EA046204B1 - COMPLEX-SELECTIVE ANTIBODIES TO proTGFβ1-GARP, METHODS AND THEIR APPLICATIONS - Google Patents

Description

List of sequences

The invention contains a sequence listing presented electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy of this listing, created on July 10, 2017, is named JBI5093_SL.txt and is 27577 bytes in size.

Field of technology

The present invention relates to monoclonal antibodies that inhibit growth factor activity, and methods for producing and using the described antibodies.

Prerequisites for creating the invention

Regulatory T cells, or Tregs, are a subset of CD4+T lymphocytes specialized in inhibiting immune responses. Insufficient Treg activity mediates autoimmune pathology, while excessive Treg activity can inhibit antitumor immune responses in cancer patients. The precise mechanisms by which Tregs inhibit immune responses are not fully understood.

Due to their immunosuppressive function, Tregs represent potential inhibitors of spontaneous or vaccine-induced immune antitumor responses. In mouse models, depletion of Tregs can improve immune responses against experimental tumors (Colombo et al. Nat. Rev. Cancer 2007, 7:880-887). Thus, targeting Tregs in humans may improve the efficacy of immunotherapies against cancer.

TGF-β 1, which plays an important role in the activation of human Tregs, but not other types of human T lymphocytes (Stockis, J. et al. Eur. J. Immunol. 2009, 39:869-882), may be of interest. However, antibodies against hTGF β1 are not considered promising. Phase 1 clinical studies were conducted for focal segmental glomerulosclerosis (FSGS), idiopathic pulmonary fibrosis (IPF), and advanced malignant melanoma or renal cell carcinoma (RCC) (Lonning S et al. Current Pharmaceutical Biotechnology 2011, 12:2176-2189) . Depending on the trial, some patients experienced side effects. The main side effects reported were the development of keratoacanthoma (KA) and squamous cell carcinoma (SCC) in patients with melanoma. It is possible that CA or SCC lesions in melanoma patients developed from precancerous cells whose proliferation is inhibited by endogenous TGF-e1 (Lonning S et al. Current Pharmaceutical Biotechnology 2011, 12:2176-2189). Therefore, one of the major concerns with the use of anti-TGF-e1 antibodies in cancer is that they may promote the emergence of new tumor lesions by inhibiting the antitumor effect provided by endogenous TGF-e1 on precancerous cells. Thus, new strategies are needed to improve cancer treatment by preventing the release of TGF-e1 from Tregs.

Brief description of the invention

The present invention includes antibodies selective for the proTGFe1-GARP complex and antigen binding fragments thereof. Also described are related polynucleotides capable of encoding the presented proTGFe1-GARP complex-selective antibodies and antigen-binding fragments, cells expressing the presented antibodies and antigen-binding fragments, as well as associated vectors and detectably labeled proTGFe1GARP complex-selective antibodies and antigen-binding fragments. The antibody or antigen-binding fragment thereof does not bind selectively to the growth factor domain TGFe1, the growth factor domain TGFe2, the growth factor domain TGFe3, proTGFe1 covalently linked to LTBP1, proTGFe1 covalently linked to LTBP3, proTGFe1 covalently linked to LRRC33, and proTGFe1, which does not associated with human GARP, as determined by OctetRed_384 under the conditions shown in Examples 4-6.

In some embodiments, the antibodies and antigen binding fragments of the present invention may have the following: (1) a dissociation constant (Kd) of no more than 1 Nm for human proTGFe1 complexed with human glycoprotein A repeats (proTGFe1-GARP complex) in solution; (2) an inhibitory concentration (IC50) of not more than 10 nM to inhibit the release of growth factor TGFe1 from the cell-associated proTGFe1-GARP complex; and (3) more than 100-fold selectivity for the proTGFe1-GARP complex relative to the growth factor domain TGFe1, growth factor domain TGFe2, growth factor domain TGFe3, proTGFe1 covalently linked to LTBP1, proTGFe1 covalently linked to LTBP3, proTGFe1 covalently linked to LRRC33, where the isolated antibodies or their antigen-binding fragments do not bind to proTGFe1, which is not associated with human GARP.

Moreover, methods of using the present antibodies selective for the proTGFe1-GARP complex and antigen-binding fragments are described. The described antibodies selective for the proTGFe1-GARP complex can be used in methods of treating various diseases or disorders associated with TGFe1 in which it is necessary to modulate the immune response, for example, various immunotherapies, for example, in cancer, vaccination and infectious disease.

In some embodiments, the present invention includes isolated antibodies and

- 1 046204 antigen-binding fragments, where the antibody or antigen-binding fragment specifically binds to human proTGFe1 in a complex with preferential repeats of human glycoprotein A (proTGFe1-GARP complex), while the complex is in solution. These proTGFe1-GARP complex-selective antibodies or their antigen-binding fragments can inhibit Treg function in vitro. In some embodiments, proTGFe1-GARP complex-selective antibodies and antigen-binding moieties inhibit TGFe1 activation. In some embodiments, proTGFe1-GARP complex-selective antibodies and antigen binding fragments bind to an epitope of human proTGFe1 modified by formation of a complex with human GARP. This proTGFe1-GARP complex-selective antibody, proTGFe1-GARP, or antigen binding fragment can bind proTGFe1 of the proTGFe1-GARP complex with a binding affinity of 880 pM or less.

Table 1

CDR sequences of human antibodies selective for the proTGFe-GARP complex (SEQ ID NO:)

ID HC-CDR1 HC-CDR2 HC-CDR3 LC-CDR1 LC-CDR2 LC-CDR3 4В1С1 DYTMH (4) LISWDGGSTYYADSVKG (5) DADDSTFDI (6) RASQSVSRNLA (7) WASTRES (8) QQYYSVPYT (9) 4В16В9 SYAIS (Yu) GIIPMFGTTNYAQKFQG (11) DREWEPAYGMDV (12) IGTSSDVGGYN YVS (13) DVSNRPS (14) SAYTVSSTWV (15)

In some embodiments, an antibody selective for the proTGFe1-GARP complex, proTGFe1-GARP, or an antigen-binding fragment thereof comprises a heavy chain comprising CDR1, CDR2, and CDR3 of any of the amino acid sequences described in Table. 1, and a light chain containing CDR1, CDR2 and CDR3 of any of the amino acid sequences described in table. 1. Antibodies selective for the proTGFe1-GARP complex of the present invention may be heavy chain variable regions of SEQ ID NOs: 16 and 18 and may contain light chain variable regions of SEQ ID NOs 17 and 19.

Antibodies selective for the proTGFe1-GARP complex described herein include antibodies with the described CDR and variable domain properties in combination with any of the IgG isotypes, including modified versions in which the Fc sequence is modified to affect various effector functions.

In addition to the described proTGFe1-GARP complex-selective antibodies and antigen-binding fragments, polynucleotide sequences capable of encoding the described proTGFe1-GARP complex-selective antibodies and antigen-binding fragments are also provided. Also provided are vectors containing the disclosed polynucleotides, as well as cells expressing antibodies selective for the proTGFe1-GARP complex or antigen binding fragments provided herein. In addition, cells capable of expressing the described vectors are described. These cells may be mammalian cells (eg, 293F cells, CHO cells), insect cells (eg, Sf9 cells), yeast cells, plant cells, or bacterial cells (eg, E. coli). The process of production of the described antibodies selective for the proTGFe1-GARP complex, or antigen-binding fragments, is also described.

The present invention also provides methods for using antibodies selective for the proTGFe1-GARP complex or antigen binding fragments. Antibodies selective for the proTGFe1-GARP complex for use in the methods described in this section include those having the set of CDRs described for the antibodies in Table. 1. For example, the key role played by TGFe1 in the immune response makes it an attractive target for immunotherapy, including by inducing or enhancing an immune response against any weakly immunogenic antigen, including tumor antigens. Thus, antibodies selective for the proTGFe1-GARP complex are used in the treatment of various forms of cancer and infectious diseases.

In one embodiment, antibodies selective for the proTGFe1-GARP complex are administered to block the release of TGFe1 from Tregs and thereby prevent regulatory T cells from inhibiting T cell effector activity. Analysis of such inhibition can be done in a variety of ways known to those skilled in the art, including, for example, by monitoring T cell proliferation, expression of known activation markers, or cytokine secretion. In another embodiment, a proTGFe1-GARP complex-selective antibody, proTGFe1-GARP, is administered to a subject to reduce the level of regulatory T cells, such as the level of tumor regulatory T cells. In yet another embodiment, effector T cell activity is induced or enhanced by administration of antibodies selective for the proTGFe1GARP complex, as described herein.

The present invention provides kits, including disclosed antibodies, selective for the proTGFe1-GARP complex, or antigen-binding fragments thereof. The described kits can be used to implement methods of using proTGFe1GARP complex selective antibodies or antigen binding fragments provided herein or other methods.

- 2 046204 bobs known to specialists in this field. In some embodiments, the described kits may include proTGFe1-GARP complex-selective antibodies or antigen-binding fragments described herein and reagents for use in determining the presence of the proTGFe1-GARP complex in a biological sample and optionally a vessel containing the proTGFe1-GARP complex antibody. proTGFe1-GARP complex or fragment in the absence of use, instructions for use of the proTGFe1-GARP complex-selective antibody or fragment, proTGFe1-GARP complex-selective antibody or fragment attached to a solid support and/or labeled for detection form of an antibody selective for the proTGFe1-GARP complex or fragment.

Brief description of graphic materials

In fig. 1 shows that addition of 4B1C1 and 4B16B9 to T cell co-cultures inhibits T regulatory cell activity through enhanced growth of effector T cells.

In fig. Figure 2 shows that 4B1C1 and 4B16B9 inhibit TGFe1 activation as assessed through SMAD signaling.

In fig. Figure 3 shows the dose-dependent inhibition of TGFe1 activity by 4B1C1 and 4B16B9.

Fig. 4. Octet affinity results for proTGFe1-GARP complex selective antibody candidates demonstrate specificity by binding to the human proTGFe1-GARP complex, but not to other proTGFb1 complexes or soluble forms of TGFb1, 2 or 3.

Detailed Description of Illustrative Embodiments

Definitions.

Throughout the present specification and claims, various terms are used to relate to aspects of the specification. Such terms shall have their usual meaning in the art unless otherwise noted. Other specifically defined terms should be construed as defined herein.

When used in this specification and in the accompanying claims, the singular number includes the plural unless the content of the text clearly indicates otherwise. Thus, for example, a reference to a cell includes a combination of two or more cells and the like.

As used herein, the term about, when indicating a measurable quantity such as quantity, duration in time, etc., is considered to cover deviations of up to ±10% from the specified value, as such deviations are acceptable for implementation of the described methods. Unless otherwise indicated, all numbers expressing quantities of ingredients, characteristics such as molecular weight, reaction conditions, etc., used in the description and claims are to be understood as modified in all cases by the term about. Accordingly, unless otherwise stated, the numerical parameters set forth in the following description and the accompanying claims are approximate values that may vary depending on the desired properties that are desired to be obtained by the present invention. As a last resort, and not as an attempt to limit the application of the theory of equivalents to the scope of the claims, each numerical parameter should at least be considered in light of the number of significant figures presented and using standard rounding techniques.

Although the numerical ranges and parameters defining the broad scope of the invention are approximate, the numerical values indicated in the specific examples are presented as accurately as possible. However, any numerical value by its nature contains certain errors, inevitably resulting from the standard deviation found in the corresponding test measurements.

The term isolated means that a biological component (eg, a nucleic acid, peptide or protein) has been substantially separated from, obtained separately from, or purified from other biological components of the organism in which the component occurs naturally, i.e. from other chromosomal and extrachromosomal DNA, RNA and proteins. Thus, the nucleic acids, peptides and proteins that have been isolated include nucleic acids and proteins purified by standard purification methods. Isolated nucleic acids, peptides and proteins can be part of a composition and still be considered isolated if such composition is not part of the original environment of the nucleic acid, peptide or protein. The term also includes nucleic acids, peptides and proteins produced by recombinant expression in a host cell, as well as chemically synthesized nucleic acids. As used herein, the term isolated antibody or antigen-binding fragment means an antibody or antigen-binding fragment that is substantially free of other antibodies or antigen-binding fragments having different antigen specificities (e.g., an isolated antibody that is an antibody selective for the proTGFe1GARP complex, proTGFe1-GARP by essentially does not contain antibodies that are not antibodies selective for the proTGFe1-GARP complex).

As used herein, the term transforming growth factor beta 1 and TGFe1 specifically binds to human TGFe 1 protein. In scientific literature, TGFe1 is also known as TGFbeta1 and

- 3 046204

TGFB1. The growth factor TGFei is synthesized in conjunction with a prodomain, for example, as described in NCBI exemplary sequence GenBank™ accession number AK291907: NP_000651.3.1 and UniProtKB/Swiss-Prot accession number P01137.2 (see also Derynck et al. 1985, Nature 316, 701-705). In a specific embodiment, the translated TGFe1 protein is a human protein with the amino acid sequence SEQ ID NO: 2. TGFe1, which includes both prodomain and growth factor elements, is called proTGFel· ¹ . In some embodiments, proTGFe 1 includes prodomain and growth factor components that have been proteolytically separated but which remain associated through one or more chemical interactions. Such chemical interactions may include, but are not limited to, hydrophobic bonds, interactions due to van der Waals forces, polar and ionic interactions, hydrogen bonds, and non-covalent bonds.

As used herein, the terms glycoprotein A dominant repeats and GARP refer to total GARP. GARP is otherwise known as leucine-rich repeat containing protein 32 (lrrc32) and harpin. Exemplary sequence NP_001122394.1 and NP_005503.1 in NCBI provide exemplary human GARP amino acid sequences. In a specific embodiment, the GARP is human GARP of SEQ ID NO: 1.

Antibody refers to all isotypes of immunoglobulins (IgG, IgA, IgE, IgM, IgD and IgY), including various monomeric, polymeric and chimeric forms, unless otherwise specified. In particular, the term antibody includes polyclonal antibodies, monoclonal antibodies (mAbs), and antibody-like polypeptides such as chimeric antibodies and humanized antibodies.

Antigen-binding moieties are any protein structure capable of exhibiting binding affinity for a specific antigen. Antigen-binding fragments include fragments obtained by any known method, such as enzymatic digestion, peptide synthesis and recombinant methods. Some antigen-binding fragments are composed of portions of intact antibodies that retain the antigen-binding specificity of the original antibody molecule. For example, antigen binding fragments may contain at least one variable region (heavy chain or light chain variable region) or one or more antibody CDR regions known to bind to a particular antigen. Examples of suitable antigen binding fragments include, but are not limited to, diabodies and single chain molecules, as well as Fab, F(ab')2, Fc, Fabc and Fv molecules, single chain (Sc) antibodies, single antibody light chains, single antibody heavy chains, chimeric fusions of antibody chains or CDRs with other proteins, protein scaffolds, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, monovalent fragment consisting of VL, VH, CL and CH1 domains, or a monovalent antibody described in WO2007059782, divalent fragments containing two Fab fragments connected by a disulfide bridge in the hinge region, an Fd fragment consisting essentially of V.sub.H and C.sub.H1 domains; An Fv fragment consisting essentially of the VL and VH domains of one arm of an antibody, a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a VH domain and is also called a domain antibody (Holt etal; Trends Biotechnol. 2003 Nov.;21(11):484-90); camel-type antibody (camelid) or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan.; 5(1):lll-24); selected complementarity determining regions (CDRs), etc. All antibody isotypes can be used to obtain antigen-binding fragments. In addition, antigen binding fragments may include non-antibody protein scaffolds into which polypeptide segments can be successfully inserted in an orientation conferring affinity for a given antigen of interest, such as protein scaffolds. Antigen-binding fragments can be produced recombinantly or by enzymatic or chemical digestion of intact antibodies. The phrase antibody or antigen binding fragment thereof may be used to indicate that the antigen binding fragment comprises one or more amino acid segments of an antibody referred to in the phrase.

