CN101218257A - GITR binding molecules and uses thereof - Google Patents

Abstract

The present invention provides binding molecules that specifically bind to GITR, e.g., human GITR (hGITR), on T cells and dendritic cells. Binding molecules of the invention are characterized by binding to hGITR with high affinity, in the presence of a stimulating agent, e.g., CD3, are agonistic, and abrogate the suppression of Teff cells by Treg cells. Various aspects of the invention relate to binding molecules, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such binding molecules. Methods of using a binding molecule of the invention to detect human GITR or to modulate human GITR activity, either in vitro or in vivo, are also encompassed by the invention.

Description

GITR binding molecules and uses thereof

related application

This application is entitled to U.S. Provisional Patent Application 60/665,322, filed March 25, 2005, entitled "GITR Binding Molecules and Uses Thereof," and to U.S. Provisional Patent Application No. 60/665,322, filed June 3, 2005, entitled "GITR Binding Molecules and Uses Thereof." Priority to US Provisional Patent Application 60/687,265, both applications are hereby incorporated by reference in their entirety.

Background technique

Members of the tumor necrosis factor and TNF receptor (TNFR) superfamily regulate a variety of biological functions, including cell proliferation, differentiation and survival. Using differential display to identify T cell mRNAs induced by the synthetic glucocorticoid dexamethasone, Nocentini et al. ((1997) Proc. Natl. Acad. Sci. USA 94:6216-6221997) identified a novel gene encoding the TNFR family Member mouse cDNA. Its corresponding gene is named GITR, which stands for Glucocorticoid-Inducible TNFR Family-Related Gene (also known as TNFRSF18). Like other TNFRs, the extracellular domain of this predicted CITR protein contains homocysteine repeats. In addition, the intracellular domain of GITR is highly homologous to those of mouse and human TNFR, 4-1BB, and CD27. Nocentini et al. ((1997) Proc. Natl. Acad. Sci. USA 94:6216-6221997) demonstrated that the GITR gene is induced in T cells by dexamethasone and other cell-activating stimuli. GITR expression was able to protect T cells from apoptosis induced by anti-CD3 antibody treatment, but not by other apoptotic agents.

Shimizu et al. ((2002) Nat Immunol 3:135-42) found that GITR is mainly expressed on CD4+CD25+ regulatory T cells. However, GITR is also expressed on conventional CD4+ and CD8+ T cells, and its expression increases rapidly after activation. In vitro studies have shown that GITR plays an important role in peripheral immune tolerance mediated by these cells and can eliminate the suppressive function of CD4+CD25+ regulatory T cells (Shimizu et al. (2002) Nat Immunol 3:135-42; McHughet al. (2002) Immunity 16:311-23).

It would be highly beneficial to develop reagents that can be used to modulate signaling through GITR.

Summary of the invention

The present invention provides binding molecules that are capable of specifically binding GITR on cells such as T cells and dendritic cells, such as human GITR (hGITR). The binding molecule of the present invention is characterized in that it can bind hGITR with high affinity; it has antagonism in the presence of stimulators (such as CD3), and can eliminate the effector T (Teff) cells of regulatory T (Treg) cells. inhibition.

One aspect of the invention provides a binding molecule comprising the amino acid sequence of SEQ ID NO: 1, optionally further comprising a leader sequence.

In another aspect, the invention provides a binding molecule comprising the amino acid sequence of SEQ ID NO: 66, optionally further comprising a leader sequence.

In another aspect, the invention provides a binding molecule comprising the amino acid sequence of SEQ ID NO: 2, optionally further comprising a leader sequence.

In another aspect, the invention provides a binding molecule comprising the amino acid sequence of SEQ ID NO: 58, optionally further comprising a leader sequence.

One aspect of the invention provides a binding molecule comprising the amino acid sequence of SEQ ID NO: 59, optionally further comprising a leader sequence.

In another aspect, the present invention provides a binding molecule comprising the amino acid sequence of SEQ ID NO: 60, optionally further comprising a leader sequence.

One aspect of the invention provides a binding molecule comprising the amino acid sequence of SEQ ID NO: 61, optionally further comprising a leader sequence.

In another aspect, the invention provides a binding molecule comprising the amino acid sequence of SEQ ID NO: 62, optionally further comprising a leader sequence.

One aspect of the present invention provides a binding molecule comprising the amino acid sequence of SEQ ID NO: 63, optionally further comprising a leader sequence.

Yet another aspect of the present invention provides such binding molecule, described molecule comprises at least one complementarity determining region (CDR) selected from SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO:19 and SEQ ID NO.5 ) amino acid sequence. In one embodiment, the binding molecule comprises at least two complementarity determining region (CDR) amino acid sequences selected from SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO:19 and SEQ ID NO.5. In another embodiment, the binding molecule comprises at least three complementarity determining region (CDR) amino acid sequences selected from SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO:19 and SEQ ID NO.5.

Another aspect of the present invention provides such a binding molecule, said molecule comprising at least one complementarity determining region (CDR) amino acid sequence selected from SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8. In one embodiment, the binding molecule comprises at least two complementarity determining region (CDR) amino acid sequences selected from SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8. In another embodiment, said binding molecule comprises at least three complementarity determining region (CDR) amino acid sequences selected from SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.

Another aspect of the present invention provides binding molecules comprising the CDRs shown in SEQ ID NO: 3, 4, 5, 6, 7 and 8. Another aspect of the invention provides binding molecules comprising the CDRs set forth in SEQ ID NO: 3, 19, 5, 6, 7 and 8.

One aspect of the present invention provides a binding molecule comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. Another aspect of the invention provides a binding molecule comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:66, and further comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO:2. In more than one embodiment, the binding molecule comprises human or substantially human heavy and light chain framework regions. In another embodiment, one or more human framework region amino acid residues are mutated to the corresponding mouse amino acid residues. In another embodiment, the constant region comprises an IgG2b heavy chain constant region. In another embodiment, the constant region comprises a human, eg, human IgGl heavy chain constant region. In another embodiment, the binding molecule is altered so as to reduce effector function and/or glycosylation. In one embodiment, the binding molecule binds human GITR. In one embodiment, the binding molecule does not induce apoptosis. In another embodiment, the binding molecule is unable to block the primary mixed lymphocyte reaction. In yet another embodiment, the binding molecule is capable of counteracting the suppression of effector T cells by regulatory T cells. In one embodiment, the binding molecule is capable of modulating the proliferation of effector T cells. In one embodiment, the binding molecule is from a mouse. In another embodiment, the binding molecule comprises a murine IgG2b heavy chain. In one embodiment, the binding molecule is a humanized antibody. In yet another embodiment, the binding molecule is a chimeric antibody. In yet another embodiment, the binding molecule is capable of modulating the activity of human GITR. In another embodiment, the binding molecule attenuates the degradation of I-κB in T cells.

Another aspect of the present invention provides binding molecules capable of binding to GITR on human T cells and human dendritic cells and having a binding constant (Kd) of 1×10 ⁻⁹ or lower. In one embodiment, the binding molecule counteracts the suppression of effector T cells by regulatory T cells. In another embodiment, the binding molecule is a humanized antibody.

In yet another aspect the invention provides a composition comprising a binding molecule of the invention and a pharmaceutically acceptable carrier. In one embodiment, the composition further comprises at least one other therapeutic agent for treating cancer in the subject. In one embodiment, the composition further comprises at least one other therapeutic agent for treating the viral infection in the subject. In another embodiment, the composition further comprises at least one tumor antigen for treating cancer in the subject. In yet another embodiment, the composition further comprises at least one antigen from a pathogen.

One aspect of the invention provides a method of eliminating the suppression of effector T cells by regulatory T cells, the method comprising contacting human immune cells with a binding molecule of the invention, thereby allowing regulatory T cells to suppress effector T cells is offset.

Another aspect of the present invention provides a method of modulating T cell receptor-induced signal transduction in effector T cells, the method comprising contacting said cells with a binding molecule of the present invention, thereby causing T cells in effector T cells to Induced receptor signaling is modulated. In one embodiment, the method modulates the degradation of I-κB. In one embodiment, the T cells are Th1 cells. In another embodiment, the T cells are CD4+ cells. In yet another embodiment, said T cells are CD8+ cells.

Yet another aspect of the present invention provides a method for increasing the immune response of a subject, the method comprising contacting cells with the binding molecule of the present invention, so that the immune response of the subject is enhanced.

Another aspect of the invention provides a method of treating cancer in a subject, the method comprising contacting a cell with a binding molecule of the invention, whereby the cancer in the subject is treated. In one embodiment, the type of cancer is selected from the group consisting of: pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer cancer), prostate cancer, gastric cancer, ovarian cancer, urethral bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer (peripheral nervous system cancer), esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer ( liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid cancer ), adrenal gland cancer, osteosarcoma, chondrosarcoma, and cancer of hematopoietic tissues.

Another aspect of the present invention provides a method of treating an infection caused by a pathogen in a subject, the method comprising contacting cells with the binding molecule of claim 1, so that the infection caused by the pathogen in the subject is obtained treat. In one embodiment, the pathogen is a virus, for example selected from: hepatitis A virus (hepatitis type A), hepatitis B virus (hepatitis type B), hepatitis C virus (hepatitis type C), influenza virus (influenza), varicella virus (varicella virus) ), adenovirus (adenovirus), type I herpes simplex virus (herpes simplex type I, HSV I), type II herpes simplex virus (HSV II), rinderpest virus (rinderpest), rhinovirus (rhinovirus), Eike virus ( echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus ( arbovirus, hantavirus, coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, I Human immunodeficiency virus type I (HIV I) and type II (HIV II), any picornaviridae, enteroviruses, caliciviridae, nord Any of the Norwalk group of viruses, togaviruses (such as alphaviruses), flaviviruses, coronaviruses, rabies virus, Malaria Marburg viruses, ebola viruses, parainfluenza virus, orthomyxoviruses, bunya viruses, arenaviruses, reoviruses ( reoviruses), rotaviruses, orbiviruses, human T cell leukemia virus type I, human T cell leukemia virus type II, simian immunodeficiency virus, lentivirus ( lentiviruses), polyomaviruses, parvoviruses, Epstein Barr virus, human herpesvirus 6, cercopithecine hernesvirus 1 (B virus) and pox Viruses (poxviruses). In one embodiment, the method is used to treat chronic viral infections.

In another embodiment, the pathogen is a bacterium, for example selected from: Neisseria spp, Streptococcus spp, Streptococcus mutans (S. mutans), Haemophilus spp. , Moraxella spp, Bordetella spp, Mycobacterium spp, Legionella spp, Escherichia spp, Arc Vibrio spp., Yersinia spp., Campylobacter spp., Salmonella spp., Listeria spp., Helicobacter spp. , Pseudomonas spp, Staphylococcus spp., Enterococcus spp, Clostridium spp., Bacillus spp, Corynebacterium spp.), Borrelia spp., Ehrlichia spp., Rickettsia spp., Chlamydia spp., Leptospira spp. ), Treponema spp.

Another aspect of the present invention provides a method for modulating GITR function, comprising contacting human GITR with the binding molecule of the present invention in the presence of an immune activator, thereby modulating GITR function.

One aspect of the present invention is embodied in such binding molecule, it comprises at least one selected from SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO:19, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO .7 and the CDR amino acid sequence of SEQ ID NO.8. In one embodiment, the composition further comprises at least one therapeutic agent for treating cancer in the subject. In another embodiment, the binding molecule comprises at least one CDR derived from a 6C8 binding molecule. In another embodiment, the binding molecule comprises at least two CDRs derived from a 6C8 binding molecule. In another embodiment, the binding molecule comprises at least three CDRs derived from a 6C8 binding molecule. In another embodiment, the binding molecule comprises at least four CDRs derived from a 6C8 binding molecule. In another embodiment, the binding molecule comprises at least five CDRs derived from the 6C8 binding molecule. In another embodiment, the binding molecule comprises at least six CDRs derived from a 6C8 binding molecule.

Another aspect of the present invention is embodied in a binding molecule comprising six CDRs shown in SEQ ID NOs.3, 4 or 19, 5, 6, 7 and 8.

Another aspect of the present invention is embodied in such binding molecule, which comprises the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, and also comprises the light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In one embodiment, the binding molecule comprises human or substantially human heavy and light chain framework regions. In another embodiment, the binding molecule of the invention comprises a human framework region, wherein one or more human framework region amino acid residues are backmutated to the corresponding mouse amino acid residue, or mutated to another amino acid residue base. In another embodiment, a binding molecule of the invention comprises a constant region of an immunoglobulin molecule, such as an IgG2b heavy chain constant region. In yet another embodiment, the binding molecule is capable of binding human GITR (hGITR). In one embodiment, the binding molecule does not induce apoptosis. In another embodiment, the binding molecule does not block the primary mixed lymphocyte reaction. In yet another embodiment, the binding molecule counteracts the suppression of effector T cells by regulatory T cells. In one embodiment, the binding molecule enhances the proliferation of effector T cells. In another embodiment, the binding molecule neutralizes the activity of human GITR. In yet another embodiment, the binding molecule attenuates degradation of I-κB in T cells.

In one aspect, the invention is embodied in binding molecules that bind GITR on human T cells and human dendritic cells and have a binding constant (Kd) of 1 x 10 ^-9 or less. In one embodiment, the binding molecule counteracts the suppressive effect of regulatory T cells. In another embodiment, the binding molecule is of murine origin or comprises murine CDRs. In yet another embodiment, the binding molecule comprises an IgG2b heavy chain. In one embodiment, the binding molecule is a humanized antibody. In a further embodiment, the binding molecule is a chimeric antibody.

Another aspect of the invention provides a composition comprising a binding molecule of the invention and a pharmaceutically acceptable carrier. In one embodiment, the composition further comprises at least one other therapeutic agent for treating cancer in the subject.

One aspect of the present invention provides a method of eliminating the inhibitory effect of regulatory T cells on T effector cells, the method comprising contacting human immune cells with a binding molecule of the present invention, thereby eliminating the inhibitory effect of regulatory T cells.

Another aspect of the present invention provides a method for modulating the transduction of T cell receptor-induced signals in effector T cells, the method comprising contacting the cells with the binding molecules of the present invention, so that T cell receptors in effector T cells induce signal transduction is regulated. In one embodiment, the method modulates the degradation of I-κB. In one embodiment, the T cells are Th1 cells.

Yet another aspect of the present invention provides a method of enhancing an immune response in a subject, the method comprising contacting a cell with a binding molecule of the present invention, thereby increasing the immune response of the subject.

Another aspect of the invention provides a method of treating cancer in a subject, the method comprising contacting a cell with a binding molecule of the invention, whereby the cancer is treated. In one embodiment, the type of cancer is selected from: pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colon cancer, prostate cancer, gastric cancer, ovarian cancer, urethral bladder cancer, brain or central nervous system cancer, peripheral nerve cancer Systemic cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral cavity or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or adnexal cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, Chondrosarcoma and cancer of the blood-forming tissues.

Another aspect of the present invention provides a method for inhibiting the function of GITR, the method comprising contacting human GITR with the binding molecule of the present invention in the presence of a stimulator, so that the function of GITR is inhibited.

One aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain variable region comprising the nucleotide sequence SEQ ID NO: 9, optionally further comprising a leader sequence. Another aspect of the present invention provides an isolated nucleic acid molecule, which comprises a nucleotide sequence encoding a heavy chain variable region comprising the nucleotide sequence SEQ ID NO: 67, optionally further comprising a leader sequence.

Another aspect of the present invention provides an isolated nucleic acid molecule, which comprises a nucleotide sequence encoding a light chain variable region comprising the nucleotide sequence SEQ ID NO: 10, optionally further comprising a leader sequence.

Another aspect of the present invention provides an isolated nucleic acid molecule comprising at least one nucleotide encoding a CDR selected from SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO:65 and SEQ ID NO.13 sequence. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding at least two CDRs derived from a 6C8 binding molecule. In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding at least three CDRs derived from a 6C8 binding molecule.

Another aspect of the present invention provides an isolated nucleic acid molecule comprising at least one nucleotide sequence selected from the group consisting of SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16 encoding CDRs. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding at least two CDRs derived from a 6C8 binding molecule. In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding at least three CDRs derived from a 6C8 binding molecule.

One aspect of the present invention provides an isolated nucleic acid molecule comprising the nucleotide sequences shown in SEQ ID NO: 11-16 and SEQ ID NO: 65.

One aspect of the present invention provides a recombinant expression vector comprising the nucleic acid molecule of the present invention. In one embodiment, provided recombinant expression vectors comprise a nucleic acid molecule having a nucleotide sequence encoding a binding molecule of the invention. In another embodiment, the present invention provides host cells introduced with the recombinant expression vector of the invention. Another aspect of the present invention provides a method for preparing a binding molecule capable of binding to human GITR, the method comprising culturing the host cell of the invention in a culture medium until the cell produces a binding molecule capable of binding to human GITR.

Brief description of the drawings

Figure 1 shows SDS-PAGE blots of purified mouse and human GITR binding molecules. Twelve micrograms of protein were added to each well.

Figure 2 shows the results of size exclusion chromatography (SE-HPLC) of purified human GITR binding molecules. 50 micrograms of protein were injected onto the SE-HPLC column at a flow rate of 0.6 ml/min. Binding molecules purified by SE-HPLC yielded a population of bound molecules of which 99.8% were in monomeric form and 0.2% were aggregates.

Fig. 3 shows the results of FACS analysis of staining of L-M cells (mouse fibroblasts) transfected with GITR gene with 50 μl of the supernatant of GITR-expressing hybridoma cells. GITR-binding molecules stained GITR-transfected cells, but not non-transfected L-M cells.

Figure 4 shows FACS analysis showing that GITR is mainly expressed on activated lymphocytes. 6C8 binding molecules stained CD4+, CD8+, CD25+ lymphocytes, and weakly stained CD103+ cells.

Figure 5 shows the binding saturation curve of the 6C8 binding molecule, as assessed by titration of biotin-labeled 6C8 on CD3-activated lymphocytes.

Figure 6 shows that 6C8 binding molecules have co-stimulatory activity on T lymphocytes stimulated with sub-optimal OKT3 (anti-CD3; 0.01 μg/ml) and incubated with anti-CD28 or anti-GITR Cell. An isotype control (IgG2b) was also used.

Figures 7A and 7B show that 6C8 binding molecules do not induce apoptosis. Lymphocytes were activated with PHA for 3 days, and then 10 μg/ml YTH655 (an anti-CD2 antibody known to induce apoptosis on activated lymphocytes; Friend, P. et al. (1987) Transplant. Proc. 19: 4317 ), 6C8, or isotype control (IgG2b). Apoptosis was determined by cell viability (A), annexin V staining (B), flow cytometry.

Figure 8 demonstrates that 6C8 binding molecules do not block the primary mixed lymphocyte reaction (MLR). Lymphocytes from allogeneic donors were pooled in the presence of different concentrations of TRX1 (anti-human CD4), 6C8 or MOPC (isotype control for TRX1). Cells were incubated for 3 days, pulsed with ³ H-thymidine for 18 hours, harvested and counted.

Figure 9 shows that 6C8-binding molecules block regulatory T cell (Treg)-induced suppression of T effector cells. CD4+/CD25+ cells were added to CD4+/CD25- cells at various ratios. Cells were stimulated with anti-CD3 and anti-CD28 bound to microplates. At a ratio of 1:1, the proliferation of CD4+/CD25- cells was inhibited. Adding two different dilutions of 6C8 could block the inhibitory effect of CD4+/CD25+regulatory T cells on CD4+T effector cells.

Figure 10 shows that 6C8 binding molecules have co-stimulatory activity even when T cells are stimulated with anti-CD3 in the absence of anti-CD28. CD4+/CD25+ cells were incubated with CD4+/CD25- cells at different cell ratios. Cells were stimulated only with anti-CD3 bound to the microplate. 6C8 was added to the cells, where co-stimulatory activity was shown.

Figure 11 shows the effect of anti-GITR on the degradation of I-κB in CD3-activated T cells.

Figure 12 shows the effect of anti-GITR on the phosphorylation of I-κB in CD3-activated T cells.

Figure 13 shows the effect of anti-GITR on the degradation of I-κB in CD3+CD28 activated T cells.

Figure 14 shows the effect of anti-GITR on the phosphorylation of I-κΒ in CD3+CD28 activated T cells.

Figure 15 shows that antibodies from 6C8 and R&D Systems (Minneapolis, MN) recognize unique epitopes. Competition experiments were performed on lymphocytes activated with OKT3 and concanavalin (Con A). 1 μg/ml 6C8 was used with varying amounts of a competing R&D Systems antibody (GITT/TNFRSF18 monoclonal antibody). Some competition was observed at the highest concentration of antibody, but this was most likely due to steric hindrance.

Figure 16 shows the kinetic analysis of 6C8 anti-GITR antibody relative to R&D Systems GITR antibody.

Figure 17 shows the survival rate of mice injected with mitomycin C-treated B16 cells after treatment with an anti-GITR antibody (2F8 rat anti-mouse GITR binding molecule).

Figures 18A-18D show the nucleic acid and amino acid sequences of the variable heavy chain (VHD) (A and B, respectively) and variable light chain (VKA) (C and D, respectively) of the 6C8 binding molecule. Leader sequences are shown in bold; framework sequences are underlined; CDR sequences are in italics.

Figures 19A and 19B show that 2F8 and 2F8 F(ab') ₂ fragments enhance the humoral response to HA.

Figures 20A and 20B show that 2F8 and 2F8 F(ab') ₂ fragments enhance the humoral response to Ova.

Detailed description of the invention

The present invention provides binding molecules capable of specifically binding to GITR on T cells and dendritic cells, such as human GITR (hGITR). The binding molecules of the present invention are characterized by the ability to bind hGITR with high affinity, and in the presence of stimulators (such as CD3), they have an antagonistic effect and can counteract the inhibitory effect of Treg cells on T effector (Teff) cells. Various aspects of the invention relate to binding molecules, pharmaceutical compositions thereof, as well as nucleic acids encoding the binding molecules, recombinant expression vectors and host cells for making the binding molecules. Methods for detecting human GITR in vitro or in vivo or regulating human GITR activity using the binding molecules of the present invention also fall within the scope of the present invention.

In order to facilitate the understanding of the present invention, some terms are defined first.

I. Definition

The term "glucocorticoid-induced TNF receptor" (abbreviated herein as "GITR"), also known as TNF receptor superfamily 18 (TNFRSF18), as used herein refers to the tumor necrosis factor/nerve growth factor receptor family member. It is a type I transmembrane protein of 241 amino acids, characterized by three cysteine pseudorepeats in the extracellular domain, which specifically protects cells from T cell receptor-induced apoptosis, although it does not protect Cells are protected from other apoptotic signals, including Fas induction, dexamethasone treatment, or UV radiation (Nocentini, G, et al. (1997) Proc. Natl. Acad. Sci. USA94:6216-622). The nucleic acid sequence of human GITR (hGITR) is shown in SEQ ID NO: 17, and its amino acid sequence is shown in SEQ ID NO.18.

The term "binding molecule" as used herein includes molecules comprising at least one antigen binding site capable of specifically binding GITR. By "specific binding" is meant that the binding molecule exhibits substantially only background binding to non-GITR molecules. However, an isolated binding molecule that specifically binds GITR may be cross-reactive with GITR molecules from other species.

Binding molecules of the invention may comprise any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), type (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecules. type of immunoglobulin heavy chain. Binding molecules can have both heavy and light chains. As used herein, the term binding molecule also includes antibodies (including full length antibodies); monoclonal antibodies (including full length monoclonal antibodies); polyclonal antibodies; multispecific antibodies (e.g., bispecific antibodies); Antibodies or chimeric antibodies; and antibody fragments, such as Fab fragments, F(ab') fragments, Fab expression library-generated fragments, epitope-binding fragments of all of the above, and engineered forms of antibodies, such as scFv molecules, so long as they exhibit the desired activity, such as being able to bind GITR.

