RU2797464C2 - Tgf-beta receptor ectodomain functions and their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: following are presented: polypeptide constructs capable of binding and neutralizing ligands of transforming growth factor beta (TGF-β), methods for their preparation and the use of these polypeptides for the treatment of disorders associated with the expression or activation of TGF-beta, such as cancer and fibrotic disorders. Also nucleic acid molecules encoding any polypeptide constructs, vectors containing nucleic acid molecules, compositions for inhibiting TGF-β, transgenic cellular hosts for the production of a polypeptide construct are presented.

EFFECT: invention makes it possible to obtain polypeptide constructs capable of binding and effectively using them for the treatment of disorders associated with the expression or activation of TGF-beta.

26 cl, 15 dwg, 6 tbl

Description

Field of invention

The present invention relates to TGF-β (transforming growth factor beta) receptor ectodomain related molecules and their uses. More specifically, the present invention relates to related molecules of the TGF-β superfamily receptor ectodomain and their use in neutralizing a TGF-β ligand.

Prior Art

TGF-β is part of a superfamily of over 30 ligands that regulate several physiological processes including cell proliferation, migration and differentiation. Returbation of their levels and/or signaling leads to serious pathological effects. For example, TGF-β and activin ligands play significant pathogenic roles in many diseases, including cancer (Hawinkels & Ten Dijke, 2011; Massague et al., 2000; Rodgarkia-Dara et al, 2006). In particular, TGF-β is considered a critical regulator of tumor development and is overexpressed by most tumor types. It promotes oncogenesis by inducing in part an epithelial-mesenchymal transition (EMT) in epithelial tumor cells, leading to aggressive metastasis (Thiery et al, 2009). TGF-β also promotes tumorigenesis by acting as a potent suppressor of the immune response in the tumor microenvironment (Li et al, 2006). In fact, TGF-β is recognized as one of the most effective immunosuppressive factors present in the tumor microenvironment. TGF-β interferes with the differentiation, proliferation, and survival of many types of immune cells, including dendritic cells, macrophages, NK cells (natural killer cells), neutrophils, B cells, and T cells; thus, it modulates both innate and adaptive immunity (Santarpia et al, 2015; Yang et al, 2010). The importance of TGF-β in the tumor microenvironment is supported by evidence that in some types of tumors (including melanoma, lung cancer, pancreatic cancer, colorectal cancer, liver and breast cancer), elevated levels of TGF-β ligand correlate with progression and recurrence. disease, metastasis and mortality. Consequently, significant efforts have been invested in the development of antitumor therapeutic approaches that involve TGF-β inhibition (Arteaga, 2006; Mourskaia et al, 2007; Wojtowicz-Praga, 2003). These approaches include the use of linked TGF-β receptor ectodomain polypeptides that bind or "capture" the TGF-β ligand (see WO 0183525; WO 2005028517; WO 2008113185; WO 2008157367; WO 2010003118; WO 2010099219; WO 2012071649; WO 2012142515; WO 2013000234; US5693607; US20050203022; US20070244042; US8318135; US8658135; US8815247; US20150225483; and US20150056199).

One approach to develop therapeutics that inhibit TGF-β function has been to use antibodies or soluble decoy receptors (also called receptor ectodomain-based (ECD) ligand decoys) to bind and sequester the ligand, which blocks the access of the ligand to its receptors. on the cell surface (Zwaagstra et al, 2012). In general, ECD decoy receptors are a class of therapeutic agents that are able to sequester a wide range of ligands and that can be optimized using protein engineering techniques (Economides et al, 2003; Holash et al, 2002; Jin et al, 2009) .

Previously, a novel protein engineering strategy (Zwaagstra et al, 2012) [WO 2008113185; WO 2010031168]. In this case, bivalence was achieved by covalently linking the two TβRII ectodomains using internally disordered regions (IDRs) that flank the structured TβRII-ECD ligand-binding domain. An example of these single-stranded bivalent traps, T22d35, demonstrated approximately 100-fold higher TGF-β neutralization efficiencies than the monovalent non-engineered TβRII ectodomain, while the bivalent trap did not neutralize the TGF-β2 isotype and had a relatively short circulating half-life.

It would be useful to provide TβRII-ECD based traps with improved properties such as increased efficiency.

Brief description of the invention

The present invention provides a polypeptide construct with increased efficacy for TGFβ inhibition.

The polypeptide construct of the present invention contains a first region and a second region, where the first region contains the first and/or second ectodomain of the TGFβ receptor (ECD); and where the second region contains the second constant domain ( _CH 2) and/or the third constant domain ( _CH 3) of the heavy chain of the antibody. In a preferred non-limiting embodiment, the C-terminus of the first region is linked to the N-terminus of the second region. In a preferred non-limiting embodiment, the first region of the polypeptide construct contains a first TβRII-ECD (ECD1) and/or a second TβRII-ECD (ECD2), where ECD1 and ECD2 are connected in series.

A polypeptide construct is provided wherein the first region contains a (TβRII-ECD)-(TβRII-ECD) doublet linked at its C-terminus to an antibody constant domain that inhibits TGFβ activity with neutralization at least 600 times more effective than a similar construct, having a single TβRII-ECD linked at its C-terminus to the constant domain of an antibody (i.e. when there is no second ECD, also referred to herein as a singlet).

Provided is a polypeptide construct comprising a second TGFβ receptor ectodomain ECD coupled in series to the first ECD, wherein the polypeptide construct (i.e., the doublet ECD construct) coupled to an antibody constant domain exhibits TGFβ neutralization (inhibition) that is at least 100 200, 300, 400, 500, 600, 700, 800, or 900 times greater than a similar construct in which the antibody constant domain is absent (i.e., an ECD doublet construct, also referred to herein as a non-Fc coupled doublet) .

In connection with TβRII-ECD and the potency with which they inhibit TGF-β activity, it has been found that unexpectedly increased efficiencies may be the result of careful selection of their components. This occurs when certain TβRII-ECDs are connected in series and their C-terminus is linked to the N-terminus of an antibody constant domain (Fc). When they are linked in a dimeric form containing two such polypeptides cross-linked via a cysteine bridge between the constant domain(s) of each polypeptide, the resulting so-called "Fc-linked" having two TβRII-ECDs (i.e. ECD "doublet") on each of the two "branches", can show inhibitory activity that is more than 600 times greater for TGF-β1 and more than 20 times greater for TGF-β3 compared to "Fc-related", having one an ectodomain on each of the two "branches", as demonstrated by the inhibition of TGF-β1 and β3-induced IL-11 secretion, among others, by human non-small cell lung cancer (NSCLC) A549 cells. The improvement in efficacy is evident when compared to counterparts that either lack an Fc region or lack a second ECD (ie, a "singlet" ECD). The efficiency increase is at least 100, 200, 300, 400, 500, or 600 times greater for an Fc-bound doublet compared to an Fc-bound singlet. The increase in activity is at least 100, 200, 300, 400, 500, 600, 700, 800, 900 times and about 1000 times greater for the Fc-bound doublet compared to the Fc-bound doublet. The increase in potency is evident when compared to analogues that either lack the Fc region (the Fc-bound T22d35-Fc doublet is 972-fold and 243-fold better for TGF-β1 and TGF-β3, respectively, than the non-Fc-bound doublet) or in which there is no second ECD (ECD "singlet"; the Fc-bound T22d35-Fc doublet is 615-fold and 24-fold higher for TGF-β1 and TGF-β3, respectively, than the non-Fc-bound doublet). More specifically, the Fc-linked doublet (T22d35-Fc) shows a potency enhancement that is at least 970-fold greater for TGF-β1 and at least 240-fold greater for TGF-β3 compared to the ECD construct. doublet associated with Fc. In addition, the Fc-linked doublet (T22d35-Fc) exhibits an enhancement efficiency that is at least 600-fold greater for TGF-β1 and more than 20-fold greater for TGF-β3 compared to the Fc-linked singlet (T2m -Fc).

In a general aspect, a polypeptide construct is provided comprising at least two TβRII-ECDs connected in series (i.e., an ECD doublet) and an antibody constant domain comprising at least a second constant domain ( _CH 2) and/ or a third constant domain ( _CH 3 ) of the antibody heavy chain, wherein the C-terminus of the ectodomains is linked to the N-terminus of the antibody constant domain. In this form, the construct is a single chain polypeptide. Thus, an antibody constant domain may contain only a C _H 2 domain, or it may contain a C _H 2 domain and a C _H 3 domain.

In embodiments, the polypeptides are provided from linked dimeric polypeptides containing two single chain polypeptides and covalently linking crosslinkers.

In other embodiments, the two ectodomains are the same in terms of their overall binding targets and/or their types of targets.

In a preferred embodiment, the first region contains two TGF-β receptor ectodomains (TGFβR-ECD or "TβR-ECD"). In a preferred embodiment, the TβR-ECD is the ectodomain of the TGF-β type II receptor (TβRII-ECD). In a preferred embodiment, the TβR-ECD contains SEQ ID NO: 1 and a sequence essentially identical to it.

The second region may contain a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 24 and a sequence substantially identical thereto.

In a preferred embodiment, the second region of the polypeptide construct of the present invention may further comprise C _H 1. In embodiments, the constructs are monofunctional.

The polypeptide constructs of the present invention use the heavy chain of an antibody of human origin. In a preferred embodiment, the antibody heavy chain is selected from the group consisting of human IgG1 (SEQ ID NO: 15) and IgG2 (SEQ ID NO: 24).

Thus, in another aspect, a polypeptide construct according to the present invention is provided, wherein the construct is a dimeric polypeptide; where the dimeric polypeptide contains:

(i) a first single chain polypeptide comprising a second region containing a second constant domain (CH ₂ ) and a third constant domain ( _CH 3) of the antibody heavy chain, and a first region containing two TGF-β receptor ectodomains (TβRII-ECD ), where the C-terminus of the first region is connected to the N-terminus of the second region, and

(ii) a second single chain polypeptide comprising a second region containing a second constant domain (CH ₂ ) and a third constant domain ( _CH 3) of the heavy chain of the antibody, and a first region containing two ectodomains of the TGF-β receptor (TβRII-ECD ) connected in tandem, wherein the C-terminus of the first region is linked to the N-terminus of the second region, and the first single chain polypeptide is cross-linked to the second single chain polypeptide.

Also provided is a nucleic acid molecule encoding any polypeptide construct of the present invention. Also provided is a vector containing a nucleic acid molecule according to the present invention.

Also provided is a composition comprising one or more independently selected polypeptide constructs of the present invention and a pharmaceutically acceptable carrier, diluent or excipient.

Also provided is a transgenic cellular host containing a nucleic acid molecule or a vector according to the present invention. The transgenic cellular host may further comprise a second nucleic acid molecule or a second vector encoding a second polypeptide construct, when the second polypeptide construct is the same as or different from the first polypeptide construct. A second nucleic acid molecule or a second vector is necessarily present when the two polypeptide constructs are different (heterodimeric), but not necessary when the constructs are the same (homodimeric).

Also provided is the use of a polypeptide construct of the present invention for the treatment of a medical condition, disease or disorder; where the medical condition, disease, or disorder includes, but is not limited to, cancer, eye disease, fibrotic disease, or genetic disorders of the connective tissue, and immune disorders.

The polypeptide construct of the present invention may contain C _H 2 and C _H 3 or only C _H 2 from the heavy chain of an antibody of human origin. For example, without limitation, the heavy chain of an antibody can be selected from the group consisting of human IgG1 and IgG2. In embodiments, the constant domain in the constructs is C _H 2 per se or C _H 3 per se or C _H 2- _CH 3. Accordingly, the antibody heavy chain component provides disulfide crosslinking between single chain polypeptide constructs that are the same or different. It is also expedient that the heavy chain of the antibody provides for the isolation of a dimeric polypeptide based on Protein A, which is produced by the host cells.

In embodiments, the ectodomain region of the receptor contains two independently selected ectodomains that are connected in series, that is, in a linear manner. In some embodiments, the ectodomains are the same in sequence, or at least the same with respect to their target ligand.

The present invention also provides a nucleic acid molecule encoding polypeptide constructs as described herein. A vector containing the nucleic acid molecule just described is also included in the invention. The invention also includes a transgenic cellular host comprising a nucleic acid molecule or a vector as described herein; the cell host may further include a second nucleic acid molecule or a second vector encoding a second polypeptide construct other than the first polypeptide construct. The systems used to produce the polypeptides of the present invention may be secretion systems, especially when dimerization through disulfide bridges is required, and the expression polynucleotides thus encode secretion signals that are cleaved by the host upon secretion into the culture medium.

Compositions containing one or more independently selected polypeptide constructs described herein and a pharmaceutically acceptable carrier, diluent or excipient are also included in the present invention.

These and other features of the invention will now be described by way of example with reference to the accompanying drawings, where:

Brief description of the drawings

Fig. 1 A/B is a schematic representation of the TGF-β type II receptor ectodomain (TβRII-ECD; also abbreviated T2m) and one chain of linked T2m domains (also abbreviated T22d35) (FIG. 1A); On FIG. 1B shows the corresponding amino acid sequences, where the natural linker sequences (SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8) are underlined and the TβRII structural domain sequence (SEQ ID NO: 4) is in bold.

Fig. 2A-G, where FIG. 2A/B/C is a schematic representation of T2m and T22d35 modules linked to the N-terminus of the heavy chains of the IgG2 Fc region (2A) to generate T2m-Fc (2B) and T22d35-Fc (2C) fusion proteins; On FIG. 2D shows the amino acid sequence of the T2m-Fc and T22d35-Fc fusion proteins (SEQ ID NO: 9, SEQ ID NO: 10). On FIG. 2E shows an aligned sequence of additional variants of the linker region between the Fc region and the ECD in the T22d35-Fc fusion constructs. On FIG. 2F and 2G show the amino acid sequence of T22d35-Fc linker variants using human IgG1 Fc (FIG. 2F; SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20) and the human IgG2 Fc region. (Fig. 2G; SEQ ID NO: 23). Natural linker sequences (SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8) are underlined, TβRII structured domain sequence (SEQ ID NO: 4) is shown in bold, and human Ig G Fc sequence variants (SEQ ID NO: : 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 24) are shown in bold italics.