The terms CDR and the plural of CDR refer to the complementarity determining region (CDR), with three such regions defining the binding nature of the light chain variable region (CDRL1, CDRL2 and CDRL3), and three defining the binding nature of the heavy chain variable region (CDRH1, CDRH2 and CDRH3). CDRs contribute to the functional activity of the antibody molecule and are separated by amino acid sequences that include backbone or framework regions. The precise definition of the boundaries and extent of the CDR depends on various classification and numbering systems. Therefore, CDRs can be distinguished using Kabat, Chothia, contact or any other boundary definitions. Despite their different boundaries, each of these systems is characterized by a certain degree of overlap in that part that constitutes the so-called hypervariable region within the variable sequences. Thus, CDR definitions under such systems may differ in length and boundary regions relative to the adjacent frame region. See, for example, Kabat et al., Sequences of Proteins

- 4 046204 of Immunological Interest, 5th ed. NIH Publication No. 91-3242 (1991); Chothia et al., Canonical Structures for the Hypervariable Regions of Immunoglobulins, J. Mol. Biol. 196:901 (1987); and MacCallum et al., Antibody-Antigen Interactions: Contact Analysis and Binding Site Topography, J. Mol. Biol. 262:732 (1996)), each of which is incorporated herein by reference in its entirety.

Typically, CDRs form a loop structure, which can be classified as a canonical structure. The term canonical structure refers to the backbone conformation adopted by the antigen-binding loops (CDRs). As a result of comparative structural studies, five of the six antigen-binding loops were found to have only a limited set of available conformations. Each canonical structure can be characterized by the torsion angles of the polypeptide backbone. Therefore, the corresponding loops between antibodies may have very similar three-dimensional structures, despite the high level of amino acid sequence variability in most regions of the loops (Chothia et al., Canonical Structures For the Hypervariable Regions of Immunoglobulins, J. Mol. Biol. 196: 901 (1987) ; Chothia et al., Conformations of Immunoglobulin Hypervariable Regions, I 342:877 (1989); Martin and Thornton, Structural Families in Loops of Homologous Proteins: Automatic Classification, Modeling and Application to Antibodies, J. Mol. Biol. 263:800 (1996)), each of which is incorporated by reference in its entirety. Moreover, there is a certain relationship between the formed loop structure and the amino acid sequences surrounding it. The conformation of a particular canonical class is determined by the chain length and the amino acid residues located at key positions in the loop, as well as within the conserved framework (i.e., outside the loop). Therefore, assignment to a particular canonical class can be made based on the presence of such key amino acid residues.

The term polypeptide is used interchangeably with the term protein and in its broadest sense refers to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. Subunits can be linked by peptide bonds. In another embodiment, the subunit may be linked by other linkages, such as ester, ether, etc. As used herein, the term amino acid refers to natural and/or artificial or synthetic amino acids, including glycine, as well as D and L optical isomers, amino acid analogues and peptidomimetics. A peptide consisting of three or more amino acids is usually called an oligopeptide if the peptide chain is short enough. If the peptide chain is long, the peptide is usually called a polypeptide or protein.

The terms specifically bind, or specifically bind, or derivatives thereof, when used with antibodies and antibody fragments, represent binding, through domains encoded by immunoglobulin genes or gene fragments, to one or more epitopes of the protein of interest and, preferably, the absence of binding to other molecules in the sample. containing a mixed population of molecules. Typically, an antibody binds to a cognate antigen with a K _d of less than about 1 x 10 ^-8 M, as measured by a surface plasmon resonance or cell binding assay. In a preferred embodiment, binding specificity is measured using biolayer interferometry. Phrases such as [antigen]specific antibody mean that said antibody specifically binds to said antigen.

The term polynucleotide, which is synonymous with the term nucleic acid molecule, nucleotides or nucleic acids, refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Polynucleotides include, but are not limited to, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is a mixture of single- and double-stranded regions, hybrid molecules containing DNA and RNA, which may be single-stranded or, more typically, double-stranded, or a mixture of single- and double-stranded regions. In addition, a polynucleotide refers to three-stranded regions containing RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA with the backbones modified for stability or other purposes. Modified bases include, for example, tritylated bases and atypical bases such as inosine. Various modifications can be made to DNA and RNA; therefore, the term polynucleotide covers the chemically, enzymatically or metabolically modified forms of polynucleotides commonly found in nature, as well as the chemical forms of DNA and RNA found in viruses and cells. The term polynucleotide also covers relatively short chains of nucleic acids, often called oligonucleotides.

A vector is a replicon, such as a plasmid, phage, cosmid or virus, into which another nucleotide segment can be operably inserted so that replication or expression of the segment occurs.

As used herein, the term host cell can refer to any type of cell, such as a primary cell, a cell in culture, or a cell from a cell line. In specific

- 5 046204 embodiments, the term host cell refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such cell. The descendants of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, for example due to mutations or environmental effects that may occur in subsequent generations, or integration of the nucleic acid molecule into the genome of the host cell. The terms expression and production are used interchangeably herein to refer to the biosynthesis of a gene product. These terms cover the transcription of a gene into RNA. These terms also cover the translation of RNA into one or more polypeptides and further cover all naturally occurring post-transcriptional and post-translational modifications. Expression or production of an antibody or antigen-binding fragment thereof may occur within the cytoplasm of a cell or in an extracellular environment, such as a cell culture growth medium. The meaning of a term essentially the same may differ depending on the context in which it is used. Because variation is possible in the natural sequence of the light and heavy chains and the genes encoding them, it is expected that a certain level of variation within the amino acid sequences or genes encoding the antibodies or antigen-binding fragments described herein may be encountered with little or no effect on them. unique binding properties (e.g. specificity and affinity). This expectation is partly due to the degeneracy of the genetic code, as well as the evolutionary success of conservative variants of amino acid sequences that do not significantly change the nature of the encoded protein. Accordingly, in the context of nucleotide sequences, essentially the same means at least 65% identity between two or more sequences. Preferably, the term refers to at least 70% identity between two or more sequences, more preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identical, more preferably at least 97% identical, more preferably at least 98% identical, and more preferably at least 99% or more identical. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences (i.e. % homology = number of identical positions/total number of positions x 100), taking into account the number of gaps and the length of each gap introduced for optimal alignment of two sequences. Percent identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm described in E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988), which is built into the ALIGN program (version 2.0), using the PAM120 residue weight table with a gap length penalty of 12 and a gap penalty of 4. In addition, the percentage identity of two amino acid sequences can be determined using the algorithm , described in Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

The degree of variation possible within the amino acid sequence of a protein without significantly affecting the function of the protein is much lower than in a nucleotide sequence, since the principles of degeneracy do not apply to amino acid sequences. Accordingly, in the context of an antibody or antigen binding fragment, substantially the same refers to an antibody or antigen binding fragments having 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with the described antibodies or antigen binding fragments. Other embodiments include proTGFe1-GARP complex-selective antibodies or antigen-binding fragments that have a framework, backbone, or other nonbinding regions that do not share significant identity with the proTGFe1-GARP complex-selective antibodies and antigen-binding fragments described in herein, but necessarily include one or more CDRs or other sequences necessary to effect binding that have 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the sequences described herein document.

Binding affinity generally refers to the strength of the total sum of noncovalent interactions between one binding site of a molecule (eg, antibody) and its binding partner (eg, antigen). Unless otherwise specified, the term binding affinity as used herein refers to intrinsic binding affinity, which reflects the 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can usually be determined by the dissociation constant (KD). Affinity can be measured and/or expressed in a variety of ways known to those skilled in the art, including, without limitation, by the equilibrium dissociation constant (KD) and the equilibrium association (binding) constant (K _A ). KD is calculated from the ratio k _off /k _on , while KA is calculated from the ratio

- 6 046204 nia kon/koff. kon refers to the rate constant of association, eg antibody to antigen, and koff refers to the rate constant of dissociation, eg antibody to antigen. kon and koff can be determined by methods known to those skilled in the art, for example, biolayer interferometry.

The term subject means human and non-human animals, including all vertebrates, such as mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians and reptiles. In many embodiments of the described methods, the subject is a human.

Antibodies selective for the proTGFe 1-GARP complex and antigen-binding fragments.

Disclosed herein are isolated monoclonal antibodies or antigen binding fragments that are antibodies selective for the proTGFe1-GARP complex. As used herein, the term proTGFe1-GARP complex-selective antibody, proTGFe 1-GARP, refers to an antibody with excellent affinity, specificity and activity. Antibodies selective for the proTGFe1-GARP complex may have the following: (1) a dissociation constant (Kd) of no more than 1 nM for human proTGFe1 if proTGFe1 is complexed with human GARP in solution (e.g., as measured using an assay not including cells); (2) an inhibitory concentration (IC50) of no more than 10 nM to inhibit the release of growth factor TGFe1 from cell-associated proTGFe1-GARP complexes; (3) greater than 100-fold selectivity (as measured by binding affinity, i.e., Kd value) for the proTGFe1-GARP complex over the entire TGFe1 growth factor domain, TGFe2 growth factor domain, TGFe3 growth factor domain, proTGFe1 covalently bound with LTBP1, proTGFe1 covalently linked to LTBP3, and proTGFe 1 covalently linked to LRRC33; and (4) lack of affinity for proTGFe1 unless complexed with GARP. In some cases, an antibody selective for the proTGFe 1GARP complex, proTGFe 1-GARP, also has greater than 100-fold selectivity for the proTGFe 1GARP complex over all proTGFe 1 covalently bound to LTBP2 and/or LTBP4. The general structure of an antibody molecule contains an antigen-binding domain, which includes heavy and light chains, and an Fc domain, which performs various functions, including complement fixation and binding to antibody receptors.

The described antibodies selective for the proTGFe1-GARP complex, or antigen-binding fragments, include all isotypes, IgA, IgD, IgE, IgG and IgM, and synthetic multimers of the four-chain structure of immunoglobulins. The disclosed antibodies or antigen binding fragments also include the IgY isotype substantially found in chicken or turkey serum and in the yolk of chicken or turkey eggs.

Antibodies selective for the proTGFe 1-GARP complex and antigen-binding fragments can be obtained from any species using recombinant methods. For example, the antibodies or antigen binding fragments may be murine, rat, goat, equine, porcine, bovine, chicken, rabbit, camel, donkey, human or chimeric variants thereof. For the purpose of administration to humans, antibodies or antigen-binding fragments obtained from non-human sources can be genetically or structurally modified to be less antigenic when administered to a human patient.

In some embodiments, the antibodies or antigen binding fragments are chimeric. As used herein, the term chimeric means an antibody or antigen-binding fragment thereof comprising at least some portion of at least one variable domain derived from the amino acid sequence of a non-human mammal, rodent or reptile antibody, while the remaining portions of the antibody or antigen-binding fragment thereof fragments are of human origin.

In some embodiments, the antibodies are humanized antibodies. Humanized antibodies may be chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab', F(ab')2, or other antigen-binding antibody subsequences) containing a minimal sequence derived from a non-human animal immunoglobulin. In general, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementarity determining region (CDR) of the recipient are replaced by residues from the complementarity determining region of a non-human species (donor antibody), such as a mouse, rat or rabbit having the desired specificity, affinity and ability. Typically, a humanized antibody will contain substantially all of at least one, and usually two, variable domains in which all or substantially all of the CDR regions correspond to those in an immunoglobulin from a non-human animal, and all or substantially all of the frame regions correspond to those of the human immunoglobulin sequence. The humanized antibody may comprise at least a portion of an immunoglobulin constant region (Fc), typically human immunoglobulin.

Antibodies or antigen binding fragments described herein may have

- 7 046204 various forms, but will include one or more antibody CDRs as shown in table. 1.

In some embodiments, the proTGFe1-GARP complex-selective antibodies or antigen-binding fragments are human IgG or derivatives thereof. Although the proTGFe1-GARP complex-selective antibodies or antigen binding fragments exemplified herein are human, the exemplified antibodies or antigen binding fragments can be chimerized.

In some embodiments, antibodies selective for the proTGFe1-GARP complex contain a heavy chain comprising CDR1, CDR2, and CDR3 of any one of the antibodies described in Table 1. 1, and a light chain containing CDR1, CDR2 and CDR3 of any one of the antibodies described in table. 1.

In some embodiments, proTGFe1-GARP complex selective antibodies and antigen binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 4, a heavy chain CDR2 comprising SEQ ID NO: 5, a heavy chain CDR3 comprising SEQ ID NO: 6, A light chain CDR1 containing SEQ ID NO: 7, a light chain CDR2 containing SEQ ID NO: 8, and a light chain CDR3 containing SEQ ID NO: 9. This proTGFe1-GARP complex selective antibody, proTGFe1GARP or an antigen binding fragment may contain human frame sequences. This proTGFe1-GARP complex-selective antibody, proTGFe1-GARP, or antigen-binding fragment can bind to proTGFe1 from the proTGFe1-GARP complex with an affinity of 880 pM or less, can inhibit Treg function in vitro, and can inhibit TGFe1 activation. In some embodiments, proTGFe1-GARP complex-selective antibodies and antigen binding fragments comprise a heavy chain substantially the same or identical to SEQ ID NO: 16 and a light chain substantially the same or identical to SEQ ID NO: 17. In some embodiments In embodiments, the proTGFβ1-specific antibodies and antigen binding fragments comprise a heavy chain variable region substantially the same or identical to the amino acid sequence 1-118 of SEQ ID NO: 16, and a light chain variable region substantially the same or identical to the amino acid sequence 1-107 of SEQ ID NO: 17. The heavy chain and light chain variable regions of the antibodies described in this paragraph are suitable for inclusion in bispecific constructs in which one arm is the arm of an antibody selective for the proTGFe1GARP complex.

In some embodiments, proTGFe1-GARP complex selective antibodies and antigen binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 10, a heavy chain CDR2 comprising SEQ ID NO: 11, a heavy chain CDR3 comprising SEQ ID NO: 12, Light chain CDR1 containing SEQ ID NO: 13, light chain CDR2 containing SEQ ID NO: 14, and light chain CDR3 containing SEQ ID NO: 15. This antibody is selective for the proTGFe1-GARP complex, proTGFe1-GARP or antigen binding fragment may contain human framework sequences. This proTGFe1-GARP complex-selective antibody, proTGFe1-GARP, or antigen-binding fragment can bind to proTGFe1 from the proTGFe1-GARP complex with an affinity of 880 pM or less, can inhibit Treg function in vitro, and can inhibit TGFe1 activation. In some embodiments, proTGFe1-GARP complex-selective antibodies and antigen binding fragments comprise a heavy chain substantially the same or identical to SEQ ID NO: 18 and a light chain substantially the same or identical to SEQ ID NO: 19. In some embodiments In embodiments, the proTGFβ1-specific antibodies and antigen binding fragments comprise a heavy chain variable region substantially the same or identical to the amino acid sequence 1-121 of SEQ ID NO: 18, and a light chain variable region substantially the same or identical to the amino acid sequence 1-110 of SEQ ID NO: 19. The heavy chain and light chain variable regions of the antibodies described in this paragraph are suitable for inclusion in bispecific constructs in which one arm is an antibody arm selective for the proTGFe1-GARP complex.

