Antagonistic anti-tumor necrosis factor receptor superfamily antibodies

Abstract

Antagonistic TNFR superfamily polypeptides, such as antibodies and antigen-binding fragments thereof, and uses of these polypeptides for inhibiting proliferation of regulatory T cells (T-regs) are disclosed. For example, antibodies of the invention include antagonistic TNFR2 antibodies and antigen-binding fragments thereof, and can be used to inhibit T-reg-mediated inactivation of tumor-reactive T lymphocytes, as well as to treat various cancers and infectious diseases.

Description

Technical Field

[0001] This invention relates to polypeptides, such as antibodies and their antigen-binding fragments, capable of antagonizing members of the tumor necrosis factor receptor superfamily, such as tumor necrosis factor receptor 2. The polypeptides of this invention can be used to modulate the activity of regulatory T cells, for example, in the field of immunotherapy for treating cell proliferation disorders and infectious diseases. Background Technology

[0002] The use of naturally occurring and genetically engineered T lymphocytes is a prominent example of their application to improve various human pathologies. For instance, while traditional treatment platforms for cancer include surgical resection of tumor masses, radiotherapy, and the administration of chemotherapy agents (Shewach, Chem. Rev. [Chemical Review], 109:2859-2861, 2009), the last decade has witnessed a resurgence of adoptive immunotherapy in cancer treatment regimens. With the advent of chimeric antigen receptor (CAR-T) therapy, new methods have emerged for infusing patients with autologous and allogeneic tumor-reactive T cells (June, J. Clin. Invest. [Journal of Clinical Research], 117:1466-1476, 2007). CAR-T therapy utilizes the resources of acquired immune responses to promote cytotoxicity in cancer cells and eradicate tumor material. A common theme in adoptive immunotherapy is the use of T cells that exhibit the ability to selectively enhance cytotoxicity in cells displaying different tumor antigens. Examples of this technique include tumor-infiltrating lymphocytes (Dudley et al., J. Immunother. [Journal of Immunotherapy], 26:332-342, 2003) and the administration of autologous or allogeneic T cells that have been genetically reengineered to exhibit responsiveness to tumor-specific antigens (Yee et al., PNAS. [Proceedings of the National Academy of Sciences of the United States of America], 99:16168-16173, 2002).

[0003] Despite the promise of T-lymphocyte-based cancer immunotherapy, the development of this therapeutic platform is hampered by the immune system's natural tendency to suppress the immune attack on its own cells. Like all nucleated human cells, cancer cells express class I major histocompatibility complex (MHC) proteins that distinguish these cells from foreign cells. To prevent cell-on-cell killing, regulatory T cells (T-reg cells) have evolved, which suppress the activity of T cells that react to “self” MHC antigens. T-reg cells represent a heterogeneous type of T cell, distinguishable based on their unique surface protein presentation. The best-understood populations of T-reg cells include CD4+, CD25+, FoxP3+ T-reg cells, and CD17+ T-reg cells. The exact mechanisms by which these cells mediate the suppression of autoreactive T cells are the subject of ongoing research, although it has been shown that certain types of T-reg cells suppress the production of the proliferation-inducing cytokine IL-2 in target T cells and can further isolate IL-2 from autoreactive cells by means of the affinity of CD25 (a subdomain of the IL-2 receptor) for IL-2 (Josefowicz et al., Ann. Rev. Immun. [Annual Review of Immunology], 30:531-564, 2012).

[0004] Although T-reg cells play a crucial role in maintaining peripheral tolerance, the same biochemical characteristics that constitute these cells' ability to regulate the activity of autoreactive T cells also disrupt adoptive immunotherapy and innate immune responses by inhibiting the activity of tumor-reactive T lymphocytes. The development of chemical modulators of T-reg cell activity has been the subject of much pharmacological research, as obtaining agents capable of inhibiting T-reg-mediated T cell suppression could significantly improve the scope and efficacy of adoptive cancer immunotherapy and enhance the immune system's ability to eradicate pathogens causing infectious diseases.

[0005] Tumor necrosis factor receptor (TNFR) isoforms 1 and 2 have been identified as cell fate-determining signaling molecules on the surface of T-reg cells. For example, activation of TNFR1 enhances the caspase signaling cascade and terminates T-reg apoptosis, while activation of TNFR2 induces signal transduction via the mitogen-activated protein kinase (MAPK) signaling pathway, which orchestrates signal transduction through the transcription of genes mediated by TRAF2 / 3 and NFκB, promoting apoptosis escape and cell proliferation. Due to its role in guiding cell survival and growth, TNFR2 represents an attractive target for the immunoassay of blocking tumor-reactive T lymphocytes. Therefore, there is a need for therapies capable of blocking T-reg cell survival and proliferation for the treatment of cell proliferation disorders such as cancer and a wide variety of infectious diseases. Summary of the Invention

[0006] This invention provides antagonistic tumor necrosis factor receptor 2 (TNFR2) antibodies and their antigen-binding fragments, such as those that specifically bind to epitopes containing one or more residues of the KCRPG sequence (SEQ ID NO: 19) in human TNFR2 or equivalent epitopes in TNFR2 of non-human primates (e.g., bison or cattle, as described herein), and those that do not specifically bind to epitopes containing the KCSPG sequence (SEQ ID NO: 12) in human TNFR2 or equivalent epitopes in TNFR2 of non-human primates. Furthermore, this invention is characterized by antibodies and their antigen-binding fragments that inhibit the activity of other TNFR superfamily members, such as antibodies and their antigen-binding fragments that bind these proteins in an antiparallel dimeric configuration. The antibodies and their antigen-binding fragments described herein can be used to treat a variety of conditions, including cancer and infectious diseases.

[0007] This article discloses peptides (such as single-chain peptides, antibodies, or their antigen-binding fragments) capable of specifically binding to human TNFR2, wherein the peptides comprise a complementarity-determining region-heavy chain 1 (CDR-H1), a CDR-H2 derived from a neutral TNFR2 antibody, and a CDR-H3 having the following amino acid sequence: JZ 1 JZ 2 Z 4 JZ 3 JZ 5 (J)2Z 5 Z 2 Z 5 JZ 1 JZ 2 Z 4 Z 3 Z 5 (J)2Z 5 Z 2 Z 5 (J)2, JRJDGJSJY(J)2FDJ(SEQ ID NO:278), JRJDGSY(J)2FD(J)3(SEQ ID NO:279), QZ 1 VZ 2 Z 4 YZ 3 SZ 5 WYZ 5 Z 2 Z 5 (SEQ ID NO:265) or AZ 1 DZ 2 Z 4 Z 3 Z 5 SPZ 5 Z 2 Z5 WG (SEQ ID NO:266), where

[0008] Each J is an naturally occurring amino acid;

[0009] Each Z 1 These are naturally occurring amino acids that contain cationic side chains at physiological pH, such as lysine, arginine, and histidine.

[0010] Each Z 2 These are naturally occurring amino acids that contain anionic side chains at physiological pH, such as aspartic acid and glutamic acid.

[0011] Each Z 3 These are naturally occurring amino acids that, independently, contain polar, uncharged side chains at physiological pH, such as serine, threonine, asparagine, and glutamine.

[0012] Each Z 4 It is either glycine or alanine; and

[0013] Each Z 5 Independently, these are naturally occurring amino acids containing hydrophobic side chains, such as alanine, valine, leucine, isoleucine, proline, methionine, tryptophan, phenylalanine, and tyrosine. As used herein in the context of polypeptide formulas, the numeric characters in the subscripts indicate the amount of the preceding amino acid present in the formula, and the numeric characters in the superscripts indicate the type of the preceding amino acid present in the formula.

[0014] This article discloses polypeptides, such as single-chain polypeptides, antibodies, or antigen-binding fragments thereof, capable of specifically binding to human TNFR2. The polypeptides (e.g., antibodies and their antigen-binding fragments) may contain one or more or all of the following CDRs:

[0015] (a) Has an amino acid sequence Z 4 JZ 3 Z 5 (J)2Z 5 or Z 4 JZ 3 Z 5 (J)2Z 5 J's CDR-H1;

[0016] (b) Has the amino acid sequence (J)3Z 4 Z 3 J or (J)5Z 4 Z 3 J's CDR-H2;

[0017] (c) Possesses the amino acid sequence JZ 1 JZ2 Z 4 JZ 3 JZ 5 (J)2Z 5 Z 2 Z 5 or JZ 1 JZ 2 Z 4 Z 3 Z 5 (J)2Z 5 Z 2 Z 5 (J)2's CDR-H3;

[0018] (d) Has an amino acid sequence (J)9Z 5 Or (J)5Z 5 CDR-L1;

[0019] (e) has the amino acid sequence (J)6Z 3 or (J)2Z 3 CDR-L2; and

[0020] (f) Has an amino acid sequence (J)5Z 5 (J)2Z 3 or (J)3Z 5 (J)4Z 3 CDR-L3;

[0021] Each J is an independently naturally occurring amino acid;

[0022] Each Z 1 These are naturally occurring amino acids that contain cationic side chains at physiological pH, such as lysine, arginine, and histidine.

[0023] Each Z 2 These are naturally occurring amino acids that contain anionic side chains at physiological pH, such as aspartic acid and glutamic acid.

[0024] Each Z 3 These are naturally occurring amino acids that, independently, contain polar, uncharged side chains at physiological pH, such as serine, threonine, asparagine, and glutamine.

[0025] Each Z 4 It is either glycine or alanine; and

[0026] Each Z 5 These are naturally occurring amino acids that contain hydrophobic side chains, such as alanine, valine, leucine, isoleucine, proline, methionine, tryptophan, phenylalanine, and tyrosine.

[0027] Antibodies or their antigen-binding fragments may contain non-natural constant regions (e.g., human constant regions), lack all or part of the Fc domain, lack all or part of the natural Fc domain, or completely lack the Fc domain.

[0028] Furthermore, this article discloses polypeptides (e.g., single-chain polypeptides, antibodies, and their antigen-binding fragments) capable of specifically binding to human TNFR2, wherein the polypeptide (e.g., antibody or its antigen-binding fragment) contains one or more or all of the following CDRs:

[0029] (a) CDR-H1 having the amino acid sequence GJTF(J)2Y (SEQ ID NO:276) or GJTF(J)2YJ (SEQ ID NO:277);

[0030] (b) CDR-H2 with amino acid sequences (J)3GSJ or (J)5GSJ;

[0031] (c) CDR-H3 having the amino acid sequence JRJDGJSJY(J)2FDJ (SEQ ID NO:278) or JRJDGSY(J)2FD(J)3 (SEQ ID NO:279);

[0032] (d) CDR-L1 with amino acid sequences (J)9Y or (J)5Y;

[0033] (e) CDR-L2 having the amino acid sequence (J)6S or (J)2S; and

[0034] (f) CDR-L3 with amino acid sequences (J)5Y(J)2T or (J)3Y(J)4T;

[0035] Each J is an independently naturally occurring amino acid. Antibodies or their antigen-binding fragments may contain non-natural constant regions (e.g., human constant regions), lack all or part of the Fc domain, lack all or part of the natural Fc domain, or completely lack the Fc domain.

[0036] A further feature of this invention is the ability to specifically bind to human TNFR2 peptides (e.g., single-chain peptides, antibodies, and their antigen-binding fragments). The peptide (e.g., an antibody or its antigen-binding fragment) may contain the following CDRs:

[0037] (a) Has an amino acid sequence Z 4 FZ 3 Z 5 SSZ 5 or Z 4 YZ 3 Z 5 TDZ 5 X's CDR-H1;

[0038] (b) Has the amino acid sequence SSGZ 4 Z 3 Y (SEQ ID NO:263) or VDPEYZ 4 Z 3 T(SEQ ID NO:264) CDR-H2;

[0039] (c) Possesses the amino acid sequence QZ 1 VZ 2 Z 4 YZ 3 SZ 5 WYZ 5 Z 2 Z 5 (SEQ ID NO:265) or AZ 1 DZ 2 Z 4 Z 3 Z 5 SPZ 5 Z 2 Z 5 CDR-H3 of WG (SEQ ID NO:266);

[0040] (d) Has the amino acid sequence SASSSVYYMZ 5 (SEQ ID NO:267) or QNINKZ 5 CDR-L1 of (SEQ ID NO:268);

[0041] (e) Has the amino acid sequence STSNLAZ 3 (SEQ ID NO:269), TYZ 3 Or YTZ 3 CDR-L2; and

[0042] (f) Has the amino acid sequence QQRRNZ 5 PYZ 3 (SEQ ID NO:270) or CLQZ 5 VNLXZ 3 CDR-L3 of (SEQ ID NO:271);

[0043] Each Z 1 Amino acids that independently contain cationic side chains at physiological pH;

[0044] Each Z 2 Amino acids that independently contain anionic side chains at physiological pH;

[0045] Each Z 3Amino acids that independently contain polar, uncharged side chains at physiological pH;

[0046] Each Z 4 It can be either glycine or alanine.