Fig. 3 A/B/C shows the preparative SEC elution profile for the T2m-Fc fusion protein (3A); Fractions (fr.) 6-11 were combined and concentrated. The protein integrity of the purified SEC material was then assessed by UPLC-SEC profile (3B) and SDS-PAGE evaluation under non-reducing (NR) and reducing (R) conditions (3C).

Fig. 4 A/B/C shows the preparative SEC elution profile for the T22d35-Fc fusion protein (4A); Fr. 7-10 were combined and concentrated. The protein integrity of the purified SEC material was then assessed by UPLC-SEC profile (4B) and SDS-PAGE evaluation under non-reducing (NR) and reducing (R) conditions (4C).

On FIG. 5 A/B shows SDS-PAGE analysis of protein A purified T22d35-Fc, T22d35-Fc-IgG2-v2(CC), T22d35-Fc-IgG1-v1(CC), T22d35-Fc-IgG1-v2(SCC), T22d35 -Fc-IgG1-v3(GSL-CC), hIgG1FcΔK(C)-T22d35, hIgG1FcΔK(CC)-T22d35 and hIgG2FcΔK(CC)-T22d35 variants under non-reducing (A) and reducing (B) conditions.

Fig. 6 A/B/C represents the percentage of intact monomer (A), aggregates (B), and fragments (C) of various fusion proteins, indicating the advantage of expressing the T22d35 doublet at the N-terminus of the IgG Fc portion. The table shows the numerical differences of the parameters that were analyzed.

Fig. 7 A/B/C presents a functional evaluation of the T2m-Fc and T22d35-Fc fusion proteins compared to the non-Fc single chain T22d35 decoy in the IL-11 A549 release assay. Neutralization of TGF-β1 (5A), -β2 (5B), -β3 (5C) was assessed and calculated as % of the TGF-β control (average value in a triplicate experiment +/- SD). The table shows the calculated IC ₅₀ values calculated in Graphpad Prism (4-PL algorithm (logarithm (inhibitor) versus response - variable slope (four parameters)).

On FIG. 8 shows the functional evaluation of T22d35-Fc, T22d35-Fc-IgG2-v2 (CC), T22d35-Fc-IgG1-v1 (CC), T22d35-Fc-IgG1-v2 (SCC) and T22d35-Fc-IgG1-v3 (GSL -CC) compared to C-terminal variants of the T22d35 trap associated with Fc in the A549 IL-11 release assay. Neutralization of TGF-β 1 was assessed and calculated as % of the control TGF-β 1 (mean of a three-fold experiment +/- SD). The table shows the calculated IC ₅₀ values calculated in Graphpad Prism (4-PL algorithm (logarithm (inhibitor) vs response - variable slope (four parameters)).

On FIG. 9 shows a functional evaluation of TGF-β1, -β2 and -β3 neutralization by the T22d35-Fc-IgG1-v1 (CC) variant in the A549 IL-11 release assay. Neutralization of TGF-β was assessed and calculated as % of the control TGF-β (mean of a three-fold experiment +/- SD). The table shows the calculated IC ₅₀ values calculated in Graphpad Prism (4-PL algorithm (logarithm (inhibitor) vs response - variable slope (four parameters)).

Fig. 10. In vivo functional evaluation of the T22d35-Fc fusion protein in the MC-38 mouse syngeneic colon carcinoma model. (A) Tumor volumes were calculated as described and plotted as mean tumors +/- SD per group. Two-way ANOVA analysis was used to analyze statistically significant differences between the calculated mean tumor volumes in the T22d35-Fc and CTL IgG treatment cohorts over time. In addition, MC-38 tumor growth (estimated volume) was plotted for each mouse for CTL IgG (B) and T22d35-Fc (C) treated cohorts.

Additional aspects and advantages of the present invention will become apparent in view of the following description. The detailed descriptions and examples, while indicating preferred embodiments of the invention, are given by way of illustration only, as various changes and modifications within the scope of the invention will become apparent to those skilled in the art.

Detailed description of the invention

Currently, polypeptide constructs are provided that bind and neutralize all isoforms of transforming growth factor beta (TGF-β1, -β2 and -β3). These polypeptides use TGF-β receptor ectodomains to capture or sequester various TGFβ species, including TGF-β1 and TGF-β3, and to some extent including TGF-β2. The efficiency with which the present polypeptide constructs neutralize TGF-β1 and -β3 is unexpectedly much higher than that of related constructs, as demonstrated herein. For this reason, the present constructs are expected to be particularly useful as pharmaceuticals for the treatment of medical conditions such as cancer, fibrotic diseases, and certain immune disorders.

The present polypeptide constructs contain two TβR-ECDs, such as TβRII-ECD, which are connected in series (C-terminus to N-terminus) and additionally contain an antibody constant domain that contains at least a second constant domain (C _H 2) and/ or a third constant domain ( _CH 3) of the antibody heavy chain. The constant domain of an antibody (Fc) is linked at its N-terminus to the C-terminus of the ectodomain. The presence of the ectodomain as a doublet and the presence of this doublet bound to the N-terminus of the constant domain of the antibody provides a "trap" with a 615-fold increased neutralization efficiency for TGFβ1 and 24-fold for TGFβ3 compared to a construct having one ECD associated with N the end of the constant domain of the antibody.

As used herein, the term TβRII-ECD refers to the extracellular region of the type II TGFβ receptor that binds to a TGFβ ligand. In a preferred embodiment of the present constructs, the TGFβ-RII ectodomain is a TGFβ-R (i.e., TβRII-ECD) species ectodomain containing a sequence that forms a stable three-dimensional folded structure:

QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF (SEQ ID NO:4).

In bound form, which contains a flexible natural flanking sequence, ECD can include underlined structures, as shown below:

IPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPD (SEQ ID NO:1)

This sequence binds to the TGFβ ligand isotypes designated TGF-β1 and TGF-β3. The binding affinity for TGF-β2 is less.

In the present polypeptide constructs, two TβRII-ECDs may contain the same sequence. Two ectodomains are connected in series, resulting in a linear polypeptide in which the C-terminus of one ectodomain is linked to the N-terminus of the other ectodomain.

The two ectodomains can be linked by direct linkage so that no additional amino acid residues are introduced. Alternatively, additional amino acid residues may form a linker that connects the two receptor ectodomains in series. In the protein construct of the present invention, the first and second regions of the polypeptide construct of the present invention are also connected. The term "connected" means that the two regions are covalently linked. The chemical bond may be achieved by a chemical reaction or may be the product of recombinant expression of two regions in the same polypeptide chain. In a specific non-limiting example, the C-terminus of the first region is linked directly to the N-terminus of the second region, i.e. there are no additional "linker" amino acids between the two regions. In the case where the linker is absent, that is, when the two regions are directly linked, there will be a direct link between the C-terminus of the complete ectodomain and the N-terminus of the constant regions of the C _H 2-CH ₃ antibody. For example, in the Fc-linked variant ( SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 24) with SEQ ID NO: 1 via an internal misorder linker with SEQ ID NO: 8, which is part of TβRII-ECD, having SEQ ID NO: 1 (i.e. no additional "linker" amino acids added), one links aspartic acid at the last position of SEQ ID NO: 1 to the glutamic acid, threonine, valine, or valine found at the first position of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, or SEQ ID NO: 24, respectively.

Common practice in the manufacture of fusion constructs is to introduce glycine or glycine-serine linkers (GSL) such as GGGGS or [G4S] _n (where n is 1, 2, 3, 4 or 5 or more, for example 10, 25 or 50) between related components. As indicated in the paragraph above, the fusion polypeptides of the present invention can be obtained by direct linkage without the use of any additional amino acid sequence, except for those present in the Fc region and in the region of the ectodomain of the receptor. Thus, the use of foreign sequences as linkers can be refrained from, providing an advantage due to their potential undesirable immunogenicity and their added molecular weight. Entropy factors are also a potential responsibility of glycine and glycine-serine linkers, which are very flexible and can become partially restricted when binding to a target, resulting in a loss of entropy unfavorable for binding affinity. Therefore, only the flexible, internally random N-terminal regions of TβRII-ECD have been used as natural linkers in embodiments of the present invention. However, the specific amino acid compositions and lengths of these substantially random linkers (e.g., SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8) did not accurately predict whether the resulting direct link constructs would have the desired geometry and favorable molecular interactions for proper binding to their putative dimeric ligands. In other embodiments, the fusion polypeptide may include a flexible artificial GSL as illustrated in the construct in SEQ ID NO: 20, wherein the GS linker of SEQ ID NO: 21 is inserted between the aspartic acid (D) at the last position of the TβRII-ECD having SEQ ID NO: : 1, and threonine (T) at the first position of the Fc region variant having SEQ ID NO: 15.

The first and second regions of the polypeptide construct, in embodiments, are linked by natural polypeptide internally randomized linkers selected from the group consisting of SEQ ID NOs: 8, 13, 16, 19, 25 and a sequence substantially identical to them. In other embodiments, the regions of the polypeptide constructs are connected by flexible linkers selected from the group consisting of SEQ ID NO: 21 and SEQ ID NO: 22, and a sequence substantially identical to them.

In this embodiment, one region of the present polypeptide constructs contains a TβRII-ECD doublet containing the first and second receptor ectodomains connected in series by a natural internally random polypeptide linker of SEQ ID NO: 6 and having an amino acid sequence containing:

IPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETV CHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPD (SEQ ID NO:5).

The present constructs also contain an antibody constant domain containing region containing at least a second constant domain ( _CH 2 ) and/or a third constant domain ( _CH 3 ) of the heavy chain of the antibody. The antibody constant domain is linked at its N-terminus to the C-terminus of the ectodomain doublet so that the orientation of the construct is one strand TβRII-ECD (link) TβRII-ECD (link) C _H 2-C _H 3.

The constant domain of the antibody provides cross-linking between two of the presented polypeptide constructs. This is achieved when the expressed polypeptide constructs are secreted by their expressing host. Thus, the production of a single chain polypeptide provides a construct in dimeric form in which two constructs are cross-linked via disulfide bridges that include one or more cysteine residues in each of the antibody constant domains present in each of the constructs.

The constant domain of the antibody present in the construct is desirably derived from the constant region of IgG, and especially from the constant domain of either IgG1 or IgG2.

The provided constructs are monofunctional in the sense that the constant region itself may not have any particular activity other than to act as a structure through which dimers of the polypeptide constructs can be formed. These minimum constant regions can also be modified to provide some advantage by including the appropriate hinge regions and optionally changing the composition of the cysteine residues. Thus, some or all of the cysteine residues included in the bridge between two Fc fragments or used naturally to bridge between the heavy and light chains of a full-length antibody may be replaced or removed. One advantage of minimizing the number of cysteine residues is to reduce the tendency to scramble disulfide bonds, which can promote aggregation. For example, these cysteine residues and their modification are seen in natural or non-natural linker sequences located around the junction of the first and second regions of the polypeptide constructs and which are listed below:

SEEYNTSNPDTHTCPPCPAPE (SEQ ID NO:16), SEEYNTSNPDVEPKSSDKTHTCPPCPAPE (SEQ ID NO:19), SEEYNTSNPDGGGSGGGSGGGTHTCPPCPAPE (SEQ ID NO:22) including human IgG1 hinge sequence variations; and SEEYNTSNPDERKCCVECPPCPAPP (SEQ ID NO:13) and SEEYNTSNPDVECPPCPAPP (SEQ ID NO:25) comprising human IgG2 hinge sequence variations; and a sequence essentially identical to them.

Not all naturally occurring inter-hinge disulfide bonds need to form for Fc homodimerization, and it should be noted that the stability of the Fc homodimer may depend on the number of intermolecular disulfide bridges.

As used herein, an "antibody", also referred to in the art as an "immunoglobulin" (Ig), refers to a protein constructed from paired heavy and light polypeptide chains. The structure of the antibody and each of the domains is well known and well known to those skilled in the art, although summarized herein. When an antibody is properly folded, each chain folds into a series of individual globular domains connected by more linear polypeptide sequences; the light chain of an immunoglobulin folds into a variable ((V _L ) and constant (C _L ) domain, while the heavy chain folds into a variable (V _H ) and three constant (CH ₁ , C _H 2, C _H 3) domains. After pairing, the interaction of the variable domains of the heavy and light chains (V _H and V _L ) and the first constant domain (C _L and C _H 1) leads to the formation of a Fab (fragment, antigen-binding) containing the binding region (Fv); the interaction of the two heavy chains results in to the pairing of the _CH 2 and _CH 3 domains, resulting in the formation of Fc (fragment, crystallizing) The characteristics described here for the CH ₂ and _CH 3 domains also apply to Fc.

In the present invention and specific embodiments thereof, polypeptide constructs that exhibit significantly improved performance include the following:

T22d35-Fc:

IPPHVQKSVNNMDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPD ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:10)

T22d35-Fc-IgG2-v2 (CC):

IPPHVQKSVNNMDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPD VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:23)

T22d35-Fc-IgG1-v1 (CC):

IPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPD THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:14)

T22d35-Fc-IgG1-v2 (SCC):

IPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPD VEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:17); and

T22d35-Fc-IgG1-v3 (GSL-CC):

IPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPDGGGSGGGSGGG THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:20)

In a particular embodiment, the polypeptide construct comprises a polypeptide of the present invention that exhibits significantly improved efficacy, e.g., a polypeptide having significantly improved efficacy may comprise SEQ ID NO: 10, SEQ ID NO.14, SEQ ID NO: 17, SEQ ID NO : 20 or SEQ ID NO: 23. In another specific embodiment, the polypeptide construct may be a homodimer comprising two polypeptides that exhibit significantly increased potency; for example, if the polypeptide construct that exhibits significant increased efficacy is SEQ ID NO: 14, the polypeptide construct is a homodimer containing two polypeptide constructs, where each polypeptide construct contains SEQ ID NO.14. Similarly, if the polypeptide that exhibits increased efficacy is SEQ ID NO: 10, 14, 17, 20, or 23, the homodimer of the present invention also contains two polypeptide constructs, where each polypeptide of the homodimer is SEQ ID NO: 10, 14. 17, 20 or 23 respectively.

As noted, these single chain polypeptide constructs will dimerize upon secretion from the producing host to form a dimeric polypeptide construct comprising two single chain polypeptides linked by disulfide bridges that form between the constant domains of the two single chain polypeptides.