The proTGFe1-GARP selective antibody complex and antigen binding fragments may have amino acid sequences that are at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96 % identical, at least 97% identical, at least 98% identical, at least 99% or more identical to the CDR amino acid sequences in SEQ ID NO: 4-15 and the variable region amino acid sequences SEQ ID NO: 16 -19.

Also described are isolated polynucleotides that encode antibodies selective for the proTGFe1-GARP complex or antigen binding fragments as described herein. Isolated polynucleotides capable of encoding variable domain segments proposed in the

- 8 046204 of the present document, can be included in the same or different vectors for the production of antibodies or antigen-binding fragments.

Polynucleotides encoding recombinant antigen-binding proteins are also included within the scope of the description. In some embodiments, the described polynucleotides (and the peptides they encode) include a leader sequence. Any leader sequence known in the art can be used. The leader sequence may include, without limitation, a restriction site or a translation initiation site.

Antibodies selective for the proTGFe1-GARP complex or antigen binding fragments described herein include variants having single or multiple amino acid substitutions, deletions or additions that retain the biological properties (e.g., binding affinity or immune effector activity) of the described antibodies. selective for the proTGFe1-GARP complex, or antigen-binding fragments. Such variants may include: (a) variants in which one or more amino acid residues are replaced by conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to or removed from the polypeptide, (c) variants in wherein one or more amino acids include a substituent group, and (d) variants in which the polypeptide is fused to another peptide or polypeptide, such as a fusion partner, a protein tag, or other chemical functional group capable of conferring beneficial properties on the polypeptide, such as an epitope for an antibody, polyhistidine sequence, biotin residue, etc. Antibodies or antigen binding fragments described herein may include variants in which amino acid residues of one type are replaced by corresponding residues of another type (at conserved or non-conserved positions). In other embodiments, amino acid residues at non-conserved positions are replaced with conservative or non-conservative residues. Techniques for obtaining such variants, including genetic (deletions, mutations, etc.), chemical and enzymatic, are known to those skilled in the art.

Antibodies selective for the proTGFe1-GARP complex, or antigen-binding fragments described herein, can belong to several isotypes of antibodies, such as IgM, IgD, IgG, IgA and IgE. In some embodiments, the antibody isotype is an IgGl, IgG2, IgG3, or IgG4 isotype, preferably an IgGl isotype. The specificity of an antibody or antigen-binding fragment thereof is determined in large part by the amino acid sequence and the location of the CDRs. Therefore, CDRs from one isotype can be transferred to another isotype without changing antigen specificity. In an alternative embodiment, techniques have been established to cause hybridomas to switch from producing one antibody isotype to another (isotype switching) without changing antigen specificity. Accordingly, such antibody isotypes are included within the scope of the disclosed antibodies or antigen binding fragments.

The proTGFe1-GARP complex-selective antibodies or antigen binding fragments described herein have binding affinities for proTGFe1 in the proTGFe1GARP complex that contain a dissociation constant (K _D ) of less than about 880 pM. The affinity of the disclosed proTGFe1-GARP complex-selective antibodies or antigen-binding fragments can be determined by a variety of methods known in the art, such as biolayer interferometry, surface plasmon resonance, or ELISA-based methods. Assays for measuring affinity by biolayer interferometry include assays performed using OctetRed 384, where the assay is performed at room temperature (eg, 25°C or so), with an antibody capable of binding to proTGFe1 from the proTGFe1-GARP complex fixed to biotinylated proTGFe1-GARP complex.

In addition, vectors are provided containing the polynucleotides described herein. The vectors may be expression vectors. Therefore, recombinant expression vectors containing the sequence encoding the polypeptide of interest are considered to be within the scope of this specification. The expression vector may contain one or more additional sequences, such as, but not limited to, regulatory sequences (eg, promoter, enhancer), a selection marker, and a polyadenylation signal. Vectors for transforming a wide range of host cells are widely known and include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), as well as other bacterial, yeast and viral vectors.

Recombinant expression vectors included within the scope of the disclosure include synthetic, genomic or cDNA-derived nucleic acid fragments that encode at least one recombinant protein that can be operably linked to suitable regulatory elements. Such regulatory elements may include a transcription promoter, sequences encoding suitable sites for binding of mRNA to the ribosome, and sequences that control the termination of transcription and translation. Expression vectors, oso

- 9 046204 generic mammalian expression vectors may also include one or more non-transcribed elements, for example an origin of replication, a suitable promoter and enhancer associated with the gene to be expressed, other 5' or 3' flanking non-transcribed sequences, 5' or 3' non-translated sequences (eg, essential ribosome binding sites), polyadenylation site, splice donor and acceptor sites, or transcription termination sequences. In addition, an origin of replication may be inserted to provide the ability to replicate in the host cell.

Transcription and translation control sequences in expression vectors intended for transformation of vertebrate cells can be obtained from viral sources. Examples of vectors that can be constructed are described in Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

In some embodiments, the sequence encoding the antibody or antigen binding fragment is placed under the control of an effective constitutive promoter, such as, for example, the promoters of the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta actin, human myosin, human hemoglobin, human muscle creatine and others. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use in the described embodiments. Such viral promoters include, but are not limited to, cytomegalovirus (CMV) immediate-early promoter, SV40 early and late promoters, mouse mammary tumor virus (MMTV) promoter, Maloney leukemia virus (LTR) long terminal repeats, human immunodeficiency virus (HIV) , Epstein-Barr virus (EBV), Rous sarcoma virus (RSV) and other retroviruses and the herpes simplex virus thymidine kinase promoter. In one embodiment, the coding sequence of a proTGFe1-GARP complex-selective antibody or an antigen-binding fragment thereof is placed under the control of an inducible promoter, e.g., a metallothionein promoter, a tetracycline-inducible promoter, a doxycycline-inducible promoter, promoters containing one or more interferon-stimulated response elements (ISRE), promoters of protein kinase R 2',5'-oligoadenylate synthases, Mx, ADAR1 genes, etc.

The vectors described herein may contain one or more internal ribosome entry sites (IRES). Incorporation of an IRES sequence into fusion vectors may be useful for enhancing the expression of certain proteins. In some embodiments, the vector system may include one or more polyadenylation sites (eg, SV40), which may be upstream or downstream of any of the above nucleic acid sequences. The vector components may be ligated together or positioned to provide optimal spatial separation for expression of the gene products (ie, by introducing spacer nucleotides between open reading frames (ORFs)), or otherwise positioned. Regulatory elements, such as the IRES motif, can also be positioned to provide optimal spacing for expression.

Vectors may contain selectable markers well known in the art. Selectable markers include positive and negative selection markers, antibiotic resistance genes (eg, neomycin resistance gene, hygromycin resistance gene, kanamycin resistance gene, tetracycline resistance gene, penicillin resistance gene), glutamate synthase genes, HSV-TK , HSV-TK derivatives for ganciclovir selection, or the bacterial purine nucleoside phosphorylase gene for b-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000); the nucleotide sequence encoding the selection marker or cloning site may be located upstream or downstream the nucleotide sequence encoding the polypeptide of interest or cloning site.

The vectors described herein can be used to transform various cells with genes encoding the described antibodies or antigen binding fragments. For example, vectors can be used to generate cells that produce an antibody selective for the proTGFe1-GARP complex, proTGFe1-GARP, or an antigen-binding fragment. Accordingly, another aspect of the invention describes host cells transformed with vectors containing a nucleotide sequence encoding an antibody or antigen binding fragment thereof that specifically binds to proTGFe1 of the proTGFe1-GARP complex, for example, the antibodies or antigen binding fragments described and exemplified herein.

Numerous methods for introducing foreign genes into cells are known in the art and can be used to construct recombinant cells to implement the described methods in accordance with the various embodiments described and exemplified herein. The technique used must ensure stable transfer of the heterologous gene sequence into the host cell in such a way that the heterologous gene sequence can be inherited and expressed by the progeny of the cell, and in such a way that the necessary development and physiological functions of the recipient cells are not impaired. To the methods

- 10 046204 which can be used include, without limitation, chromosome transfer (e.g. cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer), physical methods (e.g. transfection, spheroplast fusion, microinjection, electroporation, liposome carrier), viral vector transfer (for example, recombinant DNA viruses, recombinant RNA viruses), etc. (described in Cline, 29 Pharmac. Ther. 69-92 (1985); calcium phosphate precipitation and polyethylene glycol (PEG)-induced fusion of bacterial protoplasts with mammalian cells can also be used to transform cells.

Cells suitable for use in the expression of proTGFe1-GARP complex-selective antibodies or antigen-binding fragments described herein preferably include eukaryotic cells, more preferably cells derived from a plant, rodent or human, such as, but not limited to, cell lines NSO, CHO, CHOK1, perC.6, Tk-ts13, BHK, HEK293 cells, COS-7, T98G, CV-1/EBNA, L cells, C127, 3T3, HeLa, NS1, Sp2/0 myeloma cells, BHK and so on. In addition, antibody expression can be achieved using hybridoma cells. Methods for producing hybridomas are well known in the art.

Cells transformed with the expression vectors described herein can be selected or screened for recombinant expression of the antibodies or antigen binding fragments described herein. Recombination-positive cells are expanded and screened for subclones that exhibit a desired phenotype, such as high expression levels, enhanced growth characteristics, or the ability to produce proteins with desired biochemical properties, for example, due to protein modification or altered post-translational modifications. These phenotypes may be due to intrinsic properties of a given subclone or due to mutation. Mutations can be accomplished by the use of chemical agents, UV light, radiation, viruses, insertional mutagens, inhibition of DNA mismatch repair, or a combination of these methods.

Methods of using antibodies selective to the proTGFe1-GARP complex for treatment.

Provided herein are antibodies selective for the proTGFe1-GARP complex, or antigen-binding fragments thereof, for use in therapy. In particular, these antibodies or antigen binding fragments can be used in the treatment of cancer. As described above, active TGFe1 released from Tregs inhibits the actions of other T cells. Thus, inhibition of TGFe1-mediated immunosuppressive function represents an attractive approach to enhance the immune response against various forms of cancer. Antibodies selective for the proTGFe1GARP complex can be used to treat both solid tumors and hematological cancers, including leukemia.

Antibodies for use in these methods include antibodies described above herein, such as antibodies selective for the proTGFe1-GARP complex, or an antigen binding fragment with the properties described in table. 1, for example with CDR or variable domain sequences, which are also presented in the further description of these antibodies.

In some embodiments described herein, the immunoeffector properties of antibodies selective for the proTGFe1-GARP complex can be modulated by Fc modifications using techniques known to those skilled in the art. For example, Fc fragment effector functions such as Clq binding, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), down-regulation of cell surface receptors (eg, B-cell receptor; BCR) etc., can be provided and/or controlled by modifying residues in the Fc fragment responsible for these actions.

Antibody-dependent cell-mediated cytotoxicity, or ADCC, is a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRc) (such as natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.

The ability of monoclonal antibodies to induce ADCC can be enhanced by engineering their oligosaccharide component. Human IgG1 or IgG3 undergoes N-glycosylation at Asn297 by most glycans in the well-known 2-antennary forms G0, G0F, G1, G1F, G2 or G2F. Antibodies produced by unengineered CHO cells typically have a fucose glycan content of at least about 85%. Removal of central fucose from the 2-antennary complex type oligosaccharides attached to Fc regions enhances antibody ADCC through improved Fc.gamma.RSha binding without altering antigen binding or CDC activity. Such mAbs can be produced by a variety of methods that have been shown to result in the successful expression of relatively highly defucosylated antibodies bearing Fco-oligosaccharides of the 2-antennary complex type, such as by controlling the osmolality of the culture medium (Konno et al., Cytotechnology 64 :249-65, 2012), use as a host cell line of the variant CHO Lec13 line (Shields et al., J Biol Chem 277:26733-26740, 2002), use as

- 11 046204 host cell line of the variant cell line CHO EB66 (Olivier et al., MAbs; 2(4), 2010; electronic edition before print edition; PMID:20562582), using the rat hybridoma cell line YB2/0 as the cell line -hosts (Shinkawa et al., J Biol Chem 278:3466-3473, 2003), introduction of small interfering RNAs, especially to the alpha-1,6-fucosyltransferase gene (FUT8) (Mori et al., Biotechnol Bioeng 88:901- 908, 2004) or coexpression of beta-1,4-N-acetylglucosaminyltransferase III and Golgi alpha-mannosidase II or the effective alpha-mannosidase I inhibitor, kifunensin (Ferrara et al., J Biol Chem 281:5032-5036, 2006, Ferrara et al. , Biotechnol Bioeng 93:851-861, 2006; Xhou et al., Biotechnol Bioeng 99:652-65, 2008).

In some embodiments described herein, ADCC caused by antibodies selective for the proTGFe1-GARP complex can also be enhanced by introducing certain substitutions into the Fc region of the antibody. Examples of substitutions are, for example, substitutions at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378 or 430 (residue numbering according to the EC index), as described in US Pat. No. 6,737,056.

Pharmaceutical compositions and administration.

The pharmaceutical compositions provided herein contain: a) an effective amount of a proTGFe1-GARP complex-selective antibody or antibody fragment of the present invention and b) a pharmaceutically acceptable carrier, which may be inert or physiologically active. In preferred embodiments, the proTGFe1-GARP complex-selective antibody, proTGFe1-GARP, is a proTGFe1-GARP complex-selective antibody, proTGFe1-GARP, or an antigen-binding fragment thereof, as described above. As used herein, the term pharmaceutically acceptable carriers includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like, which are physiologically compatible. Examples of suitable carriers, diluents and/or excipients include one or more of water, saline, phosphate-buffered saline (PBS), dextrose, glycerol, ethanol, and the like, and any combination thereof. In many cases it will be preferable to include isotonic agents such as sugars, polyhydric alcohols or sodium chloride in the composition. In particular, suitable examples of an acceptable carrier include: (1) Dulbecco's phosphate-buffered saline solution, pH about 7.4, containing or not containing from about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9% saline (0.9% w/v sodium chloride (NaCl)) and (3) 5% w/v dextrose; and it may also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween 20®.

The compositions described herein may also contain an additional therapeutic agent as necessary for the particular disorder being treated. It is preferred that antibodies selective for the proTGFe1-GARP complex, or an antibody fragment, and the additional active compound have complementary activities that do not negatively interfere with each other. In a preferred embodiment, the additional therapeutic agent is cytarabine, an anthracycline, histamine dihydrochloride, or interleukin 2. In a preferred embodiment, the additional therapeutic agent is a chemotherapeutic agent.

