met Leu Val Ala

450 455 460

Ile Asp Glu Leu Met Gln Ser Leu Asn His Asn Gly Glu Thr Leu Arg

465 470 475 480

Gln Lys Pro Pro Val Gly Glu Ala Asp Pro Tyr Arg Val Lys Met Lys

485 490 495

Leu Cys Ile Leu Leu His Ala Phe Ser Thr Arg Val Val Thr Ile Asn

500 505 510

Arg Val Met Gly Tyr Leu Ser Ser Ala Gly Gly Gly Gly Ser Gly Gly

515 520 525

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

530 535 540

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

545 550 555 560

Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Asp

565 570 575

Asp Ala Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Tyr Cys Val Lys Gly Leu Tyr Ser Asn Pro Arg Gly Gly Ala Phe Asp

645 650 655

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Gly

660 665 670

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val His

675 680 685

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

690 695 700

Gln Thr Ala Thr Ile Thr Cys Gly Gly Asn Asn Ile Gly Thr Lys Ser

705 710 715 720

Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val

725 730 735

Tyr Ala Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Val Ser Gly

740 745 750

Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala

755 760 765

Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Arg Ser Asp

770 775 780

His Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

785 790 795

<210> 219

<211> 788

<212> PRT

<213> Artificial Sequence

<220>

<223> Human IL-12sc-A10m3

<400> 219

Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr

1 5 10 15

Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu

20 25 30

Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly

35 40 45

Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Gly

50 55 60

Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu

65 70 75 80

Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys

85 90 95

Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys

100 105 110

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

115 120 125

Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln

130 135 140

Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly

145 150 155 160

Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala

165 170 175

Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala

180 185 190

Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg

195 200 205

Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu

210 215 220

Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp

225 230 235 240

Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln

245 250 255

Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr

260 265 270

Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala

275 280 285

Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro

290 295 300

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

305 310 315 320

Pro Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu

325 330 335

Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe

340 345 350

Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp

355 360 365

Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn

370 375 380

Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser

385 390 395 400

Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser

405 410 415

Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met

420 425 430

Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln

435 440 445

Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn

450 455 460

Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr

465 470 475 480

Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg

485 490 495

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

500 505 510

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

515 520 525

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

530 535 540

Gly Leu Ile Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser

545 550 555 560

Gly Ile Thr Phe Asp Asp Ala Val Met His Trp Val Arg Gln Ala Pro

565 570 575

Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Ser Ser Asn Ser Gly Tyr

580 585 590

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

595 600 605

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

610 615 620

Asp Thr Ala Val Tyr Tyr Cys Val Lys Gly Leu Tyr Ser Asn Pro Arg

625 630 635 640

Gly Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

645 650 655

Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

660 665 670

Gly Gly Ser Val His Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser Val

675 680 685

Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Thr Cys Gly Gly Asn Asn

690 695 700

Ile Gly Thr Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala

705 710 715 720

Pro Val Leu Val Val Tyr Ala Asp Ser Asp Arg Pro Ser Gly Ile Pro

725 730 735

Glu Arg Val Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile

740 745 750

Ser Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp

755 760 765

Asp Ser Arg Ser Asp His Leu Trp Val Phe Gly Gly Gly Thr Lys Leu

770 775 780

Thr Val Leu Gly

785

<210> 220

<211> 938

<212> PRT

<213> Artificial Sequence

<220>

<223> hIL15RA-A10m3-mIL-12sc

<400> 220

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Arg Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Ala