[0047] Each Z 5 It is an amino acid that contains a hydrophobic side chain independently;

[0048] Each X is independently a leucine or isoleucine. Antibodies or their antigen-binding fragments may contain non-natural constant regions (e.g., human constant regions), lack all or part of the Fc domain, lack all or part of the natural Fc domain, or completely lack the Fc domain.

[0049] On the other hand, the present invention is characterized by a polypeptide capable of specifically binding to human TNFR2, such as a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof, wherein the polypeptide (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof) contains the following CDR:

[0050] (a) CDR-H1 having the amino acid sequences GFTFSSY (SEQ ID NO:23), GYTFTDYX (SEQ ID NO:257), or an amino acid sequence having up to two amino acid substitutions (e.g., one or two amino acid substitutions, such as conserved amino acid substitutions) relative to these sequences;

[0051] (b) CDR-H2 having the amino acid sequences SSGGSY (SEQ ID NO:24), VDPEYGST (SEQ ID NO:258), or an amino acid sequence having up to two amino acid substitutions (e.g., one or two amino acid substitutions, such as conserved amino acid substitutions) relative to these sequences;

[0052] (c) CDR-H3 having the amino acid sequences QRVDGYSSYWYFDV (SEQ ID NO:25), ARDDGSYSPFDYWG (SEQ ID NO:259), or an amino acid sequence having up to two amino acid substitutions (e.g., one or two amino acid substitutions, such as conserved amino acid substitutions) relative to these sequences;

[0053] (d) CDR-L1 having the amino acid sequences SASSSVYYMY (SEQ ID NO:26), QNINKY (SEQ ID NO:260), or an amino acid sequence having up to two amino acid substitutions (e.g., one or two amino acid substitutions, such as conserved amino acid substitutions) relative to these sequences;

[0054] (e) CDR-L2 having the amino acid sequences STSNLAS (SEQ ID NO:27), TYS, YTS, or an amino acid sequence having up to two amino acid substitutions (e.g., one or two amino acid substitutions, such as conserved amino acid substitutions) relative to SEQ ID NO:27; and

[0055] (f) CDR-L3 having the amino acid sequences QQRRNYPYT (SEQ ID NO:28), CLQYVNLXT (SEQ ID NO:261), or an amino acid sequence having up to two amino acid substitutions (e.g., one or two amino acid substitutions, such as conserved amino acid substitutions) relative to these sequences;

[0056] Each X is independently a leucine or isoleucine. The antibody or its antigen-binding fragment may contain a non-natural constant region (e.g., a human constant region), lack all or part of the Fc domain, lack all or part of the natural Fc domain, or completely lack the Fc domain. In some embodiments, the amino acid substitution is a conserved substitution. In some embodiments, the amino acid substitution is a non-conserved substitution.

[0057] In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof) comprises one or more of the following CDRs:

[0058] (a) CDR-L1 with the amino acid sequence SASSSVYYMY (SEQ ID NO:26);

[0059] (b) CDR-L2 having the amino acid sequence STSNLAS (SEQ ID NO:27); and

[0060] (c) CDR-L3 having the amino acid sequence QQRRNYPYT (SEQ ID NO:28).

[0061] In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof) comprises one or more of the following CDRs:

[0062] (a) CDR-L1 with the amino acid sequence QNINKY (SEQ ID NO:260);

[0063] (b) CDR-L2 with the amino acid sequence TYS or YTS; and

[0064] (c) CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID NO:261).

[0065] In some embodiments, the antibody or its antigen-binding fragment CDR-L2 has the amino acid sequence TYS. In some embodiments, the antibody or its antigen-binding fragment CDR-L2 has the amino acid sequence YTS. In some embodiments, CDR-L3 has the amino acid sequence CLQYVNLLT (SEQ ID NO:272). In some embodiments, CDR-L3 has the amino acid sequence CLQYVNLIT (SEQ ID NO:273).

[0066] In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof) comprises one or more of the following CDRs:

[0067] (a) CDR-H1 with the amino acid sequence GFTFSSY (SEQ ID NO:23);

[0068] (b) CDR-H2 having the amino acid sequence SSGGSY (SEQ ID NO:24); and

[0069] (c) CDR-H3 having the amino acid sequence QRVDGYSSYWYFDV (SEQ ID NO:25).

[0070] In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof) comprises one or more of the following CDRs:

[0071] (a) CDR-H1 having the amino acid sequence GYTFTDYX (SEQ ID NO:257);

[0072] (b) CDR-H2 having the amino acid sequence VDPEYGST (SEQ ID NO:258); and

[0073] (c) CDR-H3 having the amino acid sequence ARDDGSYSPFDYWG (SEQ ID NO:259).

[0074] In some embodiments, CDR-H1 has the amino acid sequence GYTFTDYL (SEQ ID NO:274). In some embodiments, CDR-H1 has the amino acid sequence GYTFTDYI (SEQ ID NO:275).

[0075] In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof) contains:

[0076] (a) CDR-L1 with the amino acid sequence SASSSVYYMY (SEQ ID NO:26);

[0077] (b) CDR-L2 having the amino acid sequence STSNLAS (SEQ ID NO:27); and

[0078] (c) CDR-L3 having the amino acid sequence QQRRNYPYT (SEQ ID NO:28).

[0079] The polypeptide (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof) may comprise:

[0080] (a) CDR-H1 with the amino acid sequence GFTFSSY (SEQ ID NO:23);

[0081] (b) CDR-H2 having the amino acid sequence SSGGSY (SEQ ID NO:24); and

[0082] (c) CDR-H3 having the amino acid sequence QRVDGYSSYWYFDV (SEQ ID NO:25).

[0083] In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof) comprises:

[0084] (a) CDR-L1 with the amino acid sequence QNINKY (SEQ ID NO:260);

[0085] (b) CDR-L2 with the amino acid sequence TYS or YTS; and

[0086] (c) CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID NO:261).

[0087] In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof) contains:

[0088] (a) CDR-H1 having the amino acid sequence GYTFTDYX (SEQ ID NO:257);

[0089] (b) CDR-H2 having the amino acid sequence VDPEYGST (SEQ ID NO:258); and

[0090] (c) CDR-H3 having the amino acid sequence ARDDGSYSPFDYWG (SEQ ID NO:259).

[0091] In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof) includes a frame region containing an amino acid sequence LLIR (SEQ ID NO: 262) that binds to the N-terminus of the CDR-L2 region. In some embodiments, the polypeptide (e.g., an antibody or an antigen-binding fragment thereof) includes a frame region containing an amino acid sequence TLE that binds to the C-terminus of the CDR-L2 region.

[0092] The invention is further characterized by a polypeptide (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof) that specifically binds to TNFR2 and contains a light chain amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO:4 and a non-natural constant region. In some embodiments, the polypeptide (e.g., an antibody, or an antigen-binding fragment thereof) contains a light chain amino acid sequence having at least 90% (e.g., 95%, 97%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO:4. The polypeptide (e.g., an antibody, or an antigen-binding fragment thereof) may additionally contain a heavy chain sequence having at least 85% sequence identity with SEQ ID NO:2. In some embodiments, the polypeptide (e.g., an antibody, or an antigen-binding fragment thereof) may contain a heavy chain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2. For example, the polypeptide (e.g., an antibody, or an antigen-binding fragment thereof) may contain a light chain having at least 85% sequence identity with the amino acid sequence of SEQ ID NO:4 and a heavy chain having at least 85% sequence identity with the amino acid sequence of SEQ ID NO:2. Optionally, the polypeptide (e.g., an antibody or its antigen-binding fragment) may contain a light chain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:4 and a heavy chain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2.

[0093] On the other hand, the present invention provides constructs containing a first polypeptide domain and a second polypeptide domain, each domain containing a single-chain polypeptide of the present invention. The first and second polypeptide domains may be identical. In some embodiments, the first and second polypeptide domains may be different. The first and second polypeptide domains may be linked by a linker (such as a linker containing an amide bond or a disulfide bond). These constructs may lack a mouse Fc domain.

[0094] The embodiments of the present invention are further characterized by the following antibody or antigen-binding fragment thereof, which binds to a peptide comprising the amino acid sequence of any one of SEQ ID NO: 11, 19, 20 and 34-117 at a Kc concentration of less than about 100 nM. DThe antibody or antigen-binding fragment thereof is characterized by specifically binding to a peptide containing amino acids 56-60 (KCSPG) of SEQ ID NO:7, and containing a non-natural constant region (e.g., containing a human constant region), lacking all or part of the Fc domain, lacking all or part of the natural Fc domain, or completely lacking the Fc domain. The invention is further characterized by an antibody or antigen-binding fragment thereof capable of specifically binding to human TNFR2, wherein the antibody or antigen-binding fragment thereof specifically binds to a peptide containing one or more amino acids 142-146 (KCRPG) of SEQ ID NO:7 and does not bind to a peptide containing amino acids 56-60 (KCSPG) of SEQ ID NO:7, and contains a non-natural constant region (e.g., containing a human constant region), lacking all or part of the Fc domain, lacking all or part of the natural Fc domain, or completely lacking the Fc domain. The antibody or antigen-binding fragment thereof may bind to a peptide containing one or more amino acids 142-146 (KCRPG) of SEQ ID NO:7 at a Kc concentration of less than about 10 nM. D Specific binding. Optionally, the antibody or its antigen-binding fragment may specifically bind to the epitope within amino acids 142-149 (KCRPGFGV) of SEQ ID NO:7. Alternatively or additionally, the antibody or its antigen-binding fragment may specifically bind to the epitope within amino acids 137-144 (CAPLRKCR) of SEQ ID NO:7. In some cases, the antibody or its antigen-binding fragment may specifically bind to an epitope containing at least five discontinuous or continuous residues within amino acids 150-190 (RPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI) of SEQ ID NO:7. The antibody or its antigen-binding fragment may optionally bind to the epitope within amino acids 161-169 (CKPCAPGTF) of SEQ ID NO:7. In other cases, the antibody or its antigen-binding fragment may bind to an epitope containing at least five discontinuous or continuous residues within amino acids 75-128 of SEQ ID NO:7 (CDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCAL). Specifically, the antibody or its antigen-binding fragment may bind to one or more epitopes within amino acids 80-86 (DSTYTQL), 91-98 (PECLSCGS), and 116-123 (RICTCRPG) of SEQ ID NO:7.

[0095] In some embodiments, the antibody or its antigen-binding fragment binds to an epitope in amino acids 112-131 (REQNRICTCRPGWYCALSKQ) of SEQ ID NO:7. The antibody or its antigen-binding fragment may also bind to an epitope in amino acids 120-139 (CRPGWYCALSKQEGCRLCAP) of SEQ ID NO:7. Alternatively or additionally, the antibody or its antigen-binding fragment may bind to an epitope in amino acids 128-147 (LSKQEGCRLCAPLRKCRPGF) of SEQ ID NO:7. In some embodiments, the antibody or its antigen-binding fragment binds to an epitope in amino acids 136-155 (LCAPLRKCRPGFGVARPGTE) of SEQ ID NO:7.

[0096] The antibody and antigen-binding fragment of the present invention can inhibit TNFR2 signaling. In some embodiments, the antibody or its antigen-binding fragment reduces or inhibits the expression of one or more genes selected from the group consisting of: CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and cIAP2 / BIRC3. In some embodiments, the antibody or its antigen-binding fragment inhibits NFκB activation. For example, the antagonistic TNFR2 antibody or its antigen-binding fragment of the present invention can reduce or inhibit the expression or post-translational modification (e.g., phosphorylation) of one or more of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, or cIAP2 / BIRC3, for example, by reducing or inhibiting the expression or post-translational modification (e.g., phosphorylation) of one or more of these molecules relative to samples never treated with the antagonistic TNFR2 antibody or its antigen-binding fragment of the present invention by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. Exemplary assays for determining expression levels and phosphorylation status are known in the art and include Western blotting assays for determining protein content and quantitative reverse transcription polymerase chain reaction (RT-PCR) experiments for determining mRNA content. In a preferred embodiment, the anti-TNFR2 peptide (e.g., a single-chain peptide, an antibody, or an antigen-binding fragment thereof) is a dominant TNFR2 antagonist and therefore can inhibit TNFR2 activation even in the presence of TNFR2 agonists (such as TNFα) or growth promoters (such as IL-2).