An "antigen" is an entity (eg, a proteinaceous entity or a peptide) to which a binding molecule specifically binds.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which a binding molecule specifically binds. Epitopes can be composed of continuous amino acids, or discontinuous amino acids arranged together through the quaternary folding of the protein. Epitopes composed of contiguous amino acids are generally maintained upon exposure to denaturing solvents, whereas epitopes formed by quaternary folding are generally lost upon treatment with denaturing solvents. An epitope typically includes a unique three-dimensional conformation of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids. Methods of determining the steric conformation of an epitope include, for example, X-ray crystallography and 2-dimensional nuclear magnetic resonance. See, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

Binding molecules that recognize the same epitope can be identified by simple immunoassays that show that one antibody can block the binding of another antibody to the target antigen, ie competitive binding. Competitive binding is determined by assays in which a test binding molecule is capable of preventing specific binding of a reference binding molecule to a consensus antigen (eg, GITR). Many types of competitive binding assays are known, such as solid-phase direct or indirect radioimmunoassays (RIA); solid-phase direct or indirect enzyme immunoassays (EIA) sandwich competition assays (see Stahli et al. Methods in Enzymology 9: 242(1983)); solid phase direct biotin-avidin EIA (see Kirkland et al.J. Immunol.137:3614(1986)); solid phase direct labeling assay, solid phase direct labeling sandwich assay ( See Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); Solid phase direct labeling of RIA with I-125 labeling (see Morel et al. Mol. Immunol. 25(1): 7(1988 )); solid-phase direct biotin-avidin EIA (Cheung et al. Virology 176: 546 (1990)); and direct-labeled RIA (Moldenhauer et al. Scand. J. Immunol. 32: 77 (1990)) . Typically, such assays will involve the use of purified antigen bound to a solid surface or cells, with unlabeled test binding molecules and labeled reference binding molecules. Competitive inhibition is measured by determining the amount of label bound to the solid surface and to the cells in the presence of the binding molecule to be tested. Generally, the binding molecule to be tested is in excess. Typically, when the competing binding molecule is in excess, it inhibits the specific binding of the reference binding molecule to the consensus antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more .

Epitopes can also be recognized by immune cells, such as B cells and/or T cells. Cellular recognition of epitopes can be determined by in vitro assays measuring antigen-dependent proliferation, like ^by3H -thymidine incorporation, cytokine secretion, antibody secretion or antigen-dependent killing (cytotoxic T lymphocyte assay). Sure.

The term "monoclonal binding molecule" as used herein refers to a binding molecule obtained from a substantially homogeneous population of binding molecules. Monoclonal binding molecules are highly specific, directed against a single antigenic site. Furthermore, each monoclonal binding molecule is directed against a single determinant on the antigen, unlike polyclonal binding molecule preparations which generally contain different binding molecules directed against different determinants (epitopes). The modifier "monoclonal" indicates that such binding molecules are characterized as being obtained from a substantially homogeneous population of binding molecules and should not be construed as requiring any particular method for preparing the binding molecules. For example, monoclonal binding molecules consistent with the present invention can be prepared by the hybridoma method first described by Kohler et al. (Nature 256:495 (1975)), or by recombinant DNA methods (see, e.g., U.S. Patent 4,816,567). "Monoclonal binding molecules" can also be isolated from phage libraries using techniques such as those described in Clackson, et al. Nature 352:624-628 (1991) and Marks et al. J. Mol Biol. .

The term "chimeric binding molecule" refers to a binding molecule comprising amino acid sequences derived from different species. Chimeric binding molecules can be constructed, for example, by genetic engineering from binding molecule gene fragments from different species.

Monoclonal binding molecules herein specifically include "chimeric" binding molecules whose heavy and/or light chain portions are identical or identical to corresponding sequences in binding molecules derived from a particular species, or belonging to a particular antibody type or subtype. origin, while the rest of the chain is identical or homologous to corresponding sequences in binding molecules derived from another species or belonging to another antibody type or subtype, and fragments of such binding molecules, provided they exhibit the desired Desired biological activity (US Patent 4,816,567; and Morrison, et al. Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)), such as binding to human GITR (hGITR).

Both light and heavy chains are divided into distinct regions based on structural and functional homology. The terms "constant" and "variable" are used to describe functionality. In this regard, it should be understood that the variable domain (VL) of the light chain portion and the variable domain (VH) of the heavy chain portion determine antigen recognition and specificity. In contrast, the constant domain (CL) of the light chain and the constant domain of the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, placental crossing, Fc receptor binding, complement fixation, etc. Traditionally, constant region domains have been numbered greater the farther they are from the antigen binding site or the amino terminus of the antibody. The N-terminus is the variable region and the C-terminus is the constant region; the CH3 and CL domains actually comprise the carboxy-terminal ends of the heavy and light chains, respectively.

"Variable region" when used to describe a binding molecule refers to the amino-terminal portion of the binding molecule which facilitates binding of antigen to said binding molecule and is not a constant region. The term includes functional fragments that maintain some or all of the binding functions of the entire variable region.

The term "hypervariable region" as used herein refers to a region of the variable domain of a binding molecule that is hypervariable in sequence and/or forms loops that are structurally confined together. Hypervariable regions comprise amino acid residues from "complementarity determining regions" or "CDRs".

As used herein, the term "CDR" or "complementarity determining region" means the non-contiguous antigen binding sites that can be found in the variable regions of heavy and light chain polypeptides. Kabat, et al.J.Biol.Chem.252, 6609-6616 (1977) and Kabat, et al.Sequences of protein of immunological interest. (1991), and Chothia, et al.J.Mol.Biol.196:901 -917 (1987) and MacCallum, et al. J. Mol. Biol. 262:732-745 (1996) describe these specific regions, where definitions include overlaps or subgroups of amino acid residues when compared to each other. In general, use of any of these definitions to refer to a binding molecule or to graft a CDR of a binding molecule or a variant thereof is within the scope of the term as defined and used herein.

As used herein, the term "framework region" or "FR" means each domain in the framework separated by CDRs. Thus, the variable region framework is about 100-120 amino acids long, but only those amino acids outside the CDRs.

"Humanized" forms of non-human (eg, murine) binding molecules are chimeric antibodies that contain minimal sequence derived from the non-human binding molecule. In most cases, a humanized binding molecule is a human binding molecule (receptor/acceptor binding molecule) in which residues from the hypervariable region are replaced by a non-human species (donor binding molecule), such as mouse, rat, Rabbit or non-human primate hypervariable region residues with the desired specificity, affinity and capacity are substituted. In some instances, Fv framework region (FR) residues of the human binding molecule are altered, eg, substituted, substituted, or backmutated to corresponding non-human residues. In addition, humanized binding molecules may contain residues that are absent from either the receptor binding molecule or the donor binding molecule. Such modifications are generally intended to further optimize the properties of the binding molecule. Typically, a humanized binding molecule will comprise substantially all of at least one, typically two, variable domains, wherein all or substantially all of the hypervariable loops correspond to hypervariable loops of a non-human binding molecule, and all or substantially all of the FR Regions are FRs of the human binding molecule sequence. A humanized binding molecule optionally further comprises at least a portion of the binding molecule constant region (Fc), typically a human binding molecule. For more details, see Jones, et al. Nature 321: 522-525 (1986); Riechmann, et al. Nature 332: 323-329 (1988); Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).

Preferably, the humanized binding molecule of the present invention comprises at least one selected from SEQ ID NO.3 (GFSLSTSGMGVG (HC CDR1)), SEQ ID NO.4 (HIWWDDDKYYNPSLKS (HC CDR2N)), SEQ ID NO.5 (TRRYFPFAY ( HC CDR3)), SEQ ID NO.6 (KASQNVGTNVA (LC CDR1)), SEQ ID NO.7 (SASYRYS (LC CDR2)), SEQ ID NO.8 (QQYNTDPLT (LC CDR3)) and SEQ ID NO: 19 (HIWWDDDKYYQPSLKS (HC CDR2Q)) CDR.

The term "engineered" or "recombinant" binding molecule as used herein includes a binding molecule prepared, expressed, created or isolated by recombinant means, such as a binding molecule expressed using a recombinant expression vector transfected into a host cell, from recombinant, combined A binding molecule isolated from a library of binding molecules, a binding molecule isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor, L.D. et al. (1992) Nucl. Acids Res. 20:6287- 6295), or binding molecules prepared, expressed, created or isolated by other means involving the splicing of human binding molecule gene sequences to other DNA sequences. In some embodiments, however, such recombinant human binding molecules have been subjected to in vitro mutagenesis (or, when using transgenic animals of human Ig sequences, in vivo somatic mutation), so that the amino acid sequences of the VH and VL regions of the recombinant binding molecules although Derived from and related to human germline VH and VL sequences, may not be naturally present in the germline of the human binding molecule in vivo.

An "isolated binding molecule" as used herein refers to a binding molecule that is substantially free of other binding molecules having a different antigen specificity (e.g., an isolated binding molecule that specifically binds GITR is substantially free of specific binding molecules). binding molecules to antigens other than GITR). Furthermore, an isolated binding molecule may be substantially free of other cellular material and/or chemical substances. An "isolated" binding molecule is one that has been identified and separated and/or recovered from a component in its natural environment. Impurity components of the natural environment include, for example, substances that interfere with the diagnostic or therapeutic use of the binding molecule and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the binding molecule can be purified to the following extent: (1) as determined by the Lowry method, reaching more than 95% of the weight of the compound, most preferably more than 99% of the weight, (2) enough to undergo rotor-cup sequencing The N-terminal amino acid sequence or at least 15 residues of the internal amino acid sequence were determined by a spinning cup sequencer, or (3) SDS-PAGE and Coomassie blue or preferably silver staining under reducing or non-reducing conditions Out is homogeneous. Isolated binding molecule includes binding molecule in situ in recombinant cells since at least one component of the binding molecule's natural environment is absent. Ordinarily, however, isolated binding molecules are prepared by at least one purification step.

As used herein, the term "binding constant""(kd)" (also known as "affinity constant") is used to measure the degree of reversible association between two molecules, including actual binding affinity and apparent binding affinity . Actual binding affinities are determined by calculating the ratio of the M ^-1 S ^-1 association constant to the S ^-1 dissociation constant, expressed in "M ^-1 ". Thus, conferring or optimizing binding affinity involves altering either or both of these components to achieve the desired level of binding affinity. Apparent affinity can include, for example, affinity of a reaction. For example, bivalent heteronumber variable region binding fragments may exhibit varying or optimized binding affinities due to their valence states. Binding affinity can be determined, for example, by measuring surface plasmon resonance using the BIAcore system.

The term "nucleic acid molecule" as used herein includes DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA.

When the term "isolated nucleic acid molecule" is used herein to describe a nucleic acid encoding a binding molecule capable of binding GITR, it means that the nucleotide sequence encoding said binding molecule in the nucleic acid molecule does not contain the naturally flanking DNA in human genomic DNA. other nucleotide sequences of the nucleic acid. Such sequences optionally include 5' or 3' nucleotide sequences important for regulation or protein stability.

The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring another nucleic acid to which it has been linked. One class of vectors is a "plasmid", which refers to a circular double-stranded DNA circle into which other DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with a bacterial origin of replication, and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) are capable of integrating into the genome of the host cell after introduction into the host cell, thereby replicating along with the host genome. In addition, there are vectors capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors used in recombinant DNA techniques are usually plasmids. In this specification, "plasmid" and "vector" are used interchangeably, since the most commonly used form of vector is a plasmid. However, the invention includes other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses, and adeno-associated viruses), which can serve the same function.

The term "recombinant host cell" (or simply "host cell") as used herein refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms refer not only to that particular cell, but also to the progeny of that cell. These progeny may actually be different from the parental cells because of possible modifications due to mutations or environmental influences in subsequent generations, but are still included within the scope of the term "host cell" as used herein.

As used herein, the term "T cells" (ie, T lymphocytes) includes all cells of the T cell lineage from mammals (eg, humans), including thymocytes, immature T cells, mature T cells, and the like. Preferably, said T cells are mature T cells expressing CD4 or CD8, but not both, and the T cell receptor. The various T cell populations described herein can be defined according to their cytokine profile and function, known to those skilled in the art.

As used herein, the term "dendritic cells" refers to professional antigen-presenting cells (APCs) capable of activating naive T cells, stimulating the growth and differentiation of B cells.

As used herein, the term "naive T-cell" includes T-cells that have not been exposed to the cognate antigen and thus are not activated or have memory. Naive T cells do not cycle and human naive T cells are CD45RA+. If naive T cells recognize the antigen and receive other signals, which may be based on, but not limited to, the amount of antigen, route of administration, and timing of administration, naive T cells may proliferate and differentiate into various T cell subtypes, such as effector T cells.

As used herein, the term "effector T cell" or "Teff cell" includes T cells that function to clear antigen (eg, by producing cytokines that modulate the activation of other cells or by cytotoxic activity). The term "effector T cells" includes helper T cells (eg, Th1 and Th2 cells) as well as cytotoxic T cells. Th1 cells mediate delayed hypersensitivity and giant cell activation, while Th2 cells can assist B cells and are critical in allergic reactions (Mosmann and Coffman, 1989, Annu.Rev.Immunol.7, 145-173; Paul and Seder , 1994, Cell 76, 241-251; Arthurand Mason, 1986, J.Exp.Med.163, 774-786; Paliard, et al.1988, J.Immunol.141, 849-855; Finkelman, et al.1988 , J. Immunol. 141, 2335-2341).

As used herein, the term "type 1 helper T cell response" (Th1 response) refers to a response characterized by the production of one or more of the group consisting of IFN-γ, IL-2, TNF and lymphotoxin (LT). Cytokines, and other cytokines that are preferentially or exclusively produced by Th1 cells rather than Th2 cells. As used herein, a "type 2 helper T cell response" (Th2 response) refers to a CD4+ T cell response characterized by the production of one or more cells selected from the group consisting of IL-4, IL-5, IL-6 and IL- 10 and is associated with B cell "help" provided by efficient Th2 cells (eg, increased IgG1 and/or IgE production).

As used herein, the term "regulatory T cells" or "Treg cells" includes T cells that produce low levels of IL-2, IL-4, IL-5 and IL-12. Regulatory T cells produce TNFα, TGFβ, IFN-γ, and IL-10, but at lower levels than effector T cells. Although TGFβ is the major cytokine produced by regulatory T cells, it produces cytokines at levels lower than or equal to those produced by Th1 or Th2 cells, for example an order of magnitude lower than in Th1 or Th2 cells. Regulatory T cells can be found in CD4+CD25+ cell populations (see, eg, Waldmann and Cobbold. 2001. Immunity. 14:399). Regulatory T cells can actively suppress the proliferation and cytokine production of Th1, Th2, or naive T cells that have been stimulated in culture with activating signals (e.g., antigens and antigen-presenting cells, or with mock MHC). Antigen (for example, anti-CD3 antibody, plus anti-CD28 antibody)) stimulation.

As used herein, the term "tolerance" includes refractory to stimuli mediated by activating receptors. This refractoriness is usually antigen-specific and persists after exposure to the tolerized antigen is discontinued. For example, tolerance can be characterized by a lack of production of cytokines such as IL-2, or can be assessed using mixed lymphocyte cultures. Tolerance can occur to self or foreign antigens.

"Mixed lymphocyte culture"("MLC") is a lymphocyte proliferation assay in which lymphocytes from two individuals are cultured together and the proliferative response is measured by the uptake ^of3H -labeled thymidine ("MLC ").

As used herein, the term "apoptosis," also known as programmed cell death (PCD), is cell death characterized by, but not limited to, condensation of nuclear heterochromatin, cell shrinkage, cytoplasmic condensation, and Later in apoptosis, endonuclease-mediated cleavage of cellular DNA into specific fragments. When electrophoresis analysis is performed on the DNA of cells that have undergone apoptosis, an obvious characteristic "gradient" of DNA fragments may be formed.

"Treatment"includes both therapeutic treatment and prophylactic or preventative measures. Those who need some kind of treatment include those who already suffer from a certain disorder, and those who have not yet manifested a disorder.

A "disorder" is any condition that may benefit from treatment with a binding molecule of the invention. It includes chronic and acute disorders or diseases or pathological states associated with hyper or hypoimmune responses.

The following sections describe various aspects of the invention in further detail.

II. GITR Binding Molecules

The invention provides isolated GITR binding molecules. Exemplary binding molecules of the invention include the 6C8 antibody and the 2F8 antibody. Antibody 6C8 is an anti-GITR antibody that binds with high affinity to GITR on T cells and dendritic cells, such as human T cells and dendritic cells. Preferably, such binding molecules counteract the inhibition of Treg cells on Teff cells and are antagonistic to partially activated T cells in vitro in the presence of a stimulator (eg CD3).

In one embodiment, the VH domain of a binding molecule of the invention comprises the amino acid sequence shown in SEQ ID NO: 1 (6C8 VH domain "N", including leader sequence). It should be understood that while some sequences of the binding molecules described herein include leader sequences, it is optional that the binding molecules of the invention do not include leader sequences. For example, in one embodiment, the binding molecule of the invention comprises the amino acid sequence of the mature protein shown in SEQ ID NO: 1, such as amino acids 20-138 in SEQ ID NO: 1.

In one embodiment, the VH domain of a binding molecule of the invention comprises the amino acid sequence shown in SEQ ID NO: 66 (6C8 VH domain "Q", including leader sequence).

In one embodiment, the VL domain of the binding molecule of the invention comprises the amino acid sequence shown in SEQ ID NO: 2 (6C8 VL domain, including leader sequence).

In one embodiment, the VH domain of a binding molecule of the invention comprises amino acid residues 20-138 of SEQ ID NO: 1 (6C8 VH domain "N" without leader sequence).

In one embodiment, the VH domain of a binding molecule of the invention comprises amino acid residues 20-138 of SEQ ID NO: 66 (6C8 VH domain "Q", without leader sequence).

In one embodiment, the VL domain of a binding molecule of the invention comprises amino acid residues 21-127 in SEQ ID NO: 2 (6C8 VL domain without leader sequence).

In one embodiment of the invention, the VL chain comprises a guide and/or signal sequence, amino acid residues 1-20 of SEQ ID NO: 2 (SEQ ID NO: 59). In one embodiment, the VH chain comprises a leader and/or signal sequence, amino acid residues 1-19 of SEQ ID NO: 1 (SEQ ID NO: 64).

In one embodiment, the VH domain comprised by the binding molecule of the present invention contains the CDR shown in SEQ ID NO: 3 (6C8 VH CDR1).

In one embodiment, the binding molecule of the invention comprises a VH domain comprising the CDRs shown in SEQ ID NO: 4 (6C8 VH CDR2-"N").

In one embodiment, the VH domain comprised by the binding molecule of the present invention contains the CDR shown in SEQ ID NO: 5 (6C8 VH CDR3).

In one embodiment, the binding molecule of the present invention comprises a VH domain comprising the CDRs shown in SEQ ID NO: 19 (6C8 VH CDR2-alternate "Q").

In one embodiment, the binding molecule of the present invention comprises a VL domain comprising the CDRs shown in SEQ ID NO: 6 (6C8 VL CDR1).

In one embodiment, the binding molecule of the present invention comprises a VL domain comprising the CDRs shown in SEQ ID NO: 7 (6C8 VL CDR2).

In one embodiment, the binding molecule of the present invention comprises a VL domain comprising the CDRs shown in SEQ ID NO: 8 (6C8 VL CDR3).

The present invention also relates to nucleic acid molecules encoding the above amino acid sequences.

In one embodiment, the VH domain of the binding molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 9 (6C8 VH domain, "N", including the leader sequence).

In one embodiment, the VH domain of a binding molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 65 (6C8 VH domain, "Q", including leader sequence).

In one embodiment, the VH domain of a binding molecule of the invention comprises nucleotides 58-414 of SEQ ID NO: 9 (6C8 VH domain, "N", without leader sequence).

In one embodiment, the VH domain of a binding molecule of the invention comprises nucleotides 58-414 of SEQ ID NO: 65 (6C8 VH domain, "Q", without leader sequence).

In one embodiment, the VL domain of the binding molecule of the present invention comprises the nucleotide sequence shown in SEQ ID NO: 10 (6C8 VL domain, including leader sequence).

In one embodiment, the VL domain of a binding molecule of the invention comprises nucleotides 61-381 in SEQ ID NO: 10 (6C8 VL domain without leader sequence).

In one embodiment, the VH domain contained in the binding molecule of the present invention contains a CDR (6C8 VH CDR1) whose nucleic acid sequence is shown in SEQ ID NO: 11.

In one embodiment, the VH domain comprised by the binding molecule of the present invention contains a CDR whose nucleic acid sequence is shown in SEQ ID NO: 12 (6C8 VH CDR2-"AAT").

In one embodiment, the VH domain comprised by the binding molecule of the present invention contains a CDR (6C8 VH CDR3) whose nucleic acid sequence is shown in SEQ ID NO: 13.

In one embodiment, the VH domain comprised by the binding molecule of the present invention contains a CDR whose nucleic acid sequence is shown in SEQ ID NO: 65 (6C8 VH CDR2-alternate "CAA").

In one embodiment, the VL domain contained in the binding molecule of the present invention contains a CDR (6C8 VL CDR1) whose nucleic acid sequence is shown in SEQ ID NO: 14.

In one embodiment, the VL domain contained in the binding molecule of the present invention contains a CDR (6C8 VL CDR2) whose nucleic acid sequence is shown in SEQ ID NO: 15.

In one embodiment, the VL domain contained in the binding molecule of the present invention contains a CDR (6C8 VL CDR3) whose nucleic acid sequence is shown in SEQ ID NO: 16.

In one embodiment, the CL domain of the binding molecule of the invention comprises the amino acid sequence shown in SEQ ID NO: 20 (mouse IgG2a light chain constant region).

In one embodiment, the CH domain of the binding molecule of the invention comprises the amino acid sequence shown in SEQ ID NO: 21 (mouse IgG2a heavy chain constant region).

In one embodiment, the binding molecule of the present invention comprises the amino acid sequence shown in SEQ ID NO: 22 (chimeric-6C8VL/human CL IgG1).

In one embodiment, the binding molecule of the present invention comprises the amino acid sequence shown in SEQ ID NO: 23 (chimeric Gly-6C8 VH/human CH IgG1).

In one embodiment, the binding molecule of the present invention comprises the amino acid sequence shown in SEQ ID NO: 24 (chimeric Agly-6C8 VH/human CH IgG1).

In one embodiment, the binding molecule of the present invention comprises the amino acid sequence shown in SEQ ID NO: 44 (humanized 6C8 VL).

In one embodiment, a binding molecule of the invention comprises the amino acid sequence shown in SEQ ID NO: 53 (humanized 6C8 VH "N").

In one embodiment, a binding molecule of the invention comprises the amino acid sequence shown in SEQ ID NO: 54 (humanized 6C8 VH "Q").

In one embodiment, the CL domain of the binding molecule of the invention comprises the amino acid sequence shown in SEQ ID NO: 55 (human IgG1 Gly heavy chain constant region).

In one embodiment, the CH domain of the binding molecule of the present invention comprises the amino acid sequence shown in SEQ ID NO: 56 (human IgG1 Agly heavy chain constant region).

In one embodiment, the CL domain of the binding molecule of the invention comprises the amino acid sequence shown in SEQ ID NO: 57 (human IgG1 light chain constant region).

In one embodiment, the binding molecule of the invention comprises the amino acid sequence shown in SEQ ID NO: 58 (fully humanized 6C8 light chain).

In one embodiment, the binding molecule of the present invention comprises the amino acid sequence shown in SEQ ID NO: 60 (fully humanized 6C8 heavy chain-HuN6C8-Gly).

In one embodiment, the binding molecule of the present invention comprises the amino acid sequence shown in SEQ ID NO: 61 (fully humanized 6C8 heavy chain-HuN6C8-Agly).