100 105 110

Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

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

130 135 140

Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Arg Ser Leu Arg Leu Ser

145 150 155 160

Cys Ala Ala Ser Gly Ile Thr Phe Asp Asp Ala Val Met His Trp Val

165 170 175

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Ser Ser

180 185 190

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

195 200 205

Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Arg

210 215 220

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Lys Gly Leu Tyr

225 230 235 240

Ser Asn Pro Arg Gly Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met

245 250 255

Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly

260 265 270

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

275 280 285

Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Thr Cys

290 295 300

Gly Gly Asn Asn Ile Gly Thr Lys Ser Val His Trp Tyr Gln Gln Lys

305 310 315 320

Pro Gly Gln Ala Pro Val Leu Val Val Tyr Ala Asp Ser Asp Arg Pro

325 330 335

Ser Gly Ile Pro Glu Arg Val Ser Gly Ser Asn Ser Gly Asn Thr Ala

340 345 350

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

355 360 365

Cys Gln Val Trp Asp Ser Arg Ser Asp His Leu Trp Val Phe Gly Gly

370 375 380

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

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

405 410 415

Ser Met Trp Glu Leu Glu Lys Asp Val Tyr Val Val Glu Val Asp Trp

420 425 430

Thr Pro Asp Ala Pro Gly Glu Thr Val Asn Leu Thr Cys Asp Thr Pro

435 440 445

Glu Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln Arg His Gly Val Ile

450 455 460

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

465 470 475 480

Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Thr Leu Ser His Ser His

485 490 495

Leu Leu Leu His Lys Lys Glu Asn Gly Ile Trp Ser Thr Glu Ile Leu

500 505 510

Lys Asn Phe Lys Asn Lys Thr Phe Leu Lys Cys Glu Ala Pro Asn Tyr

515 520 525

Ser Gly Arg Phe Thr Cys Ser Trp Leu Val Gln Arg Asn Met Asp Leu

530 535 540

Lys Phe Asn Ile Lys Ser Ser Ser Ser Ser Pro Asp Ser Arg Ala Val

545 550 555 560

Thr Cys Gly Met Ala Ser Leu Ser Ala Glu Lys Val Thr Leu Asp Gln

565 570 575

Arg Asp Tyr Glu Lys Tyr Ser Val Ser Cys Gln Glu Asp Val Thr Cys

580 585 590

Pro Thr Ala Glu Glu Thr Leu Pro Ile Glu Leu Ala Leu Glu Ala Arg

595 600 605

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

610 615 620

Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Met Lys Pro Leu Lys

625 630 635 640

Asn Ser Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Ser Trp Ser Thr

645 650 655

Pro His Ser Tyr Phe Ser Leu Lys Phe Phe Val Arg Ile Gln Arg Lys

660 665 670

Lys Glu Lys Met Lys Glu Thr Glu Glu Gly Cys Asn Gln Lys Gly Ala

675 680 685

Phe Leu Val Glu Lys Thr Ser Thr Glu Val Gln Cys Lys Gly Gly Asn

690 695 700

Val Cys Val Gln Ala Gln Asp Arg Tyr Tyr Asn Ser Ser Cys Ser Lys

705 710 715 720

Trp Ala Cys Val Pro Cys Arg Val Arg Ser Gly Gly Gly Gly Ser Gly

725 730 735

Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Val Ile Pro Val Ser Gly

740 745 750

Pro Ala Arg Cys Leu Ser Gln Ser Arg Asn Leu Leu Lys Thr Thr Asp

755 760 765

Asp Met Val Lys Thr Ala Arg Glu Lys Leu Lys His Tyr Ser Cys Thr

770 775 780

Ala Glu Asp Ile Asp His Glu Asp Ile Thr Arg Asp Gln Thr Ser Thr

785 790 795 800

Leu Lys Thr Cys Leu Pro Leu Glu Leu His Lys Asn Glu Ser Cys Leu

805 810 815

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

820 825 830

Gln Lys Thr Ser Leu Met Met Thr Leu Cys Leu Gly Ser Ile Tyr Glu

835 840 845

Asp Leu Lys Met Tyr Gln Thr Glu Phe Gln Ala Ile Asn Ala Ala Leu

850 855 860

Gln Asn His Asn His Gln Gln Ile Ile Leu Asp Lys Gly Met Leu Val

865 870 875 880

Ala Ile Asp Glu Leu Met Gln Ser Leu Asn His Asn Gly Glu Thr Leu

885 890 895

Arg Gln Lys Pro Pro Val Gly Glu Ala Asp Pro Tyr Arg Val Lys Met

900 905 910

Lys Leu Cys Ile Leu Leu His Ala Phe Ser Thr Arg Val Val Thr Ile

915 920 925

Asn Arg Val Met Gly Tyr Leu Ser Ser Ala

930 935

<210> 221

<211> 938

<212> PRT

<213> Artificial Sequence

<220>

<223> mIL-12sc-A10m3-hIL15R

<400> 221

Met Trp Glu Leu Glu Lys Asp Val Tyr Val Val Glu Val Asp Trp Thr

1 5 10 15

Pro Asp Ala Pro Gly Glu Thr Val Asn Leu Thr Cys Asp Thr Pro Glu

20 25 30

Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln Arg His Gly Val Ile Gly

35 40 45

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

50 55 60

Gln Tyr Thr Cys His Lys Gly Gly Glu Thr Leu Ser His Ser His Leu

65 70 75 80

Leu Leu His Lys Lys Glu Asn Gly Ile Trp Ser Thr Glu Ile Leu Lys

85 90 95

Asn Phe Lys Asn Lys Thr Phe Leu Lys Cys Glu Ala Pro Asn Tyr Ser

100 105 110

Gly Arg Phe Thr Cys Ser Trp Leu Val Gln Arg Asn Met Asp Leu Lys

115 120 125

Phe Asn Ile Lys Ser Ser Ser Ser Ser Pro Asp Ser Arg Ala Val Thr

130 135 140

Cys Gly Met Ala Ser Leu Ser Ala Glu Lys Val Thr Leu Asp Gln Arg

145 150 155 160

Asp Tyr Glu Lys Tyr Ser Val Ser Cys Gln Glu Asp Val Thr Cys Pro

165 170 175

Thr Ala Glu Glu Thr Leu Pro Ile Glu Leu Ala Leu Glu Ala Arg Gln

180 185 190

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

195 200 205

Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Met Lys Pro Leu Lys Asn

210 215 220

Ser Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Ser Trp Ser Thr Pro

225 230 235 240

His Ser Tyr Phe Ser Leu Lys Phe Phe Val Arg Ile Gln Arg Lys Lys

245 250 255

Glu Lys Met Lys Glu Thr Glu Glu Gly Cys Asn Gln Lys Gly Ala Phe

260 265 270

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

275 280 285

Cys Val Gln Ala Gln Asp Arg Tyr Tyr Asn Ser Ser Cys Ser Lys Trp

290 295 300

Ala Cys Val Pro Cys Arg Val Arg Ser Gly Gly Gly Gly Ser Gly Gly

305 310 315 320

Gly Gly Ser Gly Gly Gly Gly Ser Arg Val Ile Pro Val Ser Gly Pro

325 330 335

Ala Arg Cys Leu Ser Gln Ser Arg Asn Leu Leu Lys Thr Thr Asp Asp

340 345 350

Met Val Lys Thr Ala Arg Glu Lys Leu Lys His Tyr Ser Cys Thr Ala

355 360 365

Glu Asp Ile Asp His Glu Asp Ile Thr Arg Asp Gln Thr Ser Thr Leu

370 375 380

Lys Thr Cys Leu Pro Leu Glu Leu His Lys Asn Glu Ser Cys Leu Ala

385 390 395 400

Thr Arg Glu Thr Ser Ser Thr Thr Arg Gly Ser Cys Leu Pro Pro Gln

405 410 415

Lys Thr Ser Leu Met Met Thr Leu Cys Leu Gly Ser Ile Tyr Glu Asp

420 425 430

Leu Lys Met Tyr Gln Thr Glu Phe Gln Ala Ile Asn Ala Ala Leu Gln

435 440 445

Asn His Asn His Gln Gln Ile Ile Leu Asp Lys Gly Met Leu Val Ala

450 455 460

Ile Asp Glu Leu Met Gln Ser Leu Asn His Asn Gly Glu Thr Leu Arg

465 470 475 480

Gln Lys Pro Pro Val Gly Glu Ala Asp Pro Tyr Arg Val Lys Met Lys

485 490 495

Leu Cys Ile Leu Leu His Ala Phe Ser Thr Arg Val Val Thr Ile Asn

500 505 510

Arg Val Met Gly Tyr Leu Ser Ser Ala Gly Gly Gly Gly Ser Gly Gly

515 520 525

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

530 535 540

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

545 550 555 560

Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Asp

565 570 575

Asp Ala Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Tyr Cys Val Lys Gly Leu Tyr Ser Asn Pro Arg Gly Gly Ala Phe Asp

645 650 655

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Gly

660 665 670

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val His

675 680 685

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

690 695 700

Gln Thr Ala Thr Ile Thr Cys Gly Gly Asn Asn Ile Gly Thr Lys Ser

705 710 715 720

Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val

725 730 735

Tyr Ala Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Val Ser Gly

740 745 750

Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala

755 760 765

Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Arg Ser Asp

770 775 780

His Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly

785 790 795 800

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

805 810 815

Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp

820 825 830

Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr

835 840 845

Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met

850 855 860

Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp

865 870 875 880

Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn

885 890 895

Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Arg Glu Cys

900 905 910

Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val

915 920 925

His Ile Val Gln Met Phe Ile Asn Thr Ser

930 935

<210> 222

<211> 938

<212> PRT

<213> Artificial Sequence

<220>

<223> mIL-12sc-A10m3-hIL15RA

<400> 222

Met Trp Glu Leu Glu Lys Asp Val Tyr Val Val Glu Val Asp Trp Thr

1 5 10 15

Pro Asp Ala Pro Gly Glu Thr Val Asn Leu Thr Cys Asp Thr Pro Glu

20 25 30

Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln Arg His Gly Val Ile Gly

35 40 45

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

50 55 60

Gln Tyr Thr Cys His Lys Gly Gly Glu Thr Leu Ser His Ser His Leu

65 70 75 80

Leu Leu His Lys Lys Glu Asn Gly Ile Trp Ser Thr Glu Ile Leu Lys

85 90 95

Asn Phe Lys Asn Lys Thr Phe Leu Lys Cys Glu Ala Pro Asn Tyr Ser

100 105 110

Gly Arg Phe Thr Cys Ser Trp Leu Val Gln Arg Asn Met Asp Leu Lys

115 120 125

Phe Asn Ile Lys Ser Ser Ser Ser Ser Pro Asp Ser Arg Ala Val Thr

130 135 140

Cys Gly Met Ala Ser Leu Ser Ala Glu Lys Val Thr Leu Asp Gln Arg

145 150 155 160

Asp Tyr Glu Lys Tyr Ser Val Ser Cys Gln Glu Asp Val Thr Cys Pro

165 170 175

Thr Ala Glu Glu Thr Leu Pro Ile Glu Leu Ala Leu Glu Ala Arg Gln

180 185 190

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

195 200 205

Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Met Lys Pro Leu Lys Asn

210 215 220

Ser Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Ser Trp Ser Thr Pro

225 230 235 240

His Ser Tyr Phe Ser Leu Lys Phe Phe Val Arg Ile Gln Arg Lys Lys

245 250 255

Glu Lys Met Lys Glu Thr Glu Glu Gly Cys Asn Gln Lys Gly Ala Phe

260 265 270

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

275 280 285

Cys Val Gln Ala Gln Asp Arg Tyr Tyr Asn Ser Ser Cys Ser Lys Trp

290 295 300

Ala Cys Val Pro Cys Arg Val Arg Ser Gly Gly Gly Gly Ser Gly Gly

305 310 315 320

Gly Gly Ser Gly Gly Gly Gly Ser Arg Val Ile Pro Val Ser Gly Pro

325 330 335

Ala Arg Cys Leu Ser Gln Ser Arg Asn Leu Leu Lys Thr Thr Asp Asp

340 345 350

Met Val Lys Thr Ala Arg Glu Lys Leu Lys His Tyr Ser Cys Thr Ala

355 360 365

Glu Asp Ile Asp His Glu Asp Ile Thr Arg Asp Gln Thr Ser Thr Leu

370 375 380

Lys Thr Cys Leu Pro Leu Glu Leu His Lys Asn Glu Ser Cys Leu Ala

385 390 395 400

Thr Arg Glu Thr Ser Ser Thr Thr Arg Gly Ser Cys Leu Pro Pro Gln

405 410 415

Lys Thr Ser Leu Met Met Thr Leu Cys Leu Gly Ser Ile Tyr Glu Asp

420 425 430

Leu Lys Met Tyr Gln Thr Glu Phe Gln Ala Ile Asn Ala Ala Leu Gln

435 440 445

Asn His Asn His Gln Gln Ile Ile Leu Asp Lys Gly Met Leu Val Ala

450 455 460

Ile Asp Glu Leu Met Gln Ser Leu Asn His Asn Gly Glu Thr Leu Arg

465 470 475 480

Gln Lys Pro Pro Val Gly Glu Ala Asp Pro Tyr Arg Val Lys Met Lys

485 490 495

Leu Cys Ile Leu Leu His Ala Phe Ser Thr Arg Val Val Thr Ile Asn

500 505 510

Arg Val Met Gly Tyr Leu Ser Ser Ala Gly Gly Gly Gly Ser Gly Gly

515 520 525

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

530 535 540

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

545 550 555 560

Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Asp

565 570 575

Asp Ala Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Tyr Cys Val Lys Gly Leu Tyr Ser Asn Pro Arg Gly Gly Ala Phe Asp

645 650 655

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Gly

660 665 670

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val His

675 680 685

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

690 695 700

Gln Thr Ala Thr Ile Thr Cys Gly Gly Asn Asn Ile Gly Thr Lys Ser

705 710 715 720

Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val

725 730 735

Tyr Ala Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Val Ser Gly

740 745 750

Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala

755 760 765

Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Arg Ser Asp

770 775 780

His Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly

785 790 795 800

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

805 810 815

Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp

820 825 830

Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr

835 840 845

Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met

850 855 860

Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp

865 870 875 880

Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn

885 890 895

Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Arg Glu Cys

900 905 910

Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val

915 920 925

His Ile Val Gln Met Phe Ile Ala Thr Ser

930 935

<210> 223

<211> 927

<212> PRT

<213> Artificial Sequence

<220>

<223> hIL15RA-A10m3-hIL-12sc

<400> 223

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Arg Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Ala