By "significantly improved potency" we mean that the effect or activity of the present polypeptide construct in this dimeric form is greater than that of a similar construct when measured in an assay suitable for evaluating the biological activity of TGF-β. Appropriate means for this determination are given as an example. For example, the N-terminal Fc-bound T22d35 doublet neutralizes TGF-β to a much greater extent than the N-terminal Fc-bound T2m- singlet, as shown by TGF-β-induced release of IL-11 by A549 cells (Fig. .7).

This type of fusion construct has been observed to have advantages over several other versions of the TβRII receptor ectodomain-based molecules, including non-Fc bivalent TGF-β receptor ectodomain constructs (such as the T22d35 doublet) and constructs in which one receptor ectodomain is bound with the N-terminus of the Fc region. In particular, currently proposed Fc-related constructs have improved processability due to the presence of the Fc region (eg, purification can be performed using protein A chromatography). The Fc region also allows for an increase in the half-life from the circulation. Importantly, the present constructs have significantly higher TGF-β neutralization values compared to the Fc-bound singlet (T2m-Fc) and the non-Fc-bound doublet (T22d35). The N-terminal constructs of the TGF-β ECD Fc-coupled doublet (T22d35-Fc) are advantageous in significantly improving TGF-β ligand neutralization efficiency (as shown, for example, in over 970-fold improvement in TGF-β1 neutralization relative to the doublet, not associated with Fc, as shown in Fig. 7). In addition, they show improved processability as evidenced by biophysical analysis showing a monomer content of greater than 99% (i.e., minimal presence of aggregates and no fragments of purified T22d35-Fc N-terminal fusion constructs) (as shown in Fig. 6). Thus, an advantage of the present invention is the unexpected high efficiency of neutralization of the TGF-β ligand, including some degree of neutralization of TGF-β2, which is not observed with T2m-Fc (Fc-bound singlet) or T22d35 (non-Fc-bound) constructs.

In specific embodiments, the second region of the polypeptide construct of the present invention is selected from the group of sequences showing variations in the N-terminal sequence as illustrated by SEQ ID NOs: 12, 15, 18, 24. They may differ in length and number of cysteine residues retained from hinge region, as a means to modulate the degree of dimerization of the Fc region and hence affect both efficiency and processability. Thus, in embodiments, the polypeptide construct contains a variation in the constant domain, wherein at least one crosslinking cysteine residue is removed or substituted. Suitable substitutions include serine or alanine, and preferably serine.

A substantially identical sequence may contain one or more conservative amino acid mutations that still fold correctly when secreted into the culture medium. It is known in the art that one or more conservative amino acid mutations in a control sequence can produce a mutant peptide without significant change in physiological, chemical, physicochemical, or functional properties compared to the control sequence; in such a case, the control and mutant sequences will be considered "substantially identical" polypeptides. A conservative amino acid substitution is defined here as the replacement of an amino acid residue with another amino acid residue with similar chemical properties (eg, size, charge, or polarity). These conservative amino acid mutations can be introduced into the framework regions while maintaining the overall structure of the constant domains; thus the Fc function is preserved.

In a specific non-limiting example, the first region of the polypeptide construct according to the present invention may contain a type II TGF-β receptor, such as:

IPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPD (SEQ ID NO:1, also referred to here as T2m).

In preferred embodiments, the polypeptide constructs comprise a TβRII-ECD "doublet" in which a TβRII-ECD is sequentially linked to another TβRII-ECD, wherein the ectodomains may be the same or different ectodomains of the TGF-β superfamily receptor, such as:

IPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF

-linker-

QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPD (SEQ ID NO:5, also referred to here as T22d35), where in one non-limiting embodiment, the linker corresponds to SEQ ID NO: 6; and

a sequence that is essentially identical to it. The term "substantially identical" is as defined above.

The ectodomain doublet may include the same or different ectodomains, both belonging to the TGFβ superfamily receptor family. In embodiments, the ectodomains bind the same target. In other embodiments, the ectodomains belong to the same kind of receptor. In other embodiments, the implementation of the ectodomains are identical and, therefore, are homomeric.

For example, the polypeptide constructs of the invention may have a TGF-β neutralizing potency selected from the group having at least 900-fold and 200-fold greater potency than the T22d35 doublet-only construct for TGF-β1 and TGF-β3, respectively. . For example, in an IL-11 release assay, a construct with a T22d35-Fc doublet was about 972 times more effective at neutralizing TGF-β1 and about 243 times more effective at neutralizing TGF-β3 compared to a single T22d35 doublet without Fc.

In another example, the construct is at least 600-fold and at least 20-fold more efficient at neutralizing TGF-β1 and TGF-β3, respectively, than a construct in which the antibody constant domain is linked to a single ectodomain rather than a doublet. The polypeptide constructs of the present invention can have at least 615 and 24 times better neutralization efficiency against TGF-β1 and TGF-β3, respectively, compared to a construct in which the antibody constant domain is linked to a single ectodomain rather than a doublet.

The neutralization efficiency can be summarized as follows: the neutralization efficiency of the Fc-linked doublet (ECD-ECD-Fc) is greater than that of the Fc-linked singlet (ECD-Fc); i.e., ECD-ECD-Fc is greater than ECD-Fc, while ECD-Fc is more effective than an Fc-free doublet (ECD-ECD), and an Fc-free doublet is more effective than an unbound singlet with Fc; those. ECD-ECD-Fc more than ECD-Fc more than ECD-ECD more than ECD). From a processability point of view, the presence of the Fc protein allows the use of protein A purification and avoids the need for cleavable labels. In addition, the location of the ECD singlet or doublet at the N-terminus of the Fc moiety prevents aggregation problems due to mismatching of cysteine residues in the hinge region of the Fc moiety. Therefore, a singlet or doublet linked to the N-terminus of the Fc moiety provides improved processability compared to C-terminal fusion constructs (N-terminal fusion constructs have a higher percentage of monomer particles, fewer aggregates, fewer fragments). In addition, there is an unexpected significant increase in TGF-β neutralization efficiency for all TGF-β isotypes for the N-terminal Fc-linked ECD doublet construct compared to the N-terminal Fc-linked T2m ECD singlet.

Furthermore, when the polypeptide constructs of the present invention include a TβRII-ECD that binds TGF-β, the polypeptide construct can neutralize all three TGF-β isotypes (i.e., TGF-β1, TGF-β2, and TGF-β3) to varying degrees.

The polypeptide constructs of the present invention have been evaluated in cellular assays to have a TGF-β neutralizing potency that is significantly higher (20-fold or more) than bivalent comparator polypeptides, i.e. non-Fc bound T22d35 (doublet only) and T2m-Fc (Fc-related singlet). In the range of polypeptide constructs of the present invention, those containing two or more copies of TβRII-ECD linked to the N-terminus of the Fc constant region have a potency that is higher than those containing only one copy as assessed by cellular assays.

The polypeptide construct of the present invention is expressed as a single chain polypeptide. Upon expression, the polypeptide construct of the present invention forms a dimer where the C _H 2 and _CH 3 domains of the respective polypeptide constructs interact to form a properly assembled Fc region that occurs when the expressed products are secreted into the culture medium.

The polypeptide construct of the present invention may also contain additional sequences to aid in the expression, detection, or purification of the recombinant antibody or fragment thereof. Any such sequences or labels known to those skilled in the art may be used. For example, and without limitation, an antibody or fragment thereof may contain a targeting or signal sequence (for example, but not limited to ompA), a detection/purification tag (for example, but not limited to c-Myc, His ₅ , His ₆ , or His _8G ) or a combination thereof. In another example, the signal peptide may be MDWTWRILFLVAAATGTHA (SEQ ID NO: 11). In a further example, the additional sequence may be a biotin recognition site such as described in [WO/1995/04069] or in [WO/2004/076670]. As is also known to those skilled in the art, linker sequences may be used in combination with additional sequences or labels, or may serve as a detection/purification label. Accordingly, the constant region contains a protein A binding site (typically located between about C _H 2 and C _H 3) that allows extraction/isolation of a single chain polypeptide using the protein A affinity approach.

The present invention also encompasses nucleic acid sequences encoding molecules as described above. Given the degeneracy of the genetic code, a number of nucleotide sequences will have the property of encoding the desired polypeptide, as will be readily understood by one skilled in the art. The nucleic acid sequence can be codon optimized for expression in various microorganisms. The present invention also encompasses vectors containing nucleic acids as just described, wherein the vectors typically contain a promoter and a signal sequence that are operably linked to a construct-encoding polynucleotide to direct its expression in a selected cellular production host. The vectors may be the same or different, provided that both result in the secretion of a dimeric polypeptide construct.

In addition, the invention covers cells, also referred to here as transgenic cellular hosts, containing a nucleic acid and/or a vector, as described, encoding a first polypeptide construct. The host cells may contain a second nucleic acid and/or a vector encoding a second polypeptide construct other than the first polypeptide construct. Co-expression of the first and second polypeptide constructs can lead to the formation of heterodimers.

The present invention also covers a composition containing one or more than one polypeptide construct, as described in this document. The composition may contain a single polypeptide construct, as described above, or may be a combination of polypeptide constructs. The composition may also contain one or more polypeptide constructs of the present invention linked to one or more cargo molecules. For example, without any limitation, a composition may comprise one or more polypeptide constructs of the present invention coupled to a cytotoxic drug to form an antibody drug conjugate (ADC) of the present invention.

The composition may also contain a pharmaceutically acceptable diluent, excipient or carrier. The diluent, excipient, or carrier may be any suitable diluent, excipient, or carrier known in the art, and must be compatible with the other ingredients in the composition, with the method of delivery of the composition, and not harmful to the recipient of the composition. The composition may be in any suitable form; for example, the composition may be provided in the form of a suspension, in the form of a powder (for example, but not limited to lyophilized or encapsulated), in the form of a capsule or tablet. For example, and without limitation, when the composition is provided as a suspension, the carrier may contain water, saline, a suitable buffer, or additives to improve solubility and/or stability; recovery to obtain a suspension is carried out in a buffer at a suitable pH value to ensure the viability of the antibody or its fragment. Dry powders may also include additives to improve stability and/or carriers to increase mass/volume; for example, and without limitation, the dry powder composition may contain sucrose or trehalose. In a specific non-limiting example, the composition may be formulated to deliver the antibody, or fragment thereof, to the subject's gastrointestinal tract. Thus, the composition may include encapsulation, slow release, or other suitable techniques for delivery of the antibody or fragment thereof. A person skilled in the art can prepare suitable compositions containing the present compounds.

The constructs of the present invention can be used to treat diseases or disorders associated with overexpression or overactivation of TGF-β superfamily ligands. The disease or disorder may be selected from, but not limited to, cancer, eye disease, fibrotic disease, or genetic disorders of the connective tissue.

In the field of cancer therapy, it has recently been demonstrated that TGF-β is a key factor inhibiting the antitumor response induced by immunotherapies such as immune checkpoint inhibitors (ICIs) (Hahn & Akporiaye, 2006). In particular, the therapeutic response to ICI antibodies is primarily due to the reactivation of tumor localized T cells. Resistance to ICI antibodies is explained by the presence of immunosuppressive mechanisms that lead to a shortage of T cells in the tumor microenvironment. Thus, it is now recognized that in order to detect responses in resistant patients, it is necessary to combine ICI antibodies with agents that can activate T cells and induce their recruitment into the tumor, i.e. return the tumor phenotype "without T-cell inflammation". One publication noted that overcoming the tumor microenvironment without T-cell inflammation is the most significant next hurdle in immuno-oncology (Gajewski, 2015).

Using a TGF-β test trap, T22d35, the authors have shown that blocking TGF-β effectively reverses the tumor phenotype “without T-cell inflammation” (Zwaagstra et al, 2012). This positions anti-TGF-β molecules as potential synergistic combinations with ICI and other immunotherapeutic agents. In support of this, a 2014 study (Holtzhausen et al., ASCO poster presentation) examined the effects of a TGF-β blocker when combined with anti-CTLA-4 antibodies in a physiologically relevant transgenic melanoma model. The study demonstrated that although anti-CTLA-4 antibody monotherapy did not suppress melanoma progression, the combination of a TGF-β antagonist and anti-CTLA-4 antibody significantly and synergistically suppressed both primary melanoma growth and melanoma metastasis. These observations correlated with a significant increase in the number of effector T cells in melanoma tissues.

This document shows that the polypeptides of the present invention, having a basic structure that is T22d35-Fc, significantly reduce tumor growth in the MC-38 syngeneic mouse colon cancer model. Thus, this allows the positioning of anti-TGF-β molecules in a potential synergistic combination with other immunotherapies.

The present constructs may be useful in the treatment of fibrotic diseases, including those that affect any organ of the body, including, but not limited to, the kidneys, lungs, liver, heart, skin, and eyes. These diseases include, but are not limited to, chronic obstructive pulmonary disease (COPD), glomerulonephritis, hepatic fibrosis, postinfarction cardiac fibrosis, restenosis, systemic sclerosis, eye surgery fibrosis, and scarring.

Connective tissue genetic disorders can also be treated and include, but are not limited to, Marfan syndrome (MFS) and osteogenesis imperfecta (OI).

The present invention will be further illustrated in the following examples. However, it should be understood that these examples are for illustrative purposes only and should not be used in any way to limit the scope of the present invention.

Materials and methods

Production and cleaning

Temporary expression CHO

Each of the various variants of the TβRII-ECD fusion construct (such as T2m-Fc and T22d35-Fc) consists of a heavy chain Fc region and includes the signal sequence MDWTWRILFLVAAATGTHA (SEQ ID NO: 11) at its N-termini. The DNA coding regions for the constructs were generated synthetically (Biobasic Inc. or Genescript USA Inc.) and cloned into the HindIII (5' end) and BamH1 (3' end) sites of the pTT5 mammalian expression plasmid vector (Durocher et al, 2002) . Fusion proteins were generated by transient transfection of Chinese Hamster Ovary (CHO) cells with a T2m or T22d35 heavy chain coupled to an IgG heavy chain construct (T2m-Hc and T22d35-HC, respectively). Briefly, T2m-HC or T22d35-HC plasmid DNA were transfected into 2.5 and 4.6 L CHO-3E7 cell cultures, respectively, in FreeStyle F17 medium (Invitrogen) containing 4 mmol/L glutamine and 0.1% Kolliphor p -188 (Sigma) and maintained at 37°C. The transfection conditions were as follows: DNA (80% plasmid construct, 15% AKT plasmid, 5% GFP plasmid): PEI(polyethyleneimine)pro (ratio 1:2.5): PEIpro (Polyplus) (ratio=1:2.5) . 24 hours after transfection, 10% trypton N1 (TekniScience Inc.) and 0.5 mmol valproic acid (VPA, Sigma) were added and the temperature was shifted to 32°C to stimulate the production and secretion of fusion proteins, and then maintained for 15 days after transfection, after which the cells were harvested. At the final harvest, cell viability was 89.6%.