In one embodiment, the binding molecule of the present invention comprises the amino acid sequence shown in SEQ ID NO: 62 (fully humanized 6C8 heavy chain-HuQ6C8-Gly).

In one embodiment, the binding molecule of the present invention comprises the amino acid sequence shown in SEQ ID NO: 63 (fully humanized 6C8 heavy chain-HuQ6C8-Agly).

In one embodiment, the binding molecules of the present invention have VL and VH sequences as shown in Figures 18A-18D; SEQ ID NO: 1 also shows the amino acid sequence of the 6C8 VH region; the amino acid sequence of the 6C8 VL region is shown in SEQ ID NO : 2 shown.在另一实施方案中，本发明的结合分子具有SEQ ID NO：20和21所示之LC和HC序列；ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE(SEQ ID NO：20)；AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK(SEQ ID NO：21)。

In one embodiment of the invention, the VL chain comprises a guide and/or signal sequence, such as amino acid residues 1-20 of SEQ ID NO:2. In one embodiment, the VH chain comprises a leader and/or signal sequence, such as amino acid residues 1-19 in SEQ ID NO: 1. In another embodiment, the binding molecules of the invention do not contain guide and/or signal sequences.

In one aspect, the invention relates to 6C8 binding molecules and other binding molecules comparable to 6C8 in properties such as high affinity binding to GITR and counteracting the inhibitory effect of Treg cells on Teff cells. Furthermore, the binding molecules of the invention do not induce apoptosis nor inhibit mixed lymphocyte reactions. Accordingly, equivalent binding molecules according to the invention are GITR antagonists, ie they are capable of inducing signal transduction via GITR. GITR is a member of the TNFR superfamily. Since members of the TNFR family are involved in cell survival and apoptosis through signaling through NF-κB, in one embodiment, binding molecules of the invention attenuate the degradation of I-κB.

In one embodiment, the invention provides an isolated hGITR binding molecule whose light chain variable region (VL) comprises the amino acid sequence of SEQ ID NO: 2, and an optional leader sequence; heavy chain variable region (VH) Comprising the amino acid sequence of SEQ ID NO: 1, and an optional leader sequence. In some embodiments, the binding molecule comprises a heavy chain constant region, such as an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. Furthermore, the binding molecule may comprise a light chain constant region, ie a kappa light chain constant region or a lambda light chain constant region. The binding molecule preferably comprises a kappa light chain constant region. In one embodiment, the binding molecule of the invention comprises the light chain constant region set forth in SEQ ID NO:20. In one embodiment, a binding molecule of the invention comprises the heavy chain constant region set forth in SEQ ID NO:21. In one embodiment, a binding molecule of the invention comprises the heavy chain constant region set forth in SEQ ID NO:55. In one embodiment, a binding molecule of the invention comprises the heavy chain constant region set forth in SEQ ID NO:56. In one embodiment, the binding molecule of the invention comprises the heavy chain constant region set forth in SEQ ID NO:57.

In another embodiment, the invention provides binding molecules having a 6C8-related VL CDR domain, e.g., binding molecules with a light chain variable region (VL) having at least one CDR structure domain, the latter comprising an amino acid sequence selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8. In another embodiment, the light chain variable region (VL) has at least two CDR domains, and the amino acid sequence of the domains is selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8. In yet another embodiment, the CDR domain of the light chain variable region (VL) comprises an amino acid sequence selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8.

In still other embodiments, the invention provides binding molecules having a 6C8-related VH CDR domain, for example, the CDR domain of the light chain variable region (VH) of the binding molecule comprises a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 3, and Amino acid sequences of NO: 4, SEQ ID NO: 5 and SEQ ID NO: 19. In another embodiment, the heavy chain variable region (VH) has at least two CDR domains comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO :19. In yet another embodiment, the heavy chain variable region (VH) has a CDR domain comprising an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 19.

In another embodiment, a binding molecule of the invention comprises at least one CDR derived from a mouse anti-human GITR binding molecule, eg, a 6C8 binding molecule. As used herein, the term "derived from" a given protein indicates the source of the polypeptide. In one embodiment, the polypeptide or amino acid sequence derived from a particular starting polypeptide is a CDR sequence or a related sequence thereof. In another embodiment, the polypeptide or amino acid sequence derived from a particular starting polypeptide is a FR sequence or a related sequence thereof. In one embodiment, the amino acid sequence derived from a particular starting polypeptide is not contiguous.

For example, in one embodiment, 1, 2, 3, 4, 5, or 6 CDRs are derived from the mouse 6C8 antibody. In one embodiment, a binding molecule of the invention comprises at least one heavy or light chain CDR of the mouse 6C8 antibody. In another embodiment, the binding molecule of the invention comprises at least two CDRs from the mouse 6C8 antibody. In another embodiment, a binding molecule of the invention comprises at least three CDRs from the mouse 6C8 antibody. In another embodiment, the binding molecule of the invention comprises at least four CDRs from the mouse 6C8 antibody. In another embodiment, a binding molecule of the invention comprises at least five CDRs from the mouse 6C8 antibody. The binding molecules of the invention comprise at least six CDRs from the mouse 6C8 antibody.

Those skilled in the art will appreciate that binding molecules of the invention may be modified to differ in amino acid sequence from the 6C8 molecule from which they are derived. For example, nucleotide or amino acid substitutions that result in conservative substitutions, or changes to non-essential amino acid residues (e.g., residues in the CDRs and/or framework) can be made and remain bound to GITR (e.g., human GITR) Ability.

In one embodiment, at least one CDR (or at least one of the more than one 6C8 CDR in the binding molecule) is modified to differ from the sequence of the native 6C8 binding molecule, but retains the ability to bind 6C8. For example, in one embodiment, one or more CDRs in the 6C8 antibody are modified to remove potential glycosylation sites. For example, because the amino acid sequence Asn-X-(Ser/Thr) is predicted to be a consensus sequence for glycosylation sites, which may affect the production of binding molecules, and the CDR2 of the 6C8 heavy chain has the sequence Asn-Pro-Ser, the heavy chain A second version of the chain was prepared in which the asparagine (Asn) at amino acid residue 62 in SEQ ID NO:53 was conservatively substituted for glutamine (Gln).

In one embodiment, a binding molecule of the invention comprises a polypeptide or amino acid sequence substantially identical to the 6C8 antibody or a portion of the antibody consisting of at least 3-5 amino acids, at least 5-10 amino acids, at least 10-20 amino acids, at least 20-30 amino acids, or at least 30-50 amino acids, or as can be seen by those skilled in the art to be derived from the starting sequence.

In another embodiment, a polypeptide or amino acid sequence derived from a particular starting polypeptide or amino acid sequence has about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity, or as can be seen by a person skilled in the art to be derived from the starting sequence.

An isolated nucleic acid molecule encoding a non-natural variant of a polypeptide may introduce one or more amino acid substitutions in the encoded protein by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence of the binding molecule , addition or deletion to produce. Mutations can be introduced by conventional techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. In one embodiment, conservative amino acid substitutions are made at one or more non-essential amino acid residues. A "conservative amino acid substitution" is the replacement of an amino acid residue with another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are well defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid, amino acids), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine amino acid, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine , isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in a binding molecule polypeptide can be replaced by another amino acid residue from the same side chain family. In another embodiment, a string of amino acids may be replaced with a string of amino acids that is similar in structure but differs in sequence and/or composition of side chain families.

Alternatively, in another embodiment, mutations may be introduced along all or part of the binding molecule coding sequence.

Preferred binding molecules of the invention comprise framework and constant region amino acid sequences derived from human amino acid sequences. However, the binding molecule may comprise framework and/or constant region sequences derived from another mammalian species. For example, primate framework regions (eg, non-human primate), heavy chain portions, and/or hinge portions can be included in the binding molecule. In one embodiment, the framework region of the binding polypeptide may contain one or more murine amino acids, for example a human or non-human primate framework amino acid sequence may comprise one or more amino acid substitutions and/or back mutations, wherein there are corresponding murine amino acid residues. Preferred binding molecules of the invention are less immunogenic than the starting 6C8 murine antibody.

The invention also provides specific chimeric and/or humanized binding molecules (ie, chimeric and/or humanized immunoglobulins) for GITR. Chimeric and/or humanized binding molecules have the same or similar binding specificity and affinity as the mouse or other non-human binding molecule provided as starting material for construction of the chimeric or humanized binding molecule.

Chimeric binding molecules are generally constructed by genetic engineering from immunoglobulin gene fragments belonging to different species into its light chain and heavy chain genes. For example, the variable (V) segment of a mouse monoclonal binding molecule gene can be linked to a human constant (C) segment (eg, IgGl or IgG4, preferably the human isotype IgGl). Thus, an exemplary chimeric binding molecule is a hybrid protein composed of a V or antigen binding domain from a mouse binding molecule and a C or effector domain from a human binding molecule.

In one embodiment, the invention relates to the variable regions of humanized 6C8 binding molecules and polypeptides comprising such humanized variable regions. In one embodiment, a binding molecule of the invention comprises at least one humanized 6C8 binding molecule variable region, such as a light chain or heavy chain variable region.

The term "humanized binding molecule" refers to a binding molecule comprising at least one chain comprising variable region framework residues derived from a human binding molecule chain (referred to as a receptor immunoglobulin or binding molecule), and at least One is derived from the complementarity determining region of a mouse binding molecule (called the donor immunoglobulin or binding molecule). Humanized binding molecules can be prepared using recombinant DNA techniques discussed below. See, e.g., Hwang, W.Y.K.et al. (2005) Methods 36:35; Queen et al.Proc.Natl.Acad.Sci.USA, (1989), 86:10029-10033; Jones et al.Nature, (1986) , 321:522-25; Riechmann et al.Nature, (1988), 332:323-27; Verhoeyen et al.Science, (1988), 239:1534-36; Orlandi et al.Proc.Natl.Acad.Sci .USA, (1989), 86:3833-37; U.S. Patents 5,225,539; 5,530,101; 5,585,089; 5,693,761; ). The constant regions, if any, are preferably also derived from human immunoglobulins.

When a preferred non-human donor binding molecule is selected for humanization, a suitable human receptor binding molecule can be obtained from, for example, a database of expressed human antibody gene sequences, germline Ig sequences, or consensus sequences of multiple human binding molecules.

In one embodiment, methods based on CDR homology are used for humanization (see, e.g., Hwang, W.Y.K. et al. (2005) Methods 36:35, which is hereby incorporated by reference in its entirety) . This approach generally involves substituting mouse CDRs into human variable domain frameworks based on similarly structured mouse and human CDRs, rather than similarly structured mouse and human frameworks. The similarity of mouse and human CDRs is generally determined by identifying human genes of the same type of chain (light or heavy) that have the same combination of canonical CDR structures as the mouse binding molecule, thereby maintaining the integrity of the CDR peptide backbone. three-dimensional conformation. Second, for each candidate variable gene with a matching canonical structure, residues and residue homology were evaluated between mouse and candidate CDRs. Finally, to generate humanized binding molecules, the CDR residues in the selected human candidate CDRs that differ from the mouse CDRs were changed to mouse sequences. In one embodiment, there are no mutations introduced into the human framework in the humanized binding molecule.

In one embodiment, the CDR homology of the human germline sequence to the CDRs of the GITR binding molecule is assessed. For example, for the mouse 6C8 antibody, all germline light chain kappa chain V genes with a 2-1-1 canonical structure in the IMGT database were compared to the 6C8 antibody sequence. All 3-1 germline heavy chain V genes were also compared to the 6C8 amino acid sequence for the heavy chain. Accordingly, in one embodiment, a binding molecule of the invention comprises a human kappa chain V-region framework having a 2-1-1 canonical structure. In another embodiment, a binding molecule of the invention comprises a human heavy chain V region framework having a 3-1 canonical structure.

We have identified the following potential human light chain germline sequences that can be incorporated into the binding molecules of the invention:

The IMGT accession number of the IGKV3-15 gene is M23090. The amino acid sequence is:

EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP (SEQ ID NO: 25).

The IMGT accession number of the IGKV3D-11 gene is X17264. The amino acid sequence is:

EIVLTQSPATLSSLSPGERATLSCRASQGVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQRSNWH (SEQ ID NO: 26).

The IGKV3-11 gene has two alleles. The IMGT accession number for allele ^* 01 of the IGKV3-11 gene is X01668. The amino acid sequence is:

EIVLTQSPATLSSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWP (SEQ ID NO: 27).

The IMGT accession number for allele ^* 02 is X02768. The amino acid sequence is:

EIVLTQSPATLSSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGRDFTLTISSLEPEDFAVYYCQQRSNWP (SEQ ID NO: 28).

The IMGT accession number of the IGKV1D-43 gene is X72817. The amino acid sequence is:

AIRMTQSPFSLSASVGDRVTITCWASQGISSYLAWYQQKPAKAPKLFIYYASSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYYSTP (SEQ ID NO: 29).

The IGKV1-39 gene has two alleles. The IMGT accession number for allele ^* 01 of the IGKV1-39 gene is X59315. The amino acid sequence is:

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP (SEQ ID NO: 30).

The IMGT accession number for allele ^* 02 of the IGKV1-39 gene is X59318. The amino acid sequence is:

DIQMTQSPSSFLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQCGYSTP (SEQ ID NO: 31).

The IMGT accession number of the IGKV1-33 gene is M64856. The amino acid sequence is:

DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLP (SEQ ID NO: 32).

The IMGT accession number of the IGKV1-27 gene is X63398. The amino acid sequence is:

DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAP (SEQ ID NO: 33).

The IGKV1-17 gene has two alleles. The IMGT accession number for allele ^* 01 of the IGKV1-17 gene is X72808. The amino acid sequence is:

DIQMTQSPSSLSASSVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYP (SEQ ID NO: 34).

The IMGT accession number for allele ^* 02 of the IGKV1-17 gene is D88255. The amino acid sequence is:

DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCLQHNSYP (SEQ ID NO: 35).

The IGKV1D-16 gene has two alleles. The IMGT accession number for allele ^* 01 of the IGKV1D-16 gene is K01323. The amino acid sequence is:

DIQMTQSPSSLSASSVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYP (SEQ ID NO: 36).

The IMGT accession number for allele ^* 02 of the IGKV1D-16 gene is J00244. The amino acid sequence is:

DIQMTQSPSSLSASVGDRVTITCRARQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYP (SEQ ID NO: 37).

The IMGT accession number for the IGKV1-16 gene is J00248. The amino acid sequence is:

DIQMTQSPSSLSASSVGDRVTITCRASQGISNYLAWFQQKPGKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYP (SEQ ID NO: 38).

The IGKV1-12 gene has two alleles. The IMGT accession number for allele ^* 01 of the IGKV1-12 gene is V01577. The amino acid sequence is:

DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFP (SEQ ID NO: 39).

The IMGT accession number for allele ^* 02 of the IGKV1-12 gene is V01576. The amino acid sequence is:

DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFP (SEQ ID NO: 40).

The IMGT accession number for the IGKV1-9 gene is Z00013. The amino acid sequence is:

DIQLTQSPSSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYP (SEQ ID NO: 41).

The IMGT accession number of the IGKV1-6 gene is M64858. The amino acid sequence is:

AIQMTQSPSSLSASSVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYP (SEQ ID NO: 42).

The IGKV1-5 gene has three alleles. The IMGT accession number for allele ^* 01 of the IGKV1-5 gene is Z00001. The amino acid sequence is:

DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYS (SEQ ID NO: 43).

We have identified the following potential human heavy chain germline sequences that can be incorporated into the binding molecules of the invention:

The IGHV2-5 gene has ten alleles. The IMGT accession number for allele ^* 01 of the IGHV2-5 gene is X62111. The amino acid sequence is:

QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLALIYWNDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYY (SEQ ID NO: 45).

The IMGT accession number of the IGHV2-26 gene is M99648. The amino acid sequence is: QVTLKESGPVLVKPTETLTLTCTVSGFLSNARMGVSWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARI (SEQ ID NO: 46).

The IGHV2-70 gene has thirteen alleles. The IMGT accession number for allele ^* 01 of the IGHV2-70 gene is L21969. The amino acid sequence is:

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMCVSWIRQPPGKALEWLALIDWDDDKYYSTSLKTRLTISKDTSKNQVVLTMTNMDPVDTATYYCARI (SEQ ID NO: 47).

The IGHV4-30-2 gene has 4 alleles. The IMGT accession number for allele ^* 01 of the IGHV4-30-2 gene is L10089. The amino acid sequence is:

QLQLQESGSGLVKPSQTLSLTCAVSGGSISSGGYSWSWIRQPPGKGLEWIGYIYHSGSTYYNPSLKSRVTISVDRSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 48).

The IGHV4-30-4 gene has six alleles. The IMGT accession number for allele ^* 01 of the IGHV4-30-4 gene is Z14238. The amino acid sequence is:

QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 49).

The IGHV4-31 gene has ten alleles. The IMGT accession number for allele ^* 01 of the IGHV4-31 gene is L10098. The amino acid sequence is:

QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGSTYYNPSLKSLVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 50).

The IGHV4-39 gene has six alleles. The IMGT accession number for allele ^* 01 of the IGHV4-39 gene is L10094. The amino acid sequence is:

QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 51).

The IGHV4-61 gene has eight alleles. The IMGT accession number for allele ^* 01 of the IGHV4-61 gene is M29811. The amino acid sequence is:

QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 52).

Each of these germline sequences can be used to provide framework regions for use with one or more 6C8 CDRs.

As used herein, the "canonical structure" is the conserved hypervariable loop conformation formed by the different CDRs by which the binding molecule makes contact with the antigen. New binding molecules can be assigned canonical structure types using public domain software.

In another embodiment, the substitution of mouse CDRs into a human variable domain framework is performed on the basis that the correct stereo-orientation of the mouse variable domain framework is maintained by identifying such a human variable domain framework. Variable Domain Frameworks These human variable domain frameworks maintain the same conformation as the mouse variable domain frameworks from which the CDRs are derived. In one embodiment, this is achieved by obtaining human variable domains in human binding molecules whose framework sequences exhibit a high degree of sequence identity to the mouse variable framework domains from which the CDRs are derived. See Kettleborough et al. Protein Engineering 4:773 (1991); Kolbinger et al. Protein Engineering 6:971 (1993) and Carter et al. WO 92/22653.

Preferably, the human acceptor binding molecule retains the canonical and interface residues of the donor binding molecule. Furthermore, said human receptor binding molecules preferably have considerable similarity in CDR loop lengths. See, Kettleborough et al. Protein Engineering 4:773 (1991); Kolbinger et al. Protein Engineering 6:971 (1993) and Carter et al. WO 92/22653.

In another embodiment, suitable human acceptor sequences can be selected on the basis of homology to the framework regions of the 6C8 binding molecule. For example, the amino acid sequence of the 6C8 binding molecule can be compared to the amino acid sequences of other known binding molecules, e.g., by comparing the FR regions or variable regions of the 6C8 amino acid sequence to public databases of known binding molecules and selecting those variable regions. The sequence with the highest percentage of amino acid identity in the region or FR region, i.e. reaching 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% %, 97%, 98%, 99%, 99.5% the same. In one embodiment, the framework series set forth in SEQ ID NO: 67 (QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYNPSLKSRLTISKDTSSNQVFLKITSVDTRDTATYYCARTRRYFPFAYWGEGTSVTVTS (SEQ ID NO: 67; framework residues in bold)) can be used. In another embodiment, the framework sequence set forth in SEQ ID NO: 68 (QVTLRESGPALVKPTQTLLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYNP SLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCATRRYFPFAYWGQGTLVTVSS (SEQ ID NO: 68; framework residues in bold)) can be used.

After identifying the complementarity determining regions of the murine donor immunoglobulin and a suitable human recipient immunoglobulin, the next step is to decide which residues in these components should be substituted, if desired, so that the properties of the resulting humanized binding molecule are obtained. optimization. In general, substitution of human amino acid residues with murine should be minimized since introducing murine residues increases the risk of binding molecules eliciting a human anti-mouse antibody (HAMA) response in humans. Art-recognized methods for determining immune responses can be used to monitor HAMA responses in specific patients or during clinical trials. Immunogenicity assessments can be performed on patients administered humanized binding molecules at the beginning and during the course of this therapy. HAMA response is measured, for example, by detecting antibodies against the humanized therapeutic agent in patient serum samples using methods known to those skilled in the art, including surface plasmon resonance technology (BIACORE) and/or solid phase ELISA assays.

If necessary, one or more residues in the human framework regions can be altered or substituted for corresponding positions in the murine antibody so as to preserve the binding affinity of the humanized antibody for the antigen. This change is sometimes called a "back mutation". Some amino acids in human variable region framework residues were selected for backmutation based on their possible impact on CDR conformation and/or antigen binding. Replacing the murine CDR regions with human variable framework regions may introduce conformational constraints that, if not corrected by substitution of some amino acid residues, lead to loss of binding affinity.

In one embodiment, the amino acid residues to undergo back mutations can be partially selected by computer model construction using techniques recognized in the art. Typically, molecular models are generated starting from already solved structures of immunoglobulin chains or domains thereof. Compare the amino acid sequence similarity of the chain to be modeled with the chain or domain of known three-dimensional structure, and select the chain or domain showing the highest sequence similarity as the starting point for molecular modeling. Chains or domains with at least 50% sequence identity, preferably at least 60%, 70%, 80%, 90% or more sequence identity are selected for modeling. The known starting structure is altered to allow for differences between the actual amino acids in the immunoglobulin chain or domain to be modeled and the amino acids in the starting structure. The altered structures are then assembled into composite immunoglobulins. Finally, the model is fine-tuned, including through energy minimization, and confirming that all atoms are within appropriate distances and that bond lengths and angles are within chemically acceptable ranges.

内)，或者(4)参与VL-VH界面，可以将人框架氨基酸用小鼠结合分子中的等同框架氨基酸来取代。Amino acid residues for substitution may also be selected in part by examining the identity of the amino acid at a particular position, or by empirically observing the likely effect of a particular amino acid substitution or mutation. For example, when the amino acid of a mouse variable region framework residue differs from selected human variable region framework residues, if the amino acid can reasonably be expected to (1) directly bind antigen non-covalently, (2) interact with the CDR region close to, (3) or interact with the CDR region (e.g., determined by computer modeling to be within about 3-6

), or (4) to participate in the VL-VH interface, the human framework amino acids can be replaced with equivalent framework amino acids in the mouse binding molecule.

Residues that "directly bind antigen non-covalently" include amino acids at framework region sites that have a high probability of interacting with amino acids on the antigen according to established chemical forces (e.g. hydrogen bonds, van der Waals forces, hydrophobic interactions, etc.) interact directly.

Residues "close to the CDR region" include amino acid residues in the primary sequence of a humanized immunoglobulin chain immediately adjacent to one or more CDRs, for example, immediately adjacent to a CDR as defined by Kabat, or Chothia at the positions of defined CDRs (see, eg, Chothia and Lesk JMB 196:901 (1987)). These amino acids are particularly likely to interact with amino acids in the CDRs and, if selected from the acceptor, may distort the donor CDR and reduce affinity. In addition, adjacent amino acids may interact directly with the antigen (Amit et al. Science, 233:747 (1986), which is hereby incorporated by reference), and it may be desirable to select these amino acids in the donor so that the original There are all antigen contacts that provide affinity in the binding molecule.

以内，并且必须含有原子能够与CDR原子根据已建立的化学力，比如上面列举的那些化学力相互作用。Residues "or interacting with a CDR region" include those residues determined by secondary structure analysis to be in a sufficient steric orientation to affect the CDR region. In one embodiment, residues that "or interact with a CDR region" are identified by analysis of a three-dimensional model (eg, a computer-generated model) of the donor immunoglobulin. The three-dimensional model, usually the three-dimensional model of the original donor-binding molecule, shows that some amino acids outside the CDR are close to the CDR, and there is a high possibility of interacting with the amino acids in the CDR through hydrogen bonds, van der Waals forces, and hydrophobic interactions. At these amino acid positions, donor immunoglobulin amino acids can be selected over acceptor immunoglobulin amino acids. Amino acids meeting this criterion usually have side chain atoms at about 3

within, and must contain, atoms capable of interacting with CDR atoms according to established chemical forces, such as those enumerated above.