[0097] The antagonistic TNFR2 peptide of the present invention (e.g., an antibody or its antigen-binding fragment) can be expressed at a Kc of no more than about 10 nM and no more than 1 nM. D Or, in specific cases, approximately 621 pM of K D Binding to TNFR2. For example, the antagonistic TNFR2 peptide of the present invention (e.g., an antibody or its antigen-binding fragment) can bind to TNFR2 or its epitopes as described herein, K D For speeds ranging from approximately 1 pM to approximately 900 pM (e.g., approximately 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 110 pM, 120 pM, 130 pM, 140 pM, 150 pM). pM, 160pM, 170pM, 180pM, 190pM, or 200pM, 300pM, 310pM, 320pM, 330pM, 340pM, 350 pM, 360pM, 370pM, 380pM, 390pM, 400pM, 410pM, 420pM, 430pM, 440pM, 450pM, 460p M, 470pM, 480pM, 490pM, 500pM, 510pM, 520pM, 530pM, 540pM, 550pM, 560pM, 570p M, 580pM, 590pM, 600pM, 610pM, 620pM, 630pM, 640pM, 650pM, 660pM, 670pM, 680pM 690pM, 700pM, 710pM, 720pM, 730pM, 740pM, 750pM, 760pM, 770pM, 780pM, 790pM, 800pM, 810pM, 820pM, 830pM, 840pM, 850pM, 860pM, 870pM, 880pM, 890pM, or 900pM). In some embodiments, the polypeptide (e.g., an antibody or its antigen-binding fragment) is at a Kc of about 44pM (e.g., 44.4pM). D Binding to TNFR2. In some cases, the TNFR2 antibody or its antigen-binding fragment of the present invention can bind to TNFR2 to at least about 10 4 M -1 s -1 k on (And in some embodiments, approximately 3.6x10 5 M -1 s -1 k on ( ) to form antibody-antigen complexes. For example, the antagonistic TNFR2 antibody of the present invention or its antigen-binding fragment can bind TNFR2, thereby achieving a concentration from about 1 x 10⁻⁶.4 M -1 s -1 Approximately 1x107M -1 s -1 k on (For example, approximately 1x10) 4 M -1 s -1 5x10 4 M -1 s -1 1x10 5 M -1 s -1 5x10 5 M -1 s -1 1x10 6 M -1 s -1 5x10 6 M -1 s -1 Or 1x10 7 M -1 s -1 This forms an antibody-antigen complex. In some embodiments, the antibody or its antigen-binding fragment can bind to TNFR2, thereby achieving a concentration of approximately 5 x 10⁻⁶. 6 M -1 s -1 (For example, 4.98x10) 6 M -1 s -1 ) of k on Antibody-antigen complexes are formed. The antibodies or antigen-binding fragments of the present invention can bind to TNFR2 to form antibody-antigen complexes, wherein the complexes contain no more than about 10... -3 s -1 k off Dissociation. In some embodiments, the complex dissociates at a concentration of no more than about 10. -4 s -1 k off (For example, approximately 3.0 x 10 -5 s -1 k off Dissociation. For example, the antagonistic TNFR2 antibody of the present invention or its antigen-binding fragment can bind to TNFR2 to form an antibody-antigen complex, wherein the complex is in the form of a fragment of about 1 x 10⁻⁶. -5 s -1 To approximately 1x10 -3 s -1 (For example, approximately 1x10) -5 s -1 5x10 -5 s -1 1x10 -4s -1 5x10 -4 s -1 Or 1x10 -3 s -1 ) of k off Dissociation. In some embodiments, the antibody or its antigen-binding fragment binds to TNFR2 to form an antibody-antigen complex, wherein the complex is no larger than about 2.3 x 10⁻⁶. -4 s - (For example, approximately 2x10) -4 s - ) of k off Dissociation. In some embodiments, the complex is approximately 2.21 x 10⁻⁶. -4 s - k off Dissociation.

[0098] The antagonistic TNFR2 peptides of the present invention (e.g., antibodies and their antigen-binding fragments) may be able to reduce or inhibit the proliferation of T-reg cell populations, and may optionally be present in the presence of a TNFR2 agonist (such as TNFα). In some embodiments, the antibody or its antigen-binding fragment is able to reduce or inhibit the proliferation of a TNFR2-expressing cancer cell population, and may optionally be present in the presence of a TNFR2 agonist (such as TNFα). For example, cancer cells may be Hodgkin lymphoma cells, cutaneous non-Hodgkin lymphoma cells, T-cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, or renal cell carcinoma cells. In some embodiments, the antibody or its antigen-binding fragment is able to reduce or inhibit the proliferation of a myeloid-derived suppressor cell population, and may optionally be present in the presence of a TNFR2 agonist (such as TNFα). In some embodiments, the antibody or its antigen-binding fragment is able to selectively reduce or inhibit the expression of CD25. 高 (CD25 Hi T-reg cell populations (such as those expressing CD25) 高 and CD45RA 低 (CD45RA Low The proliferation of T-reg cells (of which the antibody or its antigen-binding fragment can reduce the expression of CD25). 高 and CD45RA 低 The proliferation of T-reg cell populations (i.e., activated T-reg cell populations, or aT-reg cells) increased by, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, relative to the absence of CD25 expression. 高and CD45RA 低 T-reg cell populations expressing proteins, such as CD25 中 (CD25 Med ) and CD45RA 高 This refers to the T-reg cell population of the protein (i.e., the resting T-reg cell population, or rT-reg cells).

[0099] The antagonistic TNFR2 peptide of the present invention (e.g., an antibody or its antigen-binding fragment) can additionally bind to TNFR2 on the surface of cancer cells (such as tumor cells). Binding of TNFR2 to cancer cells can inhibit or reduce cancer cell proliferation or promote cancer cell apoptosis.

[0100] The antagonistic TNFR2 peptides of the present invention (such as single-chain peptides, antibodies, or antigen-binding fragments thereof) can also bind to myeloid-derived suppressor cells (MDSCs; for example, cells expressing all or subgroups of proteins and small molecules selected from the group consisting of: B7-1 (CD80), B7-H1 (PD-L1), CCR2, CD1d, CD1d1, CD2, CD31 (PECAM-1), CD43, CD44, complement component C5aR1, F4 / 80 (EMR1), FcγRIII (CD16), FcγRII (CD32), FcγRIIA (CD32a), FcγRIIB (CD32b), FcγRIIB / C (CD32b / c), FcγRIIC (CD32c), and FcγRIII. TNFR2 on the surface of A (CD16A), FcγRIIIB (CD16b), galactagogue-3, GP130, Gr-1 (Ly-6G), ICAM-1 (CD54), IL-1RI, IL-4Rα, IL-6Rα, integrin α4 (CD49d), integrin αL (CD11a), integrin αM (CD11b), M-CSFR, MGL1 (CD301a), MGL1 / 2 (CD301a / b), MGL2 (CD301b), nitric oxide, PSGL-1 (CD162), L-selectin (CD62L), salivary lectin-3 (CD33), transferrin receptor (TfR), VEGFR1 (Flt-1), and VEGFR2 (KDR or Flk-1). Specifically, MDSCs do not express proteins selected from the following group: B7-2 (CD86), B7-H4, CD11c, CD14, CD21, CD23 (FcεRII), CD34, CD35, CD40 (TNFRSF5), CD117 (c-kit), HLA-DR, and Sca-1 (Ly6). TNFR2 binding on MDSCs can inhibit or reduce MDSC proliferation or promote MDSC apoptosis.

[0101] The antagonistic TNFR2 peptides of this invention (such as single-chain peptides, antibodies, or antigen-binding fragments thereof) have been shown to attenuate the proliferation of T-reg cells and / or cancer cells in the presence of TNFR2 agonists (such as TNFα), agonistic TNFR2 antibodies, or growth-promoting molecules (such as IL-2). These antibodies or their antigen-binding fragments can bind to TNFR2 and stabilize the dimer, antiparallel dimer conformation of the receptor and prevent phosphorylation and other post-translational modifications that occur during NFκB signaling.

[0102] The antagonistic TNFR2 peptides of the present invention (such as single-chain peptides, antibodies or antigen-binding fragments thereof) can reduce the total amount of T-regs or cancer cells in a patient (such as a human patient) or sample (e.g., from a patient (such as a human patient) undergoing treatment for cancer or infectious disease as described herein, relative to a sample isolated from a patient who has never received such treatment, or relative to a sample isolated from a patient prior to receiving such treatment).

[0103] In some embodiments, the antagonistic TNFR2 peptide (e.g., a single-chain peptide, an antibody, or an antigen-binding fragment thereof) reduces TNFR2 expression, for example, by T-reg cells or cancer cells (such as Hodgkin lymphoma cells or cutaneous non-Hodgkin lymphoma cells, T-cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, or renal cell carcinoma cells).

[0104] The antagonistic TNFR2 peptides of the present invention (such as single-chain peptides, antibodies and their antigen-binding fragments) can inhibit the proliferation of T-reg cell populations or reduce the total number of T-reg cell populations in patients (e.g., human patients) or samples (e.g., samples isolated from human patients who have undergone treatment for cancer or infectious diseases as described herein).

[0105] The invention is further characterized by a method for identifying TNFR2 antagonist peptides (e.g., single-chain peptides, antibodies, or antigen-binding fragments thereof) using the following:

[0106] (a) Exposing a heterogeneous mixture of antibodies or fragments thereof to at least one peptide having an amino acid sequence having any one of SEQ ID NO: 11, 19, 20, and 34-117, or a peptide containing about 10 to about 30 consecutive or discontinuous amino acids between positions 80 and 130 of SEQ ID NO: 7; and

[0107] (b) Retaining antibodies or fragments thereof that specifically bind to the peptide and removing antibodies or fragments thereof that do not specifically bind to the peptide, thereby producing an enriched antibody mixture containing at least one TNFR2 antagonist antibody or its antigen-binding fragment.

[0108] In some embodiments, the amino acid sequence of one or more antibodies or their antigen-binding fragments in the enriched antibody mixture can be determined. Optionally, the polypeptide can bind to a surface, and in some embodiments, the antibody or its antigen-binding fragment can be expressed on the surface of a bacteriophage, bacterial cell, or yeast cell. Alternatively, the antibody or its antigen-binding fragment is expressed as one or more polypeptide chains that are non-covalently bound to ribosomes or covalently bound to mRNA or cDNA. In certain cases, the peptide can be conjugated to a detectable label (such as a fluorescent molecule, epitope tag, or radiolabel). In some cases, the fluorescent molecule can be green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, red fluorescent protein, phycoerythrin, allophycocyanin, Hoescht, 4',6-diamidinyl-2-phenylindole (DAPI), propidium iodide, fluorescein, coumarin, rhodamine, tetramethylrhodamine, or cyanine. In some embodiments, the epitope tag may be maltose-binding protein, glutathione S-transferase, polyhistidine tag, FLAG tag, myc- tag, human influenza hemagglutinin (HA) tag, biotin, or streptavidin. Additionally, in some embodiments, steps (a) and (b) above may be repeated sequentially once or multiple times.

[0109] The invention is further characterized by a method for generating a TNFR2 antagonist antibody or an antigen-binding fragment thereof by immunizing a non-human mammal with a peptide comprising the sequence of any one of SEQ ID NO: 11, 19, 20, and 34-117 or a peptide comprising about 10 to about 30 continuous or discontinuous amino acids between positions 80 and 130 of SEQ ID NO: 7, and collecting serum containing the TNFR2 antagonist antibody or an antigen-binding fragment thereof. Exemplary non-human mammals that can be immunized include rabbits, mice, rats, goats, guinea pigs, hamsters, horses, and sheep. In some embodiments, the peptide for immunization may contain the amino acid sequence KCRPG (SEQ ID NO: 19). In some cases, the peptide for immunization may contain the amino acid sequence CAPLRKCR (SEQ ID NO: 11). Optionally, the peptide may contain the amino acid sequence KCRPGFGV (SEQ ID NO: 20).

[0110] The present invention is characterized by antibodies or antigen-binding fragments thereof produced by any of the methods described above. In some embodiments, the antibody or antigen-binding fragment thereof may be a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a primatized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a multispecific antibody or antigen-binding fragment thereof, a dual variable immunoglobulin domain, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single-chain Fv molecule (scFv), a biantibody, a triantibody, a nanobody, an antibody-like protein scaffold, a domain antibody, an Fv fragment, a Fab fragment, an F(ab')2 molecule, and a tandem scFv (taFv). In some embodiments, the antibody or antigen-binding fragment thereof is an F(ab')2 molecule. In some embodiments, the antibody or antigen-binding fragment thereof has an isotype selected from the group consisting of IgG, IgA, IgM, IgD, and IgE.