The compositions of the invention may be presented in various forms. These forms include, for example, liquid, semi-solid and solid dosage forms, but the preferred form depends on the intended route of administration and the therapeutic use. Typical preferred compositions are in the form of solutions suitable for injection or infusion. The preferred route of administration is parenteral (eg, intravenous, intramuscular, intraperitoneal, subcutaneous). In a preferred embodiment, the compositions of the invention are administered as an intravenous bolus or as a continuous infusion over a period of time. In another preferred embodiment, they are administered intramuscularly, subcutaneously, intraarticularly, intratumorally, periatumorally, into or near diseased tissue to produce local and systemic therapeutic effects.

Sterile compositions for parenteral administration can be prepared by introducing the antibody, antibody fragment or antibody conjugate of the present invention in the desired amount into a suitable solvent, followed by sterilization by microfiltration. The solvent or carrier may be water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., or a combination thereof. In many cases it will be preferable to include isotonic agents such as sugars, polyhydric alcohols or sodium chloride in the composition. These compositions may also contain auxiliary substances, in particular wetting, isotonicizing, emulsifying, dispersing and stabilizing agents. Sterile compositions for parenteral administration can also be prepared as sterile solid compositions that are dissolved at the time of use in sterile water or any other sterile injectable medium.

Antibodies selective for the proTGFe1-GARP complex or an antibody fragment can also be administered

- 12 046204 orally. As solid compositions for oral administration, tablets, pills, powders (gelatin capsules, sachets) or granules can be used. In these compositions, the active ingredient according to the invention is mixed in a stream of argon with one or more inert diluents such as starch, cellulose, sucrose, lactose or silica. These compositions may also contain substances other than diluents, for example, one or more lubricants such as magnesium stearate or talc, a colorant, a coating (sugar-coated tablet) or a glaze.

As liquid compositions for oral administration, pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents such as water, ethanol, glycerin, vegetable oils or paraffin oil can be used. These compositions may contain substances other than diluents, such as wetting agents, sweeteners, thickeners, flavoring agents, or stabilizing agents.

Doses depend on the desired effect, duration of treatment and route of administration used; they are essentially 5 to 1000 mg per day orally for adults, with single doses ranging from 1 to 250 mg of active substance. In general, the appropriate dosage will be determined by the physician based on age, body weight, and other factors specific to the subject being treated.

In a preferred embodiment, proTGFe1-GARP complex-selective antibodies or antibody fragments of the invention are used to treat a hyperproliferative disorder in a mammal. In a more preferred embodiment, one of the above-described pharmaceutical compositions containing an antibody selective for the proTGFe1-GARP complex, proTGFe1-GARP, or an antibody fragment of the invention is used to treat a hyperproliferative disorder in a mammal. In one embodiment, the disorder is cancer. Many different cancers such as adrenal cancer, anal cancer, bladder cancer, brain tumor, glioma, breast cancer, carcinoid cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, tumor Ewing, extracranial germ cell tumor, eye cancer, gallbladder cancer, stomach cancer, germ cell tumor, gestational trophoblastic tumor, head and neck cancer, pharynx cancer, islet cell cancer, kidney cancer, laryngeal cancer, leukemia, lip and oral cavity cancer , liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, Merkel cell carcinoma, squamous cell carcinoma of the head and neck, myeloma, neoplasm, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, sinus and parathyroid cancer, penile cancer, pheochromocytoma cancer, pituitary cancer, plasma neoplasm, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cancer Carcinoma, salivary gland cancer, skin cancer, Kaposi's sarcoma, T-cell lymphoma, soft tissue sarcoma, stomach cancer, testicular cancer, thymoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer or Wilms tumor can be cured by the antibodies described herein.

In treating any of the above forms of cancer, the present treatments may be used to further suppress tumor growth, induce tumor regression, increase progression-free survival, and/or prolong the overall survival of a subject suffering from a tumor. In some embodiments, antibodies selective for the proTGFe1GARP complex may also delay or prevent the onset of metastasis. Treatment progress can be tracked in a variety of ways. For example, inhibition may reduce tumor size and/or reduce metabolic activity within the tumor. Both of these parameters can be measured through, for example, MRI or PET scans. Inhibition can also be monitored by biopsy to confirm the level of necrosis, tumor cell death, and the level of vascularization within the tumor. The extent of metastasis can be monitored by known methods. Accordingly, the pharmaceutical compositions of the invention are useful for the treatment or prevention of metastasis in various forms of cancer, including, without limitation, breast cancer, prostate cancer, endometrial cancer, bladder cancer, kidney cancer, esophageal cancer, testicular cancer, ovarian cancer, squamous cell carcinoma (e.g. squamous cell carcinoma of the head and neck - SCCHN), uveal melanoma, follicular lymphoma, cervical cancer, brain cancer, pancreatic cancer, liver cancer, lymphoma, Hodgkin's disease, multiple myeloma, gastrointestinal cancer and astrocytic cancer.

Likewise, herein further provided is a method of inhibiting the growth of selected populations of cells, comprising contacting TGFe1-expressing immune cells with an effective amount of a proTGFe1-GARP complex-selective antibody or antibody fragment of the present invention, alone or in combination with other therapeutic agents. In preferred embodiments, the proTGFe1-GARP complex-selective antibody, proTGFe1-GARP, is a proTGFe1GARP complex-selective antibody, proTGFe1-GARP, or an antigen-binding fragment thereof, as described above. In a preferred embodiment, it is carried out

- 13 046204 An additional therapeutic agent is immunotherapy, that is, an immunostimulating agent that induces or enhances an immune response. Such agents may include, for example: 1) dendritic cell activators, 2) vaccine adjuvants, 3) T cell stimulators, 4) immunological checkpoint inhibitors, and 5) inhibitors of suppressor cells, cytokines and/or enzymes. Thus, in one embodiment, the antibody is administered with the vaccine.

In clinical use, a therapeutically effective amount of a proTGFe1-GARP complex selective antibody or antigen binding fragment is administered to a subject in need of such treatment. For example, antibodies selective for the proTGFe1-GARP complex and antigen-binding fragments thereof can be used to treat cancers that contain TGFe1-positive immune cells. In preferred embodiments, the proTGFe1-GARP complex-selective antibody, proTGFe1-GARP, is a proTGFe1-GARP complex-selective antibody, proTGFe1-GARP, or an antigen-binding fragment thereof, as described above. In some embodiments, the subject is a mammal, preferably a human. In some embodiments, an antibody selective for the proTGFe1-GARP complex, proTGFe1-GARP, or an antigen-binding fragment is administered as a solution tested for sterility.

Dosage regimens for the above-described treatments and uses are adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over a period of time, or the dose may be proportionally decreased or increased as indicated by the therapeutic situation. Parenteral compositions may be formulated in unit dosage units for ease of administration and uniform dosing.

Effective dosages and dosage regimens of proTGFe1-GARP complex-selective antibodies and fragments depend on the disease or condition being treated and can be determined by one skilled in the art. A non-limiting example of a range for a therapeutically effective amount of a compound of the present invention is a range of about 0.001-10 mg/kg, for example about 0.001-5 mg/kg, for example about 0.001-2 mg/kg, for example about 0.001-1 mg/kg, for example about 0.001, about 0.01, about 0.1, about 1 or about 10 mg/kg.

A physician or veterinarian skilled in the art can readily determine and prescribe the required effective amount of the pharmaceutical composition. For example, a physician or veterinarian may begin with lower doses of the proTGFe1-GARP complex-selective antibody or fragment contained in the pharmaceutical composition than necessary to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, an acceptable daily dose of a proTGFe1-GARP complex selective antibody of the present invention will be that amount of the compound that is the lowest dose that produces a therapeutic effect. Administration may, for example, be parenteral, such as intravenous, intramuscular or subcutaneous. In one embodiment, an antibody selective for the proTGFe1-GARP complex, proTGFe1-GARP, or fragment can be administered by infusion at a weekly dose calculated in mg/m ² Such doses, for example, can be calculated based on the mg/kg doses described above. according to the following formula: dose (mg/kg) x70. Such administration can be repeated, for example, 1 to 8 times, for example 3 to 5 times. Administration can be done by continuous infusion over a period of time from 2 to 24 hours, for example from 2 to 12 hours. In one embodiment, an antibody selective for the proTGFe1-GARP complex, proTGFe1-GARP, or fragment can be administered by slow continuous infusion over a long period of time. time, for example more than 24 hours, to reduce toxic side effects.

In one embodiment, an antibody selective for the proTGFe1-GARP complex, proTGFe1GARP, or fragment can be administered in a weekly dosage calculated as a fixed dose administered up to eight times, such as four to six times, once a week. This pattern can be repeated one or more times as needed, for example after six months or twelve months. Such fixed doses, for example, may be based on the above doses in mg/kg based on an assumed body weight of 70 kg. The dose can be determined or adjusted by measuring the amount of the proTGFe1-GARP complex-selective antibody of the present invention in the blood after administration, for example, by obtaining a biological sample and using anti-idiotypic antibodies targeting the antigen-binding region of the proTGFe1-GARP complex-selective antibody of the present invention. invention.

In one embodiment, an antibody selective for the proTGFe1-GARP complex, proTGFe1GARP, or fragment can be administered as maintenance therapy, for example, once a week for a period of six months or more.

The proTGFe1-GARP selective antibody complex or fragment may also be administered prophylactically to reduce the risk of cancer, delay the onset of cancer progression, and/or reduce the risk of relapse if the cancer is in remission.

- 14 046204

Antibodies selective for the proTGFe1-GARP complex and fragments thereof described herein can also be administered as part of combination therapy, i.e. in combination with other therapeutic agents relevant for the treatment of the disease or condition. Accordingly, in one embodiment, the antibody drug is for combination with one or more additional therapeutic agents, such as a chemotherapeutic agent. In some embodiments, other therapeutic agents include, without limitation, antineoplastic agents, including alkylating agents, including: nitrogen mustards, such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU) and semustine (methyl-CCNU); Temodal™ (temozolamide), ethylenimines/methylmelamine, eg triethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkylsulfonates such as busulfan; triazines, eg dacarbazine (DTIC); antimetabolites including folic acid analogues, eg methotrexate and trimetrexate, pyrimidine analogues, eg 5-fluorouracil (5FU), fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2'-difluorodeoxycytidine, purine analogues, eg 6 -mecaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products including antimitotic drugs such as paclitaxel, vinca alkaloids including vinblastine (VLB), vincristine and vinorelbine, Taxotere, estramustine and estramustine phosphate; pipodfil actimomycotoxins, such as etoposide and teniposide; antibiotics, eg, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycin, plicamycin (mithramycin), mitomycin C and actinomycin; enzymes such as L-asparaginase; biological response modulators, such as interferon-alpha, IL-2, G-CSF and GM-CSF; various agents including platinum coordination complexes such as cisplatin and carboplatin, anthracenodiones such as mitoxantrone, substituted ureas such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocorticoid suppressants such as mitotane (o,p- DDD) and aminoglutethimide; hormones and antagonists, including adrenocorticosteroid antagonists, such as prednisone and equivalents, dexamethasone and aminoglutethimide; Gemzar™ (gemcitabine), a progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate; estrogens, such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens, such as tamoxifen; androgens, including testosterone propionate and fluoxymesterone/equivalents; antiandrogens, such as flutamide, gonadotropin-releasing hormone analogues and leuprolide; and nonsteroidal antiandrogens, such as flutamide. Therapies that target the epigenetic mechanism, including but not limited to histone deacetylase inhibitors, demethylating agents (eg, Vidaza), and transcription factor release (ATRA) therapies, can also be combined with antibodies selective for the proTGFe1-GARP complex.

Additional specific examples of chemotherapy agents include taxol, taxenes (eg, docetaxel and taxotere), modified paclitaxel (eg, Abraxane and Opaxio), doxorubicin, Avastin®, Sutent, Nexavar and other multikinase inhibitors, cisplatin and carboplatin, etoposide, gemcitabine and vinblastine . Specific inhibitors of other kinases may also be used in combination with antibodies selective for the proTGFe1-GARP complex, including, but not limited to, MAPK pathway inhibitors (eg, ERK, JNK, and p38 inhibitors), PI3 kinase/AKT inhibitors, and Pim inhibitors. Other inhibitors include Hsp90 inhibitors, proteasome inhibitors (eg, Velcade), and inhibitors of various mechanisms of action, such as Trisenox.

Such combined administration may be simultaneous, separate or sequential, in any order. When administered concomitantly, the agents may be administered as a single composition or as separate compositions, depending on the situation.

In one embodiment, an antibody selective for the proTGFe1-GARP complex, proTGFe1GARP, or a fragment thereof is combined with an antigen presenting cell stimulatory agent. Examples of such agents include various CD40 agonists, such as an anti-CD40L or CD40 antibody agonist.

Some methods include administering an antibody selective to the proTGFe1GARP complex, or a fragment thereof, with a vaccine adjuvant. Such adjuvants include, for example, IL-12 and various Tol-like receptor (TLR) agonists, including CpG (TLR 9 agonist), monophosphoryl lipid A (MPL - TLR4 agonist), PolyI:C or PolyICLC (TLR3 agonist). and resiquimod and 852A (TLR 7/8 agonists).

In other therapeutic approaches, an antibody selective for the proTGFe1-GARP complex, proTGFe1-GARP, is administered in combination with T cell growth factors such as IL-15 and/or IL-17, or activators of such molecules. In similar methods, a T cell stimulator is combined with an antibody selective for the proTGFe1-GARP complex. Such stimulants include 4-1BB agonists, such as anti-4-LBB antibody agonist and 4-1BBL.

In one embodiment, an antibody selective for the proTGFe1-GARP complex, proTGFe1

- 15 046204

GARP or a fragment of it is administered with a T-cell checkpoint inhibitor, such as molecules that send an inhibitory signal to the immune system. Examples of such agents include PD-1 or PD-L1 (B7-H1) inhibitors, such as anti-PD-1 antibodies, including nivolumab (Bristol-Myers Squibb) and pembrolizumab, also known as MK-3475 (Merck), pidilizumab (Curetech), AMP-224 (Amplimmune), and anti-PD-L1 antibodies, including MPDL3280A (Roche), MDX-1105 (Bristol Myer Squibb), MEDI-4736 (AstraZeneca), and MSB-0010718C (Merck). Other checkpoint inhibitors include CTLA-4 antagonists, such as anti-CTLA-4 antibodies. An example of an anti-CTLA4 antibody is Yervoy® (ipilimumab), offered by Bristol-Myers Squibb. Other examples of CTLA-4 antibodies include tremelimumab (Pfizer), tisilimumab (AstraZeneca), and AMGP-224 (Glaxo Smith Kline).

In other methods, an antibody selective for the proTGFe1-GARP complex, proTGFe1-GARP, or a fragment thereof is administered in combination with an enzyme inhibitor that has an immunosuppressive effect. An example is 1-methyltryptophan (1MT), which is a small molecule indoleamine 2,3-dioxygenase inhibitor.

The proTGFe1-GARP selective antibody complex or fragment thereof may also be used in combination with Amgen's T-VEC (talimogene laherparepvec).

In some embodiments, an antibody selective for the proTGFe1-GARP complex, proTGFe1-GARP, or a fragment thereof is administered in combination with a bispecific antibody. A bispecific antibody can target the host's immune system, in particular the cytotoxic activity of T cells, against cancer cells.