是其核间距的测量值，但是对于不成键的原子，3

是其范德华表面之间的距离的测量值。因此，在后一种情况中，原子核必须在约6

以内(3加上范德华半径的和)，原子才被认为能够相互作用。许多情况中，原子核是相距4或5到6

在确定氨基酸是否与CDR有相互作用时，优选不把重链CDR的最后8个氨基酸认为是CDR的一部分，因为从结构上看，这8个氨基酸更象是框架区的一部分。For atoms that may form hydrogen bonds, 3

is a measure of their internuclear spacing, but for non-bonding atoms, 3

is a measure of the distance between its van der Waals surfaces. Therefore, in the latter case the nuclei must be within about 6

within (3 plus the sum of the van der Waals radii), the atoms are considered capable of interacting. In many cases the nuclei are 4 or 5 to 6 apart

When determining whether an amino acid interacts with a CDR, it is preferable not to consider the last 8 amino acids of the heavy chain CDR as part of the CDR, because structurally, these 8 amino acids are more likely to be part of the framework region.

Amino acids capable of interacting with amino acids in CDRs can also be identified by another method. The solvent-accessible surface area for each framework amino acid was calculated in two ways: (1) in the intact binding molecule, and (2) in a pseudo molecule composed of the binding molecule with the CDRs removed. A difference of about 10 angstroms or more between these two numbers indicates that the framework amino acid is at least partially blocked from solvent contact by the CDR, and thus the amino acid has contact with the CDR. The solvent-accessible surface area of an amino acid can be calculated on the basis of a three-dimensional model of the bound molecule using algorithms known in the art (e.g., Connolly, J. Appl. Cryst. 16:548 (1983) and Lee and Richards, J . Mol. Biol. 55:379 (1971), both of which are incorporated herein by reference). A framework amino acid may occasionally interact with a CDR indirectly by affecting the conformation of another framework amino acid that is in contact with the CDR.

Amino acids at several positions in the framework are known to be capable of interacting with CDRs in many binding molecules (Chothia and Lesk, supra, Chothia et al. supra and Tramontano et al. J. Mol. Biol. 215:175( 1990), which are incorporated herein by reference). Specifically, the amino acids at positions 2, 48, 64, and 71 in the light chain, and the amino acids at positions 26-30, 71, and 94 in the heavy chain (numbering according to Kabat) are known to bind to the CDR in many binding molecules. interaction. Amino acids at position 35 of the light chain and positions 93 and 103 of the heavy chain may also interact with the CDRs. At all of these numbered positions, it is preferred to select the donor amino acid rather than the acceptor amino acid (when they are different) in the humanized immunoglobulin. On the other hand, some residues capable of interacting with the CDR region, such as the first 5 amino acids of the light chain, can sometimes be selected from the recipient immunoglobulin without losing the affinity of the humanized binding molecule.

Residues "participating in the VL-VH interface" or "stacking residues" include those located, for example, in Novotny and Haber (Proc. Natl. Acad. Sci. USA, 82:4592-66 (1985)) or Chothia et al. Residues that define the interface between VL and VH. In general, uncommon stacking residues should be retained in the humanized binding molecule if they differ from those in the human framework.

Typically, one or more amino acids meeting the above criteria are substituted. In some embodiments, all or most of the amino acids meeting the above criteria are substituted. Occasionally, it is difficult to determine whether a particular amino acid meets the above criteria, and alternative variant binding molecules will be generated, one with that particular substitution and one without. The alternative variant binding molecules thus generated can be tested for activity in any of the assays described herein to select a preferred one.

Generally, the CDR regions of the humanized binding molecule are substantially identical, or more often identical, to the corresponding CDR regions in the donor binding molecule. Although not usually required, one or more conservative amino acid substitutions can sometimes be made to a CDR residue without substantially affecting the binding affinity of the resulting humanized binding molecule. Conservative substitutions represent combinations of Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr.

Other candidates for substitution are those acceptor human framework amino acids that are uncommon or "rare" for that human immunoglobulin site. These amino acids may be substituted with amino acids at equivalent positions in the mouse donor binding molecule, or more generally in human immunoglobulins. For example, when an amino acid in a human framework region in the recipient immunoglobulin is unusual for that position and the corresponding amino acid in the donor immunoglobulin is common for that position in the human immunoglobulin sequence or when an amino acid in the recipient immunoglobulin is rare for that position and the corresponding amino acid in the donor immunoglobulin is also rare for that position in other natural sequences, a substitution may be desired . These criteria ensure that atypical amino acids in the human framework do not disrupt the structure of the binding molecule. Furthermore, by substituting amino acids from the donor binding molecule that happen to be typical for human binding molecules for uncommon human acceptor amino acids, the resulting humanized binding molecules may be less immunogenic.

The term "rare" is used herein to indicate that an amino acid occurs at that position in less than about 20%, but usually less than about 10%, of representative sequence samples, and the term "common" is used herein to indicate that an amino acid occurs in more than about 25% of representative sequence samples. %, but usually in more than about 50% of representative samples. For example, all human light and heavy chain variable region sequences are divided into "subgroups" of sequences that are particularly homologous to each other and have amino acid identity at key positions (Kabat et al., supra) . When determining whether amino acids in human acceptor sequences are "rare" or "common" for human sequences, it is often preferable to consider only human sequences in the same subgroup as acceptor sequences.

Other candidates for substitution are acceptor human framework amino acids that would be considered part of the CDR regions according to the definition proposed by Chothia et al. (supra). Additional candidates for substitution are acceptor human framework amino acids that would be considered part of the CDR regions according to AbM and/or contact definitions. In particular CDR1 in the variable heavy chain is defined to include residues 26-32.

Other options for substitution are acceptor framework residues that correspond to rare or uncommon donor framework residues. Rare or unusual donor framework residues are residues that are rare or uncommon (as defined herein) for this site in the mouse binding molecule. For mouse binding molecules, subgroups can be divided according to Kabat and residue positions identified as differing from the consensus residues. These donor-specific differences may indicate somatic mutations in the mouse sequence that confer increased activity. Uncommon residues predicted to affect binding were retained, while residues predicted to be unimportant for binding could be substituted.

Other options for substitution are non-germline residues that occur in the acceptor framework regions. For example, when aligning a receptor binding molecule chain (i.e., a human binding molecule chain with significant sequence identity to the donor binding molecule chain) and a germline binding molecule chain (also having significant sequence identity to the donor chain) , residues that do not match between the acceptor chain framework and the germline chain framework can be replaced with the corresponding residues in the germline sequence.

Except for the specific amino acid substitutions discussed above, the framework regions of a humanized binding molecule are usually substantially, or identical to, the framework regions of the human binding molecule from which it is derived. Of course, many amino acids in the framework regions make little or no direct contribution to the specificity or affinity of the binding molecule. Thus, single conservative substitutions of many framework residues can be tolerated without appreciably altering the specificity or affinity of the resulting humanized binding molecule. Thus, in one embodiment, the variable framework regions of the humanized binding molecule have at least 85% sequence identity to human variable framework region sequences or a consensus sequence of these sequences. In another embodiment, the variable framework regions of the humanized binding molecule share at least 90%, preferably 95%, more preferably 96%, 97%, 98% or 99% sequence identity. In general, however, such substitutions are undesirable.

In one embodiment, the binding molecule of the invention further comprises at least one backmutation from a human amino acid residue to a corresponding mouse amino acid residue that is an interfacial packing residue. "Interface packing residues" include residues located at the interface between VL and VH, such as defined in Novotny and Haber, Proc. Natl. Acad. Sci. USA, 82:4592-66 (1985) .

In one embodiment, the binding molecule of the invention further comprises a backmutation of at least one human amino acid residue to the corresponding mouse amino acid residue, wherein said amino acid residue is a canonical residue. "Canonical residues" are conserved framework residues that belong to canonical or structural classes known to be important for CDR conformation (Tramontano et al. J. Mol. Biol. 215: 175 (1990), which The entire text is incorporated herein by reference). Canonical residues include residues 2, 25, 27B, 28, 29, 30, 33, 48, 51, 52, 64, 71, 90, 94, and 95 in the light chain, and residues 24, 26, 27, 29, 34, 54, 55, 71 and 94. Other residues (eg, CDR structure-determining residues) can be identified according to the method of Martin and Thorton (1996) J. Mol. Biol. 263:800.

In one embodiment, the binding molecule of the invention further comprises a backmutation of at least one human amino acid residue to the corresponding mouse amino acid residue, wherein said amino acid residue is at a site capable of interacting with a CDR. In particular, amino acids at positions 2, 48, 64 and 71 of the light chain and positions 26-30, 71 and 94 of the heavy chain (numbering following Kabat) are known to interact with the CDRs in many antibodies. Amino acids at position 35 of the light chain and positions 93 and 103 of the heavy chain may also interact with the CDRs.

Based on CLUSTAL W analysis, several amino acid residues in the human framework were identified as likely to be substituted with corresponding amino acid residues in, for example, the 6C8 light chain. These include positions 1, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21, 22, 43, 45, 46, 58, 60, 63, 70, 76, 77, 78, 79, Residues at 83, 85, 87, 100 and 104.

In one embodiment, the variable light chain framework of the binding molecule of the invention further comprises a substitution of at least one human amino acid residue to a corresponding mouse amino acid residue selected from the group consisting of: E1D (i.e. comprising mouse CDR and The E of position 1 in the CDR-grafted antibody of the human FR region is mutated to D, which is the corresponding amino acid residue in the 6C8 antibody), P8Q, A9K, T10F, L11M, V13T, P15V, E17D, A19V, T20S, L21V, S22T, A43S, R45K, L46A, I58V, A60D, S63T, E70D, S76N, S77N, L78V, Q79H, F83L, V85E, Y87F, G100A and V104L.

Based on the CLUSTAL W analysis, we identified several amino acid residues in the human framework that could be substituted with, for example, the corresponding amino acid residues of the 6C8 heavy chain. They include positions 5, 10, 11, 12, 15, 19, 23, 43, 46, 68, 77, 81, 83, 84, 86, 87, 89, 90 and 92.

In one embodiment, the variable heavy chain framework of the binding molecule of the invention further comprises a substitution of at least one human amino acid residue to a corresponding mouse amino acid residue selected from the group consisting of: R5K (i.e. comprising mouse CDR and The R at position 5 in the CDR-grafted antibody of the human FR region is mutated to K, which is the corresponding amino acid residue in the 6C8 antibody), A10G, L11I, V12L, T15S, T19S, T23S, P43S, A46G, R68Q, K77R, V81F, T83K, M84I, N86S, M87V, P89T, V90A and T92A.

Preferably, the humanized binding molecule has a specific binding affinity for the antigen of at least 10 ⁷ , 10 ⁸ , 10 ⁹ or 10 ¹⁰ M ⁻¹ . Typically, the upper limit of the binding affinity of the humanized binding molecule for the antigen is within 3, 4 or 5 times the upper limit of the binding affinity of the donor immunoglobulin. The lower limit of binding affinity is also within 3, 4 or 5 times the lower limit of binding affinity of the donor immunoglobulin. Alternatively, the binding affinity can be compared to that of a humanized binding molecule without substitutions (eg, a binding molecule with a donor CDR and an acceptor FR, but no FR substitutions). In such cases, the optimized binding molecule preferably binds at least 2 to 3 times, or 3 to 4 times higher than the unsubstituted binding molecule. For comparison, the activity of various binding molecules can be determined, for example, by BIACORE (ie surface plasmon resonance with unlabeled reagents) or competition binding assays.

After theoretically selecting the CDRs and framework components of a humanized binding molecule, there are many methods available for preparing such binding molecule. Due to the degeneracy of the code, each binding molecule amino acid sequence will be coded by multiple nucleic acid sequences. Desired nucleic acid sequences can be produced by de novo solid phase DNA synthesis or by PCR mutagenesis of previously prepared variants of the desired polynucleotide.

Oligonucleotide-mediated mutagenesis is the preferred method for making substitution, deletion and insertion variants of target polypeptide DNA. See Adelman et al. (DNA 2:183 (1983)). Briefly, target polypeptide DNA is altered by hybridizing an oligonucleotide encoding the desired mutation to a single-stranded DNA template. Following hybridization, a DNA polymerase is used to synthesize the full-length second complementary strand of the template incorporating the oligonucleotide primer encoding the selected alteration of the target polypeptide DNA.

Variable fragments of binding molecules (e.g., heavy and light chain variable regions of chimeric, humanized or human binding molecules) prepared as described above will usually be linked to at least a portion of an immunoglobulin constant region (Fc), typically The constant region of a human immunoglobulin. Human constant region DNA sequences can be isolated from a variety of human cells according to well known methods, but are preferably isolated from immortalized B cells (see Kabat et al. supra and Liu et al. WO87/02671) (both referred to incorporated herein by reference in its entirety). Typically, binding molecules contain both light and heavy chain constant regions. The heavy chain constant region generally includes the CH1, hinge, CH2, CH3 and CH4 regions. The binding molecules described herein include antibodies with constant regions of all classes, including IgM, IgG, IgD, IgA, and IgE; all isotypes, including IgGl, IgG2, IgG3, and IgG4. The choice of constant region depends in part on the need for binding molecule-dependent complement and/or cell-mediated toxicity. For example, isotypes IgG1 and IgG3 have complement activity, while isotypes IgG2 and IgG4 do not. When it is desired that the binding molecule (eg, a humanized binding molecule) exhibit cytotoxic activity, the constant domain is typically a complement fixation constant domain, typically of the type IgGl. When such cytotoxic activity is not desired, the constant domain can be, for example, of the IgG2 type. The choice of isotype may also affect antibody access to the brain. The human isotype IgGl is preferred. The light chain constant region can be lambda or kappa. Humanized binding molecules may comprise sequences from more than one class or isotype. Binding molecules can be expressed as tetramers containing two light chains and two heavy chains, separate heavy chains, light chains, Fab, Fab', F(ab')2 and Fv, or as heavy chains and A single-chain binding molecule in which the light chain variable domains are linked together by a spacer arm.

III. Preparation of Binding Molecules

The invention provides binding molecules specific for GITR, eg, human GITR. Such binding molecules can be used to formulate various medical compositions of the invention, or preferably provide complement determining regions for the preparation of humanized or chimeric binding molecules (described in detail below). Preparation of non-human (eg, mouse, guinea pig, primate, rabbit or rat) monoclonal binding molecules can be accomplished, for example, by vaccinating animals with GITR or a nucleic acid molecule encoding GITR. For example, 6C8-binding molecules can be produced by inserting the gene encoding human GITR into an expression vector and inoculating animals. Longer polypeptides comprising GITR or immunogenic fragments of GITR or anti-idiotype binding molecules of GITR may also be used. (See, eg, Harlow & Lane, supra, incorporated herein by reference). Such immunogens may be obtained from natural sources, by peptide synthesis or expressed recombinantly. Optionally, the immunogen can be administered fused or otherwise complexed with a carrier protein as described below. Optionally, the immunogen can be administered with an adjuvant. The term "adjuvant" refers to a compound that, when administered in combination with an antigen, enhances an immune response to that antigen but does not elicit an immune response to that antigen when administered alone. Adjuvants can enhance the immune response through several mechanisms, including recruitment of lymphocytes, stimulation of B and/or T cells, and stimulation of macrophages. As described below, several types of adjuvants can be used. Complete Freund's adjuvant followed by incomplete Freund's adjuvant is the preferred method of immunizing experimental animals.

Rabbits or guinea pigs are often used to prepare polyclonal binding molecules. An exemplary preparation of a polyclonal binding molecule for eg passive protection may proceed as follows. Animals were inoculated with 100 μg GITR, plus adjuvant, and euthanized at 4-5 months. Blood is collected and IgG is separated from other blood components. Specific binding molecules for immunogens can be partially purified by affinity chromatography. An average of about 0.5-1.0 mg of the immunogen-specific binding molecule is obtained per animal, for a total of up to 60-120 mg.

Typically mice are used to prepare monoclonal binding molecules. The preparation of a monoclonal against a certain fragment can be performed by injecting a fragment or a longer form of GITR into mice, preparing hybridomas and screening the hybridomas to find binding molecules that specifically bind to GITR. Optionally, the binding molecules screened are capable of binding a specific region or target fragment of GITR, but not other non-overlapping fragments of GITR. The latter screen can be accomplished by determining the binding of the binding molecule to a series of deletion mutants of the GITR peptide and determining which mutants are able to bind the binding molecule. Binding can be assessed by, for example, Western blot or ELISA. The smallest fragment that exhibits specific binding to the binding molecule defines an epitope of the binding molecule. Alternatively, epitope specificity can be determined by competition assays, in which test and reference binding molecules compete for binding to GITR. If the test and reference binding molecules compete, they bind the same epitope (or bind an epitope close enough) that binding to one binding molecule interferes with binding to the other. A preferred isotype of such binding molecules is the mouse isotype IgG2a or the equivalent isotype in other species. The mouse isotype IgG2a is the equivalent of the human isotype IgGl.

In another embodiment, DNA encoding the binding molecule can be readily isolated and sequenced using conventional methods (eg, by using oligonucleotide probes that bind specifically to the genes encoding the heavy and light chains of the murine binding molecule). Isolated and subcloned hybridoma cells are a preferred source of such DNA. Once isolated, the DNA can be put into an expression vector, and then the expression vector can be transfected into those prokaryotic or eukaryotic host cells that do not originally produce immunoglobulins, such as Escherichia coli cells, monkey COS cells, Chinese hamster ovary ( CHO) cells or myeloma cells. More specifically, isolated DNA (which may be synthetic as described herein) can be used as described in U.S. Patent 5,658,570, filed January 25, 1995 by Newman et al., which is incorporated herein by reference. The constant and variable region sequences are cloned in order to prepare binding molecules. Basically, the patent describes the extraction of RNA from selected cells, conversion to cDNA, and amplification by PCR using Ig-specific primers. Primers suitable for this purpose are also described in US Patent No. 5,658,570. Transformed cells expressing the desired antibody can be produced in relatively large quantities to ensure clinical and commercial supply of the binding molecule.

Those skilled in the art will appreciate that DNA encoding binding molecules or fragments thereof (ie, antigen binding sites) can also be derived from antibody phage libraries using, for example, pd phage or Fd phagemid technologies. In e.g. EP 368 684 B1; US Patent 5,969,108, Hoogenboom, H.R. and Chames. 2000. Immunol. Today 21: 371; Nagy et al. 2002. Nat. Med. Sci. USA 98: 2682; Lui et al. 2002. J. Mol. Biol. 315: 1063 (these articles are all incorporated herein by reference). Several publications (e.g., Marks et al. Bio/Technology 10:779-783 (1992)) describe the generation of high-affinity human binding molecules by strand shuffling, combinatorial infection, and in vivo recombination as strategies for constructing large phage libraries . In another embodiment, ribosome display can be used instead of phage as a display platform (see, e.g., Hanes et al. 2000. Nat. Biotechnol. 18:1287; Wilson et al. 98:3750; or Irving et al. 2001 J. Immunol. Methods 248:31). In yet another embodiment, cell surface libraries can be screened for binding molecules (Boder et al. 2000. Proc. Natl. Acad. Sci. USA97: 10701; Daugherty et al. 2000 J. Immunol. Methods 243: 211). These procedures provide an alternative to traditional hybridoma techniques for isolating and cloning monoclonal binding molecules.

Other embodiments of the invention also encompass the production of human or substantially human binding molecules in transgenic animals (e.g., mice) that are unable to produce endogenous immunoglobulins (see, e.g., U.S. Pat. 5,589,369, each of which is incorporated herein by reference). For example, it has been described that isotypic deletion of the antibody heavy chain joining region in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of human immunoglobulin gene arrays into such germline mutant mice results in the production of human binding molecules upon challenge with antigen. Another preferred means of producing human binding molecules using SCID mice is disclosed in US Patent No. 5,811,524, which is incorporated herein by reference. It should be understood that genetic material associated with these human binding molecules can also be isolated and manipulated as described herein.

Yet another efficient method for producing recombinant binding molecules is disclosed in Newman, Biotechnology, 10:1455-1460 (1992). Specifically, this technique resulted in the generation of primatized binding molecules containing monkey variable domains and human constant sequences. This reference is hereby incorporated by reference in its entirety. Additionally, this technology is described in US Patents 5,658,570, 5,693,780, and 5,756,096, all of which are incorporated herein by reference.

In another embodiment, lymphocytes can be selected by micromanipulation and the variable genes isolated. For example, peripheral blood mononuclear cells can be isolated from a vaccinated mammal and cultured in vitro for about 7 days. Cultures were screened for specific IgG that met the screening criteria. Cells in positive wells can be isolated. Individual Ig-producing B cells can be isolated by FACS or identified in a complement-mediated hemolytic plaque assay. Ig producing B cells can be micromanipulated into test tubes to amplify the VH and VL genes using eg RT-PCR. VH and VL genes can be cloned into antibody expression vectors, transfected into cells (eg, eukaryotic or prokaryotic cells) for expression.

Furthermore, the gene sequences used to produce the polypeptides of the invention can be obtained from a variety of different sources. For example, as discussed extensively above, many human antibody genes are available in publicly available deposits. The sequences of a large number of antibodies and the genes encoding the antibodies have been published, and suitable antibody genes can be chemically synthesized from these sequences using art-recognized techniques. Oligonucleotide synthesis techniques compatible with this aspect of the invention are well known to those skilled in the art and can be performed using any commercially available automated synthesizer. Additionally, DNA sequences encoding the classes of heavy and light chains listed herein can be obtained from commercial DNA synthesis services. The genetic material obtained using any of the methods described above can then be altered or synthesized to provide a polypeptide of the invention.

Alternatively, antibody-producing cell lines can be selected and grown using techniques well known to those skilled in the art. Such techniques are described in many laboratory manuals and primary publications. In this regard, techniques suitable for use in the present invention are described in Current Protocols in Immunology, Coligan et al. Eds. Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), which is incorporated by reference The full text, including supplementary material, is incorporated herein.

RNA can be isolated from starting hybridomas or other transformed cells by conventional techniques, such as guanidine isothiocyanate extraction and precipitation, followed by centrifugation or chromatography, as is well known. If desired, mRNA can be isolated from total RNA by standard techniques, such as chromatography on oligo-dT cellulose. Suitable techniques are familiar to those skilled in the art.

In one embodiment, the cDNA encoding the light chain and the heavy chain of the binding molecule can be prepared simultaneously or separately using reverse transcriptase and DNA polymerase according to well-known methods. PCR can be performed on the basis of published heavy and light chain DNA and amino acid sequences using consensus constant region primers or more specific primers. As discussed above, PCR can also be used to isolate DNA clones encoding the light and heavy chains of the binding molecule. In this case, the library can be screened by consensus primers or large homology probes, such as mouse constant region probes.

DNA, usually plasmid DNA, can be isolated from the cells by techniques known in the art, restricted and sequenced according to standard, well-known techniques described in detail in the aforementioned literature relating to recombinant DNA technology. Of course, DNA may be synthesized according to the invention at any time during isolation or subsequent analysis.

In one embodiment, a binding molecule of the invention comprises, or consists of, an antigen-binding fragment of an antibody. The term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that is capable of binding antigen, or competes with intact antibodies (ie, with the intact antibody from which they were derived) for antigen binding (ie, specifically binds). As used herein, the term "fragment" of an antibody molecule includes antigen-binding fragments of antibodies, e.g., antibody light chain (VL), antibody heavy chain (VH), single chain antibody (scFv), F(ab')2 fragment, Fab fragments, Fd fragments, Fv fragments, and single domain antibody fragments (DAbs). These fragments can be obtained, for example, by chemical or enzymatic treatment of intact or complete antibodies or antibody chains, or by recombinant means.