Stable expression by CHO pool

Pools of CHO ^BRI/rcTA cells were generated by transfection of cells with a vector expressing a target gene encoding various Fc-linked TβRII-ECDs. The next day after transfection, the cells were centrifuged for 5 min at 250 rpm and seeded at a density of 0.5×10 ⁶ cells/ml in a selective medium (PowerCHO2 medium supplemented with 50 μmol/l methionine sulfoximine). The selection medium was replaced every 2-3 days for 14-18 days by inoculation at 0.5×10 ⁶ cells/ml. Cell count and viability were measured using a Cedex Analyzer Cedex automatic cell counter as described above. When cell viability reached more than 95%, the pools were inoculated at 0.2×10 ⁶ cells/ml in 125 or 250 ml Erlenmeyer flasks. For fed-batch culture, CHO ^BRI/rcTA cell pools were inoculated as described above. On the third day after inoculation, when the cell density reached 3.5-4.5×10 ⁶ cells/ml, expression of the recombinant protein was induced by adding 2 μg/ml of cumate. The MSX concentration was adjusted to 125 µmol/L and F12.7 (Irvine Scientific) was added followed by a temperature shift to 32°C. Cultures were supplied with 5% (v:v) F12.7 every 2-3 days and samples were taken for determination of recombinant protein concentration (pA-HPLC) and glucose (VITROS 350, Orthoclinical Diagnostics, USA). Glucose was added to maintain a minimum concentration of 17 mmol/l.

cleaning

The supernatant harvested from CHO cells was filtered (0.2 μm) and loaded onto a MabSelect Sure Protein A column (GE Healthcare). The column was washed with 2 column volumes of PBS (phosphate buffered saline) and the protein was eluted with 3 column volumes of 0.1 mol/l sodium citrate, pH 3.6. Eluted fractions were neutralized with 1 mol/L Tris, and fractions containing fusion proteins were pooled and then loaded onto a 26/60 Hi-Load Superdex S200 SEC column (GE Healthcare) equilibrated in preparation buffer ( DPBS (Dulbecco's phosphate-buffered saline) without Ca ²⁺ , without Mg ²⁺ ) The protein was eluted using 1 column volume of the buffer mixture, collected in serial fractions and detected by UV absorption at 280 nm. proteins were then pooled and concentrated.The integrity of the purified Prot-A and SEC fusion proteins in pooled fractions was further analyzed by UPLC-SEC and SDS-PAGE (4-15% polyacrylamide) under reducing and non-reducing conditions (SYPRO Ruby stain). For UPLC-SEC, 2-10 μg of protein in DPBS (Hyclone, minus Ca ²⁺ , minus Mg ²⁺ ) was injected into a Waters BEH200 SEC column (1.7 μm, 4.6×150 mm) and dissolved at a flow rate of 0. 4 ml/min. for 8.5 minutes at room temperature using a Waters Acquity UPLC H-class biosystem. Protein peaks were detected at 280 nm (Acquity PDA detector).

Cell lines

A549 human non-small cell lung cancer cells were purchased from ATCC (Cat# CCL-185, Cedarlane, Burlington, ON). Cells were cultured in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 5% fetal bovine serum (FBS). MC-38 mouse colon adenocarcinoma cells were purchased from Kerafast (Cat# ENH204, Boston, MA) and cultured in DMEM supplemented with 2 mmol/l L-glutamine and 10% fetal bovine serum. Both cell lines were maintained at 37° C. in a humidified atmosphere supplemented with 5% CO ₂ .

TGF-β induced IL-11 release assay from A549 cells

A549 human lung cancer cells were seeded in 96-well plates (5×10 ³ cells/well). The next day, 10 pmol/L TGF-β in complete medium in the absence or presence of a serial dilution of the TGF-β trap fusion protein was incubated for 30 min at room temperature before being added to the cells. After 21 h incubation (37° C., 5% CO ₂ , humidified atmosphere), conditioned medium was harvested and added to MSD Streptavidin Gold plates (Meso Scale Diagnostics, Gaithersburg, MD) that were coated with 2 μg/ml biotinylated mouse anti-human IL. -11 (MAB618, R&D Systems, Minneapolis, MN). After 18 hours (4°C), the plates were washed with PBS containing 0.02% Tween 20 and then 2 µg/mL SULFO-labeled goat anti-human IL-11 antibody (AF-218-NA, R&D Systems Minneapolis, MN) was added. ) and the plates were incubated for 1 hour at room temperature. After the final wash, the plates were read on a MESO QuickPlex SQ120 machine (Meso Scale Diagnostics, Gaithersburg, MD). IL-11 readings were expressed as percent release of IL-11 compared to control cells treated with TGF-β alone. Graphpad Prism (4-PL algorithm ((logarithm (inhibitor) vs response - variable slope (four parameters)) was used to calculate IC ₅₀ (auto burst option was used when needed).

In vivo evaluation in the MC-38 mouse subcutaneous model of syngeneic mouse colon cancer

Female C57BL/6-Elite mice (5-7 weeks old) were purchased from Charles River Laboratories (Wilmington, MA). Thirteen C57BL/6 mice on day 0 were injected with 3×10 ⁵ MC-38 cells subcutaneously in the right flank. When the tumors reached a volume of 50-100 mm ³ (day 5), the animals were divided into 2 groups and treatment was started:

- Cohort 1 (7 animals): isotype control (CTL IgG; BioxCell InVivo MAb rat IgG2b, anti-KLH; clone LTF-2, Cat# BE0090); 200 μg in 100 μl of phosphate-buffered saline (PBS), intraperitoneally (ip) on days 5, 7, 9 and 11.

- Cohort 2 (6 animals): T22d35-Fc, 5 mg/kg in 100 µl PBS, i.e. on day 5, 9, 12 and 16.

Tumors were measured twice a week using digital calipers for 15 days after the start of treatment. Tumor volumes were calculated from these measurements using the modified ellipsoidal formula (T _vol =π/6×(length×width×width)) described previously (Tomayko et al., 1986).

Results and discussion

Design of fusion structures

To design TGF-β traps of interest, we constructed a TβRII-ECD singlet (designated T2m) linked to another such singlet, thereby forming an ectodomain doublet (designated T22d35) that was linked to the N-terminus of the heavy chains of the human Fc region ( h) IgG2 and Fc regions of human IgG1. On FIG. 1 shows the schemes (FIG. 1A) and amino acid sequences (FIG. 1B) of T2m and T22d35. These modules were in a construct linked to the N-terminus of heavy chains of the IgG Fc region (Figure 2A) using several linker variations (Figure 2E) to generate T2m-Fc fusion construct variants (Figure 2B) and T22d35-Fc (Fig. 2C). The sequences of these fusion constructs are shown in FIG. 2D. The authors also developed T22d35-Fc variants that explore the number of cysteine residues in the hinge region of the Fc domain, various IgG isotypes (human IgG1 vs. IgG2), and sequences of various lengths and natures as linkers between T22d35 and the N-terminus of the Fc domain (Fig. 2E, 2F and 2G). These changes are aimed at studying and ultimately optimizing the functional and technological characteristics of the T22d35-Fc design.

Expression and purification

Purification of CHO transition material

The corresponding protein fusion constructs were transiently expressed in CHO-3E7 cells (see Table 1), after which the conditioned medium was collected and purified using a Protein A affinity column followed by preparative size exclusion chromatography (SEC). The SEC elution profiles of T2m-Fc (FIG. 3A) and T22d35-Fc (FIG. 4A) showed that these fusion proteins are relatively pure and devoid of aggregates. Fractions 6-11 (T2m-Fc) and 7-10 (T22d35-Fc) were pooled and concentrated to 5.6 mg/ml (T2m-Fc) and 6.03 mg/ml (T22d35-Fc). Final yields were 267 mg and 168 mg for T2m-Fc and T22d35-Fc, respectively. The final products (indicated SEC-pooled fractions) were shown to be over 99% pure by UPLC-SEC (FIGS. 3B and 4B). SDS-PAGE evaluation (FIGS. 3C and 4C, Sypro RUBY stain) shows T2m-Fc and T22d35-Fc bands of approximately 60 kDa and approximately 90 kDa under reducing conditions, while bands of approximately 90 kDa and 150 kDa can be detected, representing completely assembled and high-purity T2m-Fc and T22d35-Fc fusion proteins, respectively, under non-reducing conditions. An overview of the details of production and purification can be found in Table 1. Together, these results demonstrate the good workability of the T2m-Fc and T22d35-Fc fusion proteins.

Table 1: Preparation (time pools) and purification details of T2m-Fc and T22d35-Fc fusion proteins.

T2m-Fc T22d35-Fc cell line CHO-3E7 CHO-3E7 Receiving method Transition Transition Receipt volume (L) 2.5 4.6 % GFP (Green Fluorescent Protein) at 24hpt (hours post-transfection) (Cellometer K2) 38.5 41 Duration of acquisition (days after transfection) 15 15 Mean cell viability @ yield (%) 88.43 89.4 Final volume (l; after 0.2 µm filtration) 2.458 4.368 Titer (mg/ml: pA-HPLC) 139 54 Maximum Expected Output (mg) 341 235 Final yield (mg) 267 168 Recovery (%) 78.30 71.49

Purification of the material of stable AtoN pools

The N- and C-terminal variants of the Fc-linked T22d35 constructs were stably expressed in CHO ^BRI/rcTA cells to compare their level of expression and some of their biophysical properties. The coding region of each variant was ligated into mammalian cell expression plasmids, and after transfection, an enriched pool of cells was selected that stably expressed each variant. The main difference between the variants can be found in the amino acid sequence constituting the linker region that separates the T22d35 doublet from the Fc domain (in the case of N-terminal fusion constructs), while for the C-terminal Fc-containing fusion constructs, the difference between each of the variants is extreme. amino-terminus of the protein (Table 2).

Table 2: Description of amino acid variation in the linker region of the N-terminal and C-terminal Fc-related T22d35 constructs (bold: natural linker sequence; italics: artificial linker sequence). Paired cysteine residues in each of the variants are underlined.

Fusion proteins were purified by protein A affinity and 100 mmol/l citrate (pH 3.6) was used as elution buffer. Samples of the eluted fusion protein were neutralized with 1 mol/L HEPES, then subjected to buffer exchange with DPBS using Zeba spin columns (Table 3), while the integrity of some of the purified fusion proteins was assessed by SDS-PAGE (FIG. 5). The cleaning of each option was similar. Although many properties were very similar between variants, the potential for aggregation, which is indicative of misfolding, revealed some differences. Protein aggregation may be indicative of a decrease in conformational stability and may result in reduced potency, efficacy, or efficacy. HPLC size exclusion chromatography (SEC-HPLC) was used to determine the purity of each of the N- and C-terminal Fc-related constructs. This method accurately measures the percentage of intact monomer particles as well as the presence of impurities such as aggregates and/or degradation products. As shown in FIG. 6, there can be a striking difference between T22d35 variants expressed as Fc-linked N-terminal constructs and those expressed as C-terminal fusion constructs. In particular, the percentage of intact monomer (FIG. 6A) was approximately 99% for all five N-terminal variants of the fusion constructs (SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23), while it was markedly lower for the three C-terminal fusion constructs (SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28). This significant decrease in the percentage of intact monomer is the result of the accumulation of higher molecular weight aggregates seen in all Fc-linked C-terminal constructs (Fig. 6B) as well as the increase in lower molecular weight fragments in two of the three C-terminal constructs associated with Fc (Fig. 6C). In addition, evaluation of the titres of individual 500 ml products and the average titers of the N-terminal T22d35 constructs associated with Fc and the C-terminal constructs T22d35 containing Fc indicates that N-terminal variants of the T22d35 constructs containing Fc can be obtained at higher values. output compared to C-terminal fused structures (Table 4). Taken together, these results indicate that there are significant and unexpected advantages to expressing the T22d35 doublet at the N-terminus of fragments such as the Fc portion of an immunoglobulin. Taken together, these data demonstrate the increased workability of the N-terminal Fc T22d35 fusion proteins.

Table 3: Overview of protein yields of T22d35 variants after purification of 500 ml of stable pool material.