[0111] In some embodiments, the antibody or antigen-binding fragment of the present invention may be conjugated with a therapeutic agent such as a cytotoxic agent.

[0112] The invention is characterized by a polynucleotide encoding the antibody or antigen-binding fragment thereof, and a vector containing such polynucleotide. This vector may be an expression vector (e.g., a eukaryotic expression vector) or a viral vector (e.g., adenovirus (Ad, such as serotypes 5, 26, 35, or 48 adenovirus), retrovirus (e.g., gamma retrovirus or lentivirus), poxvirus, adeno-associated virus, baculovirus, herpes simplex virus, or vaccinia virus (e.g., modified vaccinia ankara (MVA)). The invention is also characterized by host cells, such as prokaryotic and eukaryotic cells (e.g., mammalian cells) containing the vector of the invention.

[0113] The invention is further characterized by a method for generating the polypeptide of the invention (e.g., a single-chain polypeptide, construct, antibody, or antigen-binding fragment) by expressing a polynucleotide encoding the single-chain polypeptide, construct, antibody, or antigen-binding fragment in a host cell and recovering the single-chain polypeptide, antibody, or antigen-binding fragment from the host cell culture medium.

[0114] The invention is further characterized by a method for inhibiting a regulatory T cell-mediated immune response by administering the single-chain polypeptide, construct, antibody, or antigen-binding fragment thereof, polynucleotide, carrier, or host cell of the invention to a person in need of treatment, and a method for treating human cell proliferation disorders. Furthermore, the invention is characterized by compositions comprising the single-chain polypeptide, construct, antibody, or antigen-binding fragment thereof, polynucleotide, carrier, or host cell of the invention for inhibiting a regulatory T cell-mediated immune response and for treating human cell proliferation disorders.

[0115] In some embodiments, cell proliferation disorders are cancers such as leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, gastric cardia cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip cancer and oral cancer, eye cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, or pharyngeal cancer. In certain circumstances, cell proliferation disorders can be cancers selected from the following group: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, extrahepatic carcinoma, Ewing sarcoma family, osteosarcoma and malignant fibrous histiocytoma, embryonal tumors of the central nervous system, germ cell tumors of the central nervous system, craniopharyngioma, ependymoma, bronchial tumor, Burkitt lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative vegetations, colon cancer. Cancer, extrahepatic bile duct carcinoma, ductal carcinoma in situ (DCIS), endometrial cancer, ependymoma, esophageal cancer, nasal glioma, extracranial germ cell tumor, gonadal germ cell tumor, fallopian tube cancer, osteofibrous histiocytoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), testicular germ cell tumor, gestational trophoblastic disease, glioma, childhood brainstem glioma, hairy cell leukemia, hepatocellular carcinoma, Langerhans cell histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, islet cell tumor, pancreatic neuroendocrine tumor, nephroblastoma and other childhood kidney tumors, Langerhans cell histiocytosis, small cell lung cancer, cutaneous T-cell lymphoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline carcinoma Tract carcinoma, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell tumor, myelodysplastic syndrome, nasal cavity and sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell ovarian cancer, low-potency ovarian cancer, pancreatic neuroendocrine tumor, papilloma-like hyperplasia, paraganglioma, sinus and nasal cavity carcinoma, parathyroid carcinoma, penile cancer, pharyngeal carcinoma, pheochromocytoma, pituitary adenoma, pleural pulmonary blastoma, primary peritoneal carcinoma, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, Kaposi's sarcoma, rhabdomyosarcoma, Cezare syndrome, small bowel cancer, soft tissue sarcoma, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer. Cancer), uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenström macroglobulinemia ( macroglobulinemia.

[0116] The invention is further characterized by a method of treating Hodgkin's lymphoma or cutaneous non-Hodgkin's lymphoma, T-cell lymphoma, ovarian cancer, colon cancer, multiple myeloma, or renal cell carcinoma by administering to a patient (e.g., a mammalian patient, such as a human patient) an antagonistic TNFR2 polypeptide of the invention (e.g., a single-chain polypeptide, construct, antibody, or antigen-binding fragment thereof), a polynucleotide, a carrier, or a host cell. For example, the invention provides a method of treating ovarian cancer by administering to a patient (e.g., a mammalian patient, such as a human patient) an antagonistic TNFR2 antibody of the invention or an antigen-binding fragment thereof. Additionally, the invention is characterized by compositions containing the single-chain polypeptide, construct, antibody, or antigen-binding fragment thereof, polynucleotide, carrier, or host cell of the invention for treating patients (e.g., human patients) with Hodgkin's lymphoma or cutaneous non-Hodgkin's lymphoma, T-cell lymphoma, ovarian cancer, colon cancer, multiple myeloma, or renal cell carcinoma.

[0117] The invention is further characterized by a method of treating an infectious disease in a patient (e.g., a human patient) by administering to a person requiring treatment a single-chain polypeptide, construct, antibody, or antigen-binding fragment thereof, polynucleotide, vector, or host cell of the invention, and a composition containing the single-chain polypeptide, construct, antibody, or antigen-binding fragment thereof, polynucleotide, vector, or host cell of the invention for treating an infectious disease in a patient (e.g., a human patient). In some embodiments, the infectious disease may be caused by a virus, bacteria, fungus, or parasite. For example,Viral infections treatable according to the method of the present invention include hepatitis C virus, yellow fever virus, Kadam virus, Khosanur forest disease virus, Langat virus, Omsk hemorrhagic fever virus, Bowasan virus, Royal Farm virus, Kashgar virus, tick-borne encephalitis virus, Newdorf virus, Sovgi virus, sheep jumping disease virus, Genshi virus, Miban virus, Somaliz Reef virus, Tyuleni virus, Arroa virus, dengue virus, Kedougu virus, Kasipaccoli virus, Kutango virus, Japanese encephalitis virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Usutu virus, West Nile virus, Yaoundé virus, Cocobera virus, Bagaza virus, Ilios virus, and Israel virus. Turkey meningitis virus, Ntaya virus, Tambusu virus, Zika virus, Banzi virus, Boboi virus, Bianshan virus, Juglar virus, Savoya virus, Sepik virus, Uganda S virus, Wessels Brown virus, Yellow fever virus, Entebbe bat virus, Yokosuka virus, Apoi virus, Bogob Ridge virus, Hutiapa virus, Modoc virus, Salbeho virus, San Palita virus, Bucarasha bat virus, Keiri Island virus, Dakar bat virus, Montana mouse-eared bat encephaloleukitis virus, Phnom Penh bat virus, Rio Bravo virus, Tamana bat virus, Cell fusion agent virus, Epivirus, Lassa virus, Lymphocytic choriomeningitis virus (LCMV) Mobara virus, Mopeya virus, Amapari virus, Flexo virus, Guanarito virus, Junin virus, Latino virus, Machupo virus, Olivas virus, Paraná virus, Pichind virus, Piritau virus, Sabya virus, Tacalibo virus, Temia virus, Whitewater Arroyo virus, Chapare virus, Luyo virus, Hantavirus, Sinopo virus, Dougby virus, Buñoro virus, Rift Valley fever virus, La Crosse virus, California encephalitis virus, Congo hemorrhagic fever (CCHF) virus, Ebola virus, Marburg virus, Venezuelan equine encephalitis virus (VEE), Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE) E), Sindbis virus, rubella virus, Semlikie forest virus, Ross River virus, Bama forest virus, Onai virus and Chikungunya virus, smallpox virus, monkeypox virus, cowpox virus, herpes simplex virus, human herpesvirus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus, Kaposi's tumor-associated herpesvirus (KSHV), influenza virus, severe acute respiratory syndrome (SARS) virus, rabies virus, vesicular stomatitis virus (VSV), human respiratory syncytial virus (RSV), Newcastle disease virus, Hendra virus, Nipah virus, measles virus, rinderpest virus, canine distemper virus, Sendai virus, human parainfluenza virus (e.g.,1, 2, 3, and 4), rhinovirus, mumps virus, poliovirus, human enteroviruses (A, B, C, and D), hepatitis A virus, Coxsackie virus, hepatitis B virus, human papillomavirus, adeno-associated virus, astrovirus, JC virus, BK virus, SV40 virus, norovirus, rotavirus, human immunodeficiency virus (HIV), human T-lymphoblastic viruses type I and II, and infectious spongiform encephalopathy (such as chronic wasting disease).

[0118] In some embodiments, bacterial infections treatable by the method according to the invention include those caused by bacteria belonging to a group selected from the following genera: Salmonella, Streptococcus, Bacillus, Listeria, Corynebacterium, Nocardia, Neisseria, Actinomyces, Moraxella, Enterobacteriacece (e.g., Escherichia coli, such as O157:H7), Pseudomonas, Escherichia, Klebsiella, Serratia, Enterobacter, Proteus, Salmonella, Shigella, Yersinia, Haemophilus, Bordetella, Legionella, Pasteurella, Francisella, Brucella, Bartonella, Clostridium, Vibrio, Campylobacter, and Staphylococcus. In addition, parasitic infections that can be treated according to the method of the present invention include those caused by: dysentery amoeba, rambezia, Cryptosporidium muris, Trypanosoma gambiae, Trypanosoma rhodesiae, Trypanosoma cruzi, Leishmania mexicanum, Leishmania brasiliensis, Leishmania tropicalis, Leishmania donovani, Toxoplasma gondii, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum, Trichomonas vaginalis, and Histomonas meleagridis. Exemplary worm parasites include Trichodina, Ascaris, Pinworm, Hookworm, Necator americanus, Strongyloides stercoralis, Nematoda and Draconis medinana, Schizothorax mansoni, Schizothorax aegyptias, Schizothorax japonicus, Fasciola gigantica, Heterophyes, Paragonimus westermani, pork tapeworm, beef tapeworm, Hymenolepis shortissimus, or Echinococcus granulosus.

[0119] The invention is further characterized by kits containing, for example, the single-chain polypeptides, constructs, antibodies, or antigen-binding fragments of the invention (e.g., antagonist TNFR2 antibody), the polynucleotides of the invention, the vectors of the invention, or the host cells of the invention. In some cases, the kits of the invention may contain instructions for transfecting the vectors of the invention into the host cells of the invention. Optionally, the kits may contain instructions for expressing the single-chain polypeptides, constructs, antibodies, or antigen-binding fragments of the invention in the host cells of the invention (and optionally, reagents that can be used). The kits of the invention may also contain instructions for administering the antibodies or antigen-binding fragments of the invention, the polynucleotides of the invention, the vectors of the invention, or the host cells of the invention to human patients. Optionally, the kits may contain instructions for manufacturing or using the antibodies or antigen-binding fragments of the invention, the polynucleotides of the invention, the vectors of the invention, or the host cells of the invention.

[0120] Additionally, the present invention is characterized by a polypeptide (e.g., a single-chain polypeptide, antibody, or antigen-binding fragment thereof) that specifically binds to a member of the tumor necrosis factor receptor superfamily (TNFRS) in an antiparallel dimeric conformation. This polypeptide (e.g., a single-chain polypeptide, antibody, or antigen-binding fragment thereof) may optionally contain a non-natural constant region. For example, the single-chain polypeptide, antibody, or antigen-binding fragment thereof may inhibit the trimerization of TNFRS members, for example, in the presence of a TNFRS member agonist (such as a homologous TNFRS member ligand). The single-chain polypeptide, antibody, or antigen-binding fragment thereof may inhibit NFκB signaling in cells (e.g., eukaryotic cells, such as mammalian cells, such as human or bovine cells). In some embodiments, the single-chain polypeptide, antibody, or antigen-binding fragment thereof reduces or inhibits the expression of one or more genes selected from the group consisting of: CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and cIAP2 / BIRC3. The TNFRS members can be selected, for example, from the group consisting of: TNFR1, TNFR2, Fas, DCR3, DR3, TRAIL-R1 (DR4), TRAIL-R2 (DR5), TRAIL-R3, TRAIL-R4, DR6, EDAR, CD271, OPG, RANK, LTβR, TWEAK-R, HVEM, CD27, CD30, CD40, CD137, OX40, GITR, BCMA, TACI, BAFFR, EDAR2, TROY, and RELT. For example, the peptides of the present invention (e.g., single-chain peptides, antibodies, or antigen-binding fragments thereof) can bind TNFR2 in an antiparallel dimeric conformation, thereby preventing TNFα-mediated trimerization and activation of this receptor.