In one embodiment, the binding molecules of the invention are engineered or modified antibodies. Engineered forms of antibodies include, for example, minibodies, diabodies, diabodies fused to CH3 molecules, tetravalent antibodies, intradiabodies (e.g., Jendreyko et al. 2003. J. Biol. Chem. 278:47813), bispecific antibodies, fusion proteins (eg, antibody cytokine fusion proteins), or bispecific antibodies. Other immunoglobulins (Ig) and some of their variants are described, for example, in U.S. Patent 4,745,055; EP 256,654; Faulkner et al. Nature 298:286 (1982); EP 120,694; EP 125,023; Morrison, J. Immun.123:793 ( 1979); Kohler et al.Proc.Natl.Acad.Sci.USA 77:2197(1980); Raso et al.Cancer Res.41:2073(1981); Morrison et al.Ann.Rev.Immunol.2:239 (1984); Morrison, Science 229: 1202 (1985); Morrison et al. Proc. Natl. Acad. Sci. USA 81: 6851 (1984); EP 25 5,694; Reassembled immunoglobulin chains are also known. See, eg, US Patent 4,444,878; WO 88/03565; and EP 68,763 and references cited therein.

In one embodiment, the modified antibody of the invention is a minibody. Minibodies are dimeric molecules composed of two polypeptide chains, each of which contains a ScFv molecule (a single polypeptide containing one or more antigen-binding sites, such as the VL domain connected to the VH structure by a flexible linker domain, which is fused to the CH3 domain via a linker peptide.

ScFv molecules can be constructed in a VH-linker-VL orientation or a VL-linker-VH orientation.

The flexible hinge linking together the VL and VH domains that make up the antigen binding site preferably comprises about 10 to 50 amino acid residues. An example of a linker peptide for this use is (Gly4Ser)3 (SEQ ID NO: 17) (Huston et al. 1988. Proc. Natl. Acad. Sci. USA 85: 5879). Other linker peptides are known in the art.

Methods for preparing single chain antibodies are well known in the art, for example Ho et al.1989. Gene77:51; Bird et al.1988 Science 242:423; Pantoliano et al.1991.Biochemistry30:10117; Research 51: 6363; Takkinen et al. 1991. Protein Engineering 4: 837.

Minibodies can be prepared by constructing a ScFv component and linking a peptide-CH3 component using methods known in the art (see, eg, US Patent 5,837,821 or WO 94/09817A1). These components can be isolated from separate plasmids as restriction fragments, ligated and recloned into suitable vectors. Correct assembly can be confirmed by restriction enzyme digestion and DNA sequence analysis.

Diabodies are similar to scFv molecules, but usually have a short (less than 10, preferably 1-5) amino acid residue linker linking the two V domains together, so the VL and VH domains of the same polypeptide chain cannot interact. Conversely, the VL and VH domains of one polypeptide chain interact with the VH and VL domains (respectively) of a second polypeptide chain (WO 02/02781). In one embodiment, the binding molecule of the invention is a diabody to which at least one heavy chain portion is fused. In a preferred embodiment, the binding molecule of the invention is a diabody fused to a CH3 domain.

Other forms of modified antibodies are also included within the scope of the invention (e.g., WO 02/02781 A1; 5,959,083; 6,476,198 B1; US 2002/0103345 A1; WO 00/06605; Byrn et al. 1990. Nature. 344:667-70 ; Chamow and Ashkenazi. 1996. Trends Biotechnol. 14:52).

In one embodiment, a binding molecule of the invention comprises an immunoglobulin constant region. Constant regions are known in the art to mediate a variety of effector functions. For example, binding of the C1 component of complement to binding molecules can activate the complement system. Activation of complement is important for the opsonization and lysis of cellular pathogens. Activation of complement also stimulates inflammatory responses and may also be involved in autoimmune hypersensitivity. In addition, the binding molecule binds to the cell via the Fc region, and the Fc receptor site of the Fc region of the binding molecule binds to the Fc receptor (FcR) of the cell. There are a number of Fc receptors that are specific for different types of binding molecules, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). The binding of binding molecules to Fc receptors on the cell surface can trigger a variety of important and different biological responses, including endocytosis and destruction of particles coated with binding molecules, clearance of immune complexes, and killing of target cells coated with binding molecules. Cell lysis (termed antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental translocation and control of immunoglobulin production.

In one embodiment, the constant region of an IgG4-binding molecule is used, which is believed to be incapable of eliminating target cells, or by converting key residues in the Fc region to effector functions using techniques known in the art (eg, U.S. Patent 5,585,097). Fc variants can be produced by gene mutations that remove or reduce effector function. For example, deletion or inactivation of constant region domains (by point mutation or other means) can reduce the binding of Fc receptors to circulating modified binding molecules, thereby improving tumor localization. In other cases, it may be that constant region modifications consistent with the present invention are capable of modulating complement fixation, thus reducing serum half-life and non-specific association with conjugated cytotoxins. Modifications of other constant regions can also be used to modify disulfide bonds or oligosaccharide moieties to allow for enhanced localization due to increased antigen specificity or flexibility of the binding molecule. In summary, those skilled in the art will appreciate that binding molecules modified as described herein can exert a variety of subtle effects that may or may not be readily recognized. However, the resulting physiological change profile, bioavailability and other biochemical effects of these modifications, such as tumor localization ability, biodistribution and serum half-life, can be easily measured and analyzed without undue experimentation using well-known immunological techniques. Quantitative.

In one embodiment, a binding molecule of the invention may be derivatized or linked to another functional molecule (eg, another peptide or protein). Accordingly, the binding molecules of the invention include derivatives and other modified forms of the anti-GITR binding molecules described herein, including immunoadhesion molecules. For example, a binding molecule of the invention may be functionally linked (by chemical conjugation, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as another binding molecule (e.g., a bispecific antibody or diabodies), detectable reagents, cytotoxic agents, agents, and/or proteins or peptides capable of mediating association of the binding molecule with another molecule (such as a streptavidin core region or a polyhistidine-tagged ).

One class of derivatized binding molecules is produced by the cross-linking of two or more binding molecules (of the same type or of different types, eg for the production of bispecific antibodies). Suitable crosslinkers include heterobifunctional, i.e., two different reactive groups separated by a suitable spacer arm (e.g., m-maleimidobenzyl-N-hydroxysuccinimidyl ester ( m-maleimidobenzoyl-N-hydroxysuccinimide ester)); or the same bifunctional (for example, disuccinimidyl suberate). These coupling agents are available from Pierce Chemical Company, Rockford, IL.

The binding molecules of the invention can be derivatized with useful detectable reagents, including fluorescent compounds. Exemplary fluorescent detectable reagents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, and the like. Binding molecules can also be derivatized with detectable enzymes, such as alkaline phospholipase, horseradish peroxidase, glucose oxidase, and the like. Where a binding molecule is derivatized with a detectable enzyme, it can be detected by adding other reagents with which the enzyme produces a detectable reaction product. For example, the addition of hydrogen peroxide and diaminobenzidine in the presence of the detectable reagent horseradish peroxidase produces a colored reaction product that can be detected. Binding molecules can also be derivatized with biotin, detected by indirect measurement of avidin or streptavidin binding.

IV. Expression of Binding Molecules

Binding molecules of the invention can be prepared by recombinant expression of immunoglobulin light and heavy chains in host cells. For recombinant expression of the binding molecule, the host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the binding molecule, such that the light and heavy chains are expressed in the host cell, And preferably secreted into the medium of the growing host cell from which the binding molecule can be recovered. Using standard recombinant DNA techniques such as Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), Ausubel, F.M. et al. (eds.) Current Protocols in Molecular Biology , Greene Publishing Associates, (1989) and the techniques described in US Patent 4,816,397 to Boss et al. to obtain antibody heavy and light chain genes, introduce these genes into recombinant expression vectors, and the latter into host cells.

To express the binding molecules of the invention, DNA encoding partial or full-length light and heavy chains can be inserted into expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" means that the binding molecule gene is linked into a vector such that the transcriptional and translational control sequences in the vector can perform their intended function of regulating the transcription and translation of the binding molecule gene . In one embodiment, the selected expression vector and expression control sequences are compatible with the expression host cell used. The binding molecule light chain gene and the binding molecule heavy chain gene can be inserted into separate vectors, more commonly both genes are inserted into the same expression vector. The binding molecule gene can be inserted into the expression vector by standard methods (eg, ligating the binding molecule gene fragment with complementary restriction sites on the vector, or blunt end ligation if no restriction site is present). The expression vector may already carry the constant region sequences of the binding molecules prior to the insertion of the light or heavy chain sequences of the binding molecules. For example, one strategy to convert the VH and VL sequences into full-length binding molecule genes is to insert them into expression vectors that already encode the constant regions of the heavy and light chains, respectively, so that the VH segment and the CH segment in the vector are operably linked, and the VL The segment is operably linked to the CL segment. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the bound molecular chain from the host cell. The binding molecular chain gene can be cloned into the vector, so that the signal peptide and the amino terminal of the binding molecular chain gene are linked together in frame. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin).

In addition to the binding molecular chain gene, the recombinant expression vector of the present invention also has a regulatory sequence capable of controlling the expression of the binding molecular chain gene in the host cell. The term "regulatory set" includes promoters, enhancers, and other expression regulatory elements (eg, polyadenylation signals) that control the transcription or translation of genes associated with molecular chains. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Those skilled in the art understand that the design of expression vectors, including the selection of regulatory sequences, may depend on some factors, such as alternative host cells for transformation, the expression level of the target protein, and the like. Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high-level expression of the protein in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer promoter), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenoviruses (e.g., Adenovirus Major Late Promoter (AdMLP), and polyomaviruses. For further descriptions of viral regulatory elements and their sequences, see Examples include US Patent 5,168,062 to Stinski, US Patent 4,510,245 to Bell et al., and US Patent 4,968,615 to Schaffner et al.

In addition to combining molecular chain genes and regulatory series, the recombinant expression vector of the present invention can carry other sequences, such as sequences capable of regulating the replication of the vector in host cells (such as the origin of replication) and selectable marker genes. A selectable marker gene can assist in the selection of those host cells into which the vector has been introduced (see, eg, US Patents 4,399,216, 4,634,665, and 5,179,017, all to Axel et al.). For example, typically a selectable marker gene confers resistance to drugs such as G418, hygromycin or methotrexate in host cells into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for dhfr-host cells, selection/amplification with methotrexate) and the neo gene (selection with G418).

To express the light and heavy chains, expression vectors encoding the binding molecule heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass a variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Binding molecules of the invention may be expressed in prokaryotic or eukaryotic host cells, most preferably in eukaryotic cells, most preferably mammalian host cells, since such eukaryotic cells, especially mammalian cells, are more prokaryotic than prokaryotic cells. Cells are better able to assemble and secrete correctly folded, immunoreactive binding molecules.

Typically, expression vectors contain a selectable marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, or neomycin resistance) to enable detection of cells transformed with the desired DNA sequence (see, e.g., Itakura et al., USA ). Patent 4,704,362).

E. coli is a particularly useful prokaryotic host for cloning the polynucleotide (eg, DNA sequence) of the invention. Other suitable microbial hosts include Bacillus, such as B. subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas. In these prokaryotic hosts, expression vectors can also be prepared, which generally contain expression control sequences (eg, origin of replication) compatible with the host cell. Furthermore, there may be any number of well known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system or the promoter system from lambda phage. A promoter will generally control expression, optionally with an operator sequence, and has ribosome binding site sequences and the like for initiating and terminating transcription and translation.

Other microorganisms, such as yeast, can also be used for expression. Saccharomyces is a preferred yeast host, with suitable vectors carrying expression control sequences (eg, promoters), origins of replication, termination sequences, etc., as required. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include those from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization, among others.

In addition to microorganisms, mammalian tissue cell cultures can also be used to express and produce polypeptides of the invention (eg, polypeptides encoding binding molecules). See Winnacker, From Genes to Clones, VCH Publishers, N.Y.N.Y. (1987). Eukaryotic cells are actually preferred, as a number of suitable host cell lines capable of secreting heterologous proteins (e.g., intact binding molecules) have been established in the art, including CHO cell lines, various Cos cell lines, HeLa cells, myeloma Cell lines, or transformed B cells or hybridomas. Preferably, said cells are not human cells. Expression vectors for these cells may include expression control sequences, such as origins of replication, promoters, and enhancers (Queen et al. Inmunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosomes Binding sites, RNA splicing sites, polyadenylation sites, and transcription termination sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al. J. Immunol. 148:1149 (1992).

Alternatively, the sequence encoding the binding molecule can be incorporated into a transgene for introduction into the genome of the transgenic animal for subsequent expression in the milk of the transgenic animal (see, e.g., Deboer et al. US 5,741,957, Rosen, US 5,304,489 and Meade et al. US 5,849,992). Suitable transgenes include coding sequences for the light and/or heavy chains operably linked to promoters and enhancers from mammalian gland-specific genes such as casein or beta-lactoglobulin.

Preferred mammalian host cells for expressing the recombinant binding molecules of the invention include Chinese hamster ovary (CHO cells) (including dhfr-CHO as described in Urlaub and Chasin, (1980) Proc.Natl.Acad.Sci.USA77:4216-4220 Cells, for use with a DHFR selectable marker, such as described by R.J. Kaufman and P.A. Shatp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. When a recombinant expression vector encoding a binding molecule gene is introduced into a mammalian host cell, the host cell is cultured for a sufficient time to allow the host cell to express the binding molecule, or more preferably, to secrete the binding molecule into the medium in which the host cell grows, to produce binding molecules. Bound molecules can be recovered from the culture medium using conventional protein purification methods.

Vectors containing polynucleotide sequences of interest (eg, binding molecule heavy and light chain coding sequences and expression control sequences) can be transferred into host cells by known methods, depending on the type of host cell. For example, calcium chloride transfection is commonly used in prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistic techniques, or virus-based transfection can be used in other host cells (see generally Sambrook et al. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed.1989) (incorporated herein in its entirety by reference). Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation Perforation and microinjection (see generally Sambrook et al. supra). To generate transgenic animals, the transgene can be microinjected into fertilized oocytes, or integrated into the genome of embryonic stem cells whose nuclei are transferred to nucleus in the oocyte.

When the heavy and light chains are cloned into separate expression vectors, the vectors are co-transfected for expression and assembly of the complete immunoglobulin. Once expressed, the entire binding molecule, dimers thereof, individual light and heavy chains, or other immunoglobulin forms of the invention can be purified by standard methods in the art, including ammonium sulfate precipitation, affinity columns, column Chromatography, HPLC purification, gel electrophoresis, etc. (see generally Scopes, Protein Purification (Springer-Verlag, N.Y. (1982)). For pharmaceutical use, a substantially pure binding compound of at least about 90-95% homogeneity is preferred. Molecules, most preferably bound molecules that are 98-99% or more homogeneous.

Host cells can also be used to produce portions of intact binding molecules, such as Fab fragments or scFv molecules. It should be understood that variations of the above methods are also within the scope of the present invention. For example, it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain (but not both) of a binding molecule of the invention. Recombinant DNA techniques can also be used to remove part or all of the DNA encoding either the light chain or the heavy chain, or both, which are not essential for binding GITR. Molecules expressed from such truncated DNA molecules are also included within the scope of the binding molecules of the present invention. In addition, bifunctional binding molecules can be prepared by cross-linking a binding molecule of the invention with a second binding molecule using conventional chemical cross-linking methods, such that one heavy chain and one light chain are binding molecules of the invention, The other heavy and light chains are specific for antigens other than GITR.

As mentioned above, another aspect of the present invention relates to nucleic acid, vector and host cell compositions that can be used to recombinantly express the binding molecules of the present invention. The nucleotide sequence encoding the variable region of the light chain of 6C8 is shown in Figure 18 and SEQ ID NO:10. The CDR1 domain of VL covers nucleotides 130-162 of SEQ ID NO: 10 (SEQ ID NO: 14), the CDR2 domain covers nucleotides 208-228 of SEQ ID NO: 10 (SEQ ID NO: 15), CDR3 The domain covers nucleotides 325-351 of SEQ ID NO: 10 (SEQ ID NO: 16). The nucleotide sequence encoding the variable region of the heavy chain of 6C8 is shown in Figure 18 and SEQ ID NO:9. The CDR1 domain of VH covers nucleotides 133-168 of SEQ ID NO:9 (SEQ ID NO:11), and the CDR2 domain covers nucleotides 211-258 of SEQ ID NO:9 (SEQ ID NO:12) , the CDR3 domain encompasses nucleotides 355-381 in SEQ ID NO: 9 (SEQ ID NO: 13). In one embodiment, the nucleotide sequence encoding the VH CDR2 comprises SEQ ID NO:12. In another embodiment, the nucleotide sequence encoding the VH CDR2 comprises SEQ ID NO: 65 (CACATTTGGTGGGATGATGATAAGTACTAT CAA CCATCCCTGAAGAGC). Those skilled in the art understand that the nucleotide sequence encoding the binding molecule related to 6C8 can be obtained from the nucleotide sequence encoding 6C8 VL and VH using the genetic code and conventional molecular biology techniques.

In one embodiment, the invention provides an isolated nucleic acid molecule encoding a polypeptide sequence comprising 6C8 CDRs, for example comprising a group selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 19, SEQ ID NO: Amino acid sequences of ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8.

In another embodiment, the present invention provides an isolated nucleic acid molecule encoding a light chain variable region of a binding molecule comprising the amino acid sequence of SEQ ID NO: 2, but those skilled in the art will understand that because genetic Due to the degeneracy of the code, other nucleic acid molecules can also encode the amino acid sequence of SEQ ID NO: 2. The nucleic acid molecule may encode only the VL or may also encode a binding molecule light chain constant region operably linked to the VL. In one embodiment, the nucleic acid molecule is in a recombinant expression vector.

In another embodiment, the present invention provides an isolated nucleic acid molecule encoding a heavy chain variable region of a binding molecule comprising the amino acid sequence of SEQ ID NO: 1, but those skilled in the art will understand that because genetic Due to the degeneracy of the code, other nucleic acid molecules can also encode the amino acid sequence of SEQ ID NO: 1. In another embodiment, the invention provides an isolated nucleic acid molecule encoding the variable region of the heavy chain of the binding molecule, said variable region comprising the amino acid sequence of SEQ ID NO: 66, but those skilled in the art would understand that because of the genetic code Due to the degeneracy, other nucleic acid molecules can also encode the amino acid sequence of SEQ ID NO: 66. The nucleic acid molecule may encode only VH or may also encode a heavy chain constant region operably linked to VH. In one embodiment, the nucleic acid molecule is in a recombinant expression vector.

The present invention also provides a recombinant expression vector encoding the heavy chain of the binding molecule and/or the light chain of the binding molecule. For example, in one embodiment, the recombinant expression vector provided by the present invention encodes:

a) a binding molecule light chain comprising a variable region comprising the amino acid sequence of SEQ ID NO: 2; and

b) a binding molecule heavy chain comprising a variable region comprising the amino acid sequence of SEQ ID NO:1.

In another embodiment, the recombinant expression vector provided by the invention encodes:

a) a binding molecule light chain comprising a variable region comprising the amino acid sequence of SEQ ID NO: 2; and

b) a binding molecule heavy chain comprising a variable region comprising the amino acid sequence of SEQ ID NO:66.

The present invention also provides host cells into which one or more recombinant expression vectors of the present invention have been introduced. Preferably, said host cell is a mammalian host cell.

Still further, the present invention provides a method for synthesizing the recombinant binding molecule of the present invention, that is, culturing the host cell of the present invention in a suitable medium until the recombinant binding molecule of the present invention is synthesized. The method may further comprise isolating the recombinant binding molecule from the culture medium.

V. Uses of the Binding Molecules of the Invention

Given their ability to bind GITR, the binding molecules of the invention can be used in conventional immunoassays such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemical assays to detect GITR (e.g., in biological samples like serum or plasma). The present invention provides a method for detecting hGITR in a biological sample, the method comprising contacting the biological sample with the binding molecule of the present invention, and detecting hGITR in the biological sample by detecting the binding molecule bound to hGITR or the unbound binding molecule. The methods can be performed in vitro or in vivo. The binding molecules are directly or indirectly labeled with a detectable substance to assist in the detection of bound or unbound binding molecules. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent substances and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phospholipase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic groups include streptavidin/biotin and avidin/ Biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or Phycoerythrin; an example of a luminescent substance is luminal; and examples of suitable radioactive substances include ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ H.

As an alternative to labeled binding molecules, hGITR in biological sample fluids can be detected by a competition immunoassay using GITR standards labeled with a detectable substance and unlabeled anti-hGITR binding molecules. In this assay, a biological sample, a labeled GITR standard, and an anti-hGITR binding molecule are combined to determine the amount of labeled GITR standard bound to the unlabeled binding molecule. The amount of hGITR in the biological sample is inversely proportional to the amount of labeled GITR standard bound to the anti-hGITR binding molecule.

The anti-GITR binding molecules of the invention can also be used to detect GITR in samples from species other than humans, particularly primates (e.g., chimpanzee, baboon, marmoset, cynomolgus) (cynomolgus) and macaque (rhesus)) GITR.

In another embodiment, the present invention provides a method of abrogating the inhibitory effect of regulatory T cells on T effector cells. Abolishing the inhibitory effect of regulatory T cells on T effector cells can be detected, for example, by measuring the ability of the binding molecule to enhance T cell effector function in the presence of effective T cells. Cytokine production (eg, IL-2 production) or cell proliferation (eg, proliferation of helper T cells) is analyzed, for example, by ^measuring3H -thymidine incorporation or FACS. For example, in the presence of regulatory T cells, the response or activity of T effector cells is low, but they are elevated even in the presence of regulatory T cells after adding GITR-binding molecules, that is, GITR-binding molecules counteract the response of regulatory T cells to Inhibition of T effector cells.

The binding molecules of the invention can also be used to attenuate the degradation of I-κB in cells. For example, attenuation of I-κB degradation in cells can be detected by treating cells with an anti-GITR binding molecule, performing Western blotting, and quantifying I-κB.

Many diseases or pathological conditions would benefit from enhanced T effector cell activity and/or downregulation of regulatory T cell activity, for example by counteracting the inhibitory effect of regulatory T cells on T effector cells. For example, immune effector cells often cannot effectively respond to cancer cells. Accordingly, when enhanced effector T cell or antibody responses are desired, the methods of the invention may be used to treat subjects with such disorders. In one embodiment, such methods comprise administering a binding molecule of the invention to a subject such that the inhibitory effect of regulatory T cells on T effector cells is abolished, thereby enhancing the immune response. Preferably, said subject is a human subject. Alternatively, the subject may be a mammal expressing a GITR that cross-reacts with a binding molecule of the invention. Still further, the subject can be a mammal into which GITR has been introduced (eg, by administering GITR or by expressing a GITR transgene). The binding molecules of the invention can be administered to a human subject for therapeutic or prophylactic purposes. For example, the subject may be diagnosed with a disease or disorder, or be predisposed or susceptible to the disease. In addition, binding molecules of the invention can be administered to non-human mammals (eg, primates) expressing GITR molecules that cross-react with the binding molecules for veterinary purposes or as animal models of human disease. For the latter purpose, such animal models can be used to evaluate the therapeutic and/or prophylactic effect of the binding molecules of the invention (eg, to assess dosage and/or duration of administration).