The proTGFe1-GARP selective antibody complex or fragment thereof may also be used in combination with various forms of targeted therapy. Examples of targeted therapy include, but are not limited to, the use of therapeutic antibodies. Examples of antibodies include, without limitation, antibodies that bind to the cell surface proteins Her2, CDC20, CDC33, mucin-like glycoprotein and epidermal growth factor receptor (EGFR) present on tumor cells, OX40, PD-1, CTLA-4, and, if necessary, induce cytostatic and/or cytotoxic effects on tumor cells displaying such proteins. Examples of such antibodies also include HERCEPTIN® (trastuzumab), which can be used to treat breast cancer and other forms of cancer, and RITUXAN® (rituximab), ZEVALIN™ (ibritumomab tiuxetan) and LYMPHOCIDE™ (epratuzumab), which can be used to treat non-Hodgkin's lymphoma and other forms of cancer. A number of other examples of antibodies also include panitumumab (VECTIBIX®), ERBITUX® (IMC-C225); BEXXAR™ (iodine-131-tositumomab); KDR (kinase domain receptor) inhibitors; anti-VEGF antibodies and antagonists (eg Avastin® and VEGAF-TRAP); antibodies to VEGF receptor and antigen-binding regions; ημή-Α^-Ι and -Ang-2 antibodies and antigen-binding regions; antibodies to Tie-2 and other Ang-1 and Ang-2 receptors; Tie-2 ligands; antibodies to Tie-2 kinase inhibitors; Hif-1a inhibitors and Campath™ (alemtuzumab). In some embodiments, the anticancer therapeutic agents are polypeptides that selectively induce apoptosis in tumor cells, including, but not limited to, the TNF-related TRAIL polypeptide.

In one embodiment, an antibody selective for the proTGFe1-GARP complex, proTGFe1GARP, or a fragment thereof, as described herein, is used in combination with one or more antiangiogenic agents that reduce angiogenesis. Some such agents include, but are not limited to, IL-8 antagonists; Campath, B-F.G.F.; FGF antagonists; Tek antagonists (Cerretti et al., U.S. Publication No. 2003/0162712; Cerretti et al., U.S. Pat. No. 6,413,932, and Cerretti et al., U.S. Pat. No. 6,521,424); anti-TWEAK agents (which include, but are not limited to, antibodies and antigen-binding regions); soluble TWEAK receptor antagonists (Wiley, U.S. Pat. No. 6,727,225); ADAM disintegrin domain as an antagonist of integrin binding to its ligands (Fanslow et al., U.S. Publication No. 2002/0042368); anti-ephrin receptor and anti-ephrin antibodies; antigen binding regions or antagonists (U.S. Pat. Nos. 5,981,245; 5,728,813; 5,969,110; 6,596,852; 6,232,447; 6,057,124); anti-VEGF agents (e.g., antibodies or antigen-binding regions that specifically bind VEGF, or soluble VEGF receptors, or their ligand-binding regions), such as Avastin® or VEGF-TRAP™, and anti-VEGF receptor agents (e.g., antibodies or antigen-binding regions) regions that specifically bind to them), EGFR inhibitory agents (e.g., antibodies or antigen-binding regions that specifically bind to them), such as panitumumab, IRESSA™ (gefitinib), TARCEVA™ (erlotinib), anti-AgD and anti- AD agents (e.g., antibodies or antigen-binding regions that specifically bind to them or their receptors, e.g., Tie-2/TEK), and anti-Tie-2 kinase inhibitory agents (e.g., antibodies or antigen-binding regions that specifically bind and inhibit the activity of growth factors, such as hepatocyte growth factor (HGF, also known as scattering factor) antagonists and antibodies or antigen binding domains that specifically bind its c-met receptor (eg, rilotumumab and AMG 337, Amgen); PDGF-BB antagonists; antibodies and antigen-binding regions to PDGF-BB ligands; and PDGFR kinase inhibitors.

Other antiangiogenic agents that may be used in combination with antibodies include:

- 16 046204 agents targeting the proTGFei-GARP complex, or a fragment thereof, include such agents as MMP-2 (matrix metalloproteinase 2) inhibitors, MMP-9 (matrix metalloproteinase 9) inhibitors and COX-II (cyclooxygenase II) inhibitors. Examples of suitable COX-II inhibitors include CELEBREX™ (celecoxib), valdecoxib and rofecoxib.

The proTGFe1-GARP selective antibody complex or fragment thereof, as described, can also be used in combination with a growth factor inhibitor. Examples of such agents include, but are not limited to, agents that can inhibit the EGF-R (epidermal growth factor receptor) response, such as EGF-R antibodies (eg, panitumumab (VECTIBIX®)), EGF antibodies and molecules that are EGF-R inhibitors; VEGF (vascular endothelial growth factor) inhibitors, for example VEGF receptors and molecules that are able to inhibit VEGF; and erbB2 receptor inhibitors, such as organic molecules or antibodies that bind to the erbB2 receptor, such as HERCEPTIN® (Genentech, Inc.). EGF-R inhibitors, which are described, for example, in US patent No. 5,747,498, WO 98/14451, WO 95/19970 and WO 98/02434.

In some therapeutic applications, particularly cancer metastasis to bone with negative effects on bone, it may be advantageous to administer an antibody selective to the proTGFe1-GARP complex, proTGFe1-GARP, or a fragment thereof with a therapeutic agent that prevents further bone loss or promotes restoration of lost bone mass. Accordingly, an antibody selective for the proTGFe1-GARP complex, proTGFe1-GARP, or a fragment thereof, may be administered together with a therapeutically effective amount of a bone mass-promoting (anabolic) agent or an anti-bone resorption agent, including but not limited to: bone morphogenetic factors with designations from BMP-1 to BMP-12; transforming growth factor-β and members of the TGF-β family; fibroblast growth factors FGF-1 to FGF-10; interleukin-1 inhibitors (including IL-1ra, anti-IL-1 antibodies and anti-IL-1 receptor antibodies); TNFa inhibitors (including etanercept, adalimumab and infliximab); RANK ligand inhibitors (including soluble RANK, osteoprotegerin and antagonistic antibodies that specifically bind RANK or RANK ligand, e.g. denosumab (XGEVA®)), Dkk-1 inhibitors (e.g. anti-Dkk-1 antibodies), parathyroid hormone, group E prostaglandins , bisphosphonates and bone-strengthening minerals such as fluoride and calcium. Anabolic agents that can be used in combination with antibodies selective for the proTGFe1-GARP complex and functional fragments thereof include parathyroid hormone and insulin-like growth factor (IGF), the latter agent being preferably used in combination with IGF-binding protein. An IL-1 receptor antagonist suitable for such combination therapy is described in WO89/11540, and a suitable soluble TNF receptor-1 is described in WO98/01555. Examples of RANK ligand antagonists are disclosed, for example, in WO 03/086289, WO 03/002713, US Patent Nos. 6,740,511 and 6,479,635.

In one embodiment, a proposed method of treating cancer comprises administering to a subject in need of such treatment a therapeutically effective amount of an antibody selective for the proTGFe1-GARP complex, as described herein, in combination with radiation therapy. Radiation therapy may involve radiation or appropriate administration of radiopharmaceuticals to the patient. The radiation source may be external or internal to the patient being treated (radiation therapy may, for example, be external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be practically used for these methods include, for example, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodine-123, iodine-131 and indium-111.

Methods for detecting the proTGFe 1 GARP complex.

Provided herein are methods for detecting the proTGFe 1 complex in a biological sample by contacting the sample with an antibody or antigen binding fragment thereof described herein. As described herein, the sample can be obtained from urine, blood, serum, plasma, saliva, ascites fluid, circulating cells, circulating tumor cells, non-tissue associated cells (i.e., free cells), tissues (eg, surgical excised tumor tissue, biopsy materials, including those obtained using fine-needle aspiration biopsy), histological preparations, etc. In some embodiments, the methods described include detecting the proTGFe 1-GARP complex in a biological sample by contacting the sample with any of the proTGFe 1-GARP complex-selective antibodies or antigen-binding fragments thereof described herein.

In some embodiments, a sample can be contacted with more than one of the proTGFe 1-GARP complex-selective antibodies or antigen-binding fragments described herein. For example, a sample may be contacted with a first proTGFe1-GARP complex-selective antibody or antigen-binding fragment thereof, and then contacted with a second proTGFe1-GARP complex-selective antibody or antigen-binding fragment thereof, wherein the first antibody or antigen-binding fragment and a second antibody or antigen binding

- 17 046204 two fragments are not the same antibody or antigen-binding fragment. In some embodiments, the first antibody or antigen-binding fragment thereof may be attached to a surface, such as the surface of a multiwell plate, chip, or similar substrate, before being contacted with a sample. In other embodiments, the first antibody or antigen-binding fragment thereof may not be attached or fixed to anything before contacting the sample.

The disclosed proTGFe1-GARP complex-selective antibodies and antigen-binding fragments can be detectably labeled. In some embodiments, labeled antibodies and antigen binding fragments may facilitate detection of the proTGFe1-GARP complex through the methods described herein. Many such marks are well known to those skilled in the art. For example, suitable tags include, but are not limited to, radioactive tags, fluorescent tags, epitope tags, biotin, chromophore tags, ECL tags, or enzymes. More specifically, the tags described include ruthenium, ^m In-DOTA, '[ndiethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and betagalactosidase, polyhistidine (HIS tag), acridine dyes, cyanine dyes, fluorone dyes, oxazine dyes, phenanthridine dyes, rhodamine dyes, Alexa Fluor® dyes, etc.

The disclosed proTGFei-GARP complex-selective antibodies and antigen-binding fragments can be used in a variety of assays to detect proTGFei in a biological sample. Some acceptable assays include, but are not limited to, Western blot, radioimmunoassay, surface plasmon resonance, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence-activated cell sorting (FACS) cytometry, or ELISA.

Kits for detection of the proTGFe i-GARP complex.

Provided herein are kits for detecting the proTGFe 1 complex in a biological sample. These kits include one or more proTGFeiGARP complex-selective antibodies described herein, or antigen binding fragments thereof, and instructions for use of the kit.

The proposed antibody selective for the proTGFei-GARP complex, proTGFei-GARP or antigen-binding fragment may be in solution; be lyophilized; attached to a substrate, carrier or tablet; or may be detectably labeled.

The described kits may also include additional components used to carry out the methods described herein. By way of example, kits may contain means for obtaining a sample from a subject, a control or a reference sample, such as a sample from a subject with slowly progressing cancer and/or a subject without cancer, one or more sample compartments, and/or instructional materials describing the implementation of the methods inventions, and tissue-specific controls or standards.

The means for determining the level of the proTGFei-GARP complex may further include, for example, buffers and other reagents for use in the assay for determining the level of the proTGFei-GARP complex. The instructions may be, for example, printed instructions for performing the assay and/or instructions for assessing the expression level of the proTGFei-GARP complex.

The described kits may also include means for isolating a sample from a subject. These means may contain one or more pieces of equipment or reagents that can be used to obtain fluid or tissue from a subject. The means for obtaining a sample from a subject may also be means for isolating blood components, such as serum, from the blood sample. Preferably, the kit is intended for use in a human subject.

Examples

The following examples are provided as a supplement to the above description and in order to better understand the subject matter of the invention described herein. These examples should not be considered limiting of the described subject matter of the invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and in light of this, those skilled in the art will recognize various modifications and changes that should be included within the scope of the invention and that can be made without departing from the scope of the invention. inventions.

Example i: discovery of antibodies selective for the proTGFei-GARP complex using phage display technology.

Antibody development activities were undertaken to develop antibodies selective for the proTGFei-GARP complex. The proprietary fully intact ChemPartner phage display library (Chempartner, Shanghai, China) was used as a source of human antibody fragments. The library was first negatively panned against the combination mixture

- i8 046204 biotinylated sGARP (SEQ ID NO: 1) and LTBPl-proTGFei (SEQ ID NO: 2 and 3) to remove scFv fragments that bind the unwanted LTBP1-proTGFb1 complex or uncomplexed sGARP protein. The unselected library output was then panned against sGARPproTGFe1 for several rounds. Twenty-five scFvs that specifically bound sGARP-proTGFe1 were selected based on unique HCDR3 sequences, desired complex selectivity, and sequence lability. The unique heavy chain V regions were cloned into human IgG4 expression vectors, the unique light chains were cloned into human kappa expression vectors, and the resulting proTGFe1GARP complex selective antibody candidates were tested for binding activity in an ELISA. The most strongly binding compounds were selected for further characterization.

Example 2: Inhibition of Treg function by 4B1c1 and 4b16b9 in vitro.

The complex-selective anti-proTGFe1-GARP antibody candidates generated in the previous example were tested for inhibition of human Treg function in an in vitro inhibition assay. Activated CD4+ CD25Hi suppressor cells were used as a source of Tregs, and CFSE-tagged CD4+ CD25 effector T cells (Teff cells) were used as targets to suppress proliferation. Cells were incubated at a ratio of 1 Treg/1 Teff with plate-bound anti-CD3, soluble anti-CD28, and in the presence or absence of candidate anti-proTGFe1-GARP complex selective antibodies. Tregs inhibited Teff cell proliferation by ~50% compared to T effectors alone. Teff proliferation levels (incubated with Tregs) were restored in the presence of 4B1C1 and partially restored by 4B16B9 compared with Treg/Teff or hIgG4 controls (Fig. 1). These results confirm the activity of TGF^1 in the immunosuppression of human Tregs and indicate that 4B1C1 and 4B16B9 can partially block this activity in vitro, similar to the neutralizing positive control antibody TGFe 1D11 (R&D Systems catalog number MAB-1835).

table 2

CDR sequences of two proTGFe1-GARP complex selective antibody candidates that have been shown to bind to the proTGFe1-GARP complex and inhibit human Treg function in vitro _______________________ (SEQ ID NO:)________________________________

ID HC-CDR1 HC-CDR2 HC-CDR3 LC-CDR1 LC-CDR2 LC-CDR3 4В1С1 DYTMH (4) LISWGGGSTYYADSVK G (5) DADDSTFDI (6) RASQSVSRNLA (7) WASTRES (8) QQYYSVPYT (9) 4V16V 9 SYAIS (Yu) GIIPMFGTTNYAQKFQ G (11) DREWEPAYGMDV (12) IGTSSDVGGYNY VS (13) DVSNRPS (14) SAYTVSSTWV (15)

The VH and VL regions for two proTGFe1-GARP complex-selective antibody candidates, anti-proTGFe1 monoclonal antibodies, are shown in Table 1 below. 3.