100 105 110

Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

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

130 135 140

Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Arg Ser Leu Arg Leu Ser

145 150 155 160

Cys Ala Ala Ser Gly Ile Thr Phe Asp Asp Ala Val Met His Trp Val

165 170 175

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Ser Ser

180 185 190

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

195 200 205

Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Arg

210 215 220

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Lys Gly Leu Tyr

225 230 235 240

Ser Asn Pro Arg Gly Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met

245 250 255

Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly

260 265 270

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

275 280 285

Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Thr Cys

290 295 300

Gly Gly Asn Asn Ile Gly Thr Lys Ser Val His Trp Tyr Gln Gln Lys

305 310 315 320

Pro Gly Gln Ala Pro Val Leu Val Val Tyr Ala Asp Ser Asp Arg Pro

325 330 335

Ser Gly Ile Pro Glu Arg Val Ser Gly Ser Asn Ser Gly Asn Thr Ala

340 345 350

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

355 360 365

Cys Gln Val Trp Asp Ser Arg Ser Asp His Leu Trp Val Phe Gly Gly

370 375 380

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

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

405 410 415

Ser Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp

420 425 430

Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro

435 440 445

Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu

450 455 460

Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala

465 470 475 480

Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu

485 490 495

Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu

500 505 510

Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala

515 520 525

Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser

530 535 540

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

545 550 555 560

Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg

565 570 575

Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser

580 585 590

Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp

595 600 605

Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile

610 615 620

Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro

625 630 635 640

Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr

645 650 655

Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val

660 665 670

Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys

675 680 685

Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg

690 695 700

Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val

705 710 715 720

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

725 730 735

Thr Pro Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu

740 745 750

Leu Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu

755 760 765

Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys

770 775 780

Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys

785 790 795 800

Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly

805 810 815

Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu

820 825 830

Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr

835 840 845

Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp

850 855 860

Gln Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe

865 870 875 880

Asn Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe

885 890 895

Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile

900 905 910

Arg Ala Val Thr Ile Asp Arg Val Thr Ser Tyr Leu Asn Ala Ser

915 920 925

<210> 224

<211> 927

<212> PRT

<213> Artificial Sequence

<220>

<223> Human IL-12sc-A10m3-hIL15R

<400> 224

Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr

1 5 10 15

Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu

20 25 30

Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly

35 40 45

Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Gly

50 55 60

Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu

65 70 75 80

Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys

85 90 95

Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys

100 105 110

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

115 120 125

Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln

130 135 140

Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly

145 150 155 160

Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala

165 170 175

Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala

180 185 190

Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg

195 200 205

Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu

210 215 220

Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp

225 230 235 240

Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln

245 250 255

Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr

260 265 270

Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala

275 280 285

Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro

290 295 300

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

305 310 315 320

Pro Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu

325 330 335

Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe

340 345 350

Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp

355 360 365

Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn

370 375 380

Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser

385 390 395 400

Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser

405 410 415

Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met

420 425 430

Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln

435 440 445

Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn

450 455 460

Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr

465 470 475 480

Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg

485 490 495

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

500 505 510

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

515 520 525

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

530 535 540

Gly Leu Ile Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser

545 550 555 560

Gly Ile Thr Phe Asp Asp Ala Val Met His Trp Val Arg Gln Ala Pro

565 570 575

Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Ser Ser Asn Ser Gly Tyr

580 585 590

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

595 600 605

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

610 615 620

Asp Thr Ala Val Tyr Tyr Cys Val Lys Gly Leu Tyr Ser Asn Pro Arg

625 630 635 640

Gly Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

645 650 655

Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

660 665 670

Gly Gly Ser Val His Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser Val

675 680 685

Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Thr Cys Gly Gly Asn Asn

690 695 700

Ile Gly Thr Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala

705 710 715 720

Pro Val Leu Val Val Tyr Ala Asp Ser Asp Arg Pro Ser Gly Ile Pro

725 730 735

Glu Arg Val Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile

740 745 750

Ser Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp

755 760 765

Asp Ser Arg Ser Asp His Leu Trp Val Phe Gly Gly Gly Thr Lys Leu

770 775 780

Thr Val Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

785 790 795 800

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val

805 810 815

Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met

820 825 830

His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys

835 840 845

Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser

850 855 860

Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile

865 870 875 880

Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser

885 890 895

Gly Cys Arg Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe

900 905 910

Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

915 920 925

<210> 225

<211> 927

<212> PRT

<213> Artificial Sequence

<220>

<223> Human IL-12sc-A10m3-hIL15RA

<400> 225

Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr

1 5 10 15

Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu

20 25 30

Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly

35 40 45

Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Gly

50 55 60

Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu

65 70 75 80

Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys

85 90 95

Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys

100 105 110

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

115 120 125

Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln

130 135 140

Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly

145 150 155 160

Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala

165 170 175

Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala

180 185 190

Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg

195 200 205

Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu

210 215 220

Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp

225 230 235 240

Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln

245 250 255

Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr

260 265 270

Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala

275 280 285

Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro

290 295 300

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

305 310 315 320

Pro Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu

325 330 335

Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe

340 345 350

Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp

355 360 365

Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn

370 375 380

Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser

385 390 395 400

Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser

405 410 415

Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met

420 425 430

Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln

435 440 445

Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn

450 455 460

Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr

465 470 475 480

Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg

485 490 495

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

500 505 510

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

515 520 525

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

530 535 540

Gly Leu Ile Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser

545 550 555 560

Gly Ile Thr Phe Asp Asp Ala Val Met His Trp Val Arg Gln Ala Pro

565 570 575

Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Ser Ser Asn Ser Gly Tyr

580 585 590

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

595 600 605

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

610 615 620

Asp Thr Ala Val Tyr Tyr Cys Val Lys Gly Leu Tyr Ser Asn Pro Arg

625 630 635 640

Gly Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

645 650 655

Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

660 665 670

Gly Gly Ser Val His Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser Val

675 680 685

Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Thr Cys Gly Gly Asn Asn

690 695 700

Ile Gly Thr Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala

705 710 715 720

Pro Val Leu Val Val Tyr Ala Asp Ser Asp Arg Pro Ser Gly Ile Pro

725 730 735

Glu Arg Val Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile

740 745 750

Ser Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp

755 760 765

Asp Ser Arg Ser Asp His Leu Trp Val Phe Gly Gly Gly Thr Lys Leu

770 775 780

Thr Val Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

785 790 795 800

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val

805 810 815

Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met

820 825 830

His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys

835 840 845

Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser

850 855 860

Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile

865 870 875 880

Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser

885 890 895

Gly Cys Arg Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe

900 905 910

Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Ala Thr Ser

915 920 925

<210> 226

<211> 131

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 226

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

1 5 10 15

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

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Lys Glu Met Arg Tyr Cys Ser Gly Gly Ser Cys Tyr Pro Tyr Ala

100 105 110

Tyr Tyr Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

Ala Ser Thr

130

<210> 227

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 227

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

1 5 10 15

Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser

20 25 30

Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu

35 40 45

Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe

50 55 60

Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser

85 90 95

Gly Asn Leu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

Leu Gly Gly

115

<210> 228

<211> 261

<212> PRT

<213> Artificial Sequence

<220>

<223>scFv

<400> 228

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

1 5 10 15

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

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Lys Glu Met Arg Tyr Cys Ser Gly Gly Ser Cys Tyr Pro Tyr Ala

100 105 110

Tyr Tyr Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

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

145 150 155 160

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

165 170 175

Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

180 185 190

Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp

195 200 205

Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr

210 215 220

Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp

225 230 235 240

Ser Ser Gly Asn Leu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val

245 250 255

Leu Gly Leu Gly Gly

260

<210> 229

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable heavy (vh) chain

<400> 229

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

1 5 10 15

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

20 25 30

Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Lys Glu Ile Ser Gly Ser Tyr Leu Gly Leu Val Ala Phe Asp Ile Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr

115 120 125

<210> 230

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable light (vl) chain

<400> 230

Val His Ser Ser Tyr Val Leu Thr Gln Pro Leu Ser Val Ser Val Ala

1 5 10 15

Pro Gly Gln Thr Ala Arg Met Thr Cys Gly Gly Asp Asn Val Gly Ala

20 25 30

Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu

35 40 45

Leu Ile Tyr Tyr Asp His Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ser Asp Val Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly Leu

100 105 110

Gly Gly

<210> 231

<211> 255

<212> PRT

<213> Artificial Sequence

<220>

<223>scFv

<400> 231

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

1 5 10 15

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

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Trp Gly Glyn Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val His Ser

130 135 140

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

145 150 155 160

Thr Ala Arg Met Thr Cys Gly Gly Asp Asn Val Gly Ala Lys Ser Val

165 170 175

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Leu Ile Tyr

180 185 190

Tyr Asp His Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

195 200 205

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Thr Arg Val Glu Ala Gly

210 215 220

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

225 230 235 240

Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly Leu Gly Gly

245 250 255

<210> 232

<211> 261

<212> PRT

<213> Artificial Sequence

<220>

<223> 4H7m scFv

<400> 232

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

1 5 10 15

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

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Lys Glu Met Arg Tyr Cys Ser Gly Gly Ser Cys Tyr Pro Tyr Ala

100 105 110

Tyr Tyr Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

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

145 150 155 160

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

165 170 175

Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

180 185 190

Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp

195 200 205

Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr

210 215 220

Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp

225 230 235 240

Ser Ser Gly Asn Leu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val

245 250 255

Leu Gly Leu Gly Gly

260

<210> 233

<211> 537

<212> PRT

<213> Artificial Sequence

<220>

<223> 4H7m scFV-A6m

<400> 233

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

1 5 10 15

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

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Lys Glu Met Arg Tyr Cys Ser Gly Gly Ser Cys Tyr Pro Tyr Ala

100 105 110

Tyr Tyr Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

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

145 150 155 160

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

165 170 175

Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

180 185 190

Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp

195 200 205

Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr

210 215 220

Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp

225 230 235 240

Ser Ser Gly Asn Leu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val

245 250 255

Leu Gly Leu Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

260 265 270

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

275 280 285

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

290 295 300

Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Asn Ser Tyr Trp Ile

305 310 315 320

Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile

325 330 335

Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly

340 345 350

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

355 360 365

Trp Ser Ser Leu Lys Ala Ser Asp Ala Ala Met Tyr Tyr Cys Ala Arg

370 375 380

Gln Arg Trp Gly Ser Ser Ser Phe Asp Ala Trp Gly Gln Gly Thr Leu

385 390 395 400

Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly

405 410 415

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

420 425 430

Pro Pro Ser Val Ser Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys

435 440 445

Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr Gln

450 455 460

Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Glu Asn Asn Lys

465 470 475 480

Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr

485 490 495

Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln Thr Gly Asp Glu Ala Asp

500 505 510

Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu Ser Ala Gly Val Phe Gly

515 520 525

Gly Gly Thr Gln Leu Thr Val Leu Gly

530 535

<210> 234

<211> 255

<212> PRT

<213> Artificial Sequence

<220>

<223> 4D9m scFV

<400> 234

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

1 5 10 15

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

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Trp Gly Glyn Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val His Ser

130 135 140

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

145 150 155 160

Thr Ala Arg Met Thr Cys Gly Gly Asp Asn Val Gly Ala Lys Ser Val

165 170 175

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Leu Ile Tyr

180 185 190

Tyr Asp His Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

195 200 205

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Thr Arg Val Glu Ala Gly

210 215 220

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

225 230 235 240

Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly Leu Gly Gly

245 250 255

<210> 235

<211> 126

<212> PRT

<213> Artificial Sequence

<220>

<223> VH

<400> 235

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

1 5 10 15

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

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr

115 120 125

<210> 236

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 236

Gly Phe Thr Phe Ser Ser Tyr Ala

fifteen

<210> 237

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 237

Ser Gly Ser Gly Gly Ser Thr

fifteen

<210> 238

<211> 24

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 238

Ala Glu Asp Thr Ala Ala Tyr Tyr Cys Ala Lys Glu Ile Ser Gly Ser

1 5 10 15

Tyr Leu Gly Leu Val Ala Phe Asp

twenty

<210> 239

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> VL

<400> 239

Val His Ser Ser Tyr Val Leu Thr Gln Pro Leu Ser Val Ser Val Ala

1 5 10 15

Pro Gly Gln Thr Ala Arg Met Thr Cys Gly Gly Asp Asn Val Gly Ala

20 25 30

Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu

35 40 45

Leu Ile Tyr Tyr Asp His Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ser Asp Val Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly Leu

100 105 110

Gly Gly

<210> 240

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 240

Asn Val Gly Ala Lys Ser

fifteen

<210> 241

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 241

Tyr Asp His

one

<210> 242

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 242

Gln Val Trp Asp Pro Ser Ser Asp Val Val

1 5 10

<210> 243

<211> 531

<212> PRT

<213> Artificial Sequence

<220>

<223> 4D9m scFV-A6m

<400> 243

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

1 5 10 15

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

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Trp Gly Glyn Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val His Ser