Exemplary uses of the binding molecules of the invention are discussed in more detail below:

immunostimulatory composition

As described in the appended examples below, the binding molecules of the present invention can be combined with antigens, for example, as immunostimulatory compositions (or vaccines) to increase the subject's immune response to target antigens (eg, protein antigens). That is, the binding molecules of the invention can act as adjuvants to enhance immune responses. For example, to stimulate an antibody or cellular immune response to a target antigen (e.g., for vaccination purposes), the antigen can be administered together (e.g., simultaneously in the same or in separate compositions) with a binding molecule of the invention. ) resulting in an enhanced immune response. The antigen of interest and the binding molecule can be formulated in the same pharmaceutical composition, or in separate compositions. In one embodiment, the antigen of interest and the binding molecule are administered to the subject simultaneously. Alternatively, in some cases it may be desirable to administer the antigen first, followed by the binding molecule, or vice versa (eg, it may be beneficial to administer the antigen alone first to elicit a response, and then administer the binding molecule alone or with the antigen). In a preferred embodiment, the GITR-binding molecule of the present invention is administered at the time of sensitization with the antigen, ie at the time of the first administration of the antigen. For example, days -3, -2, -1, 0, +1, +2, +3. A particularly preferred day of administration of the GITR binding molecule of the invention is the day before the administration of the antigen.

An antigen of interest is, for example, an antigen that is capable of conferring protection to a subject against challenge by an infectious agent from which the antigen is derived, or that is capable of affecting tumor growth and metastasis in a manner beneficial to the subject. Illustrative examples of target antigens thus include those derived from an infectious source, cancer cells, etc., where an immune response against the antigen prevents or treats the disease caused by that source. Such antigens include, but are not limited to, viral, bacterial, fungal or parasitic proteins, glycoproteins, lipoproteins, glycolipids, and the like. Antigens of interest also include those antigens that would be of benefit to subjects at risk of contracting or having been diagnosed with a tumor, and may include, for example, tumor-associated antigens that are expressed exclusively or at elevated levels in tumor cells. The subject is preferably a mammal, most preferably a human.

As used herein, the term "pathogen" or "pathogen" includes microorganisms capable of infecting or parasitizing normal hosts, eg, animals such as mammals, preferably primates such as humans. As used herein, the term also includes opportunistic pathogens, such as microorganisms capable of infecting or parasitizing abnormal hosts, such as hosts whose normal flora have been disrupted as a result of a treatment regimen, or hosts who are immunocompromised. As used herein, the term also includes microorganisms whose replication is undesirable in a subject, or toxic molecules (eg, toxins) produced by these microorganisms.

Non-limiting examples of viral antigens include, but are not limited to, the nucleoprotein (NP) of influenza virus and the Gag protein of HIV. Other heterologous antigens include, but are not limited to HIV Env proteins or components thereof: gp120 and gp41, HIV Nef proteins, and HIV Pol proteins, reverse transcriptase and protease. In addition, other viral antigens such as Ebola virus (EBOV) antigen, EBOV NP or glycoprotein (GP) (full length or GP deletion in the mucin region of the molecule can also be used (Yang Z-Y, et al. (2000) Nat Med 6:886-9, 2000)); smallpox antigen; hepatitis A, B, or C virus; human rhinoviruses, such as types 2 and 14; herpes simplex virus; poliovirus type 2 or 3; foot-and-mouth disease virus (FMDV) ; Rabies virus; Rotavirus; Influenza virus; Coxsackie virus; Human papillomavirus (HPV), such as papillomavirus type 16, its E7 protein, and fragments containing E7 protein or epitopes thereof; and monkey immunization defective virus (SIV). Antigens of interest need not be limited to antigens of viral origin. Parasite antigens, such as Plasmodium antigens are also included, as are fungal antigens, bacterial antigens, and tumor antigens, which can also be used in the compositions and methods disclosed herein. Non-limiting examples of bacterial antigens include: Bordetella pertussis (e.g., P69 protein and filamentous hemagglutinin (FHA) antigens), Vibrio cholerae, Bacillus anhracis, and Escherichia coli antigens, such as Escherichia coli Bacillus heat-labile toxin B subunit (LT-B), Escherichia coli K88 antigen and enterotoxigenic Escherichia coli antigen. Examples of other antigens include Schistosoma mansoni P28 glutathione S-transferase antigen (P28 antigen) and fluke, mycoplasma, roundworm, tapeworm, Chlamydia trachomatis ( Chlamydia trachomatis) and malaria parasites, such as those of the genus Plasmodium or babesia, such as Plasmodium falciparum, and antigens that determine the immunogenic epitopes of the above antigens peptide.

Infections, diseases or disorders that can be treated or prevented by administering the vaccines of the invention include any infection, disease or disorder that elicits an immune response in the host to prevent. Diseases, disorders or infections that may be treated or prevented by administering the immunostimulatory compositions of the present invention include, but are not limited to, any infection, disease or disorder caused by or associated with fungi, parasites, viruses or bacteria, by Diseases, disorders or infections caused by or associated with various pathogens including those used in bioterrorism, listeriosis, Ebola virus, SARS, smallpox, hepatitis A, B, C or E, human Rhinoviruses, HIV (eg, HIV-1 and HIV-2), and AIDS, herpes, polio, foot-and-mouth disease, rabies, rotavirus, influenza, coxsackievirus, human papillomavirus, SIV, malaria parasites, Cancers such as neoplasms, human herpes virus, cytomegalovirus (especially human cytomegalovirus), Epstein-Barr virus, varicella-zoster virus, hepatitis viruses (such as hepatitis B virus, hepatitis A virus, hepatitis C virus), paramyxovirus: respiratory tract Syncytial virus, parainfluenza virus, measles virus, mumps virus, human papillomavirus (such as HPV6, 11, 16, 18, etc.), flavivirus (such as yellow fever virus (Yellow Fever Virus), dengue virus ( Dengue Virus), Tick-borne encephalitis virus, Japanese Encephalitis Virus), or influenza viruses such as influenza A virus (eg, hemagglutinin (H) and neuraminidase (N) subtype), influenza B virus and influenza C virus; and related diseases or disorders caused by bacterial organisms, including Gram-positive and Gram-negative bacteria. Examples include, but are not limited to, Neisseria, including N. gonorrhea and N. meningitidis; Streptococci, including S. pneumoniae, Streptococcus pyogenes ( S.pyogenes), Streptococcus agalactiae (S.agalactiae), Streptococcus mutans (S.mutans); Haemophilus spp, including H.influenzae type B, nonspecific non typeable H. influenzae, H. ducreyi; some species of Moraxella, including M catarrhalis, also known as Branham catarrhalis Branhamella catarrhalis; Bordetella, including B. pertussis, B. parapertussis and B. bronchiseptica; Mycobacteria, including M. tuberculosis, M.bovis, M.leprae, M.avium, M.paratuberculosis, M.smegmatis; Legionella, including L.pneumophila; Escherichia, including enterotoxic E.coli, enterohemorragic E.coli, enteropathogenic E.coli (enteropathogenic E.coli); bacteria of the genus Vibrio, including V.cholera; Shigella spp, including S. sonnei, Shigella dysenteriae S. dysenteriae, S. flexnerii; Yersinia, including Y. enterocolitica, Y. pestis, pseudotuberculosis Y. pseudotuberculosis; Campylobacter spp, including C. jejuni and C. coli; Salmonella, including S. typhi , S. paratyphi, S. choleraesuis, S. enteritidis; Listeria, including L. monocytogenes; Helicobacter Genus, including Helicobacter pylori (H. pylori); Pseudomonas, including Pseudomonas aeruginosa (P.aeruginosa); Staphylococcus, including Staphylococcus aureus (S.aureus), Staphylococcus epidermidis (S. ); Enterococcus, including Enterococcus faecalis (E.faecalis), Enterococcus faecium (E.faecium); Clostridium, including Clostridium tetani (C.tetani), Clostridium botulinum (C. Clostridium difficile (C. difficile); Bacillus, including B. anthracis; Corynebacterium, including C. diphtheriae; Borrelia, including B. burgdorferi, B. garzii (B. garinii), B. afzelii, B. andersonii, B. hermsii; Ehrlichia, including E. equi and human Ehrlichiosis (Human Granulocytic Ehrlichiosis); Rickettsia, including Rickettsii (R.rickettsii); Chlamydia, including Chlamydia trachomatis (C.trachomatis), Chlamydia pneumoniae (C. .pneumoniae), Chlamydia psittac (C.psittac); Leptospira, including Leptospira interrogans (L.interrogans); Treponema, including T.pallidum, Treponema denticola (T. enticola), Treponema hyodysenteriae (T.hyodysenteriae). Preferred bacteria include, but are not limited to, Listeria, mycobacteria (such as Mycobacterium tuberculosis), Anthrax, Salmonella and Listeria monocytogenes, Bordetella pertussis, Vibrio cholerae, flukes, Chlamydia, Nematodes, tapeworms, Chlamydia trachomatis and malaria parasites.

As used herein, the term "bacterial infection" includes infection by various bacteria. In another embodiment, T cells can be removed from the patient, contacted with an anti-GITR binding molecule in vitro, optionally with an activation signal (eg, antigen plus APC or polyclonal antibody), and reintroduced into the patient.

Regulatory T cells play an important role in maintaining self-immune tolerance by suppressing the immune response to autoimmune diseases and cancer. Accordingly, in one embodiment, counteracting the suppressive effect of regulatory T cells on T effector cells would be beneficial in enhancing the immune response in cancer. Thus, the binding molecules of the invention can be used to treat malignant tumors, inhibit tumor growth or metastasis. The binding molecules can be administered systemically or locally to the tumor site.

In one embodiment, modulation of GITR function may be useful for inducing tumor immunity, ie for treating a subject suffering from a neoplastic disease or cancer. In one embodiment, a binding molecule of the invention reduces tumor size, inhibits tumor growth, and/or prolongs survival of a tumor-bearing subject. GITR-binding molecules can be administered to patients bearing tumor cells (eg, sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) in order to overcome tumor-specific resistance in the subject.

The term "tumor-associated antigen" is used herein to denote an antigen in a host organism that affects tumor growth or metastasis. A tumor-associated antigen can be an antigen expressed by tumor cells, or an antigen expressed by non-tumor cells, but in the latter case, when expressed, it promotes the growth or metastasis of tumor cells. Types of tumor antigens and tumor-associated antigens include any known or hitherto unknown tumor antigens, including but not limited to, bcr/abl antigens in leukemia; HPVE6 and E7 antigens of oncogenic viruses associated with cervical cancer; MAGE1 and MZ2-E antigens in or associated with melanoma; and MVC-1 and HER-2 antigens in or associated with breast cancer.

As used herein, the term "neoplastic disease" is characterized by malignant tumor growth or in a disease state characterized by benign proliferating and hyperplastic cells. The general medical meaning of the term "neoplasia" refers to "new cell growth" that is unresponsive to normal growth control, eg neoplastic cell growth.

As used herein, the terms "proliferative", "hyperplastic", "malignant" and "neoplastic" are used interchangeably to refer to those cells in an abnormal state or condition characterized by rapid proliferation or neoplasia. These terms are intended to include all types of hyperplastic growths, hyperplasias, cancerous growths or oncogenic processes, metastatic problems or malignantly transformed cells, tissues or organs, regardless of the histopathological type or stage of infection. However, as used herein, the terms neoplasia and hyperplasia are used interchangeably, as their names suggest, to refer broadly to cells that undergo an abnormal rate of cell growth. Neoplasias and hyperplasias include "tumors," which may be benign, precancerous, or malignant.

The terms "neoplasia," "hyperplasia," and "tumor" are all commonly referred to as "cancer," which is an umbrella term for more than 100 diseases characterized by uncontrolled, abnormal cell growth. Examples of cancers include, but are not limited to: breast cancer; colon cancer; non-small cell lung cancer, head and neck cancer; colorectal cancer; lung cancer; prostate cancer; ovarian cancer; kidney cancer; melanoma; and gastrointestinal cancer (e.g., pancreatic cancer and gastric cancer); and osteosarcoma.

In one embodiment, the cancer is selected from the group consisting of: pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colon cancer, prostate cancer, gastric cancer, ovarian cancer, urethral bladder cancer, brain or central nervous system cancer, peripheral Nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or adnexal cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma , chondrosarcoma, and cancers of the hematopoietic tissue.

Accordingly, the present invention also relates to a method of treating a neoplastic disease or cancer in a subject, preferably a human or other animal, by administering to the subject or animal an effective amount of a binding molecule of the present invention. An effective amount of a polypeptide for the purpose of treating a neoplastic disease or cancer can be determined by one skilled in the art by routine experimentation. For example, a therapeutically effective amount of a binding molecule of the invention may vary depending on factors such as disease stage (e.g., stage I versus stage IV), age, sex, medical syndrome (e.g., immunosuppressed state or disease), and The body weight of the subject, and the ability of the binding molecule to elicit a desired response in the subject. Dosage regimens can be adjusted to provide the optimum therapeutic and/or prophylactic response. For example, several divided doses may be administered daily or the dose may be reduced accordingly as indicated by the exigencies of the therapeutic situation. In general, however, effective dosages are expected to be in the range of about 0.05 to 100 mg/kg body weight per day, more preferably about 0.5 to 10 mg/kg body weight per day.

Ways to Boost Your Immune Response

The binding molecules can also be used in methods of increasing an immune response. Up-regulation of an immune response can take the form of enhancing an existing immune response, or of eliciting an initial immune response. For example, enhancing the immune response by modulating GITR can be beneficial in the setting of viral infection. Because anti-GITR binding molecules can enhance the immune response, they have medical use in those situations where faster or more complete clearance of pathogens, such as bacteria and viruses, is required. Accordingly, the anti-GITR binding molecules of the invention can be used alone or in combination with antigens or other immunostimulatory agents to treat a subject suffering from a disease or disorder, such as those infectious diseases or cancers listed above.

Anti-GITR binding molecules can also be used in vaccines against various pathogens. Immunity against pathogens (eg, viruses) can be induced by co-vaccinating viral proteins with GITR binding molecules (as described above). Alternatively, vaccination can be performed using expression vectors encoding genes for pathogen antigens and GITR binding molecules, such as vaccinia virus expression vectors engineered to express nucleic acids encoding viral proteins and nucleic acids encoding GITR binding molecules. Pathogens for which vaccines may be useful include, for example, hepatitis B, hepatitis C, Epstein-Barr virus, cytomegalovirus, HIV-1, HIV-2, influenza, tuberculosis, malaria parasites, and schistosomiasis.

The invention further relates to a therapy based on said binding molecules, which therapy comprises administering the binding molecules of the invention to an animal patient, preferably a mammal, most preferably a human, in order to treat, detect and/or prevent the mentioned diseases, One or more of a disorder or condition. Therapeutic compositions of the invention include, but are not limited to, the binding molecules of the invention (including analogs and derivatives described herein) and the anti-idiotypic binding molecules described herein. The binding molecules of the invention can be used to treat, diagnose, inhibit or prevent diseases, disorders or conditions associated with aberrant GITR activity, including, but not limited to, one or more of the diseases, disorders or conditions described herein (e.g., can The binding molecules of the invention are provided in pharmaceutically acceptable compositions known in the art or described herein).

The binding molecules of the invention can advantageously be used in combination with other monoclonal or chimeric binding molecules, or lymphokines or hematopoietic cytokines (such as, for example, IL-2, IL-3 and IL-7), e.g. Those that are the number or activity of interacting effector cells.

The binding molecules of the invention can be used alone or in combination with other types of treatment (e.g., radiotherapy, chemotherapy, hormonal therapy, immunotherapy) and antineoplastic agents, antibiotics, therapies against pathogens (such as immunizations effective against viral pathogens or bacterial antigens) therapeutic or chemotherapeutic agents) in combination with immunostimulants. The binding molecules of the invention may also be administered in combination with an antigen to which it is desired to increase the immune response, such as a vaccine or antigen from a pathogen (or an attenuated form of a virus or bacterium) or from the antigen described above. tumor antigens. In one embodiment, a binding molecule of the invention is administered to a subject with an infection as a sole or combination therapy. In another embodiment, the binding molecules of the invention are administered alone or in combination to a subject with a chronic viral infection. In yet another embodiment, the binding molecules of the invention are administered alone or in combination to a subject with cancer.

In general, it is preferred to administer a binding molecule derived from a species to a patient of the same species. Thus, in a preferred embodiment, a human binding molecule, derivative, analog or nucleic acid thereof is administered to a human patient for treatment or prophylaxis.

VI. Pharmaceutical Compositions

The binding molecules of the invention can be introduced into a pharmaceutical composition suitable for administration to a subject. Typically, the pharmaceutical composition comprises a binding molecule of the invention and a pharmaceutically acceptable carrier. As used herein, the expression "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are compatible with pharmaceutical compositions. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compounds, the present invention relates to their use in the compositions. Supplementary active compounds can also be incorporated into the compositions.

Pharmaceutical compositions of the invention are formulated in dosage forms compatible with their intended route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal or subcutaneous use may include the following ingredients: sterile diluents such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents Antibacterial agents such as benzyl alcohol or methyl parabens; Antioxidants such as ascorbic acid or sodium bisulfite; Chelating agents such as EDTA; Buffers such as acetate, citrate, or phosphoric acid Salt and agents to adjust osmotic pressure such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or suspensions and sterile powders for the extemporaneous preparation of sterile injectable solutions or suspensions. Carriers suitable for intravenous administration include physiological saline, bacteriostatic water, Cremophor EL ^™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all such cases, the composition must be sterile and should be fluid to the extent that easy syringeability is achieved. It must be stable under the conditions of manufacture and storage and must be preserved against the contamination of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, ethylene glycol, propylene glycol, and liquid polyethylene glycol, etc.), and a suitable mixture thereof. Proper fluidity is maintained by, for example, the use of coatings such as lecithin, the maintenance of the desired particle size in the case of suspensions, and the use of surfactants. Various antibacterial and antifungal agents can be employed to prevent microbial contamination, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars (sugar), polyalcohols (such as mannitol, sorbitol) and sodium chloride in the compositions. Delayed absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or more ingredients enumerated above, by sterile filtration. Generally, suspensions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the formulation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, resulting in a powder of the active ingredient plus any other desired ingredient derived from the ingredient in advance Sterile filtered solution.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compounds can be presented, together with excipients, in the form of tablets, troches or capsules. Oral compositions can also be prepared using a liquid carrier as a mouthwash, wherein the compound formulated in a liquid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binders and/or auxiliary ingredients can be part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients, or compounds of similar properties: binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or Lactose; disintegrants, such as alginic acid, Primogel, or cornstarch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavor enhancers, Examples include peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered as an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal routes. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be penetrated are used in the formulation. Such penetrants are well known in the art and include, for example, for transmucosal administration, surfactants, bile salts and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds may be formulated into ointments, salves, gels or creams as known in the art.

The compositions can also be formulated for transcolonic delivery in the form of suppositories (eg, with conventional suppository bases such as coconut oil and other glycerides) or retention enemas.

In one embodiment, the binding molecules of the invention are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also serve as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, as described in US Patent 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Unit dosage form as used herein means physically separate units suitable as unitary dosages for the subject subjects to be treated; each unit containing a predetermined quantity of active compound in association with the required pharmaceutical carrier, calculated to energy. produce the desired therapeutic effect. The characteristics of the dosage unit forms of the invention are directly dictated by the unique properties of the active compound and the particular therapeutic effect to be achieved, as well as the limitations of the prior art in combining such active compounds for individual disposition.

Toxicity and therapeutic efficacy of these compounds can be determined by standard pharmaceutical procedures in cell culture or experimental animals, eg, determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the index of efficacy and it can be expressed as the ratio LD50/ED50. Compounds exhibiting higher therapeutic indices are preferred. While it is possible to use compounds that exhibit toxic side effects, care must be taken to design delivery systems that target the compound to affected tissues so that possible damage to unaffected cells is minimized, thereby reducing side effects.

The data obtained from cell culture and animal experiments can be used in formulating a dosage for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods of the invention, the therapeutically effective dose can be initially determined from cell culture assays. A dose can be determined in animal models to achieve a circulating plasma concentration range that includes the IC50 (ie, the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Concentrations in plasma can be measured, for example, by high performance liquid chromatography.

The pharmaceutical composition may be contained in a container, pack or dispenser together with instructions for use.

VII. Administration of Binding Molecules of the Invention

The binding molecules of the invention are contacted with cells from a subject in vitro or in vivo in a biocompatible form. "Biocompatible form"means the form of the agent to be administered in which any toxic effects are outweighed by the therapeutic effect of the binding molecule.

In one embodiment, the composition is administered to a subject. Administration of a pharmaceutically active amount of the pharmaceutical composition of the present invention refers to the effective amount and each dose to achieve the desired result within a certain period of time. For example, the pharmaceutically active amount of a binding molecule may vary with factors such as disease stage, age, sex, and body weight of the individual, as well as the ability of the binding molecule to elicit a desired response in the individual. Dosage regimens can be adjusted to provide the maximal medical response. For example, several divided doses may be administered daily or the dose may be reduced accordingly as indicated by the exigencies of the therapeutic situation.

A pharmaceutical composition of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of a binding molecule of the invention. "Therapeutically effective amount" refers to an effective amount and per dosage to achieve the desired therapeutic result within the time period required. A therapeutically effective amount of a binding molecule may vary with factors such as disease stage, age, sex, and body weight of the individual, as well as the ability of the binding molecule to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the binding molecule are outweighed by its therapeutically beneficial effects. "Prophylactically effective amount" refers to an effective amount and per dose to achieve the desired prophylactic result within the desired time period. Usually, the prophylactically effective amount will be lower than the therapeutically effective amount because the prophylactic dose is administered to the subject before onset or early in the disease.

Dosage regimens can be adjusted to achieve the optimum desired response (eg, a therapeutic or prophylactic response). For example, a single bolus dose may be administered, several divided doses may be administered over time or the dose may be reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Unit dosage form as used herein means physically discrete units suitable as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired dose in association with the required pharmaceutical carrier. Treatment effect is required. The properties of the dosage unit forms of the present invention are directly determined by (a) the unique properties of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations of the prior art in the sensitivity of combining the active compound for individual treatment .

Exemplary, non-limiting ranges of therapeutically or prophylactically effective amounts of binding molecules of the invention are, for example, about 0.1-25 mg/kg, about 1.0-10 mg/kg, about 0.5-2.5 mg/kg, about 5-25 mg/kg, About 1-400 mg/kg. It should be noted that dosage values may vary with the type and severity of the condition to be relieved. It is further understood that for any particular subject, the specific dosage regimen should be adjusted at any time according to the needs of the individual and the professional judgment of the person administering or supervising the administration of the composition, and the dosage ranges given here are examples only and are not limiting The scope or intended use of the claimed composition. Furthermore, non-limiting ranges for therapeutically or prophylactically effective amounts of binding molecules of the invention are about 0.0001 to 100 mg/kg, and about 0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.) subject body weight. For example, dosages may be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg, preferably at least 1 mg/kg. Dosages intermediate to the above ranges are also within the scope of the present invention.

Such doses may be administered daily, on alternate days, weekly, or on other schedules empirically determined. Exemplary treatments entail administration in multiple doses over a period of time, eg, at least six months. Other exemplary treatment regimens provide for dosing every two weeks or monthly or every 3 to 6 months. Exemplary dosage regimens include continuous daily administration of 1-10 mg/kg or 15 mg/kg, 30 mg/kg on alternate days, or 60 mg/kg weekly.

The binding molecules of the invention can be administered multiple times. Intervals between individual doses can be, for example, daily, weekly, monthly or yearly. Intervals may also be determined to be irregular by measuring blood levels of the bound molecule in the patient.

Binding molecules of the invention are optionally administered in combination with other agents effective for the disorder or condition desired to be treated (eg, prevented or treated). Preferred other agents are those recognized in the art and routinely administered for a particular disorder.

The binding molecules can be administered in a convenient manner, such as injection (subcutaneous, intravenous, etc.), oral, inhalation, transdermal or colonic administration. Depending on the route of administration, the active compound may be coated with a material to protect the compound from enzymes, acids, and other natural conditions that would render the compound inactive. For example, when administering an agent by a non-parenteral route, it may be desirable to coat the agent, or administer it with a material that will prevent its inactivation.