T22d35-Fc-IgG2 (CCCC) T22d35-Fc-IgG2-v2 (CC) T22d35-Fc-IgG1-v1 (CC) T22d35-Fc-IgG1-v2 (SCC) T22d35-Fc-IgG1-v3 (GSL-CC) hIgG1FcΔK(C)-T22d35 hIgG1FcΔK(CC)-T22d35 hIgG2FcΔK(CC)-T22d35 cell line CHO-55E1 CHO-55E1 CHO-55E1 CHO-55E1 CHO-55E1 CHO-55E1 CHO-55E1 CHO-55E1 Production Method stable pool stable pool stable pool stable pool stable pool stable pool stable pool stable pool Volume received at the start (l) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Duration of receipt (days after induction) 10 10 10 10 10 10 10 10 Average Cell Viability at Output (%) 69.4 97 97.6 95.1 98 94.3 95.7 90.3 Final volume (l; after 0.2 µm filtration) 0.65 0.65 0.65 0.65 0.65 0.65 0.65 0.65 Titer (mg/L: pA-HPLC) 212 214 556 353 526 260 339 119 Maximum Expected Output (mg) 150 150 150 150 150 150 150 150 Final yield (mg) 67.34 62.17 81.64 81.74 74.84 70.95 81.49 48.73 Recovery (%) 44.89 41.45 54.43 54.49 49.89 47.30 54.33 32.49

Table 4: Comparison of titers of individual N- and C-terminal Fc T22d35 fusions

Fc-linked N-terminal constructs Fc-linked C-terminal constructs Titer improvement (N-/C-terminal Fc-related construct) Option Titer(mg/ml) Average titer (mg/ml) Option Titer (mg/ml) Average titer (mg/ml) hIgG1 T22d35-Fc-IgG1-v1 (CC) 556 478 hIgG1FcΔK(C)-T22d35 260 300 63% T22d35-Fc-IgG1-v2 (SCC) 353 hIgG1FcΔK(CC)-T22d35 339 T22d35-Fc-IgG1-v3 (GSL-CC) 526 hIgG2 T22d35-Fc (CCCC) 212 213 hIgG2FcΔK(CC)-T22d35 119 119 56% T22d35-Fc-IgG2-v2 (CC) 214

Functional evaluation in vitro

An IL-11 release assay from A549 cells was used to compare the TGF-β neutralization efficiency of the T2m-Fc and T22d35-Fc fusion proteins and a non-Fc single chain T22d35 decoy doublet construct, as shown in FIG. 7A/B/C. These data show that for all TGF-β isotypes, the activity of T22d35-Fc is superior to that of T2m-Fc and the non-Fc-coupled T22d35 single chain decoy construct with calculated IC ₅₀ (Table 5) of 0.003348 and 0.003908 nmol/L for TGF-β1 and TGF-β3, respectively. These values demonstrate a potency that is at least 970 times and at least 240 times higher than for T22d35 (IC ₅₀ =3.253 and 0.9491 nmol/L, for TGF-β 1 and TGF- β3, respectively), as well as 615 and 24 times higher than for T2m-Fc (IC ₅₀ =2.059 and 0.0943 nanomol/l, in relation to TGF-β1 and TGF-β3, respectively). In addition, T22d35-Fc neutralizes TGF-β2, although to a much lesser extent than TGF-β1 and -β3. In contrast, TGF-β2 neutralization is not observed for either the T2m-Fc single chain trap or T22d35. It should be noted that while T22d35-Fc trap neutralization efficiency is similar for TGF-β1 and -β3, the T2m-Fc variant showed approximately 22-fold higher neutralization efficiency for TGF-β3 compared to TGF-β1 (2.059 nmol/L and 0.0943 nanomol/l, respectively). Evaluation of additional N-terminal Fc-related T22d35 constructs [T22d35-Fc-IgG2-v2 (CC), T22d35-Fc-IgG1-v1 (CC), T22d35-Fc-IgG1-v2 (SCC) and T22d35-Fc-IgG1 -v3 (GSL-CC)] (FIG. 8, Table 6) showed that all of these fusion constructs exhibited comparable TGF-β1 neutralization efficiencies, which were very similar to those of T22d35-Fc. Additional evaluation of the T22d35-Fc-IgG1-v1 (CC) variant (FIG. 9) confirms that, according to the T22d35-Fc variant, its neutralization potency for TGF-β1 and -β3 is very similar (IC ₅₀ = 0.003327 nmol/ l and 0.003251 nmol/l, respectively), while this efficiency is much lower in relation to TGF-β2 (IC ₅₀ = 17.33 nmol/l).

Table 5: Overview of the statistical evaluation of the curves shown in FIG. 5 using the 4-PL algorithm (log (inhibitor) vs response - variable slope (four parameters)) available in Graphpad Prism.

TGF-β1 TGF-β2 TGF-β3 T22d35 T2m-Fc T22d35-Fc T22d35 T2m-Fc T22d35-Fc T22d35 T2m-Fc T22d35-Fc Incline -1.236 -1.088 -1,991 -0.08682 approximately -16.05 approximately -4.763 -1.022 -0.965 -1.318 IC50 (nanomole/l) 3.253 2.059 0.003348 No data No data approximately 10.58 0.9491 0.0943 0.003908 R square 0.9364 0.8918 0.9364 - - 0.676 0.9258 0.8634 0.9624 Outliers (excluded, Q=1%) 0 2 1 - - - - - -

Table 6: Overview of the statistical evaluation of the curves shown in FIG. 7 using the 4-PL (logarithm (inhibitor) versus response - variable slope (four parameters)) algorithm available in Graphpad Prism.

TGF-β1 T22d35-Fc T22d35-Fc-IgG2-v2 (CC) T22d35-Fc-IgG1-v1 (CC) T22d35-Fc-IgG1-v2 (SCC) T22d35-Fc-IgG1-v3 (GSL-CC) Incline -2.25 -2.13 -2.056 -2.063 -2.655 IC50 (nanomole/l) 0.002863 0.002783 0.002345 0.002128 0.002476 R square 0.9805 0.9844 0.9422 0.9729 0.9464

Functional evaluation in vivo

The T22d35-Fc fusion protein (SEQ ID NO: 10) was evaluated in vivo using the MC-38 murine syngeneic colon carcinoma model (FIG. 9). Tumor growth in animals treated with the T22d35-Fc fusion construct was compared to tumor growth in animals treated with control IgG (CTL IgG). As shown in Figure 9, no significant difference in tumor growth was observed until day 11 post-treatment, however, on day 15, a significant decrease in tumor growth in volume can be observed in animals treated with T22d35-Fc compared to CTL IgG (two-way ANOVA). These data show that T22d35-Fc administration caused significant growth inhibition of MC-38 tumors compared to the CTL IgG group, suggesting that in vivo blocking of TGF-β may abolish tumor growth in this syngeneic model of colorectal cancer.