130 135 140

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

145 150 155 160

Thr Ala Arg Met Thr Cys Gly Gly Asp Asn Val Gly Ala Lys Ser Val

165 170 175

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Leu Ile Tyr

180 185 190

Tyr Asp His Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

195 200 205

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Thr Arg Val Glu Ala Gly

210 215 220

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

225 230 235 240

Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly Leu Gly Gly Gly

245 250 255

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

260 265 270

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

275 280 285

Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly

290 295 300

Ser Gly Tyr Ser Phe Asn Ser Tyr Trp Ile Gly Trp Val Arg Gln Met

305 310 315 320

Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser

325 330 335

Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Phe Ser Val

340 345 350

Asp Lys Ser Ile Asn Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala

355 360 365

Ser Asp Ala Ala Met Tyr Tyr Cys Ala Arg Gln Arg Trp Gly Ser Ser

370 375 380

Ser Phe Asp Ala Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

385 390 395 400

Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

405 410 415

Ser Val His Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala

420 425 430

Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn

435 440 445

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

450 455 460

Pro Lys Leu Leu Ile Tyr Glu Asn Asn Lys Arg Pro Ser Gly Ile Pro

465 470 475 480

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

485 490 495

Thr Gly Leu Gln Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp

500 505 510

Asp Ser Ser Leu Ser Ala Gly Val Phe Gly Gly Gly Thr Gln Leu Thr

515 520 525

Val Leu Gly

530

<210> 244

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Gly-Ser 15

<400> 244

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 245

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<223> Whitlow linker

<400> 245

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

1 5 10 15

Lys Gly

<210> 246

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> 6paxA_1(+A)

<400> 246

Ile Arg Pro Arg Ala Ile Gly Gly Ser Lys Pro Arg Val Ala

1 5 10

<210> 247

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> +B

<400> 247

Gly Lys Gly Gly Ser Gly Lys Gly Gly Ser Gly Lys Gly Gly Ser

1 5 10 15

<210> 248

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> +C

<400> 248

Gly Gly Lys Gly Ser Gly Gly Lys Gly Ser Gly Gly Lys Gly Ser

1 5 10 15

<210> 249

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> +D

<400> 249

Gly Gly Gly Lys Ser Gly Gly Gly Lys Ser Gly Gly Gly Lys Ser

1 5 10 15

<210> 250

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> +E

<400> 250

Gly Lys Gly Lys Ser Gly Lys Gly Lys Ser Gly Lys Gly Lys Ser

1 5 10 15

<210> 251

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> +F

<400> 251

Gly Gly Gly Lys Ser Gly Gly Lys Gly Ser Gly Lys Gly Gly Ser

1 5 10 15

<210> 252

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> +G

<400> 252

Gly Lys Pro Gly Ser Gly Lys Pro Gly Ser Gly Lys Pro Gly Ser

1 5 10 15

<210> 253

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> +H

<400> 253

Gly Lys Pro Gly Ser Gly Lys Pro Gly Ser Gly Lys Pro Gly Ser Gly

1 5 10 15

Lys Pro Gly Ser

twenty

<210> 254

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> +I

<400> 254

Gly Lys Gly Lys Ser Gly Lys Gly Lys Ser Gly Lys Gly Lys Ser Gly

1 5 10 15

Lys Gly Lys Ser

twenty

<210> 255

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Gly-Ser 15

<400> 255

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

twenty

<210> 256

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> 3hsc_2(-A)

<400> 256

Ser Thr Ala Gly Asp Thr His Leu Gly Gly Glu Asp Phe Asp

1 5 10

<210> 257

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> -B

<400> 257

Gly Glu Gly Gly Ser Gly Glu Gly Gly Ser Gly Glu Gly Gly Ser

1 5 10 15

<210> 258

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> -C

<400> 258

Gly Gly Glu Gly Ser Gly Gly Glu Gly Ser Gly Gly Glu Gly Ser

1 5 10 15

<210> 259

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223>-D

<400> 259

Gly Gly Gly Glu Ser Gly Gly Gly Glu Ser Gly Gly Gly Glu Ser

1 5 10 15

<210> 260

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> -E

<400> 260

Gly Glu Gly Glu Ser Gly Glu Gly Glu Ser Gly Glu Gly Glu Ser

1 5 10 15

<210> 261

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> -F

<400> 261

Gly Gly Gly Glu Ser Gly Gly Glu Gly Ser Gly Glu Gly Gly Ser

1 5 10 15

<210> 262

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> -G

<400> 262

Gly Glu Gly Glu Ser Gly Glu Gly Glu Ser Gly Glu Gly Glu Ser Gly

1 5 10 15

Glu Gly Glu Ser

twenty

<210> 263

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Additional linkers

<400> 263

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 264

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Additional linker

<400> 264

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

twenty

<210> 265

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<223> Additional linker

<400> 265

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

1 5 10 15

Lys Gly

<210> 266

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> Additional linker

<400> 266

Pro Arg Gly Ala Ser Lys Ser Gly Ser Ala Ser Gln Thr Gly Ser Ala

1 5 10 15

Pro Gly Ser

<210> 267

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> Additional linker

<400> 267

Gly Thr Ala Ala Ala Gly Ala Gly Ala Ala Gly Gly Ala Ala Ala Gly

1 5 10 15

Ala Ala Gly

<210> 268

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial linker

<400> 268

Gly Thr Ser Gly Ser Ser Gly Ser Gly Ser Gly Gly Ser Gly Ser Gly

1 5 10 15

Gly Gly Gly

<210> 269

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Additional linker

<400> 269

Gly Lys Pro Gly Ser Gly Lys Pro Gly Ser Gly Lys Pro Gly Ser Gly

1 5 10 15

Lys Pro Gly Ser

twenty

<210> 270

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Additional linker

<400> 270

Gly Gly Gly Gly Gly Gly Ser

fifteen

<210> 271

<211> 133

<212> PRT

<213> Artificial Sequence

<220>

<223> Human hIL-2

<400> 271

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 272

<211> 411

<212> PRT

<213> Artificial Sequence

<220>

<223> Human hIL-2-A10m3

<400> 272

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

145 150 155 160

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

165 170 175

Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Asp Asp Ala Val Met

180 185 190

His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Gly Leu Tyr Ser Asn Pro Arg Gly Gly Ala Phe Asp Ile Trp Gly Gln

260 265 270

Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser

275 280 285

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val His Ser Ser Tyr Val

290 295 300

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

305 310 315 320

Ile Thr Cys Gly Gly Asn Asn Ile Gly Thr Lys Ser Val His Trp Tyr

325 330 335

Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Ala Asp Ser

340 345 350

Asp Arg Pro Ser Gly Ile Pro Glu Arg Val Ser Gly Ser Asn Ser Gly

355 360 365

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

370 375 380

Asp Tyr Tyr Cys Gln Val Trp Asp Ser Arg Ser Asp His Leu Trp Val

385 390 395 400

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

405 410

<210> 273

<211> 149

<212> PRT

<213> Artificial Sequence

<220>

<223> Mouse mIL-2

<400> 273

Ala Pro Thr Ser Ser Ser Thr Ser Ser Ser Thr Ala Glu Ala Gln Gln

1 5 10 15

Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln His Leu Glu Gln Leu Leu

20 25 30

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

35 40 45

Lys Leu Pro Arg Met Leu Thr Phe Lys Phe Tyr Leu Pro Lys Gln Ala

50 55 60

Thr Glu Leu Lys Asp Leu Gln Cys Leu Glu Asp Glu Leu Gly Pro Leu

65 70 75 80

Arg His Val Leu Asp Leu Thr Gln Ser Lys Ser Phe Gln Leu Glu Asp

85 90 95

Ala Glu Asn Phe Ile Ser Asn Ile Arg Val Thr Val Val Lys Leu Lys

100 105 110

Gly Ser Asp Asn Thr Phe Glu Cys Gln Phe Asp Asp Glu Ser Ala Thr

115 120 125

Val Val Asp Phe Leu Arg Arg Trp Ile Ala Phe Cys Gln Ser Ile Ile

130 135 140

Ser Thr Ser Pro Gln

145

<210> 274

<211> 427

<212> PRT

<213> Artificial Sequence

<220>

<223> Mouse mIL2-A10m3

<400> 274

Ala Pro Thr Ser Ser Ser Thr Ser Ser Ser Thr Ala Glu Ala Gln Gln

1 5 10 15

Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln His Leu Glu Gln Leu Leu

20 25 30

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

35 40 45

Lys Leu Pro Arg Met Leu Thr Phe Lys Phe Tyr Leu Pro Lys Gln Ala

50 55 60

Thr Glu Leu Lys Asp Leu Gln Cys Leu Glu Asp Glu Leu Gly Pro Leu

65 70 75 80

Arg His Val Leu Asp Leu Thr Gln Ser Lys Ser Phe Gln Leu Glu Asp

85 90 95

Ala Glu Asn Phe Ile Ser Asn Ile Arg Val Thr Val Val Lys Leu Lys

100 105 110

Gly Ser Asp Asn Thr Phe Glu Cys Gln Phe Asp Asp Glu Ser Ala Thr

115 120 125

Val Val Asp Phe Leu Arg Arg Trp Ile Ala Phe Cys Gln Ser Ile Ile

130 135 140

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

145 150 155 160

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

165 170 175

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

180 185 190

Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Asp Asp Ala Val Met

195 200 205

His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Gly Leu Tyr Ser Asn Pro Arg Gly Gly Ala Phe Asp Ile Trp Gly Gln

275 280 285

Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser

290 295 300

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val His Ser Ser Tyr Val

305 310 315 320

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

325 330 335

Ile Thr Cys Gly Gly Asn Asn Ile Gly Thr Lys Ser Val His Trp Tyr

340 345 350

Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Ala Asp Ser

355 360 365

Asp Arg Pro Ser Gly Ile Pro Glu Arg Val Ser Gly Ser Asn Ser Gly

370 375 380

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

385 390 395 400

Asp Tyr Tyr Cys Gln Val Trp Asp Ser Arg Ser Asp His Leu Trp Val

405 410 415

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

420 425

<210> 275

<211> 152

<212> PRT

<213> Artificial Sequence

<220>

<223> Human hIL-7

<400> 275

Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val Leu

1 5 10 15

Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly Ser

20 25 30

Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys Asp

35 40 45

Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu Arg

50 55 60

Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu Leu

65 70 75 80

Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln Val

85 90 95

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

100 105 110

Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp Leu

115 120 125

Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn Lys

130 135 140

Ile Leu Met Gly Thr Lys Glu His

145 150

<210> 276

<211> 430

<212> PRT

<213> Artificial Sequence

<220>

<223> Human hIL-7-A10m3

<400> 276

Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val Leu

1 5 10 15

Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly Ser

20 25 30

Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys Asp

35 40 45

Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu Arg

50 55 60

Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu Leu

65 70 75 80

Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln Val

85 90 95

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

100 105 110

Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp Leu

115 120 125

Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn Lys

130 135 140

Ile Leu Met Gly Thr Lys Glu His Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