[0121] Additionally, the present invention is characterized by a method for treating a disease mediated by signal transduction of TNFRS members in a patient (e.g., a human patient) by administering to a human a single-chain polypeptide, antibody, or antigen-binding fragment thereof that specifically binds to a TNFR2 member in an antiparallel dimeric conformation as described above. Furthermore, the method is characterized by comprising a composition containing a single-chain polypeptide, antibody, or antigen-binding fragment thereof that specifically binds to a TNFRS member in an antiparallel dimeric conformation for treating a disease mediated by signal transduction of TNFRS members in a patient (e.g., a human patient). In some embodiments, the TNFRS member is TNFR1, TNFR2, Fas, DCR3, DR3, TRAIL-R1 (DR4), TRAIL-R2 (DR5), TRAIL-R3, TRAIL-R4, DR6, EDAR, CD271, OPG, RANK, LTβR, TWEAK-R, HVEM, CD27, CD30, CD40, CD137, OX40, GITR, BCMA, TACI, BAFFR, EDAR2, TROY, or RELT. In some embodiments, the TNFRS member is TNFR2. Diseases mediated by signaling via a TNFRS member such as TNFR2 can be cancer. For example, the cancer can be Hodgkin lymphoma, cutaneous non-Hodgkin lymphoma, T-cell lymphoma, ovarian cancer, colon cancer, multiple myeloma, or renal cell carcinoma.

[0122] definition

[0123] As used herein, the term “about” means a value that is no more than 10% above or below the described value. For example, the term “about 5 nM” indicates a range from 4.5 nM to 5.5 nM.

[0124] As used herein, the term “antibody” (Ab) refers to an immunoglobulin molecule that specifically binds to or is immunoreactive to a particular antigen, and includes polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies (including, but not limited to, chimeric antibodies, humanized antibodies, heterologous conjugates (e.g., bispecific, trispecific, and tetraspecific antibodies, biantibodies, triantibodies, and tetraantibodies)) and antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments). Furthermore, unless otherwise stated, the term “monoclonal antibody” (mAb) means comprising the complete molecule capable of specifically binding to a target protein as well as antibody fragments (e.g., Fab and F(ab')2 fragments). Fab and F(ab')2 fragments lack the Fc fragment of the complete antibody (which is cleared more quickly from animal circulation) and may have less nonspecific tissue binding than the complete antibody (see Wahl et al., J. Nucl. Med. [Journal of Nuclear Medicine] 24:316, 1983; which is incorporated herein by reference).

[0125] As used herein, the term "antigen-binding fragment" refers to one or more antibody fragments that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Antibody fragments can be Fab, F(ab')2, scFv, SMIP, biantibodies, triantibodies, affinity antibodies, nanobodies, aptamers, or domain antibodies. Examples of binding fragments encompassing the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fab fragments, a type of antibody composed of V... L V H C L and C H (ii) a monovalent segment composed of a structural domain; (iii) a bivalent segment consisting of two Fab segments connected by disulfide bonds in the hinge region; and (iv) a segment composed of V H and C H (iv) The Fd fragment composed of a single-arm antibody; L and V H The Fv segment, composed of structural domains, contains V. H and V L dAb of the structural domain; (vi) by V H The dAb fragment composed of structural domains (Ward et al., Nature 341:544-546, 1989); (vii) composed of V H or V L The domains consist of dAb; (viii) separate complementary determinant regions (CDRs); and (ix) combinations of two or more separate CDRs, which may optionally be connected by a synthesis joint. Furthermore, although the two domains V of the Fv fragment...L and V H These two domains are encoded by independent genes, but they can be joined using recombination methods via a linker that enables them to be incorporated into the V domain. L and V H Regions pair to form single protein chains (referred to as single-chain Fvs (scFvs); see, for example, Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies. Antigen-binding fragments can be generated by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some embodiments, by chemical peptide synthesis procedures known in the art.

[0126] As used herein, the terms “anti-tumor necrosis factor receptor 2 antibody,” “TNFR2 antibody,” “anti-TNFR2 antibody fraction,” and / or “anti-TNFR2 antibody fragment” include any protein or peptide molecule containing at least a portion of an immunoglobulin molecule capable of specifically binding to TNFR2 (e.g., but not limited to at least one complementarity-determining region (CDR) or ligand-binding portion of the heavy or light chain, a variable region of the heavy or light chain, a constant region of the heavy or light chain, a framework region, or any portion thereof). TNFR2 antibodies also include antibody-like protein scaffolds (such as the tenth fibronectin type III domain). 10 Fn3) contains BC, DE and FG structural rings that are similar to the antibody CDR in structure and solvent accessibility. 10 The tertiary structure of the Fn3 domain is similar to that of the variable region of the IgG heavy chain, and by... 10 The residues of the BC, DE, and FG rings of Fn3 are replaced with residues from the CDR-H1, CDR-H2, or CDR-H3 regions of a TNFR2 monoclonal antibody. Those skilled in the art can graft, for example, the CDR of a TNFR2 monoclonal antibody onto a fibronectin scaffold.

[0127] As used herein, the terms "antagonist TNFR2 antibody" and "antagonistic TNFR2 antibody" refer to TNFR2 antibodies that can inhibit or reduce TNFR2 activation and / or attenuate one or more TNFR2-mediated signal transduction pathways. For example, antagonistic TNFR2 antibodies can inhibit or reduce the growth and proliferation of regulatory T cell populations. Antagonistic TNFR2 antibodies can inhibit or reduce TNFR2 activation by blocking TNFR2 binding to TNFα. In this way, antagonistic TNFR2 antibodies can block TNFR2 trimerization, which would otherwise be induced by interaction with TNFα, leading to inhibition of TNFR2 activity.

[0128] As used herein, the term "bispecific antibody" refers to a monoclonal, typically human or humanized antibody, that has binding specificity against at least two different antigens. In this invention, one binding specificity may be detected against TNFR2, and the other may be detected against any other antigen, such as cell surface proteins, receptors, receptor subunits, tissue-specific antigens, virus-derived proteins, virus-encoded envelope proteins, bacterial-derived proteins, or bacterial surface proteins.

[0129] As used herein, the term "chimeric" antibody refers to an antibody having a variable sequence derived from an immunoglobulin of one source organism (such as rat or mouse) and a constant region derived from immunoglobulins of a different organism (e.g., human). Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715, 4,816,567, and 4,816,397, which are incorporated herein by reference.

[0130] As used herein, the term "complementarity-determining region" (CDR) refers to a hypervariable region found in both the light and heavy chain variable domains. The more conserved portion of the variable domain is called the frame region (FR). As understood in the art, the amino acid positions describing the hypervariable region of an antibody can vary depending on the context and various definitions known in the art. Some positions within a variable domain can be considered heterozygous hypervariable positions because these positions can be considered within a set of criteria but outside of a set of criteria. One or more of these positions can also be found in extended hypervariable regions. The present invention includes antibodies containing modifications at these heterozygous hypervariable positions. The variable domains of the natural heavy and light chains each contain four frame regions predominantly employing a β-sheet configuration, linked by three CDRs that form loops connecting the β-sheet structure and, in some cases, form part of the β-sheet structure. The CDRs in each chain are tightly held together by the FR regions in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and together with CDRs from other antibody chains, contribute to the formation of the antibody's target binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD. 1987; incorporated herein by reference). As used herein, unless otherwise stated, immunoglobulin amino acid residues are numbered according to the immunoglobulin amino acid residue numbering system of Kabat et al.

[0131] As used herein, the terms “conservative mutation,” “conservative substitution,” or “conservative amino acid substitution” refer to the replacement of one or more amino acids with one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric volume. For each of the twenty naturally occurring amino acids listed in Table 1 below, the following properties are summarized.

[0132] Table 1. Representative physicochemical properties of naturally occurring amino acids

[0133]

[0134]

[0135] From this table, it should be understood that conserved amino acid families include (i) G, A, V, L, and I; (ii) D and E; (iii) C, S, and T; (iv) H, K, and R; (v) N and Q; and (vi) F, Y, and W. Therefore, a conserved mutation or substitution is the replacement of an amino acid with a member of the same amino acid family (e.g., Ser replacing Thr or Lys replacing Arg).

[0136] As used herein, the term "conjugate" refers to a compound formed by the chemical bonding of a reactive functional group of one molecule with a suitable reactive functional group of another molecule.

[0137] As used herein, the term "derived antibody" refers to an antibody that has been modified by a chemical reaction to cleave residues or add a non-natural chemical motif to an isolated antibody. Derivatized antibodies can be obtained by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by adding known chemical protecting / blocking groups, proteolytic cleavage, or ligation to cellular ligands or other proteins. Any of these chemical modifications can be performed using established procedures using known techniques, including but not limited to specific chemical cleavage, acetylation, metabolic synthesis of tunicamycin, etc. Furthermore, derivatives can be used, for example, using succinate inhibition techniques to include one or more non-natural amino acids (see, for example, U.S. Patent No. 6,964,859; incorporated herein by reference).

[0138] As used herein, the term "biantibody" refers to a bivalent antibody comprising two polypeptide chains, each of which includes a V-type peptide bonded by a linker. H and V L The structural domain, the linker is too short (e.g., a linker consisting of five amino acids) to allow V H and V L The domains associate intramolecularly on the same peptide chain. This configuration forces each domain to pair with a complementary domain on another peptide chain, thus forming a homodimeric structure. Therefore, the term "triantibody" refers to a trivalent antibody comprising three peptide chains, each containing a VH domain and a VL domain linked by a linker that is extremely short (e.g., a linker consisting of 1-2 amino acids) and does not allow the VH and VL domains to associate intramolecularly on the same peptide chain. In order to fold into its native structure, peptides configured in this way typically trimerize, thereby arranging the VH and VL domains of adjacent peptide chains spatially close to each other to allow for proper folding (see Holliger et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 90:6444-48, 1993; incorporated herein by reference).

[0139] As used herein, a "dominant antagonist" of TNFR2 is an antagonist (e.g., an antagonistic peptide, such as a single-chain peptide, antibody, or its antigen-binding fragment) that inhibits TNFR2 activation even in the presence of a TNFR2 agonist (e.g., TNFα or IL-2). For example, if the IC50 of an antagonist is significantly reduced in the presence of a TNFR2 agonist (e.g., TNFα or IL-2), then the antagonist's IC50 value is significantly reduced. 50 The IC50 of the antagonist, as measured in the same assay in the absence of TNFR2 agonists (such as TNFα or IL-2), is significantly higher than that of the antagonist. 50 An increase of less than 200% (e.g., less than 200%, 100%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less) indicates that the TNFR2 antagonist is a dominant antagonist. Inhibition of TNFR2 activation can be assessed by measuring inhibition of proliferation of TNFR2+ cell populations (e.g., T-reg cells, TNFR2-expressing cancer cells, or myeloid-derived suppressor cells) and by measuring inhibition of NFκB signaling (e.g., by monitoring a decrease in expression of one or more genes selected from the group consisting of: CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and cIAP2 / BIRC3) in a routine gene expression assay. Cell proliferation assays and gene expression assays that can be used to monitor TNFR2 activation are described herein (e.g., in Examples 9 and 12, respectively).

[0140] As used herein, “dual variable domain immunoglobulin” (“DVD-Ig”) refers to the combination of two monoclonal antibodies via a linker to target-binding variable domains to produce a tetravalent dual-targeting single-agent antibody. (Gu et al., Meth. Enzymol. [Enzymatic Methods], 502:25-41, 2012; incorporated herein by reference). Suitable connectors in the light chain of the DVD used in this invention include those identified in Table 2.1 on page 30 of Gu et al.: short K-connectors ADAAP (SEQ ID NO: 118) (mouse) and TVAAP (SEQ ID NO: 119) (human); long K-connectors ADAAPTVSIFP (SEQ ID NO: 120) (mouse) and TVAAPSVFIFPP (SEQ ID NO: 121) (human); short λ-connectors QPKAAP (SEQ ID NO: 122) (human); long λ-connectors QPKAAPSVTLFPP (SEQ ID NO: 123) (human); short-GS connector GGSGG (SEQ ID NO: 124), medium-GS connector GGSGGGGSG (SEQ ID NO: 125), and long-GS connector GGSGGGGSGGGGS (SEQ ID NO: 126) (all GS connectors are mouse or human). Suitable adapters for heavy chains used in DVDs include those identified in Table 2.1 on page 30 of Gu & Ghayur, 2012, Methods in Enzymology 502:25-41 (incorporated hereby by reference): short adapters AKTTAP (SEQ ID NO:127) (mouse) and ASTKGP (SEQ ID NO:128) (human); long adapters AKTTAPSVYPLAP (SEQ ID NO:129) (mouse) and ASTKGPSVFPLAP (SEQ ID NO:130) (human); short-GS adapter GGGGSG (SEQ ID NO:131), medium-GS adapter GGGGSGGGGS (SEQ ID NO:132), and long-GS adapter GGGGSGGGGSGGG (SEQ ID NO:133) (all GS adapters are mouse or human).