The binding molecules of the invention can be administered by a variety of methods known in the art, although for many therapeutic applications the preferred route/mode of administration is intravenous injection or instillation. Those skilled in the art will appreciate that the route and/or mode of administration will vary depending on the desired result. In some embodiments, the active compound can be prepared with carriers that will protect it against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed. Marcel Dekker, Inc. New York, 1978.

In some embodiments, the binding molecules of the invention may be orally administered, eg, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) can also be sealed in hard or soft gelatin capsules, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be combined with excipients and administered in the form of disintegrable tablets, lozenges, plasters, capsules, elixirs, suspensions, syrups, wafers and the like. When administering a compound of the invention by a non-parenteral route, it may be necessary to coat or administer the compound with a material to prevent inactivation.

Binding molecules and enzyme inhibitors can be administered together or in a suitable carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffers. Adjuvants can be used in the broadest sense including any immunostimulatory compound such as interferon. Adjuvants contemplated herein include resorcinol, nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include trypsin inhibitor, diisopropylfluorophosphate (DEEP) and aprotinin (trasylol). Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Sterna et al. (1984) J. Neuroimmunol. 7:27).

The active compounds can also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, lipid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these articles can contain a preservative to prevent the growth of microorganisms.

When the active compound is suitably protected as described above, the binding molecule can be administered orally with, for example, an inert diluent or an assimilable edible carrier.

Supplementary active compounds can also be incorporated into the compositions. In some embodiments, the binding molecules of the invention are co-formulated and/or co-administered with one or more other therapeutic agents. For example, anti-GITR binding molecules of the invention can be co-formulated and/or co-administered with one or more other antibodies that bind additional target molecules, such as those that bind other cytokines or that bind cell surface molecules. Antibody. Such combination therapy advantageously allows the use of low doses of the therapeutic agents, thereby avoiding the toxicities or syndromes associated with various individual therapies.

The invention further encompasses binding molecules conjugated to diagnostic or therapeutic agents. Binding molecules can be used diagnostically, eg, as part of clinical testing, to monitor tumor development or progression in order to confirm the efficacy of a given treatment regimen. Detection can be assisted by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radioactive substances, metals that emit positrons with various positron emission tomography, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated to the binding molecule directly or indirectly via an intermediate such as, for example, a linker known in the art using techniques known in the art. See, eg, US Pat. No. 4,741,900. Metal ions can be conjugated to binding molecules for use as diagnostic agents. Examples of suitable enzymes include horseradish peroxidase, alkaline phospholipase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic groups include streptavidin/biotin and avidin/ Biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent substance is luminol; examples of bioluminescent substances include luciferase, luciferin and aequorin; examples of suitable radioactive substances include ^I125 , ^I131 , ^I111 , ^In99Tc .

Furthermore, the binding molecule can be conjugated to a therapeutic moiety, such as a cytotoxin, e.g. a cytostatic or cytocidal agent, a therapeutic agent, a radioactive metal ion (e.g., an alpha radiation source like ²¹³ Bi), a biotoxin, a pro- Drugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, immunologically active ligands (eg, lymphokines and other antibodies). In another embodiment, the binding molecules of the invention may be conjugated to molecules capable of inhibiting tumor angiogenesis. In other embodiments, the compositions disclosed herein may comprise a binding molecule of the invention conjugated to a drug or prodrug. Still other embodiments of the invention comprise the use of binding molecules conjugated to specific biotoxins such as ricin, gelonin, Pseudomonas exotoxin or diphtheria toxin or their cytotoxic fragments. The choice of which conjugated or unconjugated binding molecule to use depends on the type and stage of cancer, whether adjuvant therapy (eg, chemotherapy or external beam radiation) is used, and the condition of the patient. We believe the selection can be readily made by one skilled in the art given the teachings herein.

Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, Tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxyanthrax Dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, Procaine, tetracaine, lidocaine, propranolol and puromycin and their analogs or homologues. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (eg, mechlorethamine, thiotepa, chlorambucil, melphalan , carnustine (BSNU) and lomustine (lomustine, CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomyces C and cis-dichlorodiamine platinum (II) (DDP, cisplatin), anthracyclines (eg, daunorubicin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly known as actinomycin), bleomycin, miteromycin, and anthramycin (AMC), and anti Mitotic agents (eg, vincristine and vinblastine).

The present invention is further illustrated by the following examples, which should not be construed as limiting the invention. All references, patents and published patent applications mentioned in this application are hereby incorporated by reference in their entirety, as well as the drawings.

Example

The following materials and methods were used in some examples:

method

cultured T cell lines

Differentiated cell lines were generated from cells obtained from human umbilical cord blood or from peripheral blood CD4+CD45RA+ naive T cells by various methods including flow cytometry and magnetic bead separation. The starting population was >95% pure. Cells were then stimulated with CD3 and CD28 antibodies to generate differentiated cell types in RPMI 1640 containing 10% FCS and 1% human AB serum, and indicated cocktails of cytokines and anti-cytokine neutralizing antibodies. Cultured with IL12 (62U/ml) and anti-IL4 (0.2μg/ml) to produce Th1 cells; cultured with IL4 (145U/ml) and anti-IL12 (10ug/ml) and anti-IFNγ (10ug/ml) to produce Th2 cells ; Cultured with TGFβ (32U/ml), IL9 (42U/ml), anti-IL4 (10ug/ml), anti-IL12 (10ug/ml) and anti-IFNγ (10ug/ml) to produce regulatory T cells. (Note: Anti-IL12 was not used in all experiments). All cultures were supplemented with IL2 (65 U/ml) and IL15 (4500 U/ml). Divide cells into larger culture plates as needed for cell division.

Example 1: Isolation and purification of 6C8

The 6C8 antibody is IgG2b, kappa type. Purification of this antibody revealed the presence of two heavy chains (Figure 1). This can be due to taking one of the two glycosylations or contamination with another antibody (Ab). Size exclusion chromatography showed only one peak (Figure 2).

The 6C8 antibody was purified as follows:

1. Wash 20ml protein G (Pharmacia HR10/30) with 5 column volumes of dPBS

2. Load 1L (1st round) or 2L (2nd round) hGITR (6C8) supernatant

3. Wash with 10 column volumes of dPBS

4. Elute directly into 1M Tris (20-25% v; v) with 100 mM citrate, pH 2.8

5. Strip with 100mM citrate (pH 2.8), 0.3M NaCl

Example 2: Identification of 6C8

The 6C8 antibody bound to cells transfected with GITR-L-M (Figure 3) and to activated PBL (Figure 4). Saturation curves of biotinylated anti-GITR on activated lymphocytes suggested good relative affinity (Figure 5).

The 6C8 antibody had co-stimulatory activity on T lymphocytes activated with sub-optimized anti-CD3 (Figure 6). Costimulation of this antibody did not reach the same level as CD28, but was comparable to the anti-GITR commercial product (R&D).

The 6C8 antibody did not induce apoptosis in activated lymphocytes (Figure 7). Lymphocytes were activated with PHA, and antibodies were added 3 days later. Compared to YTH 655, an anti-human CD2 known to induce apoptosis in activated lymphocytes, 6C8 did not increase apoptosis in activated T lymphocytes.

The 6C8 antibody did not block the primary mixed lymphocyte reaction (MLR) (Figure 8). TRX1 (anti-human CD4) was used as a positive control for the MLR.

Example 3: 6C8 antibody counteracts the inhibitory effect of regulatory T cells on T effector cells

The 6C8 antibody was able to block the suppressive effect induced by regulatory T cells (Figure 9). CD4+/CD25+ cells were added to CD4+/CD25- cells at different ratios. The cells were stimulated with anti-CD3 and anti-CD28 bound to microwell plates. When the ratio is 1:1, CD4+/CD25+ cells are able to eliminate the proliferation of CD4+/CD25- cells. Addition of 6C8 to the cultures blocked this inhibitory effect in a dose-dependent manner.

When T cells were stimulated by anti-CD3 alone (not co-stimulation with anti-CD28), CD4+/CD25+ cells were added to CD4+/CD25- cells, no inhibitory effect was observed, and indeed the anti-GITR antibody was has a weak co-stimulatory effect (Figure 10).

Example 4: 6C8 antibody modulates signaling via NF-kB

Activation of T cells by CD3 or CD3+CD28 resulted in the activation of the I-κB signaling pathway, which was assessed by phosphorylation of I-κB (Figures 12 and 14) and subsequent degradation (Figures 11 and 13).

As shown in Figure 11, in the case of partial activation, anti-GITR had a clear effect on I-κB signaling, which was confirmed by the time-dependent degradation of I-κB. In the presence of GITR binding molecules, degradation was significantly attenuated at all time points analyzed. The above changes correlated well with the decrease of I-κB phosphorylation (Fig. 12).

Interestingly, the magnitude of response was greater for TH2 and Treg than for TH1. Furthermore, in parallel experiments, GITR expression appeared to be higher in TH1 cells (as assessed by MCF (Mean Channel Fluorescence)) compared to TH2 and Treg cells. T cells fully activated by cross-linking CD3 and CD28 lost their reactivity against anti-GITR, but fully retained I-κb activation via TNF-α.

Example 5: 6C8 Antibody Enhances Immune Response

The B16 melanoma tumor model is an aggressive melanoma model that has been used to study the role of regulatory T cells in cancer. Treatment of mice depleted of anti-CD25 antibodies or anti-CTLA-4 has shown promising results in this model. In both cases, the treatment delayed tumorigenesis as well as growth in tumor size. Since GITR is expressed on CD25+ cells and may be involved in counteracting the suppressive effects of regulatory T cells, B16 tumor-bearing mice were treated with anti-GITR binding molecules to determine whether there was an effect on tumorigenesis or tumor size. Treatment of mice with anti-GITR binding molecules one day after tumor injection resulted in delayed tumorigenesis and growth in size (Figure 17). In addition, some mice in the GITR-treated group remained tumor-free by the end of the experiment.

On day 0, all animals were injected with 104 B16 melanoma cells in the right flank. The GITR group received 2 mg, 1 mg, 0.5 mg or 0.2 mg of the anti-GITR binding molecule on day 1. Measurable tumors appeared from day 16 onwards.

Example 6: Simultaneous delivery of anti-GITR and antigen results in adjuvant effect

The adjuvant effect of anti-mGITR antibodies in humoral responses against ovalbumin (Ova) or hemagglutinin (HA) was further investigated. Mice were treated on days -1, 0 and 1 with no antibody, 0.4 mg/day of YAML (isotype control) or 0.4 mg/day of 2F8 (rat-anti-mGITR). To assess the importance of the involvement of Fc receptors in the mechanism of action of the binding molecule, an additional group of animals was treated with 6 mg/day of 2F8 F(ab')2 on days -1, 0 and 1. The dose is selected on the basis that F(ab')2 has a shorter half-life than intact antibody. On day 0, mice were immunized with Ova (100 μg) or HA (10 μg). Ova-treated mice were challenged with 100 μg Ova on day 14, then bled on days 21 and 28 to obtain serum for ELISA analysis. HA-treated mice were challenged with 5 μg HA on day 14 and bled also on days 21 and 28.

Serum concentrations of 2F8 and 2F8 F(ab')2 were monitored to assess changes in the pharmacokinetics of the conjugated molecules. On day 1, serum levels of the binding molecule were comparable in mice treated with 2F8 or the 2F8 F(ab')2 fragment. In mice treated with 2F8, binding molecules were detected until day 9, whereas mice treated with 2F8 F(ab')2 fragments were detected only until day 3, although their The dose is 15 times that of 2F8.

These results show that in the HA arm of this study, 2F8-treated mice had a 4- and 5-fold increase in anti-HA antibodies compared to non-antibody-treated mice on days 21 and 28; Anti-HA antibodies increased 18- and 20-fold, respectively, compared to mice in the mice (Fig. 19). Anti-HA titers observed when adjuvanted with anti-mGITR antibodies were comparable to those observed when HA was co-administered with incomplete Freund's adjuvant (IFA). This suggests that the response observed with the anti-mGITR antibody is comparable to one of the most potent adjuvants commonly used in immunological research.

In the Ova arm of this study, on days 21 and 28, 2F8-treated mice had 13- and 6-fold increases in anti-Ova antibodies compared to non-antibody-treated mice; Compared with that, anti-Ova antibodies increased by 17 and 8 times, respectively (Fig. 20). The effect of the 2F8 antibody in the response to Ova was comparable to that observed in the response to HA. On day 21 and day 28, the anti-Ova antibodies in mice treated with 2F8 F(ab')2 increased 4- and 3-fold, respectively, compared with mice treated with no antibody; compared with mice treated with YAML, Anti-Ova antibodies increased 6 and 5 fold, respectively (Figure 20). The dose and different pharmacokinetic changes of F(ab')2 compared to intact antibody could explain the decreased anti-Ova response when compared with 2F8-treated mice.

Taken together, these data show that the influence of the 2F8 antibody in the humoral response to antigen is largely attributable to the F(ab')2 portion of the antibody, and that the involvement of Fc receptors may not be necessary for the anti-mGITR antibody to exert its adjuvant effect .

Example 7: Preparation of Chimeric Anti-GITR Binding Molecules

The 6C8 variable light chain region was grafted onto the human light chain constant region using conventional molecular biology techniques. The IgG1 light chain constant region was used. The amino acids of the complete chimeric light chain GITR binding molecule are as follows:

DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTINNVHSEDLAEYFCQQYNTDPLTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：22)。

Using conventional molecular biology techniques, the 6C8 variable heavy chain was also grafted onto the human heavy chain constant region. The IgG1 heavy chain constant region was used. The amino acid sequence of the complete chimeric heavy chain GITR binding molecule is shown below (also referred to as "Gly"):

QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY N STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：23)。

Because the amino acid sequence NX(S/T) is a putative glycosylation site consensus sequence, which may affect the production of binding molecules, and the IgG1 constant region of the 6C8 heavy chain contains the sequence NST, we prepared the heavy chain constant region The second version of , conservatively substituted glutamine for asparagine at amino acid residue 299 of SEQ ID NO: 23 (bold and underlined). Correspondingly, a second human constant region was grafted onto the 6C8 heavy chain variable region. The amino acid sequence of the complete chimeric heavy chain GITR binding molecule is shown below (also referred to as "Agly"):

QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY A STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：24)。

Example 8: Preparation of humanized forms of 6C8 anti-GITR binding molecules

6C8 was humanized using the CDR homology-based strategy described in Hwang et al. (2005) Methods (36) 35-42. A search of the heavy and light chain amino acid sequences using public databases revealed that 6C8 has a 3-1 heavy chain canonical structure and a 2-1-1 light chain canonical structure. Thus, all germline kappa chain V genes with 2-1-1 canonical structure in the IMGT database were compared with the 6C8 antibody sequence. Also all 3-1 germline heavy chain V genes were compared to the 6C8 amino acid sequence. Only CDR sequences were compared, and frames were picked based on which germline sequence had the most matches in the CDRs. (see sequence alignment below).

For the light chain, the 3-15 ^* 01 sequence had 14 matches in the CDRs and this sequence was selected. Because CDR 3 ends with leucine and threonine, we used the Jk4 J gene fragment sequence.

Light chain V gene with 2-1-1 canonical structure

IMGT

Gene Name CDR1 CDR2 CDR3 IDs

IGKV1-5 RASQSISSWLA......DASSLES.......QQYNSYS..11

IGKV1-6 RASQGIRNDLG......AASSLSQ.......LQDYNYP..9

IGKV1-9 RASQGISSYLA......AASTLQS.......QQLNSYP..11

IGKV1-12 RASQGISSWLA......AASSLQS.......QQANSFP..11

IGKV1-16 RASQGISSWLA......AASSLQS.......QQYNSYP..12

IGKV1D-16 RARQGISSWLA......AASSLQS.......QQYNSYP..11

IGKV1-17 RASQGIRNDLG......AASSLQS.......LQHNSYP..9

IGKV1-27 RASQGISNYLA......AASTLQS......QKYNSAP..11

IGKV1-33 QASQDISNYLN......DASNLET.......QQYDNLP..9

IGKV1-39 RASQSISSYLN......AASSLQS.......QQSYSTP..9

IGKV1D-43 WASQGISSYLA......YASSLQS.......QQYYSTP..11

IGKV3-11 RASQSVSSYLA......DASNRAT.......QQRSNWP..11

IGKV3D-11 RASQGVSSYLA......DASNRAT.......QQRSNWH..10

IGKV3-15 RASQSVSSNLA......GASTRAT.......QQYNNWP..14

6C8 KASQNVGTNVA......SASYRYS.......QQYNTDP

All germline light chain kappa chain V genes with a 2-1-1 canonical structure in the IMGT database were compared to the 6C8 antibody sequence. All 3-1 germline heavy chain V genes were also compared to the 6C8 amino acid sequence.

A version of the light chain was prepared using this method:

EIVMTQSPATLSVSPGERATLSCKASQNVGTNVAWYQQKPGQAPRLLIYSASYRYSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNTDPLTFGGGTKVEIK (SEQ ID NO: 44) (CDR in italics)

For the heavy chain, the sequence 2-05 ^* 01 had 17 matches. However, the sequence around CDR3 is different from 6C8 (YYCAR vs. YYCAHR). Since it has been shown that CDR 3 is the most important for CDR recognition, it should be possible to achieve a complete match in this region. The sequence 2-70*01 had 16 matches in the CDR, and the sequence immediately preceding CDR3 was an exact match for 6C8, so we chose 2-70 ^* 01.

For the J gene segment of the heavy chain, JH4 had the most matches and was therefore selected. The amino acid sequence was then back-translated, and primers corresponding to the target nucleotide sequence were purchased from IDT (Coralville, IA).

Heavy chain V gene with 3-1 canonical structure

IMGT

Gene name CDR1 CDR2 IDs

IGHV2-5 TSGVGVG.....LIYWNDDKRYSPSLKS 17

IGHV2-26 NARMGVS.....HIFSNDEKSYSTSLKS 12

IGHV2-70 TSGMCVS.....LIDWDDDKYYSTSLKT 16

IGHV4-30-2 SGGYSWS.....YIYHSGSTYYNPSLKS 10

IGHV4-30-4 SGDYYWS.....YIYYSGSTYYNPSLKS 9

IGHV4-31 SGGYYWS.....YIYYSGSTYYNPSLKS 9

IGHV4-39 SSSYYWG.....SIYYSGSTYYNPSLKS 10

IGHV4-61 SGSYYWS.....YIYYSGSTNYNPSLKS 8

6C8 TSGMGVG.....HIWWDDDKYYNPSLKS

Using this approach, a version of the heavy chain was prepared:

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYYNPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARTRRYFPFAYWGQGTLVTVSS (SEQ ID NO: 53) (also referred to as "N") .

Because the amino acid sequence NX(S/T) is a putative consensus sequence for glycosylation sites, which may affect the production of binding molecules, and the CDR2 of the 6C8 heavy chain contains the sequence NPS, we prepared a second version of the heavy chain that would Conservative substitution of glutamine for asparagine at amino acid residue 62 in SEQ ID NO:53 (bold and underlined). Accordingly, a second version of the heavy chain was prepared:

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMG VGWIRQPPGKALEWLAHIWWDDDKYYQPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARTRRYFPFAYWGQGTLVTVSS (SEQ ID NO: 54) (also referred to as "Q" ) .

We also performed a CLUSTAL W (1.82) multiple sequence alignment of the 6C8 light chain variable region and the 3-15 ^* 01 germline light chain sequences (using the Blosum scoring array with a gap penalty of 10). The result is as follows:

6C8 DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPD

3-15 ^* 01 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPA

:****** :*.* *:*.:::*:***.*.:*:********:*: ***.** * :* :*

6C8 RFTGSGSGTDFLTINNVHSEDLAEYFCQQYNTDPLTFGAGTKLEIK

3-15*01 RFSGSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP------------

**:******:*****..::***:* *:*****.*

Based on CLUSTAL W analysis, some amino acid residues were identified in the human framework for possible substitution with amino acid residues corresponding to the 6C8 framework residues of the humanized 6C8 light chain. Specifically, E at position 1, P at position 8, A at position 9, T at position 10, L at position 11, V at position 13, P at position 15, and point 17 E for position 19, T for position 20, L for position 21, S for position 22, A for position 43, R for position 45, L for position 46, I for position 58 , A at position 60, S at position 63, E at position 70, S at position 76, S at position 77, L at position 78, Q at position 79, F at position 83, position V at point 85, Y at point 87, G at point 100, and V at point 104.

Similarly, a CLUSTAL W (1.82) multiple sequence alignment (Blosum scoring array with a gap penalty of 10) was also performed on the 6C8 heavy chain variable region and the germline heavy chain protein containing the amino acid sequence 2-70 ^* 01. The result is as follows:

6C8 QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKY

2-70*01 QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMCVSWIRQPPGKALEWLALIDWDDDKY

****:****.::**:***:***:********** *.*****.**.***** * *****

6C8 YNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSS

2-70*01 YSTSLKTRLTISKDTSKNQVVLTMTNMDPVDTATYYCARI-------------------

*..***::********:***.*.:*.:*..*:******

Based on CLUSTAL W analysis, some amino acid residues were identified in the human framework for possible substitution with amino acid residues corresponding to the 6C8 framework residues of the humanized 6C8 heavy chain. Specifically, R at position 5, A at position 10, L at position 11, V at position 12, T at position 15, T at position 19, T at position 23, T at position 43 P for position 46, R for position 68, K for position 77, V for position 81, T for position 83, M for position 84, N for position 86, M for position 87 , P at position 89, V at position 90, and/or T at position 92.

Four humanized full-length 6C8 binding molecules were prepared with the following combinations of humanized heavy and light chains:

Full-length version 1 (HuN6C8-Gly) - Humanized (Hu) 6C8 light chain (L)/humanized heavy chain with N in CDR2 ("N") and contains a constant region with N ("Gly" )

Full length version 2 (HuN6C8-Agly) - Humanized (Hu) 6C8 light chain (L)/humanized heavy chain with N ("N") in CDR2 and contains constant region with A ("Agly ")

Full length version 3 - (HuQ6C8-Gly) - humanized (Hu)6C8 light chain (L)/humanized heavy chain, with Q in CDR2 ("Q"), and contains constant region with N (" Gly")

Full length version 4 - (HuQ6C8-Agly) - humanized (Hu)6C8 light chain (L)/humanized heavy chain, with Q in CDR2 ("Q"), and contains constant region with A (" Agly")

用于产生全长结合分子的糖基化IgG1重链恒定区氨基酸序列如下：ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：55)。

用于产生全长结合分子的去糖基化IgG1重链恒定区氨基酸序列如下：ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：56)。

The IgG1 light chain constant region amino acid sequence used to prepare the full-length binding molecule is as follows: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 57).

人源化6C8轻链的完整氨基酸序列如下：EIVMTQSPATLSVSPGERATLSCKASQNVGTNVAWYQQKPGQAPRLLIYSASYRYSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNTDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：58).

The leader sequence METQSQVFVYMLLWLSGVDG (SEQ ID NO: 59) may also be included.

The complete amino acid sequences of the humanized 6C8 heavy chain versions HuN6C8-Agly, HuQ6C8-Gly and HuQ6C8-Agly are as follows:

HuN6C8-Gly

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYYNPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARTRRYFPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：60)；

HuN6C8-Agly

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYYNPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARTRRYFPFAYWGQGTLVTVS SASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLS SVVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：61)；

HuQ6C8-Gly

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYYQPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARTRRYFPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：62)；以及

HuQ6C8-Agly

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYYQPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARTRRYFPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：63).

The leader sequence MDRLTFSFLLLIVPAYVLS (SEQ ID NO: 64) may also be included.

equivalent

Those skilled in the art will be able to recognize, or ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The following claims are intended to cover such equivalents.

sequence listing

<110> Tolerx Inc. (TolerRx, Inc.)