Table 3

Heavy and light chain sequences of two proTGFe1-GARP complex-selective antibody candidates that have been shown to bind to the proTGFe1-GARP complex and inhibit human Treg function in vitro. Variable regions are underlined

mAb ID Amino acid heavy chain sequence SEQ ID NO: Amino acid light chain sequence SEQ ID NO: 4B1C1 EVQLVQSGGVWQPGGSLRLSCA 16 ETTLTQSPATLSVSPGERVTLSCRASQSVS 17 ASGFTFDDYTMHWVRQAPGKGLE RNLAWYQQKPGQPPKLLIYWASTRESGVPD WVSLISWDGGSTYYADSVKGRFT RFSGSGSGTDFTLTISSLQAEDVAVYYCQQ ISRDNSKNSLYLQMNSLRTEDTA YYSVPYTFGQGTKLEIKRTVAAPSVFIFPP LYYCAKDADDDSTFDIWGQGTMVT SDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFN VBSASTKGPSVFPLAPCSRSTSE STAALGCLVKDYFPEPVTVSWNS

- 19 046204

GALTSGVHTFPAVLQSSGLYSLS SWTVPSSSLGTKTYTCNVDHKP SNTKVDKRVESKYGPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISR T R E VT S VWD VS Q E D R E VQ FNW Y VDGVEVHNAKT KPREEQFNSTYR WSVLTVLHQDWLNGKEYKCKVS NKGLPSSIEKTIS KAKGQPREPQ VYTL P R S Q EEMT KNQVS LT CLVK GFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQK SLSLSLG RGEC 4В16В9 QMQLVQSGAEVKKPGSSVKVSCK 18 QSALTQPASVSGSPGQSITISCIGTSSDVG 19 ASGGTFSSYAISWVRQAPGQGLE WMGGIIPMFGTTNYAQKFQGRVT IIADESTSTAYMELRSLRSDDTA VYYCARDREWEPAYGMDVWGQGT TVTVSSASTKGPSVFPLAPCSRS TSESTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLY SLSSWTVPSSSLGTKTYTCNVD HKPSNTKVDKRVESKYGPPCPPC PAPEFLGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSQEDPEVQF NW YVD GVEVHNAKT KPREEQFNS TYRWSVL TVLHQDWLNGKEYKC KVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLG GYNYVSWYQQHPGKAPKLMIYDVSNRPSGV SNRFSGSKSGNTASLTISGLQAEDEAMYYC SAYTVS S TWVFGGGT KVTVLGQ P KAAP SVT LFPPSSEELQANKATLVCLISDFYPGAVTV AWKADSSPVKAGVETTTPSKQSNNКYAASS YLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECS

Example 3: TGFei bioassay.

The ability of proTGFei-GARP complex-selective antibody candidates to regulate active TGFe1 levels was measured using TMLC reporter cells with an integrated TGFe/Smad3-responsive luciferase expression unit. Briefly, HEK293 or Sw480 cells were transiently transfected with either human proTGFe1 and LTBP1 or human proTGFe1 and GARP expression plasmids. Cells were allowed to recover from transfection and express proTGFe1 in complex with LTBP1 or GARP for 24 h at 37°C before analysis. For the assay, transient transfectants were cocultured with SW480e6 cells, which stably express the TGFβ1-activating integrin αΥβ6.

To confirm that the assay worked as intended, media samples with known concentrations of the growth factor TGF-e'i were added to TMLC reporter cell cultures to generate a standard curve.

ProTGFei-GARP complex-selective antibody candidates against ProTGFei-GARP (10 μg/ml) were combined with transfected cells and added to TMLC reporter cell cultures. The dishes were then incubated at 37°C for 16 hours. Successful TGFei signaling is expected to activate the SMAD2/3 pathway, followed by luciferase expression, which can be detected by adding Bright-Glo as specified by the manufacturer (Promega) and measuring the resulting luminescence in a Biotek Synergy Hi reader (Biotek).

Antibodies 4B1C1 and 4B16B9 induced significantly reduced luciferase expression compared to control treatment, indicating that these antibodies regulate levels of the active growth factor TGFe1 and reduce TGFe1-mediated cell signaling. This effect is similar to that of the positive control neutralizing antibody against TGFP 1D11.

Example 4: Affinity measurement via bilayer interferometry.

The binding affinity of candidate proTGFe1-GARP complex-selective antibodies was measured by biolayer interferometry on an OctetRed 384 (Fortebio, Menlo Park, CA). Streptavidin biosensors (Fortebio, Cat. No. 18-5020) were loaded with biotinylated sGARP-proTGFe1 complex at a concentration of 20 μg/ml in sodium acetate buffer, pH 5, washed in the same buffer and transferred to wells containing 10 μg/ml of candidate antibodies. selective for the proTGFe1-GARP complex into the same buffer. The dissociation constant was obtained by nonlinearly fitting the responses to a steady state algorithm using

- 20 046204 using Octet software (Table 4). Similar affinities were obtained by kinetic fitting.

Table 4

Octet affinity results for proTGFe1-GARP complex selective antibody candidates binding to the human proTGFe1-GARP complex

To establish binding specificity, 4B1C1 and 4B16B9 were screened as above for binding to TGFP1, TGFe2, TGFe3, proTGFp1-LTBP1, proTGFe1-LTBP3, and proTGFe1LRRC33. As described above, antibodies were tested at a concentration of 10 dg/ml and antigens were tested at a concentration of 20 dg/ml and tested under the following conditions. These studies demonstrated no detectable antibody binding to any antigen other than the proTGFe1-GARP complex and are shown in FIG. 4.

Table 5

Assay conditions to determine antibody binding specificity

Analysis stage number Stage type Analysis time in seconds 1 Baseline 60 2 Antigen loading 180 3 Baseline 60 4 Connection 300 5 Dissociation 600

Example 5: Dose-response analysis.

Concentration-dependent regulation by candidate proTGFe1-GARP complex-selective antibodies of active TGFe1 levels was measured using TMLC reporter cells with an integrated TGFβ/Smad3-responsive luciferase expression unit as described in EXAMPLE 3, with the only difference being that the candidates Antibodies selective for the proTGFe1-GARP complex were added to experimental wells at various concentrations, demonstrating that 4B1C1 4B16B9 could inhibit and activate TGFe1 in a dose-dependent manner from 0.178 nM with IC50 and 1.9 nM, respectively (Fig. 3).

Example 6: Antibody Characterization.

Antibody candidates selective for the proTGFe1-GARP complex were measured by biolayer interferometry on an OctetRed 384 instrument (Fortebio, Menlo Park, CA) to determine selectivity for the proTGFe1-GARP complex over other protein complexes (Fig. 4). 4B1C1 and 4B16B9 exhibited a dissociation constant (Kd) of less than 1 nM for the proTGFe1-GARP complex, while they showed no detectable binding to the proTGFe1-LTBP1 or proTGFe1LTBP3 complexes. 4B1C1 and 4B16B9 also did not show binding to the growth factors TGFe1, TGFe2 or TGFe3.

Antibody binding to proTGFe1-LRRC33 complexes was also tested. The proTGFe1LRRC33 complex was expressed and purified by size exclusion chromatography (SEC), and the formation of nonaggregated complexes was confirmed by analytical SEC. Binding of 4B1C1 and 4B16B9 to the purified proTGFe1-LRRC33 complex was then tested using the OctetRed 384 assay (Fortebio, Menlo Park, CA) as described in Example 4. To do this, 4B1C1 or 4B16B9 was captured at anti-human Fc tips, and Octet detected binding to either proTGFe1 -GARP or proTGFe1-LRRC33. In contrast to proTGFe1-GARP, no binding of 4B1C1 or 4B16B9 to the proTGFe1-LRRC33 complex was detected.

- 21 046204

Brief description of the sequence list.

SEQ ID NO: Type View Description Subsequence 1 PRT Human sGARP IEA FQTASQPQAEFQLTWLDLRENKLLHFPDLAALPR LIYLNLSNNLIRLPTGPPQDSKGIHAPSEGWSA LPLSAPSGNASGRPLSQLLNLDLSYNEIELIPDSFLE HLTSLCFLNLSRNCLRTFEARRLGSLPCLMLLD LSHNALETLELGARALGSLRTLLLQGNALRDLPPYTF ANLASLQRLNLQGNRVSPCGGPDEPGPSGCVAF SGITSLRSLSLV DNEIELLRAGAFLHTPLTELDLSSN PGLEVATGALGGLEASLEVLALQGNGLMVLQVD LPCFICLKRLNLAENRLSHLPAWTQAVSLEVLDLRNN SFSLLPGSAMGGLETSLRRLYLQGNPLSCCGNG WLAAQLHQGRVDVDATQDLICRFSSQEEVSLSHVRPE DCEKGGLKNINHHHHHH 2 PRT Human proTGFpi LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQG EVPPGPLPEAVLALYNSTRDRVAGESAEPEPEP EADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFN TSELREAVPEPVLLSRAELRLLRLKLKVEQHVE LYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGWRQ

- 22 046204

WLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGF TTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRH RRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWK WIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQH NPGASAAPCCVPQALEPLPIVYYVGRKPKVEQL SNMIVRSCKCS 3 PRT Human LTBP1 fragment EINECTVNPDICGAGHCINLPVRYTCICYEGYRFSEQ QRKCVDIDECTQVQHLCSQGRCENTEGSFLCIC PAGFMASEEGTNCIDVDECLRPDVCGEGHCVNTVGAF RCEYCDSGYRMTQRGRCEDIDECLNPSTCPDEQ CVNSPGSYQCVPCTEGFRGWNGQCLDVDECLEPNVCA NGDCSNLEGSYMCSCHKGYTRTPDHKHCRDIDE CQQ GNLCVNGQCKNTEGSFRCTCGQGYQLSAAKDQCE DIDECQHRHLCAHGQCRNTEGSFQCVCDQGYRA SGLGDHCEDINECLEDKSVCQRGDCINTAGSYDCTCP DGFQLDDNKTCQDINECEHPGLCGPQGECLNTE GSFHCVCQQGFSISADGRTCEDIDECVNNTVCDSHGF CDNTAGSFRCLCYQGFQAPQDGQGCVDVNECEL LSG VCGEAFCENVEGSFLCVCADENQEYSPMTGQCRS RTSTDLDVDVDQPKEEKKECYYNLNDASLCDNV LAPNVTKQECCCTSGVGWGDNCEIFPCPVLGTAEFTE MCPKGKGFVPAGESSSEAGGENYKDADECLLFG QEICKNGFCLNTRPGYECYCKQGTYYDPVKLQCFDMD ECQDPSSCIDGQCVNTEGSYNCFCTHPM VLDAS EKRCIHHHHH 4 PRT Human 4B1C1-HCDR1 DYTMH 5 PRT Human 4B1C1-HCDR2 LISWDGGSTYYADSVKG 6 PRT Human 4B1C1-HCDR3 DADDSTFDI 7 PRT Human 4B1C1-LCDR1 RASQSVSRNLA 8 PRT Human 4B1C1-LCDR2 WASTRES 9 PRT Human 4B1C1-LCDR3 QQYYSVPYT 10 PRT Human 4B16B9-HCDR1 SYAIS eleven PRT Human 4B16B9-HCDR2 GIIPMFGTTNYAQKFQG 12 PRT Human 4B16B9-HCDR3 DREWEPAYGMDV 13 PRT Human 4B16B9-LCDR1 IGTSSDVGGYNYVS 14 PRT Human 4B16B9-LCDR2 DVSNRPS 15 PRT Human 4B16B9-LCDR3 SAYTVSSTWV 16 PRT Human Heavy chain 4В1С1 E VQ L VQ S GGVWQ PGGSLRLS CAAS G FT FD D YTMHWV RQAPGKGLEWVSLISWDGGSTYYADSVKGRFTISRDN SKNSLYLQMNSLRTEDTALYYCAKDADDDSTFDIWGQG TMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS WTVPSSSL GTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC WVD VS Q E D P E VQ FNW YVD GVE VHNAKT KPREEQFNS T YRWS VLTVLHQDWLNGKEYKCKVSNKGLP S SIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 17 PRT Human 4B1S1 light chain ETTLTQSPATLSVSPGERVTLSCRASQSVSRNLAWYQ QKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLT ISSLQAEDVAVYYCQQYYSVPYTFGQGTKLEIKRTVA APSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKV YACEVTHQGLS S PVTKS FNRGEC 18 PRT Human Heavy chain 4V16V9 QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWV RQAPGQGLEWMGGIIPMFGTTNYAQKFQGRVTIIADE STSTAYMELRSLRSDDTAVYYCARDREWEPAYGMDVW GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPSSSLGTK TYTCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPE VT C WVD VS Q E D P E VQ FNW YVD GVE VHNAKT K P RE EQ FNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLG 19 PRT Human Light chain 4ы6В9 QSALTQPASVSGSPGQSITISCIGTSSDVGGYNYVSW YQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTAS LTISGLQAEDEAMYYCSAYTVSSTWVFGGGTKVTVLG QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLT PEQWKSHRSYSCQV THEGSTVEKTVAPTECS

- 23 046204

Sequence Listing <110> SKOLAR ROCK, INC. CURWEN, GREGORY J. SCHURPF, THOMAS TURNER, KATHERINE <120> ANTIBODIES TO TGF-BETA, METHODS AND APPLICATIONS <130> JBI5093WOPCT <140> ASSIGNMENT OF RIGHTS <141> 07/14/2017 <150> 62/371,355 <151> 2016-08-05 <150 > 62/362,393 <151> 2016-07-14 <160> 19 <170> PatentIn, version 3.5 <210> 1 <211> 614 <212> Protein <213> Homo sapiens <400> 1