Sequence list

SEQID NO: Subsequence Description 1 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD TβRII-ECD including the structural domain and its natural linkers (also called T2m) 2 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF Structural domain of TβRII-ECD associated with its natural N-terminal linker 3 QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD Structural domain of TβRII-ECD associated with its natural C-terminal linker 4 QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF Structural domain of TβRII-ECD 5 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETV CHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECCNDNIIFSEEYNTSNPD Structural domains of the TβRII-ECD-TβRII-ECD fusion dimer associated with their natural linkers (also referred to as T22d35) 6 SEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKF Natural linker TβRII-ECD 7 IPPHVQKSVNNMDMIVTDNNGAVKF N-terminal natural TβRII-ECD linker 8 SEEYNTSNPD C-terminal natural TβRII-ECD linker 9 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG T2m-Fc construct from T2m associated with hIgG2Fc Fc region (CCCC) 10 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETV CHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG T22d35-Fc construct from T22d35 associated with hIgG2Fc Fc region (CCCC) eleven MDWTWRILFLVAAATGTHA Signal peptide 12 ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Fc region variant hIgG2Fc(CCCC) 13 SEEYNTSNPDERKCCVECCPPCPAPP Natural linker T22d35-Fc associated with hIgG2Fc hinge region (CCCC) 14 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETV CHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG T22d35-Fc-IgG1-v1(CC) construct from T22d35 associated with hIgG1Fc (CC) Fc region 15 THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Fc region variant hIgG1Fc(CC) 16 SEEYNTSNPDTHTCPPCPAPE Natural linker T22d35-Fc-IgG1-v1(CC) associated with hIgG1Fc hinge region (CC) 17 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETV CHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG T22d35-Fc-IgG1-v2(SCC) construct from T22d35 associated with hIgG1Fc Fc region (SCC) 18 VEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Fc region variant hIgG1Fc(SCC) 19 SEEYNTSNPDVEPKSSDKTHTCPPCPAPE Natural linker T22d35-Fc-IgG1-v2(SCC) associated with hIgG1Fc hinge region (SCC) 20 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETV CHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGSGGGSGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG T22d35-Fc-IgG1-v3(GSL-CC) construct from T22d35 bound to the hIgG1Fc (CC) Fc region also coupled to an artificial GS linker 21 GGGSGGGSGGG T22d35-Fc-IgG1-v3 fusion artificial GS linker (GSL-CC) 22 SEEYNTSNPDGGGSGGGSGGGTHTCPPCPAPE Linker T22d35-Fc-IgG1-v3(GSL-CC) including natural and artificial sequences and hIgG1Fc hinge region (CC) 23 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETV CHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG T22d35-Fc-IgG2-v2(CC) construct from T22d35 associated with hIgG2Fc (CC) Fc region 24 VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG Fc region variant hIgG2Fc(CC) 25 SEEYNTSNPDVECPPCPAPP Natural linker T22d35-Fc-IgG2-v2(CC) associated with hIgG2Fc hinge region (CC) 26 PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMS NCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD hIgG1FcΔK(C)-T22d35 27 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQ KSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD hIgG1FcΔK(CC)-T22d35 28 VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMS NCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD hIgG2FcΔK(CC)-T22d35 29 ATGGATTGGACCTGGAGAATCCTCTTCCTTGTAGCAGCAGCAACAGGTACACATGCTATCCCTCCTCATGTTCAAAAGTCCGTTAACAACGACATGATCGTCACCGATAACAACGGTGCTGTCAAGTTCCCACAACTCTGTAAGTTCTGCGATGTGTGCGTTTCTCCACATGTGATAACCAGAAGTCCTGTATGAGCAACTGCTCAATCACCTCCATCTGCGAAAAGCCACAAGAGGTA TGCGTAGCTGTATGGCGAAAGAACGATGAAAACATCACCCTGGAAACCGTCTGTCACGATCCAAAGCTCCCATACCATGATTTCATCCTGGAAGACGCAGCTTCTCCAAAGTGTATCATGAAGGAGAAGAAGAAGCCCGGTGAAACCTTCTTCATGTGCTCCTGTTCCTCAGATGAATGCAACGATAACATCATCTTCCGAGGAGTACAACCCTCCAACCCAGATATCCCTCCACACGTTCA GAAGTCCGTAAACAATGACATGATTGTGACCGACAACAACGGGGCTGTTAAGTTCCCACAGCTCTGTAAGTTTTGCGACGTTAGGTTCAGCACCTGTGATAATCAGAAGAGCTGCATGTCCAACTGCAGCATCACCAGTATTTGCGAGAAGCCTCAAGAAGTGTGTGTCGCTGTTTGGAGAAAGAACGACGAAAACATAACCCTGGAGACCGTTTGCCACGATCCAAAACTCCCATATCACG ATTTCATTCTGGAGGACGCCGCCAGTCCTAAATGTATAATGAAAGAGAAGAAGAAACCAGGGGAGACCTTCTTTATGTGCAGCTGCAGCAGCGACGAGTGTAACGATAATATAATTTTTAGCGAGGAGTATAATACAAGCAATCCCGACGAGCGCAAGTGCTGCGTCGAGTGCCTCCATGCCCTGCCCCTCCTGTTGCCGGACCTAGTGTGTTTTTGTTTCCTCCTAAACCTAA AGATACACTCATGATTAGCAGGACACCTGAGGTGACATGTGTCGTCGTGGACGTGAGTCATGAAGACCCGAAGTGTCAGTTTAATTGGTATGTCGACGGAGTCGAAGTCCATAATGCCAAAACTAAACCAAGGGAAGAACAGTTTAATTCAACTTTTCGCGTGGTCTCTGTGCTGACTGTGGTGCACCAGGACTGGCTTAATGGAAAGGAATACAAGTGTAAGGTGAGTAATA AGGGCCTGCCCGCCCCCATTGAAAAAACTATTAGTAAGACTAAAGGGCAGCCCCGAGAGCCCCAGGTGTATACTTTGCCCCCCTCTCGGGAGGAGATGACTAAAAATCAGGTGTGAGTCTTACATGTCTTGTGTGAAAGGATTTTACCCCTCTGACATTTCAGTGGAGTGGGAGTCTAATGGCCAGCCCGAGAATAATTACAAAACTACTCCCCCATGTTGGACTCTGACGGCTCAT TTTTCTTGTACTCTAAACTGACAGTGGACAAAAGTCGGTGGCAGCAGGGCAATGTGTTTTCTTGTTCAGTGATGCACGAGGCCCTGCATAATCACTATACACAGAAATCTCTGTCTCTGTCACCCGGCTGATGA Nucleic acid sequence encoding T22d35-Fc in a form that can be secreted thirty ATGGACTGGACCTGGAGAATCCTGTTCCTGGTGGCTGCTGCTACCGGAACACACGCTATCCCCTCATGTGCAGAAGTCCGTGTGAACAATGACATGATCGTGACAGATAACAATGGCGCCGTGAAGTTTCCTCAGCTGTGTGCAAGTTCTGTGACGTGAGGTTTAGCACCTGCGATAACCAGAAGTCCTGCATGAGCAATTGTTCTATCACATCCATCTGCGAGAAGCCACAGGAGG TGTGCGTGGCCGTGTGGCGGAAGAACGACGAGAATATCACCCTGGAGACAGTGTGCCACGATCTAAGCTGCCATACCATGACTTCATCCTGGAGGATGCTGCCTCTCCCAAGTGTATCATGAAGGAGAAGAAGAAGCCTGGCGAGACATTCTTCATGTGCTCCTGTTCCAGCGAGAGTGCAACGATAATATCATCTTCAGCGAGGAGTATAACCCTCTAATCCAGATATCCCACCCCACGTG CAGAAGTCTGTCAATAACGATATGATTGTCACAGATAACAATGGCTGTCTGTGAAGTTTCCCCAGCTGTGCAAATTTTGTGACGTGTGAGATTTTCCACCTGTGATAACCAGAAGAGCTGCATGTCTAATTGTTCCATCACATCTATTTGTGAAAAACCTCAGGAAGTGTGTGTGGCCGTGTGGAAAAAAAATGATGAAAACATCACCCTGGAGACAGTGTGTCCATGATCCCAAGCTGCCTTATCA CGACTTCATCCTGGAAGACGCTGCCAGCCCAAAATGCATTATGAAAGAGAAGAAGAAGCCCGGTGAGACATTCTTCATGTGCAGCTGTTCTTCTGATGAATGTAACGATAATATCATCTTTTCCGAGGAGTATAACACAAGCAATCCCGACACCCACACATGCCCTCCATGTCCAGCTCCTGAGCTGCTGGGAGGACCTAGCGTGTTCCTGTTTCCCCCTAAGCCAAAGGATACCCTGATG ATCAGCAGGACCCCCGAGGTGACATGCGTGGTGGTGGACGTGTCTCACGAGGACCCCGAGGTGAAGTTTAACTGGTACGTGGACGGCGTGGAGTGCATAATGCCAAGACCAAGCCTAGGGAGGAGCAGTACAACTCTACCTATCGGGTGGTGTCCGTGCTGACAGTGCTGCATCAGGATTGGCTGAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGCTC TGCCAGCCCCCATTGAGAAGACCATCAGCAAGGCTAAGGGCCAGCCAAGAGCCCCAGGTGTACACACTGCCACCCTCTCGCGACGAGCTGACCAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCTTCCGATATCGCTGTGGAGTGGGAGAGCAACGGACAGCCAGAGAACAATTACAAGACCACACCTCCAGTGCTGGACTCTGATGGCTCCTTCTTTCTG TATAGCAAGCTGACCGTGGACAAGTCTAGGTGGCAGCAGGGCAACGTGTTTAGCTGTTCTGTGATGCATGAGGCCCTGCACAATCATTACACACAGAAGTCCCTGAGCCTGTCTCCTGGC Nucleic acid sequence encoding T22d35-Fc-IgG1-v1 (CC) in a form that can be secreted 31 ATGGACTGGACCTGGAGAATCCTGTTCCTGGTGGCTGCTGCTACCGGAACACACGCTATCCCCCCATGTGCAGAAGTCTGTGTGAACAATGACATGATCGTGACAGATAACAATGGCGCCGTGAAGTTTCCCCAGCTGTGTGCAAGTTCTGTGACGTGAGGTTTTCCACCTGCGATAACCAGAAGTCTTGCATGTCCAATTGTAGCATCACATCTATCTGCGAGAAGCCTCAGGAGG TGTGCGTGGCCGTGTGGCGGAAGAACGACGAGAATATCACCCTGGAGACAGTGTGCCACGATCCTAAGCTGCCATACCATGACTTCATCCTGGAGGATGCTGCCAGCCCAAAGTGTATCATGAAGGAGAAGAAGAAGCCCGGCGAGACATTCTTCATGTGCTCTTGTTCCAGCGGACGAGTGCAACGATAATATCATCTTCTCCGAGGAGTATAACACCAGCAATCCTGACATCCCACCCCACGTGTG CAGAAGAGCGTCAATAACGATATGATTGTCACAGATAACAATGGCCGTGTGAAGTTTCCACAGCTGTGCAAATTTTGTGACGTGTGAGATTTTCTACCTGTGATAACCAGAAGTCCTGCATGAGCAATTGTTCTATCACATCCATCTGCGAGAAGCCACAGGAAGTGTGTGTGGCCGTGTGGAAAAAATGATGAAAACATCACCCTGGAGACAGTGTGTCCATGATCCCAAGCTGCCTTATCACG ACTTCATCCTGGAAGACGCTGCCTCCCCTAAATGCATTATGAAAGAGAAGAAGAAGCCAGGTGAGACATTCTTCATGTGCAGCTGTTCTTCTGATGAGTGCAACGATAACATCATCTTTTCTGAGGAGTACAACACATCCAATCCTGACGTGGAGCCAAAGAGCTCTGATAAGACCCACACATGCCCTCCATGTCCAGCTCCTGAGCTGCTGGGAGGACCATCCGTGTTCCTGTTTCCACC TAAGCCTAAGGACACCCTGATGATCTCCAGGACCCCAGAGGTGACATGCGTGGTGGTGGACGTGTGAGCCACGAGGACCCCGAGGTGAAGTTTAACTGGTACGTGGATGGCGTGGAGTGCATAATGCCAAGACCAAGCCAAGGGAGGAGCAGTACAACAGCACCTATCGGGTGGTGTCTGTGCTGACAGTGCTGCATCAGGACTGGCTGAACGGCAAGGAGTATA AGTGCAAGGTGTCTAATAAGGCTCTGCCAGCCCCCATCGAGAAGACCATCTCCAAGGCTAAGGGCCAGCCAAGAGAGCCCCAGGTGTACACACTGCCACCCAGCCGCGACGAGCTGACCAAGAACCAGGTGTCTCTGACATGTCTGGTGAAGGCTTCTATCCCTCTGATATCGCTGTGGAGTGGGAGTCCAACGGACAGCCTGAGAACAATTACAAGACCACCTCCAGTGCT GGACAGCGATGGCTCTTTCTTTCTGTATTCCAAGCTGACCGTGGATAAGCAGGTGGCAGCAGGGCAACGTGTTTTCCTGTAGCGTGATGCATGAGGCCCTGCACAATCATTACACACAGAAGTCTCTGTCCCTGAGCCCTGGC Nucleic acid sequence encoding T22d35-Fc-IgG1-v2 (SCC) in a form that can be secreted 32 ATGGATTGGACCTGGAGAATCCTGTTCCTGGTGGCTGCTGCTACCGGAACACACGCTATCCCCCCATGTGCAGAAGTCTGTGTGAACAATGACATGATCGTGACAGATAACAATGGCGCCGTGAAGTTTCCTCAGCTGTGTGCAAGTTCTGTGACGTGAGGTTTTCCACCTGCGATAACCAGAAGTCCTGCATGAGCAATTGTTCTATCACATCCATCTGCGAGAAGCCACAGGAGG TGTGCGTGGCCGTGTGGCGGAAGAACGACGAGAATATCACCCTGGAGACAGTGTGCCACGATCCTAAGCTGCCATACCATGACTTCATCCTGGAGGATGCTGCCAGCCCCAAGTGTATCATGAAGGAAGAAGAAGCCTGGCGAGACATTCTTCATGTGCTCTTGTTCCAGCGAGCGAGTGCAACGATAATATCATCTTCTCCGAGGAGTATAACACCAGCAATCCAGACATCCCACCCCACGTGTG CAGAAGAGCGTCAATAACGATATGATTGTCACAGATAACAATGGCGCTGTGAAGTTTCCCCAGCTGTGCAAATTTTGTGACGTGTGAGATTTTCTACCTGTGATAACCAGAAGAGCTGCATGTCTAATTGTTCCATCACATCTATTTGTGAAAAACCTCAGGAAGTGTGTGTGGCCGTGTGGAAAAAATGATGAAAACATCACCCTGGAGACAGTGTGTCCATGATCCCAAGCTGCCTTATCA CGACTTCATCCTGGAAGACGCTGCCTCCCCAAAATGCATTATGAAAGAGAAGAAGAAGCCCGGTGAGACATTCTTCATGTGCAGCTGTTCTTCTGATGAGTGCAACGATAACATCATCTTTTCTGAGGAGTACAACACATCCAATCCTGACGGAGGAGGCAGCGGAGGAGGCTCTGGAGGCGGCACCCACACATGCCCTCCATGTCCAGCTCCTGAGCTGCTGGGAGGACCTTCC GTGTTCCTGTTTCCCCCTAAGCCAAAGGACACCCTGATGATCTCCAGGACCCCCGAGGTGACATGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTTAACTGGTACGTGGATGGCGTGGAGTGCATAATGCCAAGACCAAGCCAAGGGAGGAGCAGTACAACAGCACCTATCGGGTGGTGTCTGTGCTGACAGTGCTGCATCAGGATTGGCTG AACGGCAAGGAGTATAAGTGCAAGGTGTCTAATAAGGCTCTGCCAGCCCCCATTGAGAAGACCATCTCCAAGGCTAAGGGCCAGCCAAGAGAGCCCCAGGTGTACACACTGCCACCCAGCCGCGACGAGCTGACCAAGAACCAGGTGTCTCTGACATGTCTGGTGAAGGCTTCTATCCTTCTGATATCGCTGTGGAGTGGGAGTCCAACGGACAGCCAGAGAACAATTACAA GACCACACCTCCAGTGCTGGACTCTGATGGCTCCTTCTTTCTGTATTCCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTTAGCTGTTCTGTGATGCATGAGGCCCTGCACAATCATTACACACAGAAGTCCCTGAGCCTGTCTCCTGGC Nucleic acid sequence encoding T22d35-Fc-IgG1-v3 (GSL-CC) in a form that can be secreted 33 ATGGATTGGACCTGGAGAATCCTCTTCCTTGTAGCAGCAGCAACAGGTACACATGCTATCCCTCCTCATGTTCAAAAGTCCGTTAACAACGACATGATCGTCACCGATAACAACGGTGCTGTCAAGTTCCCACAACTCTGTAAGTTCTGCGATGTGTGCGTTTCTCCACATGTGATAACCAGAAGTCCTGTATGAGCAACTGCTCAATCACCTCCATCTGCGAAAAGCCACAAGAGGTA TGCGTAGCTGTATGGCGAAAGAACGATGAAAACATCACCCTGGAAACCGTCTGTCACGATCCAAAGCTCCCATACCATGATTTCATCCTGGAAGACGCAGCTTCTCCAAAGTGTATCATGAAGGAGAAGAAGAAGCCCGGTGAAACCTTCTTCATGTGCTCCTGTTCCTCAGATGAATGCAACGATAACATCATCTTCCGAGGAGTACAACCCTCCAACCCAGATATCCCTCCACACGTTCA GAAGTCCGTAAACAATGACATGATTGTGACCGACAACAACGGGGCTGTTAAGTTCCCACAGCTCTGTAAGTTTTGCGACGTTAGGTTCAGCACCTGTGATAATCAGAAGAGCTGCATGTCCAACTGCAGCATCACCAGTATTTGCGAGAAGCCTCAAGAAGTGTGTGTCGCTGTTTGGAGAAAGAACGACGAAAACATAACCCTGGAGACCGTTTGCCACGATCCAAAACTCCCATATCACG ATTTCATTCTGGAGGACGCCGCCAGTCCTAAATGTATAATGAAAGAGAAGAAGAAACCAGGGGAGACCTTCTTTATGTGTCAGCTGCAGCAGCGACGAGTGTAACGATAATATAATTTTTAGCGAGGAGTATAATACAAGCAATCCCGACGTCGAGTGCCTCCATGCCCTGCCCCCTGTTGCCGGACCTAGTGTGTTTTTGTTTCCTCCTAAACCTAAAGATACACTCATGATTAG CAGGACACCTGAGGTGACATGTGTCGTCGTGGACGTGAGTCATGAAGACCCCGAAGTGCAGTTTAATTGGTATGTCGACGGAGTCGAAGTCCATAATGCCAAAACTAAACCAAGGGAAGAACAGTTTAATTCAACTTTTCGCGTGGTCTCTGTGCTGACTGTGGTGCACCAGGACTGGCTTAATGGAAAGGAATACAAGTGTAAGGTGAGTAATAAGGGCCTGCCCGCC CCCATTGAAAAAACTATTAGTAAGACTAAAGGGCAGCCCCGAGAGCCCCAGGTGTATACTTTGCCCCCCTCTCGGGAGGATGACTAAAAATCAGGTGAGTCTTACATGTCTTGTGAAAGGATTTTACCCCTCTGACATTTCAGTGGAGTGGGAGTCTAATGGCCAGCCCGAGAATAATTACAAAACTACTCCCCCATGTTGACTCTGACGGCTCATTTTTCTTGTACTCTAA ACTGACAGTGGACAAAAGTCGGTGGCAGCAGGGCAATGTGTTTTCTTGTTCAGTGATGCACGAGGCCCTGCATAATCACTATACACAGAAATCTCTGTCTCTGTCACCCGGCTGATGA Nucleic acid sequence encoding T22d35-Fc-IgG2-v2 (CC) in a form that can be secreted

LINKS

All patents, patent applications and publications mentioned in the application are listed below.

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De Crescenzo G, Grothe S, Zwaagstra J, Tsang M, O'Connor-McCourt MD (2001) Real-time monitoring of the interactions of transforming growth factor-beta (TGF-beta ) isoforms with latency-associated protein and the ectodomains of the TGF-beta type II and III receptors reveals different kinetic models and stoichiometries of binding. J Biol Chem 276: 29632-29643

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WO/1995/04069

WO/2004/076670

WO2008/113185

WO2010/031168

US 8815247

US 62777375

US 2015/0225483

WO 01/83525;

WO2005/028517;

WO2008/113185;

WO2008/157367;

WO2010/003118;

WO2010/099219;

W02012/071649;

WO2012/142515;

WO2013/000234;

US 5693607;

US 2005/0203022;

US 2007/0244042;

US 8318135;

US 8658135;

US 8815247;

US 2015/0225483; And

US 2015/0056199.

--->

SEQUENCE LIST

<110> NATIONAL RESECTION COUNCIL OF CANADA

LENFERINK Anne E.J.

ZWAGSTRA John K.

SULI Trayan

O'CONNOR-MCCORT Maureen D.