<120> GITR binding molecules and uses thereof

<130>TLN-029PC

<140>

<141>

<150>60/665,322

<151>2005-03-25

<150>60/687,265

<151>2005-06-03

<160>68

<170>PatentIn Ver.3.3

<210>1

<211>138

<212>PRT

<213> Mouse (Mus musculus)

<400>1

Met Asp Arg Leu Thr Phe Ser Phe Leu Leu Leu Ile Val Pro Ala Tyr

1 5 10 15

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

20 25 30

Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu

35 40 45

Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys

50 55 60

Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr

65 70 75 80

Asn Pro Ser Leu Lys Ser Gln Leu Thr Ile Ser Lys Asp Thr Ser Arg

85 90 95

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

100 105 110

Thr Tyr Tyr Cys Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp

115 120 125

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

130 135

<210>2

<211>127

<212>PRT

<213> Mouse (Mus musculus)

<400>2

Met Glu Thr Gln Ser Gln Val Phe Val Tyr Met Leu Leu Trp Leu Ser

1 5 10 15

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

20 25 30

Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

115 120 125

<210>3

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Peptides

<400>3

Gly Phe Ser Leu Ser Thr Ser Gly Met Gly Val Gly

1 5 5 10

<210>4

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Peptides

<400>4

His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210>5

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Peptides

<400>5

Thr Arg Arg Tyr Phe Pro Phe Ala Tyr

1 5

<210>6

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Peptides

<400>6

Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala

1 5 5 10

<210>7

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Peptides

<400>7

Ser Ala Ser Tyr Arg Tyr Ser

1 5

<210>8

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Peptides

<400>8

Gln Gln Tyr Asn Thr Asp Pro Leu Thr

1 5

<210>9

<211>414

<212>DNA

<213> Mouse (Mus musculus)

<400>9

atggacagac ttacattctc attcctgctg ctgattgtcc ctgcatatgt cttgtcccaa 60

gttactctaa aagagtctgg ccctgggata ttgaagccct cacagaccct cagtctgact 120

tgttctttct ctgggttttc actgagcact tctggtatgg gtgtaggctg gattcgtcag 180

ccttcaggga agggtctgga gtggctggcg cacatttggt gggatgatga taagtactat 240

aatccatccc tgaagagcca gctcacaatc tccaaggata cctccagaaa ccaggtattc 300

ctcaagatca ccagtgtgga cactgcagat gctgccactt actactgtgc tcgaactagg 360

aggtacttcc cctttgctta ctggggccaa gggaacactag tcacagtctc ctca 414

<210>10

<211>381

<212>DNA

<213> Mouse (Mus musculus)

<400>10

atggagacac agtctcaggt ctttgtatac atgttgctgt ggttgtctgg tgttgatgga 60

gacattgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga cagggtcagc 120

gtcacctgca aggccagtca gaatgtgggt actaatgtag cctggtatca acagaaacca 180

gggcaatctc ctaaagcact gatttactcg gcatcctacc ggtacagtgg agtccctgat 240

cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcaacaa tgtgcactct 300

gaagacttgg cagagtattt ctgtcaacaa tataacaccg atccgctcac gttcggagct 360

gggaccaagc tggaaatcaa a 381

<210>11

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Oligonucleotides

<400>11

gggttttcac tgagcacttc tggtatgggt gtaggc 36

<210>12

<211>48

<212>DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Oligonucleotides

<400>12

cacatttggt gggatgatga taagtactat aatccatccc tgaagagc 48

<210>13

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Oligonucleotides

<400>13

actaggaggt acttccccctt tgcttac 27

<210>14

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Oligonucleotides

<400>14

aaggccagtc agaatgtggg tactaatgta gcc 33

<210>15

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Oligonucleotides

<400>15

tcggcatcct accggtacag t 21

<210>16

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Oligonucleotides

<400>16

caacaatata acaccgatcc gctcacg 27

<210>17

<211>1214

<212>DNA

<213> Human (Homo sapiens)

<400>17

gtctacaccc cctcctcaca cgcacttcac ctgggtcggg attctcaggt catgaacggt 60

cccagccacc tccgggcagg gcgggtgagg acggggacgg ggcgtgtcca actggctgtg 120

ggctcttgaa acccgagcat ggcacagcac ggggcgatgg gcgcgtttcg ggccctgtgc 180

ggcctggcgc tgctgtgcgc gctcagcctg ggtcagcgcc ccaccggggg tcccgggtgc 240

ggccctgggc gcctcctgct tgggacggga acggacgcgc gctgctgccg ggttcacacg 300

acgcgctgct gccgcgatta cccgggcgag gagtgctgtt ccgagtggga ctgcatgtgt 360

gtccagcctg aattccactg cggagaccct tgctgcacga cctgccggca ccacccttgt 420

cccccaggcc agggggtaca gtcccagggg aaattcagtt ttggcttcca gtgtatcgac 480

tgtgcctcgg ggaccttctc cgggggccac gaaggccact gcaaaccttg gacagactgc 540

accccagttcg ggtttctcac tgtgttccct gggaacaaga cccacaacgc tgtgtgcgtc 600

ccagggtccc cgccggcaga gccgcttggg tggctgaccg tcgtcctcct ggccgtggcc 660

gcctgcgtcc tcctcctgac ctcggcccag cttggactgc acatctggca gctgaggagt 720

cagtgcatgt ggccccgaga gacccagctg ctgctggagg tgccgccgtc gaccgaagac 780

gccagaagct gccagttccc cgaggaagag cggggcgagc gatcggcaga ggagaagggg 840

cggctggggag acctgtgggt gtgagcctgg ccgtcctccg gggccaccga ccgcagccag 900

cccctcccca ggagctcccc aggccgcagg ggctctgcgt tctgctctgg gccgggccct 960

gctcccctgg cagcagaagt gggtgcagga aggtggcagt gaccagcgcc ctggaccatg 1020

cagttcggcg gccgcggctg ggccctgcag gagggagaga gagacacagt catggccccc 1080

ttcctccctt gctggccctg atggggtggg gtcttaggac gggaggctgt gtccgtgggt 1140

gtgcagtgcc cagcacggga cccggctgca ggggaccttc aataaacact tgtccagtga 1200

aaaaaaaaaa aaaa 1214

<210>18

<211>241

<212>PRT

<213> Human (Homo sapiens)

<400>18

Met Ala Gln His Gly Ala Met Gly Ala Phe Arg Ala Leu Cys Gly Leu

1 5 10 15

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

20 25 30

Gly Cys Gly Pro Gly Arg Leu Leu Leu Gly Thr Gly Thr Asp Ala Arg

35 40 45

Cys Cys Arg Val His Thr Thr Arg Cys Cys Arg Asp Tyr Pro Gly Glu

50 55 60

Glu Cys Cys Ser Glu Trp Asp Cys Met Cys Val Gln Pro Glu Phe His

65 70 75 80

Cys Gly Asp Pro Cys Cys Thr Thr Cys Arg His His Pro Cys Pro Pro

85 90 95

Gly Gln Gly Val Gln Ser Gln Gly Lys Phe Ser Phe Gly Phe Gln Cys

100 105 110

Ile Asp Cys Ala Ser Gly Thr Phe Ser Gly Gly His Glu Gly His Cys

115 120 125

Lys Pro Trp Thr Asp Cys Thr Gln Phe Gly Phe Leu Thr Val Phe Pro

130 135 140

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

145 150 155 160

Glu Pro Leu Gly Trp Leu Thr Val Val Leu Leu Ala Val Ala Ala Cys

165 170 175

Val Leu Leu Leu Thr Ser Ala Gln Leu Gly Leu His Ile Trp Gln Leu

180 185 190

Arg Ser Gln Cys Met Trp Pro Arg Glu Thr Gln Leu Leu Leu Glu Val

195 200 205

Pro Pro Ser Thr Glu Asp Ala Arg Ser Cys Gln Phe Pro Glu Glu Glu Glu

210 215 220

Arg Gly Glu Arg Ser Ala Glu Glu Lys Gly Arg Leu Gly Asp Leu Trp

225 230 235 240

Val

<210>19

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Peptides

<400>19

His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Gln Pro Ser Leu Lys Ser

1 5 10 15

<210>20

<211>105

<212>PRT

<213> Mouse (Mus musculus)

<400>20

Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln

1 5 10 15

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

20 25 30

Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln

35 40 45

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

50 55 60

Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg

65 70 75 80

His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro

85 90 95

Ile Val Lys Ser Phe Asn Arg Asn Glu

100 105

<210>21

<211>334

<212>PRT

<213> Mouse (Mus musculus)

<400>21

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly

1 5 10 15

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

20 25 30

Phe Pro Glu Ser Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Ser Val His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Met

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln

165 170 175

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

180 185 190

Thr Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys

195 200 205

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

210 215 220

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

225 230 235 240

Pro Ala Glu Gln Leu Ser Arg Lys Asp Val Ser Leu Thr Cys Leu Val

245 250 255

Val Gly Phe Asn Pro Gly Asp Ile Ser Val Glu Trp Thr Ser Asn Gly

260 265 270

His Thr Glu Glu Asn Tyr Lys Asp Thr Ala Pro Val Leu Asp Ser Asp

275 280 285

Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asn Met Lys Thr Ser Lys Trp

290 295 300

Glu Lys Thr Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly Leu Lys

305 310 315 320

Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro Gly Lys

325 330

<210>22

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic mouse/human light chain constructs

<400>22

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

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Val His Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Thr Asp Pro Leu

85 90 95

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

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

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

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>23

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Mouse/Human Heavy Chain Constructs

<400>23

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

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

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210>24

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic mouse/human complete chimeric heavy chain

<400>24

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

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

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210>25

<211>95

<212>PRT

<213> Human (Homo sapiens)

<400>25

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro

85 90 95

<210>26

<211>95

<212>PRT

<213> Human (Homo sapiens)

<400>26

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Val Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

<210>27

<211>95

<212>PRT

<213> Human (Homo sapiens)

<400>27

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

<210>28

<211>95

<212>PRT

<213> Human (Homo sapiens)

<400>28

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

<210>29

<211>95

<212>PRT

<213> Human (Homo sapiens)

<400>29

Ala Ile Arg Met Thr Gln Ser Pro Phe Ser Leu Ser Ala Ser Val Gly

1 5 10 15

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

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Ala Lys Ala Pro Lys Leu Phe Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro

85 90 95

<210>30

<211>95

<212>PRT

<213> Human (Homo sapiens)

<400>30

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro

85 90 95

<210>31

<211>95

<212>PRT

<213> Human (Homo sapiens)

<400>31

Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Cys Gly Tyr Ser Thr Pro

85 90 95

<210>32

<211>95

<212>PRT

<213> Human (Homo sapiens)

<400>32

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro

85 90 95

<210>33

<211>95

<212>PRT

<213> Human (Homo sapiens)

<400>33

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro

85 90 95

<210>34

<211>95

<212>PRT

<213> Human (Homo sapiens)

<400>34

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro

85 90 95

<210>35

<211>95

<212>PRT

<213> Human (Homo sapiens)

<400>35

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asn Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro

85 90 95

<210>36

<211>95

<212>PRT

<213> Human (Homo sapiens)

<400>36

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro

85 90 95

<210>37

<211>95

<212>PRT

<213> Human (Homo sapiens)

<400>37

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Arg Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro

85 90 95

<210>38

<211>95

<212>PRT

<213> Human (Homo sapiens)

<400>38

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

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

20 25 30

Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro

85 90 95

<210>39

<211>95

<212>PRT

<213> Human (Homo sapiens)

<400>39

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro

85 90 95

<210>40

<211>95

<212>PRT

<213> Human (Homo sapiens)

<400>40

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro

85 90 95

<210>41

<211>95

<212>PRT

<213> Human (Homo sapiens)

<400>41

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro

85 90 95

<210>42

<211>95

<212>PRT

<213> Human (Homo sapiens)

<400>42

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asp Tyr Asn Tyr Pro

85 90 95

<210>43

<211>95

<212>PRT

<213> Human (Homo sapiens)

<400>43

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

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

35 40 45

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser

85 90 95

<210>44

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Protein Constructs

<400>44

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>45

<211>96

<212>PRT

<213> Human (Homo sapiens)

<400>45

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

<210>46

<211>100

<212>PRT

<213> Human (Homo sapiens)

<400>46

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

1 5 10 15

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

20 25 30

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

35 40 45

Trp Leu Ala His Ile Phe Ser Asn Asp Glu Lys Ser Tyr Ser Thr Ser

50 55 60

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

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile

100

<210>47

<211>100

<212>PRT

<213> Human (Homo sapiens)

<400>47

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile

100

<210>48

<211>99

<212>PRT

<213> Human (Homo sapiens)

<400>48

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Arg

<210>49

<211>99

<212>PRT

<213> Human (Homo sapiens)

<400>49

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Arg

<210>50

<211>99

<212>PRT

<213> Human (Homo sapiens)

<400>50

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Arg

<210>51

<211>99

<212>PRT

<213> Human (Homo sapiens)

<400>51

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Arg

<210>52

<211>99

<212>PRT

<213> Human (Homo sapiens)

<400>52

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Arg

<210>53

<211>119

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Protein Constructs

<400>53

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

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

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>54

<211>119

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Protein Constructs

<400>54

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

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

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Gln Pro Ser

50 55 60

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

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>55

<211>330

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Protein Constructs

<400>55

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

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

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

50 55 60

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

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>56

<211>330

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Protein Constructs

<400>56

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

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

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

50 55 60

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

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>57

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Protein Constructs

<400>57

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

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

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

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

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210>58

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Protein Constructs

<400>58

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

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

85 90 95

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

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

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

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>59

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Amino Acid Leader Sequences

<400>59

Met Glu Thr Gln Ser Gln Val Phe Val Tyr Met Leu Leu Trp Leu Ser

1 5 10 15

Gly Val Asp Gly

20

<210>60

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Protein Constructs

<400>60

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

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

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210>61

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Protein Constructs

<400>61

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

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

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210>62

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Protein Constructs

<400>62

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

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

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Gln Pro Ser

50 55 60

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

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210>63

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Protein Constructs

<400>63

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

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

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Gln Pro Ser

50 55 60

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

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210>64

<211>19

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Leader Sequence Peptides

<400>64

Met Asp Arg Leu Thr Phe Ser Phe Leu Leu Leu Ile Val Pro Ala Tyr

1 5 10 15

Val Leu Ser

<210>65

<211>50

<212>DNA

<220>

<223> Description of Artificial Sequences: Synthetic Oligonucleotides

<400>65

cacatttggt gggatgatga taagtactat caaccatccc tgaagagcca 50

<210>66

<211>138

<212>PRT

<213> Mouse (Mus musculus)

<400>66

Met Asp Arg Leu Thr Phe Ser Phe Leu Leu Leu Ile Val Pro Ala Tyr

1 5 10 15

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

20 25 30

Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu

35 40 45

Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys

50 55 60

Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Tyr Tyr Cys Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp

115 120 125

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

130 135

<210>67

<211>118

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Protein Constructs

<400>67

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

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

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Asn Pro Ser Leu

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp Gly Glu Gly Thr

100 105 110

Ser Val Thr Val Thr Ser

115

<210>68

<211>118

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Protein Constructs

<400>68

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

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

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Asn Pro Ser Leu

50 55 60

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

65 70 75 80

Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

Claims (57)

1.GITR binding molecule, it comprises amino-acid residue 20-138 among the SEQ ID NO.1 or the amino-acid residue 20-138 among the SEQ ID NO:66.

2.GITR binding molecule, it comprises the amino-acid residue 21-127 among the SEQ ID NO.2.

3.GITR binding molecule, it comprises at least one and is selected from SEQ ID NO.3, complementary determining region (CDR) aminoacid sequence of SEQ ID NO.4 or SEQ ID NO:19 and SEQ ID NO.5.

4. the GITR binding molecule of claim 3, it comprises at least two CDR.

5. the GITR binding molecule of claim 3, it comprises three CDR.

6.GITR binding molecule, it comprises at least one cdr amino acid sequence that is selected from SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.

7. the GITR binding molecule of claim 6, it comprises at least two CDR.

8. the GITR binding molecule of claim 6, it comprises three CDR.

9.GITR binding molecule, it comprises the CDR shown in the SEQ ID NO:3,4 or 19,5,6,7 or 8.

10.GITR binding molecule, it comprises variable region of heavy chain and variable region of light chain, described variable region of heavy chain contains amino-acid residue 20-138 among the SEQ ID NO.1 or the amino-acid residue 20-138 among the SEQ ID NO:66, and described variable region of light chain contains the amino-acid residue 21-127 among the SEQ ID NO:2.

11. each GITR binding molecule of claim 3-10, it comprises ethnic group is heavy chain and light chain framework region.

12. the GITR binding molecule of claim 11, one or more people's framework amino acid residue is wherein become corresponding mouse amino-acid residue by reverse mutation.

13. the GITR binding molecule of claim 9, wherein constant region comprises the IgG2b CH.

14. the GITR binding molecule of claim 9, wherein said binding molecule is in conjunction with people GITR.

15. the GITR binding molecule of claim 9, wherein said binding molecule does not bring out apoptosis.

16. the GITR binding molecule of claim 9, wherein said binding molecule is not blocked elementary mixed lymphocyte reacion.

17. the GITR binding molecule of claim 9, wherein said binding molecule are offset the restraining effect of regulatory T cells to T effector cell.

18. the GITR binding molecule of claim 9, wherein said binding molecule promotes T effector cell's propagation.

19. the GITR binding molecule of claim 9, wherein said binding molecule derives from mouse.

20. the GITR binding molecule of claim 19, wherein said binding molecule comprise mouse IgG2a heavy chain.

21. the GITR binding molecule of claim 9, the activity of wherein said binding molecule mediator GITR.

22. the GITR binding molecule of claim 9, wherein said binding molecule weaken the degraded of I-κ B in the T cell.

23. the GITR binding molecule of claim 11, wherein said binding molecule is a chimeric antibody.

24.GITR binding molecule, this molecule are in conjunction with the GITR on human T-cell and the human dendritic cell, and binding constant (Kd) is 1 * 10 ^-9Or it is lower.

25. the GITR binding molecule of claim 24, wherein said binding molecule are offset the restraining effect of regulatory T cells to T effector cell.

26. the GITR binding molecule of claim 24, wherein said binding molecule is a humanized antibody.

27. composition, it comprises claim 1-10 or 13-26 each described GITR binding molecule and pharmaceutically acceptable carrier.

28. the composition of claim 27 also comprises at least a other therapeutical agent that is used for the treatment of experimenter's cancer.

29. the composition of claim 27 also comprises the therapeutical agent of at least a other virus infection that is used for the treatment of the experimenter.

30. the composition of claim 27 also comprises the tumour antigen of at least a treatment experimenter cancer.

31. the composition of claim 27 also comprises the pathogen antigen of at least a treatment experimenter's virus infection.

32. inhibiting method of eliminating regulatory T cells to T effector cell, this method comprises people's immunocyte is contacted with claim 1-10 or 13 each described GITR binding molecules, thereby eliminates the restraining effect of regulatory T cells to T effector cell.

33. regulate the method for conducting through TXi Baoshouti inductive signal among the T effector cell for one kind, this method comprises cell is contacted with claim 1-10 or 13 each described GITR binding molecules, thereby makes among the T effector cell adjusted through the conduction of TXi Baoshouti inductive signal.

34. the method for claim 33, the degraded of wherein said I-κ B is regulated.

35. the method for claim 33, wherein said T cell is the Th1 cell.

36. the method for claim 35, wherein said T cell is the CD4+ cell.

37. the method for claim 35, wherein said T cell is the CD8+ cell.

38. the immunoreactive method of enhancing experimenter, this method comprise cell is contacted with claim 1-10 or each described GITR binding molecule of 13-26, thereby experimenter's immune response is strengthened.

39. a method for the treatment of experimenter's cancer, this method comprise cell is contacted with claim 1-10 or each described GITR binding molecule of 13-26, thereby experimenter's cancer is obtained medical treatment.

40. the method for claim 39, the type of wherein said cancer are selected from the cancer of carcinoma of the pancreas, melanoma, mammary cancer, lung cancer, bronchogenic carcinoma, colorectal cancer, prostate cancer, cancer of the stomach, ovarian cancer, Urinary Bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, the esophageal carcinoma, cervical cancer, uterus or carcinoma of endometrium, oral carcinoma or pharynx cancer, liver cancer, kidney, carcinoma of testis, cholangiocarcinoma, small intestine or adnexal carcinoma, salivary-gland carcinoma, thyroid carcinoma, adrenal carcinoma, osteosarcoma, chondrosarcoma and hemopoietic tissue.

41. treat infection or the disorderly method that causes by pathogenic agent in the subject for one kind, this method comprises the described GITR binding molecule of cell and claim 1 is contacted, thereby the disease, disorder or the situation that are caused by pathogen in the subject are obtained medical treatment.

42. the method for claim 41, wherein said pathogenic agent are to be selected from following virus: HIV, herpes virus hominis, cytomegalovirus, rotavirus, Epstein-Barr virus, varicella zoster virus, hepatitis virus (such as hepatitis B virus, hepatitis A virus (HAV), hepatitis C virus and viral hepatitis type E virus), paramyxovirus: respiratory syncytial virus, parainfluenza virus, Measles virus, mumps virus, human papillomavirus, flavivirus and influenza virus.

43. the method for claim 41, wherein said pathogenic agent are to be selected from following bacterium: the Neisseria gonorrhoeae species, the suis species, Streptococcus mutans, the hemophilic bacterium species, the catarrhalis species, the Bordetella species, mycobacterium species, the legionella species, the Escherichia species, the vibrios species, the Yersinia species, crooked fungus kind, the Salmonellas species, the listeria spp species, the Helicobacter pylori species, the pseudomonas species, the staphylococcus species, the faecalis species, the clostridium species, bacillus species, rod bacillus species, the burgdorferi species, ehrlichiosis body species, the Rickettsiae species, the chlamydozoan species, the Leptospira species, the treponema species.

44. a method of regulating the GITR function, this method are included under the situation of stimulant, people GITR is contacted with claim 1-10 or each described GITR binding molecule of 13-26, thereby make the GITR function obtain adjusting.

45. an isolated nucleic acid molecule, it comprises the nucleotide sequence of encoding heavy chain variable region, and described sequence contains the Nucleotide 58-414 among the SEQ ID NO:9.

46. an isolated nucleic acid molecule, it comprises the nucleotide sequence of encoded light chain variable region, and described sequence contains the Nucleotide 61-381 among the SEQ ID NO:10.

47. an isolated nucleic acid molecule, it comprises the nucleotide sequence of at least one CDR that encodes, and described sequence is selected from SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO:65 and SEQ IDNO.13.

48. the isolated nucleic acid molecule of claim 47, it comprises the nucleotide sequence of at least two CDR of coding.

49. the isolated nucleic acid molecule of claim 47, it comprises the nucleotide sequence of three CDR that encode.

50. an isolated nucleic acid molecule, it comprises the nucleotide sequence of at least one CDR that encodes, and described sequence is selected from SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16.

51. the isolated nucleic acid molecule of claim 50, it comprises the nucleotide sequence of at least two CDR of coding.

52. the isolated nucleic acid molecule of claim 50, it comprises the nucleotide sequence of three CDR that encode.

53. an isolated nucleic acid molecule, it comprises the nucleotide sequence shown in the SEQ ID NO:11,12 or 65,13,14,15 and 16.

54. recombinant expression vector comprises each described nucleic acid molecule of claim 45-53.

55. recombinant expression vector, it comprises nucleic acid molecule, and described molecule has the nucleotide sequence of the described binding molecule of coding claim 24.

56. host cell has imported the described recombinant expression vector of claim 55.

57. a method for preparing in conjunction with the GITR binding molecule of people GITR, this method are included in the host cell of cultivating claim 56 in the substratum, until the binding molecule of described cell generation in conjunction with people GITR.

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