[0141] As used herein, the term “endogenous” describes molecules (e.g., polypeptides, nucleic acids, or cofactors) that are naturally found in a particular organism (e.g., humans) or in a particular location within an organism (e.g., organs, tissues, or cells, such as human cells).

[0142] As used herein, the term "exogenous" describes molecules (e.g., polypeptides, nucleic acids, or cofactors) that are not naturally found in a particular organism (e.g., human) or in a particular location within an organism (e.g., organ, tissue, or cell, such as human cell). Exogenous materials include those exogenous materials provided to or extracted from an organism from an external source.

[0143] As used herein, the term "frame region" or "FW region" includes the amino acid residues adjacent to the CDR. FW region residues may be present in, for example, human antibodies, rodent-derived antibodies (e.g., mouse antibodies), humanized antibodies, primate-derived antibodies, chimeric antibodies, antibody fragments (e.g., Fab fragments), single-chain antibody fragments (e.g., scFv fragments), antibody domains, and bispecific antibodies, etc.

[0144] As used herein, the term "fusion protein" refers to a protein covalently linked to another molecule. Fusion proteins can be chemically synthesized, for example, by an amide bond formation reaction between the N-terminus of one protein and the C-terminus of another protein. Alternatively, a fusion protein containing another protein covalently bound to one protein can be recombinantly expressed in cells (e.g., eukaryotic or prokaryotic cells) by, for example, expressing a polynucleotide encoding the fusion protein via a vector or cell genome. Fusion proteins may contain a protein covalently bound to a linker, which in turn covalently binds to another molecule. Examples of linkers that can be used to form fusion proteins include peptide-containing linkers, such as those containing naturally occurring or non-naturally occurring amino acids. In some embodiments, it is desirable to include D-amino acids in the linker because these residues are not present in naturally occurring proteins and are therefore more resistant to degradation by endogenous proteases. The adapter can be prepared using a variety of strategies well known in the art and depends on the reactive components of the adapter. The adapter can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under alkaline conditions, oxidation, disulfide bond reduction, nucleophilic cleavage, or organometallic cleavage (Leriche et al., Bioorg. Med. Chem. [Bioorganic Chemistry and Medicinal Chemistry], 20:571-582, 2012).

[0145] As used herein, the term "heterogenetic specific antibody" refers to a monoclonal antibody, preferably human or humanized, that has binding specificity to at least two different antigens. Traditionally, recombinant production of heterogeneous specific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, wherein the two heavy chains have different specificities (Milstein et al., Nature 305:537, 1983). Similar methods are disclosed, for example, in WO 93 / 08829; U.S. Patent Nos. 6,210,668, 6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453, 6,010,902, 5,989,530, 5,959,084, 5,959,083, 5,932,448, 5,833,985, 5,821,333, 5,807,706, 5,643,759, 5,601,819, 5,582,996, 5,496,549, 4,676,980; WO 91 / 00360; WO 92 / 00373; EP 03089; Traunecker et al., EMBO J. [Journal of the European Society for Molecular Biology] 10:3655 (1991); Suresh et al., Methods in Enzymology 121:210 (1986); cited herein. Heterospecific antibodies may include Fc mutations that enhance proper chain association in multispecific antibodies, as described by Klein et al., mAbs 4(6):653-663,2012; cited herein.

[0146] As used herein, the term "human antibody" refers to each part of a protein that is essentially present in humans (e.g., CDR, framework, C...). L C H Structural domains (e.g., C) H 1. C H 2. C H 3), Hinges, (V) L V HHuman antibodies are essentially non-immunogenic antibodies with only minor sequence changes or variations (e.g., through recombinant expression) or can be produced in human cells from non-human animals or prokaryotic or eukaryotic cells capable of functionally expressing rearranged human immunoglobulin (e.g., heavy chain and / or light chain) genes. Furthermore, when human antibodies are single-chain antibodies, they may include adaptor peptides not found in naturally occurring human antibodies. For example, an Fv may contain adaptor peptides, such as two to about eight glycine or other amino acid residues, that link the variable regions of the heavy chain and the variable regions of the light chain. Such adaptor peptides are considered to be of human origin. Human antibodies can be manufactured using a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT Publications WO 1998 / 46645, WO 1998 / 50433; WO 1998 / 24893; WO 1998 / 16654; WO 1996 / 34096; WO 1996 / 33735; and WO 1991 / 10741; which are incorporated herein by reference. Human antibodies can also be produced using transgenic mice that do not express functional endogenous immunoglobulins but can express human immunoglobulin genes. See, for example, PCT Publications WO 98 / 24893; WO 92 / 01047; WO 96 / 34096; WO 96 / 33735; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598 (incorporated herein by reference).

[0147] As used herein, the term "humanized" antibody refers to a non-human (e.g., mouse) antibody form that chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other target-binding subdomains of the antibody) containing a minimal sequence derived from the non-human immunoglobulin. Typically, a humanized antibody will contain substantially all of at least one and typically two variable domains, wherein all or substantially all of the CDR regions correspond to those in the non-human immunoglobulin. All or substantially all of the FR regions may also be those in the human immunoglobulin sequence. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically a portion of the common sequence of human immunoglobulins. Methods for antibody humanization are known in the art. See, for example, Riechmann et al., Nature 332:323-7, 1988; U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370 to Queen et al.; EP 239400; PCT Publication WO 91 / 09967; U.S. Patent Nos. 5,225,539; EP 592106; and EP 519596; which are incorporated herein by reference.

[0148] As used herein, the term "hydrophobic side chain" refers to an amino acid side chain that exhibits relatively low solubility in water due to, for example, the spatial or electronic properties of the chemical portion present within the side chain. Examples of amino acids containing hydrophobic side chains include those containing unsaturated aliphatic hydrocarbons, such as alanine, valine, leucine, isoleucine, proline, and methionine, as well as amino acids containing aromatic ring systems that are electrostatically neutral at physiological pH, such as tryptophan, phenylalanine, and tyrosine.

[0149] As used herein, the term "monoclonal antibody" refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), rather than the method of producing it.

[0150] As used herein, the term "multispecific antibody" refers to an antibody that exhibits affinity for more than one target antigen. Multispecific antibodies can have a structure similar to that of whole immunoglobulin molecules and include an Fc region, such as the IgG Fc region. Such structures can include, but are not limited to, IgG-Fv, IgG-(scFv)2, DVD-Ig, (scFv)2-(scFv)2-Fc, and (scFv)2-Fc-(scFv)2. In the case of IgG-(scFv)2, the scFv can be linked to the N-terminus or C-terminus of either the heavy or light chain. Kontermann, 2012, mAbs [Monoclonal Antibodies] 4(2):182-197; Yazaki et al., 2013, Protein Engineering, Design & Selection 26(3):187-193; and Grote et al., 2012, Proetzel and Ebersbach (eds.), Antibody Methods and Protocols, Methods in Molecular Biology, Vol. 901, Chapter 16:247-263 (incorporated herein by reference), have reviewed exemplary multispecific molecules containing an Fc region into which anti-TNFR2 antibodies or their antigen-binding fragments can be incorporated. In some embodiments, based on fragments of IgG or DVD or scFv, the antibody fragment may be a component of a multispecific molecule without an Fc region. Exemplary multispecific molecules lacking an Fc region and into which an antibody or antibody fragment can be incorporated include scFv dimers (biantibodies), trimers (triantibodies), and tetramers (tetraantibodies), Fab dimers (conjugated via adhesive peptide or protein domains), and Fab trimers (chemically conjugated); and are described in Hudson and Souriau, 2003, Nature Medicine 9:129-134; which are incorporated herein by reference.

[0151] As used herein, the term "myeloid-derived suppressor cells" or "MDSCs" refers to cells of the immune system that regulate the activity of various effector cells and antigen-presenting cells (such as T cells, NK cells, dendritic cells, and macrophages). Myeloid-derived suppressor cells are distinguished by their gene expression profiles, expressing all or some of the proteins and small molecules selected from the following group: B7-1 (CD80), B7-H1 (PD-L1), CCR2, CD1d, CD1d1, CD2, CD31 (PECAM-1), CD43, CD44, complement component C5aR1, F4 / 80 (EMR1), FcγRIII (CD16), FcγRII (CD32), FcγRIIA (CD32a), FcγRIIB (CD32b), FcγRIIB / C (CD32b / c), FcγRIIC (CD32c), FcγRIIIA (CD16A), FcγR IIIB (CD16b), galactagogue-3, GP130, Gr-1 (Ly-6G), ICAM-1 (CD54), IL-1RI, IL-4Rα, IL-6Rα, integrin α4 (CD49d), integrin αL (CD11a), integrin αM (CD11b), M-CSFR, MGL1 (CD301a), MGL1 / 2 (CD301a / b), MGL2 (CD301b), nitric oxide, PSGL-1 (CD162), L-selectin (CD62L), salivary lectin-3 (CD33), transferrin receptor (TfR), VEGFR1 (Flt-1), and VEGFR2 (KDR or Flk-1). Specifically, MDSCs do not express proteins selected from the following group, which consists of: B7-2 (CD86), B7-H4, CD11c, CD14, CD21, CD23 (FcεRII), CD34, CD35, CD40 (TNFRSF5), CD117 (c-kit), HLA-DR, and Sca-1 (Ly6).

[0152] As used herein, the term "neutral TNFR2 peptide" refers to a peptide (such as a single-chain peptide, antibody, or antibody fragment) that binds to TNFR2 and does not antagonize or agonize TNFR2 activation. For example, a TNFR2 peptide is neutral if it binds to TNFR2 and neither enhances nor inhibits TNFR2 activation, as assessed by measuring the proliferation of a TNFR2-expressing cell population (e.g., T-reg cells, TNFR2+ cancer cells, and / or MDSCs) and / or by measuring the expression of one or more NFκB target genes (e.g., CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and / or cIAP2 / BIRC3). Exemplary assays for measuring cell proliferation and gene expression are described, for example, in Examples 9 and 12, respectively.

[0153] As used herein, the term "non-natural constant region" refers to an antibody constant region derived from a source different from the antibody variable region, or an antibody constant region of an artificially generated synthetic polypeptide having an amino sequence different from the sequence of the natural antibody constant region. For example, an antibody containing a non-natural constant region may have a variable region derived from a non-human source (e.g., mouse, rat, or rabbit) and a constant region derived from a human source (e.g., a human antibody constant region).

[0154] As used herein, the term "percentage (%) sequence identity" refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence after alignment and the introduction of vacancies (if necessary) to achieve maximum percentage sequence identity (e.g., vacancies may be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). Alignment can be performed in various ways as is possible in the art for determining percentage sequence identity, such as using publicly available computer software like BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. For example, a reference sequence used for alignment against a candidate sequence may show sequence identity from 50% to 100% across the full length of the candidate sequence or selected portions of consecutive amino acid (or nucleic acid) residues of the candidate sequence. The length of a candidate sequence for comparison purposes can be at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of the length of a reference sequence. Molecules are identical at that position when a position in a candidate sequence is occupied by the same amino acid residue as the corresponding position in the reference sequence.

[0155] As used herein, the term "primate antibody" refers to an antibody comprising a framework region of an antibody derived from a primate source and other regions (such as CDRs and constant regions) of an antibody derived from a non-primate source. Methods for producing primate antibodies are known in the art. See, for example, U.S. Patent Nos. 5,658,570; 5,681,722; and 5,693,780; which are incorporated herein by reference.

[0156] As used herein, the term “effectively linked” in the context of polynucleotide fragments is intended to refer to two polynucleotide fragments being linked such that the amino acid sequence encoded by the two polynucleotide fragments is preserved within the frame.

[0157] As used in this article, the term "pharmacokinetic characteristics" refers to the absorption, distribution, metabolism, and elimination of a drug over time after it has been administered to a patient.