<120> TGF-BETA RECEPTOR ECTODOMAIN FUNCTIONS AND THEIR

APPLICATIONS

<130> 2017-074-03

<140> PCT/IB2018/051320

<141> 2018-03-01

<150> US 62/465,969

<151> 2017-03-02

<150> US 62/468,586

<151> 2017-03-08

<160> 33

<170> PatentIn version 3.5

<210> 1

<211> 136

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TRII-ECD comprising structural domain and its natural linkers

(also called T2m)

<400> 1

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp

130 135

<210> 2

<211> 126

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Structural domain of TRII-ECD with its native N-terminal

linker

<400> 2

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe

115 120 125

<210> 3

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Structural domain of TRII-ECD with its natural C-terminal

linker

<400> 3

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

1 5 10 15

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

20 25 30

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

35 40 45

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

50 55 60

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

65 70 75 80

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

85 90 95

Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

100 105 110

<210> 4

<211> 101

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TRII-ECD structural domain

<400> 4

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

1 5 10 15

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

20 25 30

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

35 40 45

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

50 55 60

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

65 70 75 80

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

85 90 95

Asp Asn Ile Ile Phe

100

<210> 5

<211> 272

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Structural domains of the TRII-ECD-TRII-ECD fusion dimer with their

natural linkers (also called T22d35)

<400> 5

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser

130 135 140

Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe

145 150 155 160

Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn

165 170 175

Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys

180 185 190

Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile

195 200 205

Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe

210 215 220

Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys

225 230 235 240

Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys

245 250 255

Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

260 265 270

<210> 6

<211> 34

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Natural TRII-ECD Linker

<400> 6

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln

1 5 10 15

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

20 25 30

Lys Phe

<210> 7

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> N-terminal natural TRII-ECD linker

<400> 7

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe

20

<210> 8

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TRII-ECD natural C-terminal linker

<400> 8

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

1 5 10

<210> 9

<211> 363

<212> PROTEIN

<213> Artificial sequence

<220>

<223> T2m-Fc construct from T2m associated with hIgG2Fc(CCCC) Fc region

<400> 9

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp Glu Arg Lys Cys Cys Val Glu Cys

130 135 140

Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe

145 150 155 160

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

165 170 175

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe

180 185 190

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

195 200 205

Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr

210 215 220

Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

225 230 235 240

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

245 250 255

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

260 265 270

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

275 280 285

Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Ser Asn Gly Gln Pro

290 295 300

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser

305 310 315 320

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

325 330 335

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

340 345 350

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

355 360

<210> 10

<211> 499

<212> PROTEIN

<213> Artificial sequence

<220>

<223> T22d35-Fc construct from T22d35 associated with hIgG2Fc Fc region

(CCCC)

<400> 10

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser

130 135 140

Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe

145 150 155 160

Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn

165 170 175

Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys

180 185 190

Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile

195 200 205

Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe

210 215 220

Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys

225 230 235 240

Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys

245 250 255

Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

260 265 270

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val

275 280 285

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

290 295 300

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

305 310 315 320

His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

325 330 335

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

340 345 350

Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn

355 360 365

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro

370 375 380

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln

385 390 395 400

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

405 410 415

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val

420 425 430

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

435 440 445

Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

450 455 460

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

465 470 475 480

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

485 490 495

Ser Pro Gly

<210> 11

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Signal peptide

<400> 11

Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly

1 5 10 15

Thr His Ala

<210> 12

<211> 227

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fc region variant hIgG2Fc(CCCC)

<400> 12

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val

1 5 10 15

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

20 25 30

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

35 40 45

His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

50 55 60

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

65 70 75 80

Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn

85 90 95

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro

100 105 110

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln

115 120 125

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

130 135 140

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val

145 150 155 160

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

165 170 175

Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

180 185 190

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

195 200 205

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

210 215 220

Ser Pro Gly

225

<210> 13

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> T22d35-Fc natural linker with hIgG2Fc hinge (CCCC)

<400> 13

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Glu Arg Lys Cys Cys Val

1 5 10 15

Glu Cys Pro Pro Cys Pro Ala Pro Pro

20 25

<210> 14

<211> 496

<212> PROTEIN

<213> Artificial sequence

<220>

<223> T22d35-Fc-IgG1-v1(CC) construct from T22d35 associated with Fc region

hIgG1Fc(CC)

<400> 14

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser

130 135 140

Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe

145 150 155 160

Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn

165 170 175

Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys

180 185 190

Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile

195 200 205

Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe

210 215 220

Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys

225 230 235 240

Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys

245 250 255

Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

260 265 270

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

275 280 285

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

290 295 300

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

305 310 315 320

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

325 330 335

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

340 345 350

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

355 360 365

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

370 375 380

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

385 390 395 400

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

405 410 415

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

420 425 430

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

435 440 445

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

450 455 460

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

465 470 475 480

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

485 490 495

<210> 15

<211> 224

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fc region variant hIgG1Fc(CC)

<400> 15

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

1 5 10 15

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

20 25 30

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

35 40 45

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

50 55 60

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

65 70 75 80

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

85 90 95

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

100 105 110

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

115 120 125

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

130 135 140

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

145 150 155 160

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

165 170 175

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

180 185 190

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

195 200 205

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

210 215 220

<210> 16

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Natural linker T22d35-Fc-IgG1-v1(CC) with hinge region

hIgG1Fc (CC)

<400> 16

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Thr His Thr Cys Pro Pro

1 5 10 15

Cys Pro Ala Pro Glu

20

<210> 17

<211> 504

<212> PROTEIN

<213> Artificial sequence

<220>

<223> T22d35-Fc-IgG1-v2(SCC) construct from T22d35 associated with Fc region

hIgG1Fc

<400> 17

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser

130 135 140

Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe

145 150 155 160

Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn

165 170 175

Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys

180 185 190

Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile

195 200 205

Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe

210 215 220

Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys

225 230 235 240

Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys

245 250 255

Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

260 265 270

Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro

275 280 285

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

290 295 300

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

305 310 315 320

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

325 330 335

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

340 345 350

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

355 360 365

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

370 375 380

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

385 390 395 400

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu

405 410 415

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

420 425 430

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

435 440 445

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

450 455 460

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

465 470 475 480

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

485 490 495

Lys Ser Leu Ser Leu Ser Pro Gly

500

<210> 18

<211> 232

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fc region variant hIgG1Fc(SCC)

<400> 18

Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

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

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

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

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly

225 230

<210> 19

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> T22d35-Fc-IgG1-v2(SCC) natural linker, hinged

hIgG1Fc (SCC)

<400> 19

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Val Glu Pro Lys Ser Ser

1 5 10 15

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

20 25

<210> 20

<211> 507

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Construct T22d35-Fc-IgG1-v3(GSL-CC) from T22d35, with

hIgG1Fc (CC) Fc region including artificial linker GS

<400> 20

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser

130 135 140

Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe

145 150 155 160

Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn

165 170 175

Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys

180 185 190

Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile

195 200 205

Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe

210 215 220

Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys

225 230 235 240

Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys

245 250 255

Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

260 265 270

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Thr His Thr Cys Pro

275 280 285

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

290 295 300

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

305 310 315 320

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

325 330 335

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

340 345 350

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

355 360 365

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

370 375 380

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

385 390 395 400

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

405 410 415

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

420 425 430

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

435 440 445

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

450 455 460

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

465 470 475 480

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

485 490 495

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

500 505

<210> 21

<211> 11

<212> DNA

<213> Artificial sequence

<220>

<223> T22d35-Fc-IgG1-v3 fusion artificial GS linker (GSL-CC)

<400> 21

gggsgggsgg g 11

<210> 22

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker T22d35-Fc-IgG1-v3(GSL-CC), including natural and artificial

hIgG1Fc sequences and hinge region (CC)

<400> 22

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Gly Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

20 25 30

<210> 23

<211> 494

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Construct T22d35-Fc-IgG2-v2(CC) from T22d35 associated with region

Fc hIgG2Fc (CC)

<400> 23

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser

130 135 140

Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe

145 150 155 160

Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn

165 170 175

Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys

180 185 190

Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile

195 200 205

Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe

210 215 220

Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys

225 230 235 240

Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys

245 250 255

Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

260 265 270

Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val

275 280 285

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

290 295 300

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

305 310 315 320

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

325 330 335

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser

340 345 350

Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

355 360 365

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

370 375 380

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

385 390 395 400

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

405 410 415

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Ser Asn

420 425 430

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser

435 440 445

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

450 455 460

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

465 470 475 480

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

485 490

<210> 24

<211> 222

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fc region variant hIgG2Fc(CC)

<400> 24

Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val

1 5 10 15

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

20 25 30

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

35 40 45

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

50 55 60

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser

65 70 75 80

Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

85 90 95

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

100 105 110

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

115 120 125

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

130 135 140

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Ser Asn

145 150 155 160

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser

165 170 175

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

180 185 190

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

195 200 205

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

210 215 220

<210> 25

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Natural linker T22d35-Fc-IgG2-v2(CC) with hinge region

hIgG2Fc (CC)

<400> 25

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Val Glu Cys Pro Pro Cys

1 5 10 15

Pro Ala Pro Pro

20

<210> 26

<211> 492

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG1FcΔK(C)-T22d35

<400> 26

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

1 5 10 15

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

20 25 30

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

35 40 45

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

50 55 60

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

65 70 75 80

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

85 90 95

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

100 105 110

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

115 120 125

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

130 135 140

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

145 150 155 160

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

165 170 175

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

180 185 190

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

195 200 205

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ile Pro Pro His

210 215 220

Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly

225 230 235 240

Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser

245 250 255

Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser

260 265 270

Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn

275 280 285

Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro

290 295 300

Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met

305 310 315 320

Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser

325 330 335

Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr

340 345 350

Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp

355 360 365

Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys

370 375 380

Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys

385 390 395 400

Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val

405 410 415

Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr

420 425 430

Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp

435 440 445

Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu

450 455 460

Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile

465 470 475 480

Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

485 490

<210> 27

<211> 498

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG1FcΔK(CC)-T22d35

<400> 27

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

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

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

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

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile

225 230 235 240

Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe

245 250 255

Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser

260 265 270

Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val

275 280 285

Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys

290 295 300

His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala

305 310 315 320

Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe

325 330 335

Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe

340 345 350

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln

355 360 365

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

370 375 380

Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys

385 390 395 400

Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys

405 410 415

Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu

420 425 430

Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His

435 440 445

Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu

450 455 460

Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp

465 470 475 480

Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn

485 490 495

Pro Asp

<210> 28

<211> 494

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG2FcΔK(CC)-T22d35

<400> 28

Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val

1 5 10 15

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

20 25 30

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

35 40 45

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

50 55 60

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser

65 70 75 80

Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

85 90 95

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

100 105 110

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

115 120 125

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

130 135 140

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Ser Asn

145 150 155 160

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser

165 170 175

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

180 185 190

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

195 200 205

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ile Pro

210 215 220

Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn

225 230 235 240

Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg

245 250 255

Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile

260 265 270

Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg

275 280 285

Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys

290 295 300

Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys

305 310 315 320

Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser

325 330 335

Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr

340 345 350

Asn Thr Ser Asn Pro Asp Ile Pro Pro His Val Gln Lys Ser Val Asn

355 360 365

Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln

370 375 380

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

385 390 395 400

Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln

405 410 415

Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu

420 425 430

Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu

435 440 445

Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro

450 455 460

Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp

465 470 475 480

Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

485 490

<210> 29

<211> 1560

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid sequence encoding T22d35-Fc in

a form that can be secreted

<400> 29

atggattgga cctggagaat cctcttcctt gtagcagcag caacaggtac acatgctatc 60

cctcctcatg ttcaaaagtc cgttaacaac gacatgatcg tcaccgataa caacggtgct 120

gtcaagttcc cacaactctg taagttctgc gatgtgcgtt tctccacatg tgataaccag 180

aagtcctgta tgagcaactg ctcaatcacc tccatctgcg aaaagccaca agaggtatgc 240

gtagctgtat ggcgaaagaa cgatgaaaac atcaccctgg aaaccgtctg tcacgatcca 300

aagctcccat accatgattt catcctggaa gacgcagctt ctccaaagtg tatcatgaag 360

gagaagaaga agcccggtga aaccttcttc atgtgctcct gttcctcaga tgaatgcaac 420

gataacatca tcttctccga ggagtacaac acctccaacc cagatatccc tccacacgtt 480

cagaagtccg taaacaatga catgattgtg accgacaaca acggggctgt taagttccca 540

cagctctgta agttttgcga cgttaggttc agcacctgtg ataatcagaa gagctgcatg 600

tccaactgca gcatcaccag tatttgcgag aagcctcaag aagtgtgtgt cgctgtttgg 660

agaaagaacg acgaaaacat aaccctggag accgtttgcc acgatccaaa actcccatat 720

cacgatttca ttctggagga cgccgccagt cctaaatgta taatgaaaga gaagaagaaa 780

ccaggggaga ccttctttat gtgcagctgc agcagcgacg agtgtaacga taatataatt 840

tttagcgagg agtataatac aagcaatccc gacgagcgca agtgctgcgt cgagtgccct 900

ccatgccctg cccctcctgt tgccggacct agtgtgtttt tgtttcctcc taaacctaaa 960

gatacactca tgattagcag gacacctgag gtgacatgtg tcgtcgtgga cgtgagtcat 1020

gaagaccccg aagtgcagtt taattggtat gtcgacggag tcgaagtcca taatgccaaa 1080

actaaaccaa gggaagaaca gtttaattca acttttcgcg tggtctctgt gctgactgtg 1140

gtgcaccagg actggcttaa tggaaaggaa tacaagtgta aggtgagtaa taagggcctg 1200

ccgccccca ttgaaaaaac tattagtaag actaaagggc agccccgaga gccccaggtg 1260

tatactttgc ccccctctcg ggaggagatg actaaaaatc aggtgagtct tacatgtctt 1320

1380

aataattaca aaactactcc cccatgttg gactctgacg gctcattttt cttgtactct 1440

1500

cacgaggccc tgcataatca ctatacacag aaatctctgt ctctgtcacc cggctgatga 1560

<210> 30

<211> 1545

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid sequence encoding T22d35-Fc-IgG1-v1

(CC) in a form that can be secreted

<400> 30

atggactgga cctggagaat cctgttcctg gtggctgctg ctaccggaac acacgctatc 60

ccccctcatg tgcagaagtc cgtgaacaat gacatgatcg tgacagataa caatggcgcc 120

gtgaagtttc ctcagctgtg caagttctgt gacgtgaggt ttagcacctg cgataaccag 180

aagtcctgca tgagcaattg ttctatcaca tccatctgcg agaagccaca ggaggtgtgc 240

gtggccgtgt ggcggaagaa cgacgagaat atcaccctgg agacagtgtg ccacgatcct 300

aagctgccat accatgactt catcctggag gatgctgcct ctcccaagtg tatcatgaag 360

gagaagaaga agcctggcga gacattcttc atgtgctcct gttccagcga cgagtgcaac 420

gataatatca tcttcagcga ggagtataac acctctaatc cagatatccc accccacgtg 480

cagaagtctg tcaataacga tatgattgtc acagataaca atggcgctgt gaagtttccc 540

cagctgtgca aattttgtga cgtgagattt tccacctgtg ataaccagaa gagctgcatg 600

aaacctcagg aagtgtgcgt ggccgtgtgg 660

agaaaaaatg atgaaaacat caccctggag acagtgtgcc atgatcccaa gctgccttat 720

cacgacttca tcctggaaga cgctgccagc ccaaaatgca ttatgaaaga gaagaagaag 780

cccggtgaga cattcttcat gtgcagctgt tcttctgatg aatgtaacga taatatcatc 840

ttttccgagg agtataacac aagcaatccc gacacccaca catgccctcc atgtccagct 900

cctgagctgc tgggaggacc tagcgtgttc ctgtttcccc ctaagccaaa ggataccctg 960

atgatcagca ggacccccga ggtgacatgc gtggtggtgg acgtgtctca cgaggacccc 1020

gaggtgaagt ttaactggta cgtggacggc gtggaggtgc ataatgccaa gaccaagcct 1080

agggaggagc agtacaactc tacctatcgg gtggtgtccg tgctgacagt gctgcatcag 1140

gattggctga acggcaagga gtataagtgc aaggtgtcca ataaggctct gccagccccc 1200

attgagaaga ccatcagcaa ggctaagggc cagccaagag agccccaggt gtacacactg 1260

ccaccctctc gcgacgagct gaccaagaac caggtgtccc tgacatgtct ggtgaagggc 1320

ttctatcctt ccgatatcgc tgtggagtgg gagagcaacg gacagccaga gaacaattac 1380

aagaccacac ctccagtgct ggactctgat ggctccttct ttctgtatag caagctgacc 1440

gtggacaagt ctaggtggca gcagggcaac gtgtttagct gttctgtgat gcatgaggcc 1500

ctgcacaatc attacacaca gaagtccctg agcctgtctc ctggc 1545

<210> 31

<211> 1569

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid sequence encoding T22d35-Fc-IgG1-v2