His Gln Asp Lys Val Pro Cys Lys Met Val Asp Lys Lys Val Ser Cys 1 5 1015

Gln Val Leu Gly Leu Leu Gln Val Pro Ser Val Leu Pro Pro Asp Thr 20 2530

Glu Thr Leu Asp Leu Ser Gly Asn Gln Leu Arg Ser Ile Leu Ala Ser 35 4045

Pro Leu Gly Phe Tyr Thr Ala Leu Arg His Leu Asp Leu Ser Thr Asn 50 5560

Glu Ile Ser Phe Leu Gln Pro Gly Ala Phe Gln Ala Leu Thr His Leu 65 70 7580

Glu His Leu Ser Leu Ala His Asn Arg Leu Ala Met Ala Thr Ala Leu

9095

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

100 105110

Ser Gly Asn Ser Leu Tyr Ser Gly Leu Leu Glu Arg Leu Leu Gly Glu

115 120125

Ala Pro Ser Leu His Thr Leu Ser Leu Ala Glu Asn Ser Leu Thr Arg

- 24 046204

130

135

140

Leu Thr Arg His Thr Phe Arg Asp Met Pro Ala Leu Glu Gln Leu Asp

145 150 155160

Leu His Ser Asn Val Leu Met Asp Ile Glu Asp Gly Ala Phe Glu Gly

165 170175

Leu Pro Arg Leu Thr His Leu Asn Leu Ser Arg Asn Ser Leu Thr Cys

180 185190

Ile Ser Asp Phe Ser Leu Gln Gln Leu Arg Val Leu Asp Leu Ser Cys

195 200205

Asn Ser Ile Glu Ala Phe Gln Thr Ala Ser Gln Pro Gln Ala Glu Phe

210 215220

Gln Leu Thr Trp Leu Asp Leu Arg Glu Asn Lys Leu Leu His Phe Pro

225 230 235240

Asp Leu Ala Ala Leu Pro Arg Leu Ile Tyr Leu Asn Leu Ser Asn Asn

245 250255

Leu Ile Arg Leu Pro Thr Gly Pro Pro Gln Asp Ser Lys Gly Ile His

260 265270

Ala Pro Ser Glu Gly Trp Ser Ala Leu Pro Leu Ser Ala Pro Ser Gly

275 280285

Asn Ala Ser Gly Arg Pro Leu Ser Gln Leu Leu Asn Leu Asp Leu Ser

290 295300

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

305 310 315320

Ser Leu Cys Phe Leu Asn Leu Ser Arg Asn Cys Leu Arg Thr Phe Glu

325 330335

Ala Arg Arg Leu Gly Ser Leu Pro Cys Leu Met Leu Leu Asp Leu Ser

340 345350

His Asn Ala Leu Glu Thr Leu Glu Leu Gly Ala Arg Ala Leu Gly Ser

355 360365

Leu Arg Thr Leu Leu Leu Gln Gly Asn Ala Leu Arg Asp Leu Pro Pro

370 375380

- 25 046204

Tyr Thr Phe Ala Asn 385

Leu Ala Ser Leu Gln 390

Arg Leu Asn Leu Gln 395

Asn Arg Val Ser Pro

405

Cys Gly Gly Pro Asp

410

Glu Pro Gly Pro Ser

415

Cys Val Ala Phe Ser

420

Gly Ile Thr Ser Leu

425

Arg Ser Leu Ser Leu

430

Asp Asn Glu Ile Glu 435

Leu Leu Arg Ala Gly

440

Ala Phe Leu His Thr

445

Leu Thr Glu Leu Asp

450

Leu Ser Ser Asn Pro

455

Gly Leu Glu Val Ala

460

Gly Ala Leu Gly Gly 465

Leu Glu Ala Ser Leu 470

Glu Val Leu Ala Leu 475

Gly Asn Gly Leu Met

485

Val Leu Gln Val Asp

490

Leu Pro Cys Phe Ile

495

Leu Lys Arg Leu Asn

500

Leu Ala Glu Asn Arg

505

Leu Ser His Leu Pro

510

Trp Thr Gln Ala Val 515

Ser Leu Glu Val Leu

520

Asp Leu Arg Asn Asn 525

Phe Ser Leu Leu Pro

530

Gly Ser Ala Met Gly

535

Gly Leu Glu Thr Ser 540

Arg Arg Leu Tyr Leu 545

Gln Gly Asn Pro Leu 550

Ser Cys Cys Gly Asn 555

Trp Leu Ala Ala Gln

565

Leu His Gln Gly Arg

570

Val Asp Val Asp Ala

575

Gln Asp Leu Ile Cys

580

Arg Phe Ser Ser Gln

585

Glu Glu Val Ser Leu

590

His Val Arg Pro Glu

595

Asp Cys Glu Lys Gly 600

Gly Leu Lys Asn Ile 605

Gly 400

Gly

Val

Pro

Thr

Gln 480

Cys

Ala

Ser

Leu

Gly 560

Thr

Ser

Asn

His His His His His

610 <210> 2 <211> 361 <212> Protein

- 26 046204 <213> Homo sapiens <400> 2

Leu Ser Thr Cys Lys 1 5

Thr Ile Asp Met Glu

Leu Val Lys Arg Lys

Ile Glu Ala Ile Arg

Gly Gln Ile Leu Ser 25

Lys Leu Arg Leu Ala

Pro Pro Ser Gln Gly 35

Glu Val Pro Pro Gly 40

Pro Leu Pro Glu Ala 45

Leu Ala Leu Tyr Asn 50

Ser Thr Arg Asp Arg 55

Val Ala Gly Glu Ser 60

Glu Pro Glu Pro Glu 65

Pro Glu Ala Asp Tyr 70

Tyr Ala Lys Glu Val 75

Arg Val Leu Met Val 85

Glu Thr His Asn Glu

Ile Tyr Asp Lys Phe

Gln Ser Thr His Ser

100

Ile Tyr Met Phe Phe

105

Asn Thr Ser Glu Leu

110

Glu Ala Val Pro Glu

115

Pro Val Leu Leu Ser

120

Arg Ala Glu Leu Arg 125

Leu Arg Leu Lys Leu

130

Lys Val Glu Gln His

135

Val Glu Leu Tyr Gln

140

Tyr Ser Asn Asn Ser 145

Trp Arg Tyr Leu Ser 150

Asn Arg Leu Leu Ala 155

Ser Asp Ser Pro Glu

165

Trp Leu Ser Phe Asp

170

Val Thr Gly Val Val

175

Gln Trp Leu Ser Arg

180

Gly Gly Glu Ile Glu

185

Gly Phe Arg Leu Ser

190

His Cys Ser Cys Asp

195

Ser Arg Asp Asn Thr

200

Leu Gln Val Asp Ile

205

Gly Phe Thr Thr Gly 210

Arg Arg Gly Asp Leu 215

Ala Thr Ile His Gly

220

Asn Arg Pro Phe Leu 225

Leu Leu Met Ala Thr 230

Pro Leu Glu Arg Ala 235

Arg

Ser

Val

Ala

Thr 80

Lys

Arg

Leu

Lys

Pro 160

Arg

Ala

Asn

Met

Gln 240

- 27 046204

His Leu Gln Ser Ser Arg His Arg Arg

245

Phe Ser Ser Thr Glu Lys Asn Cys Cys

260 265

Phe Arg Lys Asp Leu Gly Trp Lys Trp

275 280

His Ala Asn Phe Cys Leu Gly Pro Cys

290 295

Thr Gln Tyr Ser Lys Val Leu Ala Leu

305 310

Ala Ser Ala Ala Pro Cys Cys Val Pro

325

Ile Val Tyr Tyr Val Gly Arg Lys Pro

340 345

Met Ile Val Arg Ser Cys Lys Cys Ser

355 360 <210> 3 <211> 640 <212> Protein <213> Homo sapiens <400> 3

Glu Ile Asn Glu Cys Thr Val Asn Pro

5

Cys Ile Asn Leu Pro Val Arg Tyr Thr

25

Arg Phe Ser Glu Gln Gln Arg Lys Cys

40

Gln Val Gln His Leu Cys Ser Gln Gly

55

Ser Phe Leu Cys Ile Cys Pro Ala Gly 65 70

Thr Asn Cys Ile Asp Val Asp Glu Cys 85

Glu Gly His Cys Val Asn Thr Val Gly

Leu Asp Thr Asn Tyr Cys

255

Arg Gln Leu Tyr Ile Asp

270

His Glu Pro Lys Gly Tyr

285

Tyr Ile Trp Ser Leu Asp 300

Asn Gln His Asn Pro Gly

315 320

Ala Leu Glu Pro Leu Pro

335

Val Glu Gln Leu Ser Asn

350

Ile Cys Gly Ala Gly His 15

Ile Cys Tyr Glu Gly Tyr 30

Asp Ile Asp Glu Cys Thr 45

Cys Glu Asn Thr Glu Gly 60

Met Ala Ser Glu Glu Gly 75 80

Arg Pro Asp Val Cys Gly 95

Phe Arg Cys Glu Tyr Cys

- 28 046204

100 105

110

Asp Ser Gly Tyr Arg Met Thr Gln Arg

115 120

Glu Cys Leu Asn Pro Ser Thr Cys Pro

130 135

Pro Gly Ser Tyr Gln Cys Val Pro Cys

145 150

Asn Gly Gln Cys Leu Asp Val Asp Glu

165

Ala Asn Gly Asp Cys Ser Asn Leu Glu

180 185

His Lys Gly Tyr Thr Arg Thr Pro Asp

195 200

Asp Glu Cys Gln Gln Gly Asn Leu Cys

210 215

Thr Glu Gly Ser Phe Arg Cys Thr Cys 225 230

Ala Ala Lys Asp Gln Cys Glu Asp Ile

245

Leu Cys Ala His Gly Gln Cys Arg Asn

260 265

Val Cys Asp Gln Gly Tyr Arg Ala Ser

275 280

Asp Ile Asn Glu Cys Leu Glu Asp Lys

290 295

Cys Ile Asn Thr Ala Gly Ser Tyr Asp

305 310

Gln Leu Asp Asp Asn Lys Thr Cys Gln

325

Pro Gly Leu Cys Gly Pro Gln Gly Glu

340 345

Arg Cys Glu Asp Ile Asp

125

Glu Gln Cys Val Asn Ser

140

Glu Gly Phe Arg Gly Trp

155 160

Leu Glu Pro Asn Val Cys

175

Ser Tyr Met Cys Ser Cys

190

Lys His Cys Arg Asp Ile

205

Asn Gly Gln Cys Lys Asn 220

Gln Gly Tyr Gln Leu Ser

235 240

Glu Cys Gln His Arg His

255

Glu Gly Ser Phe Gln Cys

270

Leu Gly Asp His Cys Glu

285

Val Cys Gln Arg Gly Asp

300

Thr Cys Pro Asp Gly Phe

315 320

Ile Asn Glu Cys Glu His

335

Leu Asn Thr Glu Gly Ser

350

- 29 046204

Phe His Cys Val Cys Gln Gln Gly Phe Ser Ile Ser Ala Asp Gly Arg

355 360365

Thr Cys Glu Asp Ile Asp Glu Cys Val Asn Asn Thr Val Cys Asp Ser

370 375380

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

385 390 395400

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

405 410415

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

420 425430

Val Glu Gly Ser Phe Leu Cys Val Cys Ala Asp Glu Asn Gln Glu Tyr

435 440445

Ser Pro Met Thr Gly Gln Cys Arg Ser Arg Thr Ser Thr Asp Leu Asp

450 455460

Val Asp Val Asp Gln Pro Lys Glu Glu Lys Lys Glu Cys Tyr Tyr Asn

465 470 475480

Leu Asn Asp Ala

Ser Leu Cys Asp Asn Val Leu Ala Pro Asn Val Thr

485 490495

Lys Gln Glu Cys

Cys Cys

500

Thr Ser Gly Val Gly Trp Gly Asp Asn Cys

505 510

Glu Ile Phe Pro Cys Pro Val Leu Gly Thr Ala Glu Phe Thr Glu Met

515 520525

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

530 535540

Ala Gly Gly Glu Asn Tyr Lys Asp Ala Asp Glu Cys Leu Leu Phe Gly

545 550 555560

Gln Glu Ile Cys Lys Asn Gly Phe Cys Leu Asn Thr Arg Pro Gly Tyr

565 570575

Glu Cys Tyr Cys Lys Gln Gly Thr Tyr Tyr Asp Pro Val Lys Leu Gln

580 585590

Cys Phe Asp Met Asp Glu Cys Gln Asp Pro Ser Ser Cys Ile Asp Gly

595 600605

- 30 046204

Gln Cys Val Asn Thr Glu Gly Ser

610 615

Tyr Asn Cys Phe Cys Thr His Pro

620

Met Val Leu Asp Ala Ser Glu Lys Arg Cys Ile His His His His

625 630 635 640 <210> 4 <211> 5 <212> Protein <213> Homo sapiens <400> 4

Asp Tyr Thr Met His 1 5 <210> 5 <211> 17 <212> Protein <213> Homo sapiens <400> 5

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

5 10 15

Gly <210> 6 <211> 9 <212> Protein <213> Homo sapiens <400> 6

Asp Ala Asp Asp Ser Thr Phe Asp Ile

5 <210> 7 <211> 11 <212> Protein <213> Homo sapiens <400> 7

Arg Ala Ser Gln Ser Val Ser Arg Asn Leu Ala

5 10 <210> 8 <211> 7 <212> Protein <213> Homo sapiens <400> 8

Trp Ala Ser Thr Arg Glu Ser

5

- 31 046204 <210> 9 <211> 9 <212> Protein <213> Homo sapiens <400> 9

Gln Gln Tyr Tyr Ser Val Pro Tyr Thr 1 5 <210> 10 <211> 5 <212> Protein <213> Homo sapiens <400> 10

Ser Tyr Ala Ile Ser 1 5 <210> 11 <211> 17 <212> Protein <213> Homo sapiens <400> 11