165 170 175

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

180 185 190

Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Asp Asp

195 200 205

Ala Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Cys Val Lys Gly Leu Tyr Ser Asn Pro Arg Gly Gly Ala Phe Asp Ile

275 280 285

Trp Gly Glyn Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Gly Gly

290 295 300

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val His Ser

305 310 315 320

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

325 330 335

Thr Ala Thr Ile Thr Cys Gly Gly Asn Asn Ile Gly Thr Lys Ser Val

340 345 350

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

355 360 365

Ala Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Val Ser Gly Ser

370 375 380

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

385 390 395 400

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Arg Ser Asp His

405 410 415

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

420 425 430

<210> 277

<211> 129

<212> PRT

<213> Artificial Sequence

<220>

<223> Mouse mIL-7

<400> 277

Glu Cys His Ile Lys Asp Lys Glu Gly Lys Ala Tyr Glu Ser Val Leu

1 5 10 15

Met Ile Ser Ile Asp Glu Leu Asp Lys Met Thr Gly Thr Asp Ser Asn

20 25 30

Cys Pro Asn Asn Glu Pro Asn Phe Phe Arg Lys His Val Cys Asp Asp

35 40 45

Thr Lys Glu Ala Ala Phe Leu Asn Arg Ala Ala Arg Lys Leu Lys Gln

50 55 60

Phe Leu Lys Met Asn Ile Ser Glu Glu Phe Asn Val His Leu Leu Thr

65 70 75 80

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

85 90 95

Lys Asn Val Lys Glu Gln Lys Lys Asn Asp Ala Cys Phe Leu Lys Arg

100 105 110

Leu Leu Arg Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Lys Gly Ser

115 120 125

ile

<210> 278

<211> 407

<212> PRT

<213> Artificial Sequence

<220>

<223> Mouse mIL-7-A10m3

<400> 278

Glu Cys His Ile Lys Asp Lys Glu Gly Lys Ala Tyr Glu Ser Val Leu

1 5 10 15

Met Ile Ser Ile Asp Glu Leu Asp Lys Met Thr Gly Thr Asp Ser Asn

20 25 30

Cys Pro Asn Asn Glu Pro Asn Phe Phe Arg Lys His Val Cys Asp Asp

35 40 45

Thr Lys Glu Ala Ala Phe Leu Asn Arg Ala Ala Arg Lys Leu Lys Gln

50 55 60

Phe Leu Lys Met Asn Ile Ser Glu Glu Phe Asn Val His Leu Leu Thr

65 70 75 80

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

85 90 95

Lys Asn Val Lys Glu Gln Lys Lys Asn Asp Ala Cys Phe Leu Lys Arg

100 105 110

Leu Leu Arg Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Lys Gly Ser

115 120 125

Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

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

145 150 155 160

Ser Gly Gly Gly Leu Ile Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys

165 170 175

Ala Ala Ser Gly Ile Thr Phe Asp Asp Ala Val Met His Trp Val Arg

180 185 190

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Ser Ser Asn

195 200 205

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

210 215 220

Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Arg Leu

225 230 235 240

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Lys Gly Leu Tyr Ser

245 250 255

Asn Pro Arg Gly Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val

260 265 270

Thr Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly

275 280 285

Ser Gly Gly Gly Gly Ser Val His Ser Ser Tyr Val Leu Thr Gln Pro

290 295 300

Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Thr Cys Gly

305 310 315 320

Gly Asn Asn Ile Gly Thr Lys Ser Val His Trp Tyr Gln Gln Lys Pro

325 330 335

Gly Gln Ala Pro Val Leu Val Val Tyr Ala Asp Ser Asp Arg Pro Ser

340 345 350

Gly Ile Pro Glu Arg Val Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr

355 360 365

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

370 375 380

Gln Val Trp Asp Ser Arg Ser Asp His Leu Trp Val Phe Gly Gly Gly

385 390 395 400

Thr Lys Leu Thr Val Leu Gly

405

<210> 279

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Human hIL-12sc

<400> 279

Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr

1 5 10 15

Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu

20 25 30

Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly

35 40 45

Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Gly

50 55 60

Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu

65 70 75 80

Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys

85 90 95

Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys

100 105 110

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

115 120 125

Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln

130 135 140

Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly

145 150 155 160

Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala

165 170 175

Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala

180 185 190

Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg

195 200 205

Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu

210 215 220

Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp

225 230 235 240

Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln

245 250 255

Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr

260 265 270

Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala

275 280 285

Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro

290 295 300

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

305 310 315 320

Pro Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu

325 330 335

Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe

340 345 350

Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp

355 360 365

Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn

370 375 380

Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser

385 390 395 400

Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser

405 410 415

Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met

420 425 430

Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln

435 440 445

Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn

450 455 460

Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr

465 470 475 480

Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg

485 490 495

Ala Val Thr Ile Asp Arg Val Thr Ser Tyr Leu Asn Ala Ser

500 505 510

<210> 280

<211> 788

<212> PRT

<213> Artificial Sequence

<220>

<223> Human hIL-12sc-A10m3

<400> 280

Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr

1 5 10 15

Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu

20 25 30

Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly

35 40 45

Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Gly

50 55 60

Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu

65 70 75 80

Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys

85 90 95

Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys

100 105 110

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

115 120 125

Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln

130 135 140

Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly

145 150 155 160

Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala

165 170 175

Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala

180 185 190

Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg

195 200 205

Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu

210 215 220

Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp

225 230 235 240

Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln

245 250 255

Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr

260 265 270

Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala

275 280 285

Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro

290 295 300

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

305 310 315 320

Pro Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu

325 330 335

Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe

340 345 350

Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp

355 360 365

Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn

370 375 380

Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser

385 390 395 400

Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser

405 410 415

Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met

420 425 430

Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln

435 440 445

Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn

450 455 460

Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr

465 470 475 480

Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg

485 490 495

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

500 505 510

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

515 520 525

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

530 535 540

Gly Leu Ile Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser

545 550 555 560

Gly Ile Thr Phe Asp Asp Ala Val Met His Trp Val Arg Gln Ala Pro

565 570 575

Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Ser Ser Asn Ser Gly Tyr

580 585 590

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

595 600 605

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

610 615 620

Asp Thr Ala Val Tyr Tyr Cys Val Lys Gly Leu Tyr Ser Asn Pro Arg

625 630 635 640

Gly Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

645 650 655

Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

660 665 670

Gly Gly Ser Val His Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser Val

675 680 685

Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Thr Cys Gly Gly Asn Asn

690 695 700

Ile Gly Thr Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala

705 710 715 720

Pro Val Leu Val Val Tyr Ala Asp Ser Asp Arg Pro Ser Gly Ile Pro

725 730 735

Glu Arg Val Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile

740 745 750

Ser Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp

755 760 765

Asp Ser Arg Ser Asp His Leu Trp Val Phe Gly Gly Gly Thr Lys Leu

770 775 780

Thr Val Leu Gly

785

<210> 281

<211> 157

<212> PRT

<213> Artificial Sequence

<220>

<223> Human hIL-18-A10m3

<400> 281

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 282

<211> 435

<212> PRT

<213> Artificial Sequence

<220>

<223> Human hIL-18-A10m3

<400> 282

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp Gly Gly Gly

145 150 155 160

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

165 170 175

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

180 185 190

Leu Ile Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

195 200 205

Ile Thr Phe Asp Asp Ala Val Met His Trp Val Arg Gln Ala Pro Gly

210 215 220

Lys Gly Leu Glu Trp Val Ala Gly Ile Ser Ser Asn Ser Gly Tyr Ile

225 230 235 240

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

245 250 255

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

260 265 270

Thr Ala Val Tyr Tyr Cys Val Lys Gly Leu Tyr Ser Asn Pro Arg Gly

275 280 285

Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

290 295 300

Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

305 310 315 320

Gly Ser Val His Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser

325 330 335

Val Ala Pro Gly Gln Thr Ala Thr Ile Thr Cys Gly Gly Asn Asn Ile

340 345 350

Gly Thr Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

355 360 365

Val Leu Val Val Tyr Ala Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu

370 375 380

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

385 390 395 400

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp

405 410 415

Ser Arg Ser Asp His Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr

420 425 430

Val Leu Gly

435

<210> 283

<211> 435

<212> PRT

<213> Artificial Sequence

<220>

<223> Mouse mIL-18-A10m3

<400> 283

Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Gln Lys Arg Val Pro Gly His Asn Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser Gly Gly Gly

145 150 155 160

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

165 170 175

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

180 185 190

Leu Ile Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

195 200 205

Ile Thr Phe Asp Asp Ala Val Met His Trp Val Arg Gln Ala Pro Gly

210 215 220

Lys Gly Leu Glu Trp Val Ala Gly Ile Ser Ser Asn Ser Gly Tyr Ile

225 230 235 240

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

245 250 255

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

260 265 270

Thr Ala Val Tyr Tyr Cys Val Lys Gly Leu Tyr Ser Asn Pro Arg Gly

275 280 285

Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

290 295 300

Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

305 310 315 320

Gly Ser Val His Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser

325 330 335

Val Ala Pro Gly Gln Thr Ala Thr Ile Thr Cys Gly Gly Asn Asn Ile

340 345 350

Gly Thr Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

355 360 365

Val Leu Val Val Tyr Ala Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu

370 375 380

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

385 390 395 400

Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp

405 410 415

Ser Arg Ser Asp His Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr

420 425 430

Val Leu Gly

435

<210> 284

<211> 133

<212> PRT

<213> Artificial Sequence

<220>

<223> Human hIL-21

<400> 284

Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile

1 5 10 15

Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu

20 25 30

Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser

35 40 45

Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu

50 55 60

Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser

65 70 75 80

Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys

85 90 95

Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys

100 105 110

Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Ser Arg Thr His

115 120 125

Gly Ser Glu Asp Ser

130

<210> 285

<211> 411

<212> PRT

<213> Artificial Sequence

<220>

<223> Human hIL-21-A10m3

<400> 285

Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile

1 5 10 15

Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu

20 25 30

Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser

35 40 45

Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu

50 55 60

Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser

65 70 75 80

Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys

85 90 95

Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys

100 105 110

Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Ser Arg Thr His

115 120 125

Gly Ser Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

145 150 155 160

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

165 170 175

Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Asp Asp Ala Val Met

180 185 190

His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Gly Leu Tyr Ser Asn Pro Arg Gly Gly Ala Phe Asp Ile Trp Gly Gln