[0158] As used herein, a “recessive antagonist” of TNFR2 is an antagonist (e.g., an antagonistic peptide, such as a single-chain peptide, antibody, or its antigen-binding fragment) that inhibits TNFR2 activation to a significantly lower degree in the presence of a TNFR2 agonist (such as TNFα or IL-2), relative to the degree of inhibition of the same antagonist as measured in the absence of a TNFR2 agonist (such as TNFα or IL-2). For example, if the IC50 of an antagonist in the presence of a TNFR2 agonist (e.g., TNFα) or IL-2 is significantly lower... 50 The IC50 of the antagonist, as measured in the same assay in the absence of TNFR2 agonists (such as TNFα or IL-2), is significantly higher than that of the antagonist. 50 Increases of, for example, by 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more, indicate that the TNFR2 antagonist is a latent antagonist. Inhibition of TNFR2 activation can be assessed by measuring inhibition of proliferation of TNFR2+ cell populations (such as T-reg cells, TNFR2-expressing cancer cells, or myeloid-derived suppressor cells) and by measuring inhibition of NFκB signaling (e.g., by monitoring a decrease in expression of one or more genes selected from the group consisting of: CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and cIAP2 / BIRC3). Cell proliferation assays and gene expression assays that can be used to monitor TNFR2 activation are described herein (e.g., in Examples 9 and 12, respectively).

[0159] As used herein, the term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, CA, 1990); which is incorporated herein by reference.

[0160] As used herein, the term "scFv" refers to a single-chain Fv antibody in which variable domains from the heavy and light chains of the antibody have been linked together to form a single chain. An scFv fragment contains a single polypeptide chain comprising variable regions (VL) of the antibody light chain (e.g., CDR-L1, CDR-L2, and / or CDR-L3) and variable regions (VH) of the antibody heavy chain (e.g., CDR-H1, CDR-H2, and / or CDR-H3), separated by linkers. The linkers connecting the VL and VH regions of the scFv fragment can be peptide linkers composed of proteogenic amino acids. Alternative linkers can be used to increase the resistance of scFv fragments to proteolytic degradation (e.g., linkers containing D-amino acids), to enhance the solubility of scFv fragments (e.g., hydrophilic linkers, such as linkers containing polyethylene glycol or peptides containing repeating glycine and serine residues), to improve the biophysical stability of molecules (e.g., linkers containing cysteine ​​residues that form intramolecular or intermolecular disulfide bonds), or to reduce the immunogenicity of scFv fragments (e.g., linkers containing glycosylation sites). scFv molecules are known in the art and described, for example, in: US Patent 5,892,019; Flo et al., (Gene 77:51, 1989); Bird et al., (Science 242:423, 1988); Pantoliano et al., (Biochemistry 30:10117, 1991); Milenic et al., (Cancer Research 51:6363, 1991); and Takkinen et al., (Protein Engineering 4:837, 1991). The VL and VH domains of the scFv molecule can be derived from one or more antibody molecules. Those skilled in the art will also understand that the variable regions of the scFv molecules of the present invention can be modified so that they differ in amino acid sequence from those of the antibody molecules from which they are derived. For example, in one embodiment, nucleotide or amino acid substitutions resulting in conserved substitutions or alterations at amino acid residues (e.g., in CDR and / or framework residues) can be made. Alternatively or additionally, CDR amino acid residues may be mutated using techniques recognized in the art to optimize antigen binding. scFv fragments are described, for example, in WO2011 / 084714; which is incorporated herein by reference.

[0161] As used herein, the phrase "specific binding" refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules, which are specifically recognized, for example, by an antibody or its antigen-binding fragment. Antibodies or their antigen-binding fragments that specifically bind to an antigen will bind at a Kc concentration of less than 100 nM. DBinding to antigens. For example, antibodies or antigen-binding fragments that specifically bind to antigens will have a K+ concentration up to 100 nM (e.g., between 1 pM and 100 nM). D Binding to antigens. Antibodies or antigen-binding fragments that do not show specific binding to a particular antigen or its epitope will show a K+ greater than 100 nM (e.g., greater than 500 nm, 1 μM, 100 μM, 500 μM, or 1 mM) for that particular antigen or epitope. D Various immunoassays can be used to select antibodies that specifically react with a particular protein or carbohydrate. For example, solid-phase ELISA is routinely used to select antibodies that specifically react with a protein or carbohydrate. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), which describe immunoassays and conditions that can be used to determine specific immunoreactivity.

[0162] As used herein, the terms “subject” and “patient” refer to an organism receiving treatment for a specific disease or condition (such as cancer or an infectious disease) as described herein. Examples of subjects and patients include mammals receiving treatment for diseases or conditions (such as cell proliferation disorders, such as cancer or infectious diseases), such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the Bovidae family (such as domestic cattle, bison, buffalo, elk, and yaks), cattle, sheep, horses, and bison.

[0163] As used herein, the term “transfection” refers to any of a variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipid transfection, calcium phosphate precipitation, DEAE-glucan transfection, etc.

[0164] As used herein, the term "treat" or "treatment" refers to therapeutic treatment aimed at preventing or slowing (reducing) the progression of undesirable physiological changes or disorders, such as cell proliferation disorders (e.g., cancer or infectious diseases). Beneficial or desired clinical outcomes include, but are not limited to, reduction of symptoms, lessening of the severity of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. Those requiring treatment include those who already have the condition or disorder, those who are susceptible to the condition or disorder, and those who intend to prevent the condition or disorder.

[0165] As used herein, the terms “tumor necrosis factor receptor superfamily,” “TNFR superfamily,” or “TNFRS” refer to type I transmembrane proteins (characterized by a common cysteine-rich domain (CRD)) possessing a carboxyl-terminal intracellular domain and an amino-terminal extracellular domain. The TNFR superfamily comprises receptors that mediate cell signaling as a result of binding to one or more ligands within the TNF superfamily. The TNFR superfamily can be divided into two subgroups: receptors containing an intracellular death domain and receptors lacking this domain. The death domain is an 80-amino acid motif that propagates the apoptosis signaling cascade upon receptor activation. Exemplary TNFR superfamily members containing an intracellular death domain include TNFR1, while TNFR2 represents a TNFR superfamily protein lacking this domain. Members of this TNFR superfamily include TNFR1, TNFR2, RANK, CD30, CD40, lymphotoxin β receptor (LT-βR), OX40, Fas receptor, decoy receptor 3 (DCR3), CD27, 4-1BB, death receptor 4 (DR4), death receptor 5 (DR5), decoy receptor 1 (DCR1), decoy receptor 2 (DCR2), osteoprotein, TWEAK receptor, TACI, BAFF receptor, herpesvirus entry mediator, nerve growth factor receptor, B cell maturation antigen, glucocorticoid-induced TNFR, TROY, death receptor 6 (DR6), death receptor 3 (DR3), and ectoderm developmental abnormality protein A2 receptor.

[0166] As used herein, the term "variable region CDR" includes amino acids in a CDR or complementarity-determining region, as identified using sequence- or structure-based methods. As used herein, the term "CDR" or "complementarity-determining region" refers to a discontinuous antigen-binding site found within the variable region of both heavy and light chain polypeptides. These specific regions have been described below: Kabat et al., J. Biol. Chem. 252:6609-6616, 1977; and Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991; Chothia et al. (J. Mol. Biol. 196:901-917, 1987); and MacCallum et al. (J. Mol. Biol. 262:732-745, 1996); wherein the definitions described include overlaps or subgroups of amino acid residues when compared with each other. In some embodiments, the term “CDR” is the CDR defined by Kabat based on sequence comparison.

[0167] As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors, plasmids, RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). Various vectors have been developed for delivering polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are disclosed, for example, in WO 1994 / 11026; incorporated herein by reference. The expression vectors of the present invention contain polynucleotide sequences and additional sequence elements, for example, for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Some vectors that can be used to express antibodies and antibody fragments of the present invention include plasmids containing regulatory sequences (such as promoter and enhancer regions) that guide gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to guide efficient transcription of the genes carried on the expression vector. The expression vector of the present invention may also contain polynucleotides that encode markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding antibiotic resistance (such as ampicillin, chloramphenicol, kanamycin, or norsinotherapy).

[0168] As used herein, the term "VH" refers to the variable region of the immunoglobulin heavy chain (including the heavy chains of Fv, scFv, or Fab) of an antibody. The reference to "VL" refers to the variable region of the immunoglobulin light chain (including the light chains of Fv, scFv, dsFv, or Fab). Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins with the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules lacking target specificity. Natural antibodies and immunoglobulins are typically heterotetrameric glycoproteins of about 150,000 Daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain of a natural antibody has a variable domain (VH) at the amino terminus, followed by multiple constant domains. Each light chain of a natural antibody has a variable domain (VL) at the amino terminus and a constant domain at the carboxyl terminus. Attached Figure Description

[0169] Figure 1A and 1B The DNA and amino acid sequences of the heavy and light chains of the antagonistic TNFR2 antibody TNFRAB1 are shown. Figure 1A The DNA sequence of the heavy chain encoding TNFRAB1 is shown in the top image (top image) and the amino acid sequence of the heavy chain is shown in the bottom image. The amino acid sequences of the three complementarity-determining regions (CDRs) are shown in bold. Figure 1BThe DNA sequence encoding the light chain of TNFRAB1 is shown in the top image (top) and the amino acid sequence of the light chain is shown in the bottom image (bottom). The amino acid sequences of the three CDRs are shown in bold.

[0170] Figure 2A and 2B The amino acid sequence of human TNFR2 (SEQ ID NO:7) is shown. Notably, human TNFR2 is numbered in this paper starting with an N-terminal methionine residue at position 1 and ending with a C-terminal serine residue at position 461 (SEQ ID NO:7). Figure 2A and 2B In the context of the TNFR2 numbering scheme shown, all references to the amino acid positions within TNFR2 are described. Figure 2A The shaded residue KCRPGFGV (SEQ ID NO: 20) defines the epitope to which the antagonistic TNFR2 antibody TNFRAB1 specifically binds. Notably, the ability of TNFRAB1 to selectively bind residues within this region without binding the underlined residue KCSPG (SEQ ID NO: 12) promotes antagonism of TNFR2 signaling. The poor (or lack thereof) affinity of the antibodies of the present invention for residues in or near the underlined residue region is consistent with the antagonistic activity of these antibodies, as binding to epitopes containing underlined residues has been associated with a decrease in inhibitory activity between TNFR2 antibodies. TNFRAB1 also binds to an epitope containing the shaded residue CKPCAPGTF (SEQ ID NO: 21). Although these residues are not contiguous in the primary sequence with the KCRPG motif, they may be spatially close to the three-dimensional tertiary structure of TNFR2 and can be suitably positioned for interaction with the antagonistic TNFR2 antibody of the present invention (see [link to original text]). Figure 4 ). ( Figure 2B The shaded residue CAPLRKRCR (SEQ ID NO:11) defines the epitope to which the antagonistic TNFR2 antibody TNFRAB2 specifically binds. TNFRAB2 may additionally bind to one or more regions containing residues DSTYTQL (SEQ ID NO:8), PECLSCGS (SEQ ID NO:9), and RICTCRPG (SEQ ID NO:10), which may be portions of discontinuous epitopes. Although these residues are not contiguous in the primary sequence, they may be spatially close to the three-dimensional tertiary structure of TNFR2 and can be suitably positioned to interact with the antagonistic TNFR2 antibody of the present invention.

[0171] Figure 3A and 3BThis is a table showing raw data obtained from enzyme-linked immunosorbent assay (ELISA) experiments, performed to determine the affinity of TNFRAB1 and TNFRAB2 for various continuous and discontinuous epitopes within TNFR2 (see Example 1). Raw luminescence values ​​are shown in the fourth column (right) of the table. The peptide sequences shown represent those containing a portion of a conformational epitope within TNFR2, which is associated with TNFRAB1 (…). Figure 3A ) or TNFRAB2 ( Figure 3B Interactions. Amino acid residues with a single numerical code "2" represent cysteine ​​residues chemically protected at the thiol position by an acetaminomethyl (ACM) moiety during peptide synthesis. These residues do not react with electrophilic reagents containing bromomethyl groups and therefore are not crosslinked during the cyclization and bicyclization stages of peptide synthesis. The third column in the table represents the general structure of the peptide scaffold. "CYS.S" indicates a 27-residue peptide, where positions 1-11 and 17-27 represent 11-residue peptides derived from TNFR2 containing cysteine ​​residues that form disulfide bonds in the native protein (based on information available for UniProt entry P20333). The sequence Gly-Gly-Ser-Gly-Gly is incorporated into positions 12-16 of this group of peptides. Native Cys residues that do not form disulfide bonds are protected with an acetaminomethyl (ACM) protecting group and are represented by a single numerical code "2".