(SCC) in a form that can be secreted

<400> 31

atggactgga cctggagaat cctgttcctg gtggctgctg ctaccggaac acacgctatc 60

ccccctcatg tgcagaagtc tgtgaacaat gacatgatcg tgacagataa caatggcgcc 120

gtgaagtttc cccagctgtg caagttctgt gacgtgaggt tttccacctg cgataaccag 180

aagtcttgca tgtccaattg tagcatcaca tctatctgcg agaagcctca ggaggtgtgc 240

gtggccgtgt ggcggaagaa cgacgagaat atcaccctgg agacagtgtg ccacgatcct 300

aagctgccat accatgactt catcctggag gatgctgcca gcccaaagtg tatcatgaag 360

gagaagaaga agcccggcga gacattcttc atgtgctctt gttccagcga cgagtgcaac 420

gataatatca tcttctccga ggagtataac accagcaatc ctgacatccc acccccgtg 480

cagaagagcg tcaataacga tatgattgtc acagataaca atggcgctgt gaagtttcca 540

cagctgtgca aattttgtga cgtgagattt tctacctgtg ataaccagaa gtcctgcatg 600

agcaattgtt ctatcacatc catctgcgag aagccacagg aagtgtgcgt ggccgtgtgg 660

agaaaaaatg atgaaaacat caccctggag acagtgtgcc atgatcccaa gctgccttat 720

cacgacttca tcctggaaga cgctgcctcc cctaaatgca ttatgaaaga gaagaagaag 780

ccaggtgaga cattcttcat gtgcagctgt tcttctgatg agtgcaacga taacatcatc 840

ttttctgagg agtacaacac atccaatcct gacgtggagc caaagagctc tgataagacc 900

cacacatgcc ctccatgtcc agctcctgag ctgctgggag gaccacatccgt gttcctgttt 960

ccacctaagc ctaaggacac cctgatgatc tccaggaccc cagaggtgac atgcgtggtg 1020

gtggacgtga gccacgagga ccccgaggtg aagtttaact ggtacgtgga tggcgtggag 1080

gtgcataatg ccaagaccaa gccaagggag gagcagtaca acagcaccta tcgggtggtg 1140

tctgtgctga cagtgctgca tcaggactgg ctgaacggca aggagtataa gtgcaaggtg 1200

tctaataagg ctctgccagc ccccatcgag aagaccatct ccaaggctaa gggccagcca 1260

agagagcccc aggtgtacac actgccaccc agccgcgacg agctgaccaa gaaccaggtg 1320

tctctgacat gtctggtgaa gggcttctat ccctctgata tcgctgtgga gtgggagtcc 1380

aacggacagc ctgagaacaa ttacaagacc acacctccag tgctggacag cgatggctct 1440

ttctttctgt attccaagct gaccgtggat aagagcaggt ggcagcaggg caacgtgttt 1500

tcctgtagcg tgatgcatga ggccctgcac aatcattaca cacagaagtc tctgtccctg 1560

agccctggc 1569

<210> 32

<211> 1578

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid sequence encoding T22d35-Fc-IgG1-v3

(GSL-CC) in a form that can be secreted

<400> 32

atggattgga cctggagaat cctgttcctg gtggctgctg ctaccggaac acacgctatc 60

ccccctcatg tgcagaagtc tgtgaacaat gacatgatcg tgacagataa caatggcgcc 120

gtgaagtttc ctcagctgtg caagttctgt gacgtgaggt tttccacctg cgataaccag 180

aagtcctgca tgagcaattg ttctatcaca tccatctgcg agaagccaca ggaggtgtgc 240

gtggccgtgt ggcggaagaa cgacgagaat atcaccctgg agacagtgtg ccacgatcct 300

aagctgccat accatgactt catcctggag gatgctgcca gccccaagtg tatcatgaag 360

gagaagaaga agcctggcga gacattcttc atgtgctctt gttccagcga cgagtgcaac 420

gataatatca tcttctccga ggagtataac accagcaatc cagacatccc accccacgtg 480

cagaagagcg tcaataacga tatgattgtc acagataaca atggcgctgt gaagtttccc 540

cagctgtgca aattttgtga cgtgagattt tctacctgtg ataaccagaa gagctgcatg 600

aaacctcagg aagtgtgcgt ggccgtgtgg 660

agaaaaaatg atgaaaacat caccctggag acagtgtgcc atgatcccaa gctgccttat 720

cacgacttca tcctggaaga cgctgcctcc ccaaaatgca ttatgaaaga gaagaagaag 780

cccggtgaga cattcttcat gtgcagctgt tcttctgatg agtgcaacga taacatcatc 840

ttttctgagg agtacaacac atccaatcct gacggaggag gcagcggagg aggctctgga 900

ggcggcaccc acacatgccc tccatgtcca gctcctgagc tgctgggagg accttccgtg 960

ttcctgtttc cccctaagcc aaaggacacc ctgatgatct ccaggacccc cgaggtgaca 1020

tgcgtggtgg tggacgtgag cccacgaggac cccgaggtga agtttaactg gtacgtggat 1080

ggcgtggagg tgcataatgc caagaccaag ccaagggagg agcagtacaa cagcacctat 1140

cgggtggtgt ctgtgctgac agtgctgcat caggattggc tgaacggcaa ggagtataag 1200

tgcaaggtgt ctaataaggc tctgccagcc cccattgaga agaccatctc caaggctaag 1260

ggccagccaa gagagcccca ggtgtacaca ctgccaccca gccgcgacga gctgaccaag 1320

aaccaggtgt ctctgacatg tctggtgaag ggcttctatc cttctgatat cgctgtggag 1380

tgggagtcca acggacagcc agagaacaat tacaagacca cacctccagt gctggactct 1440

gatggctcct tctttctgta ttccaagctg accgtggaca agagcaggtg gcagcaggggc 1500

aacgtgttta gctgttctgt gatgcatgag gccctgcaca atcattacac acagaagtcc 1560

ctgagcctgt ctcctggc 1578

<210> 33

<211> 1545

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid sequence encoding T22d35-Fc-IgG2-v2

in a form that can be secreted

<400> 33

atggattgga cctggagaat cctcttcctt gtagcagcag caacaggtac acatgctatc 60

cctcctcatg ttcaaaagtc cgttaacaac gacatgatcg tcaccgataa caacggtgct 120

gtcaagttcc cacaactctg taagttctgc gatgtgcgtt tctccacatg tgataaccag 180

aagtcctgta tgagcaactg ctcaatcacc tccatctgcg aaaagccaca agaggtatgc 240

gtagctgtat ggcgaaagaa cgatgaaaac atcaccctgg aaaccgtctg tcacgatcca 300

aagctcccat accatgattt catcctggaa gacgcagctt ctccaaagtg tatcatgaag 360

gagaagaaga agcccggtga aaccttcttc atgtgctcct gttcctcaga tgaatgcaac 420

gataacatca tcttctccga ggagtacaac acctccaacc cagatatccc tccacacgtt 480

cagaagtccg taaacaatga catgattgtg accgacaaca acggggctgt taagttccca 540

cagctctgta agttttgcga cgttaggttc agcacctgtg ataatcagaa gagctgcatg 600

tccaactgca gcatcaccag tatttgcgag aagcctcaag aagtgtgtgt cgctgtttgg 660

agaaagaacg acgaaaacat aaccctggag accgtttgcc acgatccaaa actcccatat 720

cacgatttca ttctggagga cgccgccagt cctaaatgta taatgaaaga gaagaagaaa 780

ccaggggaga ccttctttat gtgcagctgc agcagcgacg agtgtaacga taatataatt 840

tttagcgagg agtataatac aagcaatccc gacgtcgagt gccctccatg ccctgcccct 900

cctgttgccg gacctagtgt gtttttgttt cctcctaaac ctaaagatac actcatgatt 960

agcaggacac ctgaggtgac atgtgtcgtc gtggacgtga gtcatgaaga ccccgaagtg 1020

1080

gaacagttta attcaacttt tcgcgtggtc tctgtgctga ctgtggtgca ccaggactgg 1140

cttaatggaa aggaatacaa gtgtaaggtg agtaataagg gcctgcccgc ccccattgaa 1200

aaaactatta gtaagactaa aggcagccc cgagagcccc aggtgtatac tttgcccccc 1260

tctcgggagg agatgactaa aaatcaggtg agtcttacat gtcttgtgaa aggatttac 1320

ccctctgaca tttcagtgga gtgggagtct aatggccagc ccgagaataa ttacaaaact 1380

actcccccca tgttggactc tgacggctca ttttctcttgt actctaaact gacagtggac 1440

aaaagtcggt ggcagcaggg caatgtgttt tcttgttcag tgatgcacga ggccctgcat 1500

aatcactata cacagaaatc tctgtctctg tcacccggct gatga 1545

<---

Claims (34)

1. A polypeptide construct effective to inhibit the effect of a transforming growth factor beta (TGF-β) isotype, comprising: the first region containing the first ectodomain of the TGF-β receptor (TβR-ECD) containing the amino acid sequence of SEQ ID NO: 1, connected in series with the second TβR-ECD containing the amino acid sequence of SEQ ID NO: 1, where the C-terminus of the first ectodomain is linked to the N-terminus of the second ectodomain, and a second region containing a second constant domain ( _CH 2) and/or a third constant domain ( _CH 3) of the antibody heavy chain selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18 , SEQ ID NO: 24 and sequences essentially identical to them; where the N-terminus of the second region is connected to the C-terminus of the first region. 2. The polypeptide construct of claim 1, wherein said TβR-ECD contains the ectodomain of the type II TGF-β receptor (TβRII-ECD). 3. The polypeptide construct of claim 1, wherein the first TβR-ECD and the second TβR-ECD bind the same TGF-β isotype. 4. The polypeptide construct of claim 3, wherein each of the first TβR-ECD and the second TβR-ECD binds both TGF-β1 and TGF-β3. 5. The polypeptide construct according to claim 1, wherein the first TβR-ECD has an amino acid sequence consisting of SEQ ID NO: 1 and the second TGF-β receptor ectodomain has an amino acid sequence consisting of SEQ ID NO: 1. 6. Polypeptide design according to any one of paragraphs. 1-5, where the second region is the amino acid sequence of SEQ ID NO: 15. 7. A polypeptide construct according to claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23 and the sequence , essentially identical to them. 8. A homodimeric polypeptide construct effective for inhibiting the effect of the transforming growth factor beta (TGF-β) isotype, containing the first polypeptide and the second polypeptide constructs linked through the corresponding constant domains of the antibody by at least one disulfide bridge, where the homodimeric polypeptide contains: the first single-chain polypeptide containing the first and second regions; where the second region contains the second constant domain (CH ₂ ) and the third constant domain ( _CH 3) of the heavy chain of the antibody and the variable region of the heavy chain of this antibody; where the first region contains two TβRII-ECD; where the N-terminus of the second region is connected to the C-terminus of the first region; And a second single chain polypeptide comprising a polypeptide of said first single chain polypeptide, wherein the first single chain polypeptide and the second single chain polypeptide are selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23 and sequences essentially identical to them. 9. Encoding nucleic acid molecule encoding any polypeptide construct according to any one of paragraphs. 1-8. 10. An expression vector containing the nucleic acid molecule according to claim 9. 11. A nucleic acid molecule encoding any polypeptide construct according to any one of paragraphs. 1-8 in a form that can be secreted by the chosen expression host. 12. A nucleic acid molecule according to claim 11 encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, and SEQ ID NO :23, or a sequence essentially identical to them. 13. A nucleic acid molecule according to claim 12, having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33 . 14. Composition for inhibiting the activity of TGF-β, containing an effective amount of the polypeptide construct according to any one of paragraphs. 1-8 and a pharmaceutically acceptable carrier, diluent or excipient. 15. Transgenic cell host for the production of a polypeptide construct according to any one of paragraphs. 1-8 containing the nucleic acid molecule according to claim 9 or the vector according to claim 10, wherein the transgenic cellular host comprises Chinese hamster ovary (CHO) cells. 16. A transgenic cell host according to claim 15, further comprising a second nucleic acid molecule or a second vector encoding a second polypeptide construct, the same as the first polypeptide construct. 17. A method for obtaining a homodimeric polypeptide, including culturing the host and isolating the homodimeric polypeptide construct according to claim 8 from the environment caused by the growth of the specified host. 18. The use of a polypeptide construct according to any one of paragraphs. 1-8 for cancer treatment. 19. A polypeptide construct according to claim 8, wherein the polypeptide construct contains the amino acid sequence of SEQ ID NO: 14. 20. A pharmaceutical composition for inhibiting TGF-β activity, containing an effective amount of a polypeptide construct according to claim 19. 21. The use of a polypeptide construct according to claim 19 for the treatment of cancer. 22. A nucleic acid molecule encoding a polypeptide construct according to claim 19. 23. A nucleic acid molecule encoding a polypeptide construct according to claim 19, in a form that can be secreted by a selected expression host, thereby obtaining a homodimer having two copies of a polypeptide construct having an amino acid sequence containing SEQ ID NO: 14 associated with Fc . 24. An expression vector containing the nucleic acid molecule according to claim 22. 25. A transgenic cell host for producing the polypeptide construct of claim 19, comprising the nucleic acid molecule of claim 22, wherein the transgenic host comprises Chinese hamster ovary (CHO) cells. 26. A method for producing a homodimeric polypeptide construct, comprising culturing a host according to claim 25 and obtaining a homodimeric polypeptide construct containing two copies of a polypeptide construct containing SEQ ID NO: 14 associated with Fc.

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