260 265 270

Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser

275 280 285

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val His Ser Ser Tyr Val

290 295 300

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

305 310 315 320

Ile Thr Cys Gly Gly Asn Asn Ile Gly Thr Lys Ser Val His Trp Tyr

325 330 335

Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Ala Asp Ser

340 345 350

Asp Arg Pro Ser Gly Ile Pro Glu Arg Val Ser Gly Ser Asn Ser Gly

355 360 365

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

370 375 380

Asp Tyr Tyr Cys Gln Val Trp Asp Ser Arg Ser Asp His Leu Trp Val

385 390 395 400

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

405 410

<210> 286

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> Mouse mIL-21

<400> 286

Pro Asp Arg Leu Leu Ile Arg Leu Arg His Leu Ile Asp Ile Val Glu

1 5 10 15

Gln Leu Lys Ile Tyr Glu Asn Asp Leu Asp Pro Glu Leu Leu Ser Ala

20 25 30

Pro Gln Asp Val Lys Gly His Cys Glu His Ala Ala Phe Ala Cys Phe

35 40 45

Gln Lys Ala Lys Leu Lys Pro Ser Asn Pro Gly Asn Asn Lys Thr Phe

50 55 60

Ile Ile Asp Leu Val Ala Gln Leu Arg Arg Arg Leu Pro Ala Arg Arg

65 70 75 80

Gly Gly Lys Lys Gln Lys His Ile Ala Lys Cys Pro Ser Cys Asp Ser

85 90 95

Tyr Glu Lys Arg Thr Pro Lys Glu Phe Leu Glu Arg Leu Lys Trp Leu

100 105 110

Leu Gln Lys Met Ile His Gln His Leu Ser

115 120

<210> 287

<211> 400

<212> PRT

<213> Artificial Sequence

<220>

<223> Mouse mIL-21-A10m3

<400> 287

Pro Asp Arg Leu Leu Ile Arg Leu Arg His Leu Ile Asp Ile Val Glu

1 5 10 15

Gln Leu Lys Ile Tyr Glu Asn Asp Leu Asp Pro Glu Leu Leu Ser Ala

20 25 30

Pro Gln Asp Val Lys Gly His Cys Glu His Ala Ala Phe Ala Cys Phe

35 40 45

Gln Lys Ala Lys Leu Lys Pro Ser Asn Pro Gly Asn Asn Lys Thr Phe

50 55 60

Ile Ile Asp Leu Val Ala Gln Leu Arg Arg Arg Leu Pro Ala Arg Arg

65 70 75 80

Gly Gly Lys Lys Gln Lys His Ile Ala Lys Cys Pro Ser Cys Asp Ser

85 90 95

Tyr Glu Lys Arg Thr Pro Lys Glu Phe Leu Glu Arg Leu Lys Trp Leu

100 105 110

Leu Gln Lys Met Ile His Gln His Leu Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

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

145 150 155 160

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe

165 170 175

Asp Asp Ala Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

180 185 190

Glu Trp Val Ala Gly Ile Ser Ser Asn Ser Gly Tyr Ile Gly Tyr Ala

195 200 205

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

210 215 220

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

225 230 235 240

Tyr Tyr Cys Val Lys Gly Leu Tyr Ser Asn Pro Arg Gly Gly Ala Phe

245 250 255

Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr

260 265 270

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val

275 280 285

His Ser Ser Tyr Val Leu Thr Gln Pro Ser Val Ser Val Ala Pro

290 295 300

Gly Gln Thr Ala Thr Ile Thr Cys Gly Gly Asn Asn Ile Gly Thr Lys

305 310 315 320

Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val

325 330 335

Val Tyr Ala Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Val Ser

340 345 350

Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu

355 360 365

Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Arg Ser

370 375 380

Asp His Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

385 390 395 400

<210> 288

<211> 209

<212> PRT

<213> Artificial Sequence

<220>

<223> Human hIL-27sc

<400> 288

Arg Lys Gly Pro Pro Ala Ala Leu Thr Leu Pro Arg Val Gln Cys Arg

1 5 10 15

Ala Ser Arg Tyr Pro Ile Ala Val Asp Cys Ser Trp Thr Leu Pro Pro

20 25 30

Ala Pro Asn Ser Thr Ser Pro Val Ser Phe Ile Ala Thr Tyr Arg Leu

35 40 45

Gly Met Ala Ala Arg Gly His Ser Trp Pro Cys Leu Gln Gln Thr Pro

50 55 60

Thr Ser Thr Ser Cys Thr Ile Thr Asp Val Gln Leu Phe Ser Met Ala

65 70 75 80

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

85 90 95

Ser Phe Val Pro Phe Ile Thr Glu His Ile Ile Lys Pro Asp Pro Pro

100 105 110

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

115 120 125

Trp Glu Pro Pro Gly Ser Trp Pro Phe Pro Glu Ile Phe Ser Leu Lys

130 135 140

Tyr Trp Ile Arg Tyr Lys Arg Gln Gly Ala Ala Arg Phe His Arg Val

145 150 155 160

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

165 170 175

Ala Arg Tyr Tyr Val Gln Val Ala Ala Gln Asp Leu Thr Asp Tyr Gly

180 185 190

Glu Leu Ser Asp Trp Ser Leu Pro Ala Thr Ala Thr Met Ser Leu Gly

195 200 205

Lys

<210> 289

<211> 716

<212> PRT

<213> Artificial Sequence

<220>

<223> Human hIL-27sc-A10m3

<400> 289

Lys Gly Pro Pro Ala Ala Leu Thr Leu Pro Arg Val Gln Cys Arg Ala

1 5 10 15

Ser Arg Tyr Pro Ile Ala Val Asp Cys Ser Trp Thr Leu Pro Pro Pro Ala

20 25 30

Pro Asn Ser Thr Ser Pro Val Ser Phe Ile Ala Thr Tyr Arg Leu Gly

35 40 45

Met Ala Ala Arg Gly His Ser Trp Pro Cys Leu Gln Gln Thr Pro Thr

50 55 60

Ser Thr Ser Cys Thr Ile Thr Asp Val Gln Leu Phe Ser Met Ala Pro

65 70 75 80

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

85 90 95

Phe Val Pro Phe Ile Thr Glu His Ile Ile Lys Pro Asp Pro Pro Glu

100 105 110

Gly Val Arg Leu Ser Pro Leu Ala Glu Arg Gln Leu Gln Val Gln Trp

115 120 125

Glu Pro Pro Gly Ser Trp Pro Phe Pro Glu Ile Phe Ser Leu Lys Tyr

130 135 140

Trp Ile Arg Tyr Lys Arg Gln Gly Ala Ala Arg Phe His Arg Val Gly

145 150 155 160

Pro Ile Glu Ala Thr Ser Phe Ile Leu Arg Ala Val Arg Pro Arg Ala

165 170 175

Arg Tyr Tyr Val Gln Val Ala Ala Gln Asp Leu Thr Asp Tyr Gly Glu

180 185 190

Leu Ser Asp Trp Ser Leu Pro Ala Thr Ala Thr Met Ser Leu Gly Lys

195 200 205

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Phe

210 215 220

Pro Arg Pro Pro Gly Arg Pro Gln Leu Ser Leu Gln Glu Leu Arg Arg

225 230 235 240

Glu Phe Thr Val Ser Leu His Leu Ala Arg Lys Leu Leu Ser Glu Val

245 250 255

Arg Gly Gln Ala His Arg Phe Ala Glu Ser His Leu Pro Gly Val Asn

260 265 270

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

275 280 285

Phe Gln Ala Trp Arg Arg Leu Ser Asp Pro Glu Arg Leu Cys Phe Ile

290 295 300

Ser Thr Thr Leu Gln Pro Phe His Ala Pro Leu Gly Gly Leu Gly Thr

305 310 315 320

Gln Gly Arg Trp Thr Asn Met Glu Arg Met Gln Leu Trp Ala Met Arg

325 330 335

Leu Asp Leu Arg Asp Leu Gln Arg His Leu Arg Phe Gln Val Leu Ala

340 345 350

Ala Gly Phe Asn Leu Pro Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu

355 360 365

Glu Glu Glu Arg Lys Gly Leu Leu Pro Gly Ala Leu Gly Ser Ala Leu

370 375 380

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

385 390 395 400

Leu Leu His Ser Leu Glu Leu Val Leu Ser Arg Ala Val Arg Glu Leu

405 410 415

Leu Leu Leu Ser Lys Ala Gly His Ser Val Trp Pro Leu Gly Phe Pro

420 425 430

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

435 440 445

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

450 455 460

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

465 470 475 480

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

485 490 495

Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Lys Gly Leu Tyr Ser Asn Pro Arg Gly Gly Ala Phe Asp Ile Trp Gly