Gly Ile Ile Pro Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 1 5 10 15

Gln

Gly <210> 12 <211> 12 <212> Protein <213> Homo sapiens <400> 12

Asp Arg Glu Trp Glu Pro Ala Tyr Gly Met Asp Val

5 10 <210> 13 <211> 14 <212> Protein <213> Homo sapiens <400> 13

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

5 10 <210> 14 <211> 7 <212> Protein <213> Homo sapiens <400> 14

Asp Val Ser Asn Arg Pro Ser 1 5

- 32 046204 <210> 15 <211> 10 <212> Protein <213> Homo sapiens <400> 15

Ser Ala Tyr Thr Val Ser Ser Thr Trp Val

5 10 <210> 16 <211> 444 <212> Protein <213> Homo sapiens <400> 16

Glu Val Gln Leu Val Gln Ser Gly 1 5

Gly Val Val Val Gln Pro Gly Gly

15

Ser Leu Arg Leu Ser Cys Ala Ala 20

Ser Gly Phe Thr Phe Asp Asp Tyr 25 30

Thr Met His Trp Val Arg Gln Ala

40

Pro Gly Lys Gly Leu Glu Trp Val 45

Ser Leu Ile Ser Trp Asp Gly Gly

55

Ser Thr Tyr Tyr Ala Asp Ser Val 60

Lys Gly Arg Phe Thr Ile Ser Arg 65 70

Asp Asn Ser Lys Asn Ser Leu Tyr

80

Leu Gln Met Asn Ser Leu Arg Thr 85

Glu Asp Thr Ala Leu Tyr Tyr Cys

95

Ala Lys Asp Ala Asp Asp Ser Thr

100

Phe Asp Ile Trp Gly Gln Gly Thr

105 110

Met Val Thr Val Ser Ser Ala Ser

115 120

Thr Lys Gly Pro Ser Val Phe Pro

125

Leu Ala Pro Cys Ser Arg Ser Thr

130 135

Ser Glu Ser Thr Ala Ala Leu Gly

140

Cys Leu Val Lys Asp Tyr Phe Pro

145 150

Glu Pro Val Thr Val Ser Trp Asn

155 160

Ser Gly Ala Leu Thr Ser Gly Val

165

His Thr Phe Pro Ala Val Leu Gln

170 175

Ser Ser Gly Leu Tyr Ser Leu Ser

180

Ser Val Val Thr Val Pro Ser Ser

185 190

- 33 046204

Ser

Asn

Pro 225

Phe

Val

Phe

Pro

Thr 305

Val

Ala

Gln

Gly

Pro 385

Ser

Glu

His

Leu Gly Thr Lys Thr

195

Thr Lys Val Asp Lys 210

Pro Cys Pro Ala Pro

230

Pro Pro Lys Pro Lys

245

Thr Cys Val Val Val

260

Asn Trp Tyr Val Asp

275

Arg Glu Glu Gln Phe 290

Val Leu His Gln Asp

310

Ser Asn Lys Gly Leu

325

Lys Gly Gln Pro Arg 340

Glu Glu Met Thr Lys 355

Phe Tyr Pro Ser Asp 370

Glu Asn Asn Tyr Lys

390

Phe Phe Leu Tyr Ser

405

Gly Asn Val Phe Ser

420

Tyr Thr Gln Lys Ser

Tyr Thr Cys Asn Val

200

Arg Val Glu Ser Lys 215

Glu Phe Leu Gly Gly

235

Asp Thr Leu Met Ile

250

Asp Val Ser Gln Glu 265

Gly Val Glu Val His 280

Asn Ser Thr Tyr Arg 295

Trp Leu Asn Gly Lys

315

Pro Ser Ser Ile Glu

330

Glu Pro Gln Val Tyr 345

Asn Gln Val Ser Leu

360

Ile Ala Val Glu Trp 375

Thr Thr Pro Pro Val

395

Arg Leu Thr Val Asp

410

Cys Ser Val Met His 425

Leu Ser Leu Ser Leu

Asp His Lys Pro Ser 205

Tyr Gly Pro Pro Cys 220

Pro Ser Val Phe Leu

240

Ser Arg Thr Pro Glu

255

Asp Pro Glu Val Gln 270

Asn Ala Lys Thr Lys

285

Val Val Ser Val Leu 300

Glu Tyr Lys Cys Lys

320

Lys Thr Ile Ser Lys

335

Thr Leu Pro Pro Ser

350

Thr Cys Leu Val Lys

365

Glu Ser Asn Gly Gln 380

Leu Asp Ser Asp Gly

400

Lys Ser Arg Trp Gln

415

Glu Ala Leu His Asn

430

Gly

- 34 046204

435

440 <210> 17 <211> 214 <212> Protein <213> Homo sapiens <400> 17

Glu Thr Thr Leu Thr 1 5

Gln Ser Pro Ala Thr

Leu Ser Val Ser Pro

Glu Arg Val Thr Leu

Ser Cys Arg Ala Ser

Gln Ser Val Ser Arg 30

Leu Ala Trp Tyr Gln 35

Gln Lys Pro Gly Gln 40

Pro Pro Lys Leu Leu 45

Tyr Trp Ala Ser Thr 50

Arg Glu Ser Gly Val 55

Pro Asp Arg Phe Ser 60

Ser Gly Ser Gly Thr 65

Asp Phe Thr Leu Thr 70

Ile Ser Ser Leu Gln 75

Glu Asp Val Ala Val 85

Tyr Tyr Cys Gln Gln

Tyr Tyr Ser Val Pro

Thr Phe Gly Gln Gly

100

Thr Lys Leu Glu Ile

105

Lys Arg Thr Val Ala

110

Pro Ser Val Phe Ile

115

Phe Pro Pro Ser Asp

120

Glu Gln Leu Lys Ser 125

Thr Ala Ser Val Val

130

Cys Leu Leu Asn Asn

135

Phe Tyr Pro Arg Glu 140

Lys Val Gln Trp Lys 145

Val Asp Asn Ala Leu 150

Gln Ser Gly Asn Ser 155

Glu Ser Val Thr Glu

165

Gln Asp Ser Lys Asp

170

Ser Thr Tyr Ser Leu

175

Ser Thr Leu Thr Leu

180

Ser Lys Ala Asp Tyr

185

Glu Lys His Lys Val

190

Ala Cys Glu Val Thr 195

His Gln Gly Leu Ser

200

Ser Pro Val Thr Lys

205

Gly

Asn

Ile

Gly

Ala 80

Tyr

Ala

Gly

Ala

Gln 160

Ser

Tyr

Ser

Phe Asn Arg Gly Glu Cys

210

- 35 046204 <210> 18 <211> 447 <212> Protein <213> Homo sapiens <400> 18

Gln Met Gln Leu Val

5

Gln Ser Gly Ala Glu

Val Lys Lys Pro Gly

Ser Val Lys Val Ser 20

Cys Lys Ala Ser Gly

Gly Thr Phe Ser Ser 30

Ala Ile Ser Trp Val 35

Arg Gln Ala Pro Gly 40

Gln Gly Leu Glu Trp 45

Gly Gly Ile Ile Pro 50

Met Phe Gly Thr Thr 55

Asn Tyr Ala Gln Lys 60

Gln Gly Arg Val Thr 65

Ile Ile Ala Asp Glu 70

Ser Thr Ser Thr Ala 75

Met Glu Leu Arg Ser

Leu Arg Ser Asp Asp

Thr Ala Val Tyr Tyr

Ala Arg Asp Arg Glu

100

Trp Glu Pro Ala Tyr

105

Gly Met Asp Val Trp

110

Gln Gly Thr Thr Val 115

Thr Val Ser Ser Ala

120

Ser Thr Lys Gly Pro

125

Val Phe Pro Leu Ala

130

Pro Cys Ser Arg Ser

135

Thr Ser Glu Ser Thr

140

Ala Leu Gly Cys Leu 145

Val Lys Asp Tyr Phe 150

Pro Glu Pro Val Thr 155

Ser Trp Asn Ser Gly

165

Ala Leu Thr Ser Gly

170

Val His Thr Phe Pro

175

Val Leu Gln Ser Ser

180

Gly Leu Tyr Ser Leu

185

Ser Ser Val Val Thr

190

Pro Ser Ser Ser Leu

195

Gly Thr Lys Thr Tyr 200

Thr Cys Asn Val Asp

205

Lys Pro Ser Asn Thr

210

Lys Val Asp Lys Arg

215

Val Glu Ser Lys Tyr

220

Ser

Tyr

Met

Phe

Tyr 80

Cys

Gly

Ser

Ala

Val 160

Ala

Val

His

Gly

- 36 046204

Pro Pro Cys Pro Pro 225

Cys Pro Ala Pro Glu 230

Phe Leu Gly Gly Pro 235

Val Phe Leu Phe Pro

245

Pro Lys Pro Lys Asp

250

Thr Leu Met Ile Ser

255

Thr Pro Glu Val Thr

260

Cys Val Val Val Asp

265

Val Ser Gln Glu Asp

270

Glu Val Gln Phe Asn

275

Trp Tyr Val Asp Gly 280

Val Glu Val His Asn

285

Lys Thr Lys Pro Arg

290

Glu Glu Gln Phe Asn

295

Ser Thr Tyr Arg Val

300

Ser Val Leu Thr Val 305

Leu His Gln Asp Trp 310

Leu Asn Gly Lys Glu 315

Lys Cys Lys Val Ser

325

Asn Lys Gly Leu Pro

330

Ser Ser Ile Glu Lys

335

Ile Ser Lys Ala Lys

340

Gly Gln Pro Arg Glu

345

Pro Gln Val Tyr Thr

350

Pro Pro Ser Gln Glu

355

Glu Met Thr Lys Asn 360

Gln Val Ser Leu Thr

365

Leu Val Lys Gly Phe

370

Tyr Pro Ser Asp Ile

375

Ala Val Glu Trp Glu 380

Asn Gly Gln Pro Glu 385

Asn Asn Tyr Lys Thr 390

Thr Pro Pro Val Leu 395

Ser Asp Gly Ser Phe

405

Phe Leu Tyr Ser Arg

410

Leu Thr Val Asp Lys

415

Arg Trp Gln Glu Gly

420

Asn Val Phe Ser Cys

425

Ser Val Met His Glu

430

Ser 240

Arg

Pro

Ala

Val

Tyr 320

Thr

Leu

Cys

Ser

Asp 400

Ser

Ala

Leu His Asn His Tyr 435

Thr Gln Lys Ser Leu 440

Ser Leu Ser Leu Gly 445 <210> 19 <211> 216 <212> Protein <213> Homo sapiens <400> 19

- 37 046204

Gln 1

Ser

Asn

Met

Ser 65

Gln

Ser

Pro

Leu

Pro 145

Ala

Ala

Arg

Thr

Ser Ala Leu Thr Gln

Ile Thr Ile Ser Cys 20

Tyr Val Ser Trp Tyr 35

Ile Tyr Asp Val Ser 50

Gly Ser Lys Ser Gly

Ala Glu Asp Glu Ala 85

Thr Trp Val Phe Gly 100

Lys Ala Ala Pro Ser

115

Gln Ala Asn Lys Ala 130

Gly Ala Val Thr Val

150

Gly Val Glu Thr Thr

165

Ala Ser Ser Tyr Leu 180

Ser Tyr Ser Cys Gln

195

Val Ala Pro Thr Glu 210

Pro Ala Ser Val Ser

Gly Ser Pro Gly Gln

Ile Gly Thr Ser Ser 25

Gln Gln His Pro Gly 40

Asn Arg Pro Ser Gly 55

Asn Thr Ala Ser Leu

Met Tyr Tyr Cys Ser 90

Gly Gly Thr Lys Val

105

Val Thr Leu Phe Pro

120

Thr Leu Val Cys Leu 135

Ala Trp Lys Ala Asp

155

Thr Pro Ser Lys Gln

170

Ser Leu Thr Pro Glu

185

Val Thr His Glu Gly

200

Cys Ser 215

Asp Val Gly Gly Tyr 30

Lys Ala Pro Lys Leu 45

Val Ser Asn Arg Phe 60

Thr Ile Ser Gly Leu

Ala Tyr Thr Val Ser

Thr Val Leu Gly Gln

110

Pro Ser Ser Glu Glu

125

Ile Ser Asp Phe Tyr 140

Ser Ser Pro Val Lys

160

Ser Asn Asn Lys Tyr

175

Gln Trp Lys Ser His

190

Ser Thr Val Glu Lys

205

-

Claims (24)

CLAIM 1. An antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment specifically binds to the human proTGFe 1 complex with predominant human glycoprotein A repeats (proTGFe 1-GARP complex), wherein the antibody or antigen binding fragment comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO : 4, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 6, light chain CDR1 having the amino acid sequence of SEQ ID NO: 7, light chain CDR2 having the amino acid sequence SEQ ID NO: 8, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 9. 2. An antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment specifically binds to a human proTGFe 1 complex with predominant human glycoprotein A repeats (proTGFe 1-GARP complex), wherein said antibody or antigen-binding fragment comprises a heavy chain CDR1 having the amino acid sequence SEQ ID NO: 10, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 11, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 12, light chain CDR1 having the amino acid sequence SEQ ID NO: 13, light chain CDR2, having the amino acid sequence of SEQ ID NO: 14, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 15. 3. An antibody or antigen binding fragment according to claim 1 or 2, wherein the antibody or antigen binding fragment inhibits Treg function in vitro. 4. An antibody or antigen-binding fragment thereof according to any of the preceding claims, wherein the antibody or antigen-binding fragment thereof inhibits TGFe1 activation. 5. The antibody or antigen binding fragment thereof as claimed in any one of the preceding claims, wherein the antibody or antigen binding fragment thereof binds to a human proTGFe1 epitope modified by formation of a human glycoprotein A repeat complex (GARP). 6. An antibody or antigen-binding fragment as claimed in any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof binds to a polypeptide having the amino acid sequence of SEQ ID NO: 2 in the presence of a polypeptide having the amino acid sequence of SEQ ID NO: 1. 7. An antibody or antigen binding fragment as claimed in any one of the preceding claims, wherein the antibody or antigen binding fragment thereof specifically binds to human proTGFe 1 with a binding affinity of at least 880 pM, as measured by a biolayer interferometric assay where streptavidin biosensors are loaded with biotinylated sGARP-proTGFe 1 complex at 20 μg/ml in sodium acetate buffer, pH 5, washed in the same buffer and transferred to wells containing 10 μg/ml proTGFe1-GARP complex-selective antibody candidates in the same buffer and the dissociation constant was measured by nonlinear fitting responses to the steady state algorithm. 8. The antibody or antigen-binding fragment thereof as claimed in any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof binds to human proTGFe1 with a dissociation constant (Kd) of less than or equal to 1 nM for the human proTGFe 1 complex with predominant repeats of human glycoprotein A (proTGFe1 complex -GARP), as determined by surface plasmon resonance, where the indicated proTGFe 1-GARP complex is in solution. 9. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment specifically binds to the human proTGFe 1 complex with predominant human glycoprotein A repeats (proTGFe 1-GARP complex), comprising: (i) a heavy chain variable region having amino acids 1-118 of amino acid sequence SEQ ID NO: 16 and a light chain variable region having amino acids 1-107 of amino acid sequence SEQ ID NO: 17; or (ii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region having the amino acid sequence of SEQ ID NO: 17. 10. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment specifically binds to the human proTGFe 1 complex with predominant human glycoprotein A repeats (proTGFe 1-GARP complex), comprising: (i) a heavy chain variable region having amino acids 1-121 of amino acid sequence SEQ ID NO: 18 and a light chain variable region having amino acids 1-110 of amino acid sequence SEQ ID NO: 19; or (ii) a heavy chain region of the amino acid sequence SEQ ID NO: 18 and a light chain region of the amino acid sequence SEQ ID NO: 19. - 39 046204 11. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment specifically binds to the human proTGFe1-GARP complex and contains a heavy chain variable region at least 90% identical to amino acids 1-118 of amino acid sequence SEQ ID NO: 16, and a variable region light chain having at least 90% identity to amino acids 1-107 of amino acid sequence SEQ ID NO: 17. 12. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment specifically binds to the human proTGFe1-GARP complex and contains a heavy chain variable region at least 90% identical to amino acids 1-121 of amino acid sequence SEQ ID NO: 18, and a variable region light chain having at least 90% identity to amino acids 1-110 of amino acid sequence SEQ ID NO: 19. 13. An antibody or antigen binding fragment as claimed in any one of the preceding claims, wherein the antigen binding fragment is a Fab fragment, a Fab2 fragment or a single chain antibody. 14. The antibody or antigen binding fragment according to any of the preceding claims, wherein the antibody or antigen binding fragment is recombinant. 15. An antibody or antigen binding fragment according to any of the preceding claims, wherein the antibody or antigen binding fragment is of an IgG1, IgG2, IgG3 or IgG4 isotype. 16. The antibody or antigen-binding fragment of claim 15, wherein the antibody is of the IgG4 isotype. 17. The antibody or antigen-binding fragment thereof as claimed in any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof binds to the human proTGFe1-GARP complex when said complex is in solution and the antibody or antigen-binding fragment thereof has no detectable binding as determined by biolayer interferometry analysis , where streptavidin biosensors are loaded with a biotinylated sGARP-proTGFe1 complex at a concentration of 20 μg/ml in sodium acetate buffer, pH 5, washed in the same buffer and transferred to wells containing 10 μg/ml proTGFe1-GARP complex-selective antibody candidates the same buffer, and measuring the dissociation constant by nonlinearly fitting the responses to the steady state algorithm for any of the following agents: growth factor domain TGFe1; growth factor domain TGFP2; growth factor domain TGFP3; proTGFe1, covalently linked to LTBP1; proTGFe1, covalently linked to LTBP3; proTGFe1, covalently linked to LRRC33; and proTGFe1, not related to human GARP; and further, the antibody or antigen binding fragment thereof has an inhibitory concentration (IC50) of less than or equal to 10 nM for inhibiting the release of growth factor TGFe1 from the cell associated proTGFe1-GARP complex, measured in TMLC cells with an integrated TGFe/Smad3 luciferase expression unit at 37°C. 18. A polynucleotide encoding an antibody or antigen-binding fragment according to any of the previous paragraphs. 19. A vector containing a polynucleotide according to claim 18. 20. A host cell containing the vector according to claim 19. 21. A method for producing an antibody or antigen-binding fragment, comprising: culturing the host cell according to claim 20 under conditions allowing expression of the antibody or antigen-binding fragment, and isolating the antibody or antigen-binding molecule from the culture. 22. A pharmaceutical composition containing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 17 and a pharmaceutically acceptable carrier. 23. A kit for determining the presence of the proTGFe1-GARP complex in a biological sample, comprising an antibody or an antigen-binding fragment according to any one of claims 1 to 17 and one or more of: (a) reagents for detecting the presence of the proTGFe1-GARP complex in a biological sample; (b) means for obtaining a sample from the subject; (c) control or reference samples; (d) one or more sample compartments; as well as its packaging. 24. The kit according to claim 23, additionally containing instructions for use. -