565 570 575

Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly Gly

580 585 590

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val His Ser Ser Tyr

595 600 605

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

610 615 620

Thr Ile Thr Cys Gly Gly Asn Asn Ile Gly Thr Lys Ser Val His Trp

625 630 635 640

Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Ala Asp

645 650 655

Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Val Ser Gly Ser Asn Ser

660 665 670

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

675 680 685

Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Arg Ser Asp His Leu Trp

690 695 700

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

705 710 715

<210> 290

<211> 210

<212> PRT

<213> Artificial Sequence

<220>

<223> Mouse mIL-27sc

<400> 290

Tyr Thr Glu Thr Ala Leu Val Ala Leu Ser Gln Pro Arg Val Gln Cys

1 5 10 15

His Ala Ser Arg Tyr Pro Val Ala Val Asp Cys Ser Trp Thr Pro Leu

20 25 30

Gln Ala Pro Asn Ser Thr Arg Ser Thr Ser Phe Ile Ala Thr Tyr Arg

35 40 45

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

50 55 60

Pro Gln Ala Ser Arg Cys Thr Ile Pro Asp Val His Leu Phe Ser Thr

65 70 75 80

Val Pro Tyr Met Leu Asn Val Thr Ala Val His Pro Gly Gly Ala Ser

85 90 95

Ser Ser Leu Leu Ala Phe Val Ala Glu Arg Ile Ile Lys Pro Asp Pro

100 105 110

Pro Glu Gly Val Arg Leu Arg Thr Ala Gly Gln Arg Leu Gln Val Leu

115 120 125

Trp His Pro Pro Ala Ser Trp Pro Phe Pro Asp Ile Phe Ser Leu Lys

130 135 140

Tyr Arg Leu Arg Tyr Arg Arg Arg Gly Ala Ser His Phe Arg Gln Val

145 150 155 160

Gly Pro Ile Glu Ala Thr Thr Phe Thr Leu Arg Asn Ser Lys Pro His

165 170 175

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

180 185 190

Lys Pro Ser Asp Trp Ser Leu Pro Gly Gln Val Glu Ser Ala Pro His

195 200 205

Lys Pro

210

<210> 291

<211> 709

<212> PRT

<213> Artificial Sequence

<220>

<223> Mouse mIL-27sc-A10m3

<400> 291

Tyr Thr Glu Thr Ala Leu Val Ala Leu Ser Gln Pro Arg Val Gln Cys

1 5 10 15

His Ala Ser Arg Tyr Pro Val Ala Val Asp Cys Ser Trp Thr Pro Leu

20 25 30

Gln Ala Pro Asn Ser Thr Arg Ser Thr Ser Phe Ile Ala Thr Tyr Arg

35 40 45

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

50 55 60

Pro Gln Ala Ser Arg Cys Thr Ile Pro Asp Val His Leu Phe Ser Thr

65 70 75 80

Val Pro Tyr Met Leu Asn Val Thr Ala Val His Pro Gly Gly Ala Ser

85 90 95

Ser Ser Leu Leu Ala Phe Val Ala Glu Arg Ile Ile Lys Pro Asp Pro

100 105 110

Pro Glu Gly Val Arg Leu Arg Thr Ala Gly Gln Arg Leu Gln Val Leu

115 120 125

Trp His Pro Pro Ala Ser Trp Pro Phe Pro Asp Ile Phe Ser Leu Lys

130 135 140

Tyr Arg Leu Arg Tyr Arg Arg Arg Gly Ala Ser His Phe Arg Gln Val

145 150 155 160

Gly Pro Ile Glu Ala Thr Thr Phe Thr Leu Arg Asn Ser Lys Pro His

165 170 175

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

180 185 190

Lys Pro Ser Asp Trp Ser Leu Pro Gly Gln Val Glu Ser Ala Pro His

195 200 205

Lys Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

210 215 220

Ser Phe Pro Thr Asp Pro Leu Ser Leu Gln Glu Leu Arg Arg Glu Phe

225 230 235 240

Thr Val Ser Leu Tyr Leu Ala Arg Lys Leu Leu Ser Glu Val Gln Gly

245 250 255

Tyr Val His Ser Phe Ala Glu Ser Arg Leu Pro Gly Val Asn Leu Asp

260 265 270

Leu Leu Pro Leu Gly Tyr His Leu Pro Asn Val Ser Leu Thr Phe Gln

275 280 285

Ala Trp His His Leu Ser Asp Ser Glu Arg Leu Cys Phe Leu Ala Thr

290 295 300

Thr Leu Arg Pro Phe Pro Ala Met Leu Gly Gly Leu Gly Thr Gln Gly

305 310 315 320

Thr Trp Thr Ser Ser Glu Arg Glu Gln Leu Trp Ala Met Arg Leu Asp

325 330 335

Leu Arg Asp Leu His Arg His Leu Arg Phe Gln Val Leu Ala Ala Gly

340 345 350

Phe Lys Cys Ser Lys Glu Glu Glu Asp Lys Glu Glu Glu Glu Glu Glu

355 360 365

Glu Glu Glu Glu Lys Lys Leu Pro Leu Gly Ala Leu Gly Gly Pro Asn

370 375 380

Gln Val Ser Ser Gln Val Ser Trp Pro Gln Leu Leu Tyr Thr Tyr Gln

385 390 395 400

Leu Leu His Ser Leu Glu Leu Val Leu Ser Arg Ala Val Arg Asp Leu

405 410 415

Leu Leu Leu Ser Leu Pro Arg Arg Pro Gly Ser Ala Trp Asp Ser Gly

420 425 430

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

435 440 445

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

450 455 460

Gly Gly Leu Ile Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala

465 470 475 480

Ser Gly Ile Thr Phe Asp Asp Ala Val Met His Trp Val Arg Gln Ala

485 490 495

Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Ser Ser Asn Ser Gly

500 505 510

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

515 520 525

Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Arg Leu Arg Ala

530 535 540

Glu Asp Thr Ala Val Tyr Tyr Cys Val Lys Gly Leu Tyr Ser Asn Pro

545 550 555 560

Arg Gly Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val

565 570 575

Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

580 585 590

Gly Gly Gly Ser Val His Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser

595 600 605

Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Thr Cys Gly Gly Asn

610 615 620

Asn Ile Gly Thr Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln

625 630 635 640

Ala Pro Val Leu Val Val Tyr Ala Asp Ser Asp Arg Pro Ser Gly Ile

645 650 655

Pro Glu Arg Val Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr

660 665 670

Ile Ser Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val

675 680 685

Trp Asp Ser Arg Ser Asp His Leu Trp Val Phe Gly Gly Gly Thr Lys

690 695 700

Leu Thr Val Leu Gly

705

<210> 292

<211> 144

<212> PRT

<213> Artificial Sequence

<220>

<223> Human GM-CSF

<400> 292

Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile

1 5 10 15

Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His

20 25 30

Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp

35 40 45

Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe

50 55 60

Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys

65 70 75 80

Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met

85 90 95

Met Ala Ser His Tyr Lys Gln His Cys Pro Thr Pro Glu Thr Ser

100 105 110

Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys

115 120 125

Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu

130 135 140

<210> 293

<211> 422

<212> PRT

<213> Artificial Sequence

<220>

<223> Human GM-CSF-A10m3

<400> 293

Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile

1 5 10 15

Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His

20 25 30

Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp

35 40 45

Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe

50 55 60

Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys

65 70 75 80

Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met

85 90 95

Met Ala Ser His Tyr Lys Gln His Cys Pro Thr Pro Glu Thr Ser

100 105 110

Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys

115 120 125

Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

145 150 155 160

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

165 170 175

Gly Gly Gly Leu Ile Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala

180 185 190

Ala Ser Gly Ile Thr Phe Asp Asp Ala Val Met His Trp Val Arg Gln

195 200 205

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Ser Ser Asn Ser

210 215 220

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

225 230 235 240

Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Arg Leu Arg

245 250 255

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Lys Gly Leu Tyr Ser Asn

260 265 270

Pro Arg Gly Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr

275 280 285

Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

290 295 300

Gly Gly Gly Gly Ser Val His Ser Ser Tyr Val Leu Thr Gln Pro Pro

305 310 315 320

Ser Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Thr Cys Gly Gly

325 330 335

Asn Asn Ile Gly Thr Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly

340 345 350

Gln Ala Pro Val Leu Val Val Tyr Ala Asp Ser Asp Arg Pro Ser Gly

355 360 365

Ile Pro Glu Arg Val Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu

370 375 380

Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln

385 390 395 400

Val Trp Asp Ser Arg Ser Asp His Leu Trp Val Phe Gly Gly Gly Thr

405 410 415

Lys Leu Thr Val Leu Gly

420

<210> 294

<211> 166

<212> PRT

<213> Artificial Sequence

<220>

<223> Human IFN-?

<400> 294

Cys Asp Leu Pro Glu Thr His Ser Leu Asp Asn Arg Arg Thr Leu Met

1 5 10 15

Leu Leu Ala Gln Met Ser Arg Ile Ser Pro Ser Ser Cys Leu Met Asp

20 25 30

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45

Gln Lys Ala Pro Ala Ile Ser Val Leu His Glu Leu Ile Gln Gln Ile

50 55 60

Phe Asn Leu Phe Thr Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Asp

65 70 75 80

Leu Leu Asp Lys Phe Cys Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu

85 90 95

Glu Ala Cys Val Met Gln Glu Glu Arg Val Gly Glu Thr Pro Leu Met

100 105 110

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

115 120 125

Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140

Arg Ala Glu Ile Met Arg Ser Leu Ser Leu Ser Thr Asn Leu Gln Glu

145 150 155 160

Arg Leu Arg Arg Lys Glu

165

<210> 295

<211> 444

<212> PRT

<213> Artificial Sequence

<220>

<223> Human IFN-?-A10m3

<400> 295

Cys Asp Leu Pro Glu Thr His Ser Leu Asp Asn Arg Arg Thr Leu Met

1 5 10 15

Leu Leu Ala Gln Met Ser Arg Ile Ser Pro Ser Ser Cys Leu Met Asp

20 25 30

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45

Gln Lys Ala Pro Ala Ile Ser Val Leu His Glu Leu Ile Gln Gln Ile

50 55 60

Phe Asn Leu Phe Thr Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Asp

65 70 75 80

Leu Leu Asp Lys Phe Cys Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu

85 90 95

Glu Ala Cys Val Met Gln Glu Glu Arg Val Gly Glu Thr Pro Leu Met

100 105 110

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

115 120 125

Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140

Arg Ala Glu Ile Met Arg Ser Leu Ser Leu Ser Thr Asn Leu Gln Glu

145 150 155 160

Arg Leu Arg Arg Lys Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

165 170 175

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

180 185 190

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

195 200 205

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

210 215 220

Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Lys Gly Leu Tyr Ser Asn Pro Arg Gly Gly Ala Phe Asp Ile Trp Gly

290 295 300

Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly Gly

305 310 315 320

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val His Ser Ser Tyr

325 330 335

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

340 345 350

Thr Ile Thr Cys Gly Gly Asn Asn Ile Gly Thr Lys Ser Val His Trp

355 360 365

Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Ala Asp

370 375 380

Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Val Ser Gly Ser Asn Ser

385 390 395 400

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

405 410 415

Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Arg Ser Asp His Leu Trp

420 425 430

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

435 440

<210> 296

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-PD-L1 10D12, Variable Heavy Domain

<400> 296

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

1 5 10 15

Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ser Arg Ser Ser Met Ala Gln His Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr

115

<210> 297

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR1

<400> 297

Gly Phe Thr Phe Ser Asp Tyr Tyr

fifteen

<210> 298

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR2

<400> 298

Ile Ser Ser Ser Gly Ser Thr Ile

fifteen

<210> 299

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223>vhCDR3

<400> 299

Ala Ser Arg Ser Ser Met Ala Gln His

fifteen

<210> 300

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Variable Light Domain

<400> 300

Ser Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly

1 5 10 15

Lys Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser

20 25 30

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

35 40 45

Val Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser

65 70 75 80

Gly Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp

85 90 95

Ser Asn Asn Gln Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly

100 105 110

<210> 301

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR1

<400> 301

Ser Gly Ser Ile Ala Ser Tyr Tyr

fifteen

<210> 302

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR2

<400> 302

Glu Asp Asn

one

<210> 303

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223>vlCDR3

<400> 303

Gln Ser Tyr Asp Ser Asn Asn Gln Val

fifteen

<210> 304

<211> 245

<212> PRT

<213> Artificial Sequence

<220>

<223>scFv

<400> 304

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ser Arg Ser Ser Met Ala Gln His Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asn Phe Met Leu Thr Gln Pro His Ser Val

130 135 140

Ser Glu Ser Pro Gly Lys Thr Val Thr Ile Ser Cys Thr Arg Ser Ser

145 150 155 160

Gly Ser Ile Ala Ser Tyr Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly

165 170 175

Ser Ala Pro Thr Thr Val Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly

180 185 190

Val Pro Asp Arg Phe Ser Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala

195 200 205

Ser Leu Thr Ile Ser Gly Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr

210 215 220

Cys Gln Ser Tyr Asp Ser Asn Asn Gln Val Phe Gly Gly Gly Thr Lys

225 230 235 240

Val Thr Val Leu Gly

245

<210> 305

<211> 303

<212> PRT

<213> Artificial Sequence

<220>

<223> linker-ABD-linker

<400> 305

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

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

20 25 30

Gly Gly Gly Leu Ile Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala

35 40 45

Ala Ser Gly Ile Thr Phe Asp Asp Ala Val Met His Trp Val Arg Gln

50 55 60

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Ser Ser Asn Ser

65 70 75 80

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

85 90 95

Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Arg Leu Arg

100 105 110

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Lys Gly Leu Tyr Ser Asn

115 120 125

Pro Arg Gly Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr

130 135 140

Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Gly Gly Gly Gly Ser Val His Ser Ser Tyr Val Leu Thr Gln Pro Pro

165 170 175

Ser Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Thr Cys Gly Gly

180 185 190

Asn Asn Ile Gly Thr Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly

195 200 205

Gln Ala Pro Val Leu Val Val Tyr Ala Asp Ser Asp Arg Pro Ser Gly

210 215 220

Ile Pro Glu Arg Val Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu

225 230 235 240

Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln

245 250 255

Val Trp Asp Ser Arg Ser Asp His Leu Trp Val Phe Gly Gly Gly Thr

260 265 270

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

275 280 285

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

290 295 300

<210> 306

<211> 303

<212> PRT

<213> Artificial Sequence

<220>

<223> linker-ABD-linker

<400> 306

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

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

20 25 30

Gly Gly Gly Leu Ile Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala

35 40 45

Ala Ser Gly Ile Thr Phe Asp Asp Ala Val Met His Trp Val Arg Gln

50 55 60

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Ser Ser Asn Ser

65 70 75 80

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

85 90 95

Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Arg Leu Arg

100 105 110

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Lys Gly Leu Tyr Ser Asn

115 120 125

Pro Arg Gly Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr

130 135 140

Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Gly Gly Gly Gly Ser Val His Ser Ser Tyr Val Leu Thr Gln Pro Pro

165 170 175

Ser Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Thr Cys Gly Gly

180 185 190

Asn Asn Ile Gly Thr Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly

195 200 205

Gln Ala Pro Val Leu Val Val Tyr Ala Asp Ser Asp Arg Pro Ser Gly

210 215 220

Ile Pro Glu Arg Val Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu

225 230 235 240

Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln

245 250 255

Val Trp Asp Ser Arg Ser Asp His Leu Trp Val Phe Gly Gly Gly Thr

260 265 270

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

275 280 285

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

290 295 300

<210> 307

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> ABD linker

<400> 307

Gly Gly Gly Gly Ser

fifteen

<210> 308

<211> 2

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker

<400> 308

Gly Ser

one

<210> 309

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker

<400> 309

Gly Ser Gly Gly Ser

fifteen

<210> 310

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker

<400> 310

Gly Gly Gly Ser

one

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Claims (22)

1. Fusion protein with an albumin-binding domain (ABD) with improved in vivo pharmacokinetics, containing an ABD attached to one or more fusion partners, while the specified ABD contains a heavy chain variable region containing vhCDR1, vhCDR2 and vhCDR3 from the heavy variable region chain having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region containing vlCDR1, vlCDR2 and vlCDR3 from the light chain variable region having the amino acid sequence of SEQ ID NO: 29. 2. An ABD fusion protein according to claim 1, wherein said heavy chain variable region has the amino acid sequence of SEQ ID NO: 25 and said light chain variable region has the amino acid sequence of SEQ ID NO: 29. 3. Fusion protein with ABD according to claim 1 or 2, characterized in that said one or more fusion partners are selected from the group consisting of: IL-2, IL-7, IL-12, IL-15, IL-18 , IL-21, GM-CSF, IFN-α, anti-TGFβ scFv, anti-PD-L1 scFv, anti-TNF scFv, anti-IL-1 scFv, anti-IL-6 scFv, anti-IL-8 scFv , anti-IL-17 (AF) scFv and anti-IL-23 scFv, and combinations thereof. 4. Fusion protein with ABD according to any one of paragraphs. 1-3, characterized in that said one or more fusion partners are selected from the group consisting of: IL-12 and IL-15, IL-18 and IL-12, IL-18 and GM-CSF, GM-CSF and IL-12, IL12-ABD-IL12 and IL7-ABD-IL15. 5. Fusion protein with ABD according to any one of paragraphs. 1-4, characterized in that said one or more fusion partners are covalently attached to said ABD via a linker. 6. An ABD fusion protein according to claim 4, wherein said one or more fusion partners comprise IL-18 and IL-12. 7. ABD fusion protein according to claim 6, characterized in that said fusion protein has an N-terminal to C-terminal formula represented by (IL-18)-L1-(ABD)-L2-(IL-12), where L1 and L2 are the first and second linkers, respectively. 8. An ABD fusion protein according to claim 4, wherein said one or more fusion partners comprise IL-18 and GM-CSF. 9. ABD fusion protein according to claim 8, characterized in that said fusion protein has an N-terminal to C-terminal formula represented by (IL-18)-L1-(ABD)-L2-(GM-CSF), where L1 and L2 are the first and second linkers, respectively. 10. An ABD fusion protein according to claim 4, wherein said one or more fusion partners comprise GM-CSF and IL-12. 11. ABD fusion protein according to claim 10, characterized in that said fusion protein has an N-terminal to C-terminal formula represented by (GM-CSF)-L1-(ABD)-L2-(IL-12), where L1 and L2 are the first and second linkers, respectively. 12. An ABD fusion protein according to claim 4, wherein said one or more fusion partners comprise IL-12 and IL-15. 13. ABD fusion protein according to claim 12, characterized in that said fusion protein has an N-terminal to C-terminal formula represented by (IL-12)-L1-(ABD)-L2-(IL-15), where L1 and L2 are the first and second linkers, respectively. 14. An ABD fusion protein according to claim 4, wherein said one or more fusion partners comprise IL-12. 15. ABD fusion protein according to claim 14, characterized in that said fusion protein has an N-terminal to C-terminal formula represented by (IL-12)-L1-(ABD)-L2-(IL-12), where L1 and L2 are the first and second linkers, respectively. 16. An ABD fusion protein according to claim 4, wherein said one or more fusion partners comprise IL-7 and IL-15. 17. An ABD fusion protein according to claim 16, characterized in that said fusion protein has an N-terminal to C-terminal formula represented by (IL-7)-L1-(ABD)-L2-(IL-15), where L1 and L2 are the first and second linkers, respectively. 18. An ABD fusion protein according to claim 3 or 4, wherein IL-15 is a variant of IL-15 containing one or more amino acid substitutions selected from the group consisting of K86A, K86R, N112A, N112S, N112Q, K86A/N112A, K86R/N112A, K86A/N112S, K86R/N112S, K86A/N112Q, K86R/N112Q, K86A/N112A/N79A, K86R/N112A/N79A, K86A/N112A/N79D, K86R/N112A/N79D, K86A/N71, K86R/N112A/N79Q K86A/N112A/N71D K86R/N112A/N71D K86A/N112A/N71Q K86R/N112A/N71Q K86A/N112A/N71D/N79A K86A/N112A/N71D/N79/N11A N71Q/N79A K86A/N112A/N71Q/N79D K86R/N112A/N71D/N79A K86R/N112A/N71D/N79D K86R/N112A/N71D/N79Q N79D and K86R/N112A/N71Q/N79Q, compared to parental IL-15. 19. An ABD fusion protein according to claim 3 or 4, wherein the IL-15 is wild-type IL-15 fused to the IL-15 receptor alpha (IL-15Rα). 20. Nucleic acid encoding a fusion protein with ABD according to any one of paragraphs. 1-19 containing an ABD attached to one or more fusion partners, wherein said ABD contains a heavy chain variable region containing vhCDR1, vhCDR2, and vhCDR3 from a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25, and a light chain variable region a chain containing vlCDR1, vlCDR2 and vlCDR3 from the light chain variable region having the amino acid sequence of SEQ ID NO: 29. 21. The host cell to obtain a fusion protein with ABD according to any one of paragraphs. 1-19 containing the specified nucleic acid according to claim 20. 22. The method of obtaining a fusion protein with ABD according to any one of paragraphs. 1-19, comprising culturing said host cell according to claim 21 under conditions in which an ABD-based fusion protein is produced, and recovering said ABD-based fusion protein.

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