[0172] Figure 4 This diagram illustrates the conformational epitopes within TNFR2 that can interact with TNFR2 antagonist antibodies (such as TNFRAB1 and TNFRAB2), as well as the residues that do not interact with TNFR2 antagonist antibodies. The KCSPG motif is shown in magnified form in the upper left corner of the diagram; the KCRPG motif is shown in magnified form on the right side of the diagram. The outer surface of the protein represents the van der Waals surface of TNFR2. Figure 4 X-ray crystal structure of TNFR2 monomer isolated is provided (PDB ID: 3ALQ, Mukai et al., Sci. Signal. [Science Signal], 3:ra83, 2010).

[0173] Figure 5This graph shows the inhibition of T-reg cell growth by TNFRAB1 in vitro. The values ​​represent the fraction of viable T-reg cells relative to untreated control cells retained in the culture after exposure to specific conditions. The bars shown on the far left represent T-reg cells treated with IL-2 (control), TNFα (20 ng / ml), TNFR2 agonist (2.5 μg / ml), TNFRAB1 (2.5 μg / ml), or TNFRAB2 (2.5 μg / ml). The second bar from the left shows the dose-dependent change in T-reg viability when treated with TNFRAB1. The second bar from the right shows the ability of TNFRAB1 to inhibit the growth-promoting activity of TNFα. T-reg cells were treated with constant concentrations of TNFα (20 ng / ml) and different concentrations of TNFRAB1 (from 0.0008 to 25 μg / ml). TNFRAB1 inhibited TNFα-induced proliferation in a concentration-dependent manner, indicating that TNFRAB1 antagonizes TNFR2. The bars shown on the far right illustrate the effect of TNFα on T-reg cell growth. Incubation of T-reg cells with 20 ng / ml TNFα resulted in approximately 130% proliferation relative to untreated cells.

[0174] Figure 6A This is a graph showing the ability of TNFα to induce T-reg cell proliferation in a dose-dependent manner. Figure 6B This figure shows the ability of IL-2 to induce aT-reg cell proliferation in a dose-dependent manner. Freshly isolated human CD4+ cells were incubated with TNFα and IL-2 (200 U / ml) for up to 48 hours, which induced T-reg cell expansion in a dose-dependent manner, and the presence of IL-2 was important for promoting T-reg expansion. Figure 6C This is a graph showing the effects of TNFα and the dominant antagonistic TNFR2 antibodies TNFRAB1 and TNFRAB2 on T-reg cell proliferation. The values ​​on the x-axis represent the percentage change in the amount of T-reg cells relative to the sample treated with IL-2. Figure 6D and 6E This is a graph showing the results of a repeat experiment conducted to determine the effect of TNFRAB2 on T-reg cell proliferation. Figure 6F and 6G This is a graph showing the results of a repeat experiment conducted to determine the effect of TNFRAB1 on T-reg cell proliferation.

[0175] Figure 7AThis is a graph showing the effect of TNFα on the proliferation of CD4+ T-reg cells. The values ​​on the x-axis represent the percentage change in the number of T-reg cells compared to treatment with IL-2. Figure 7B This is a graph showing the ability of TNFRAB1 to primarily inhibit T-reg cell proliferation in the presence and absence of TNFα. Figure 7C This is a graph showing the ability of TNFRAB2 to inhibit T-reg cell proliferation in the presence and absence of TNFα. Figure 7D and 7E This is a graph showing the effect of a dominant TNFR2 antagonist on T-reg cell proliferation in the presence and absence of TNFα.

[0176] Figure 8A This is a graph showing the ability of TNFRAB1 and TNFRAB2 to normally inhibit the proliferation of T-reg cells. Figure 8B This is a graph showing the effects of TNFRAB1 and TNFRAB2 on T-reg cell proliferation relative to the effects induced by IL-2 treatment. Figure 8C This is a graph showing the effects of TNFRAB1 and TNFRAB2 on the total amount of T-reg. Figure 8D This is a graph showing the effects of TNFRAB1 and TNFRAB2 on total T-reg levels relative to the effects induced by IL-2 treatment. Figure 8E This indicates that TNFRAB1 and TNFRAB2 express CD25 (CD25) in a high-affinity state. 高 ) and CD45RA expressed in a low affinity state (CD45RA 低 A diagram showing the effect of activated T-reg cells (aT-reg cells) on T-reg cells. Figure 8F This shows that TNFRAB1 and TNFRAB2 affect the expression of CD45RO 高 and CD25 高 A diagram illustrating the role of the T-reg cell population. Figure 8G This shows that TNFRAB2 affects the expression of CD25. 高 The diagram shows the effect of CD4+ T cells. Figure 8H This indicates that TNFRAB2 affects CD25 expression. 高 The figure shows the effect on T-reg cells. These data indicate that while treatment with TNFRAB1 or TNFRAB2 inhibited the proliferation of activated cells (aT-reg) and resting cells (rT-reg), the antagonistic TNFR2 antibody preferentially inhibited the proliferation of aT-reg cells.

[0177] Figure 9AThis is a series of two-dimensional flow cytometry plots showing the effects of agents guiding T-reg cell growth (e.g., TNFα, IL-2, and TNFR2 agonists) and the TNFR2 antagonist TNFRAB2 on the proliferation of T-reg cells with various phenotypes. Treatment with a TNFR2 antagonist antibody preferentially inhibits CD25 expression. 高 The proliferation of T-reg cells was observed. Incubation with IL-2 (200 U / ml), TNFα (20 ng / ml), or TNFR2 antagonist antibody (12.5 μg / ml) alone for up to 48 hours was shown to increase the proliferation of TNFR2-expressing CD4+ and CD25+ cells. H1 + Cell ratio. Figure 9B This is a graph showing the effect of TNFRAB1 on the secretion of TNFR2, displayed in pg / ml. Figure 9C These are a series of one-dimensional flow cytometry plots showing the effect of TNFRAB2 on CD8+ T cell counts, as measured by carboxyfluorescein (CFSE) labeling.

[0178] Figure 10A This is a graph showing the ability of full-length TNFRAB1 (IgG) and the F(ab')2 fragment of TNFRAB1 to inhibit the proliferation of T-reg cells. Figure 10B This figure shows the ability of full-length TNFRAB2 (IgG) and the F(ab')2 fragment of TNFRAB2 to inhibit T-reg cell proliferation. These data demonstrate that the specific binding of the Fab region of these antagonistic TNFR2 antibodies to TNFR2 may be responsible for regulating T-reg cell growth, rather than the non-specific binding of the Fc region of these antibodies. Incubation of freshly isolated CD4+ cells with IL-2 (200 U / ml) plus either the full-length antibody or the F(ab')2 fragment of TNFRAB1 or TNFRAB2 for up to 48 hours produced similar dose-dependent inhibition of T-reg cells in the presence or absence of TNFα (20 ng / ml). Figure 10C This is a graph showing the results of a dose-response assay, in which T-reg cells were treated with TNFRAB1 in the presence of anti-IgG antibody. Figure 10DThis is a graph showing the results of a dose-response assay, where T-reg cells were treated with TNFRAB2 in the presence of anti-IgG antibodies. The dose-dependent inhibition of T-reg cell growth induced by TNFR2 antagonist antibodies was unaffected by the presence of anti-IgG molecules, indicating that non-specific cross-linking mediated by TNFRAB1 or TNFRAB2 is not responsible for the inhibitory effect of these antibodies on T-reg cell proliferation. Co-incubation of cross-linked antibodies (2.5 μg / ml) with TNFR2 antagonist antibodies (0.02–25 μg / ml) did not affect the ability of the antibodies to inhibit T-reg proliferation in a dose-dependent manner. The data are presented as a single representative example.

[0179] Figure 11A This is an image of a polyacrylamide gel showing the results of an SDS-PAGE analysis performed after TNFRAB1 expression. Figure 11B This is an image of a polyacrylamide gel showing the results of an SDS-PAGE analysis performed after TNFRAB2 expression. Analysis of reduced and non-reduced TNFR2 antagonist antibody (2.5 μg) is shown. Figure 11C This is an image of a polyacrylamide gel showing the results of an SDS-PAGE analysis performed after expression of the F(ab')2 fragment of TNFRAB1. Figure 11D This is an image of a polyacrylamide gel showing the results of an SDS-PAGE analysis performed after expression of the F(ab')2 fragment of TNFRAB2. Analysis of the TNFR2 antagonist antibody is shown before and after digestion during F(ab')2 fragment preparation.

[0180] Figure 12A This is a graph showing the effect of TNFR2 antagonist antibodies on TNFα and lymphotoxin expression. Figure 12B This is a graph showing the effect of TNFR2 antagonist antibody on FoxP3 and CD25 expression. Figure 12C This figure shows the effect of TNFR2 antagonist antibodies on the expression of genes that promote NFκB activation: conserved helix-loop-helix protein kinase (CHUK), nuclear factor of the κ light polypeptide gene enhancer in B-cell inhibitor ε (NFKBIE), nuclear factor of the κ light polypeptide gene enhancer in B-cell inhibitor α (NFKBIA), mitogen-activated protein kinase 11 (MAP3K11), TNFα receptor-associated factor 2 (TRAF2), TNFα receptor-associated factor 3 (TRAF3), transcription factor relB, and baculovirus IAP repeat sequence containing protein 3 (cIAP2 / BIRC3). RNA isolated from fresh CD4+ cells was detected using real-time PCR analysis after 3 hours of co-culturing with IL-2 (50 U / ml) and TNFα (20 ng / ml) or a TNFR2 antagonist (2.5 μg / ml). Figure 12D This is a graph showing the ability of TNFRAB1 to inhibit NFκB activation, as measured using a cell-based ELISA assay. Figure 12E This graph shows the ability of TNFRAB2 to inhibit NFκB activation as measured using a cell-based ELISA assay. Phosphorylated RelA / NFκB p65 was used as a marker of NFκB activity. Treatment of T-reg cells with an antagonistic TNFR2 antibody resulted in attenuated NFκB activation compared to treatment with TNFα. Phosphorylated RelA / NFκB p65 was measured using a cell-based ELISA after incubating fresh CD4+ cells for 10 minutes with IL-2 (200 U / ml) and various concentrations of TNFα (0.2–20 ng / ml) or TNFR2 antagonist antibody (0.02–25 μg / ml). Phosphorylation of RelA / NFκB p65 was induced by TNFα and inhibited by TNFR2 antagonist mAb in a dose-dependent manner.

[0181] Figure 13A This is a table showing the kinetic and thermodynamic parameters of the binding of TNFRAB1 and TNFRAB2 with TNFR2. The association rate constant is expressed in M... -1 s -1 Displayed in units of s, the dissociation rate constant is expressed in seconds. -1 The values ​​are displayed in units, and the balance constant is displayed in units of M. Figure 13B This is a table showing the affinity of TNFRAB1 and TNFRAB2 for various linear peptide sequences within human TNFR2. Relative affinity is represented by a series of "+" symbols, with a higher number of these symbols indicating an increased affinity value. Raw data from ELISA binding experiments are provided in the "Read" column. Figure 13C This table shows the effects of TNFα on the affinity of TNFRAB1 and TNFRAB2 for various linear peptide sequences within human TNFR2. Relative affinity is represented by a series of "+" symbols, with a higher number of these symbols indicating an increased affinity value. Raw data from ELISA binding experiments are provided in the "Read" column.

[0182] Figure 14A and 14B This is a graph showing the results of a repeat experiment conducted to determine the effect of a latent TNFR2 antagonist antibody (latent antagonist TNFR2 antibody A) that mildly inhibits TNFR2 activity on T-reg cell proliferation. Figure 14C and 14DThis is a graph showing the results of repeated experiments conducted to determine the effect of a secondary cryptic TNFR2 antagonist (cryptic antagonist TNFR2 antibody B) that mildly inhibits TNFR2 activity on T-reg cell proliferation. These cryptic antagonist antibodies target the outer region of TNFR2 to prevent TNFR2 trimerization that leads to NFκB activation.

[0183] Figure 15A It is a structural model showing the three-dimensional structure of antiparallel dimers and parallel dimers of human TNFR2. Figure 15B This is a structural model showing the three-dimensional structure of the TNFα-TNFR2 complex. Shaded residues represent amino acids within human TNFR2 bound by TNFRAB1. The protein is represented as a band beneath the van der Waals surface. The data presented clearly demonstrate that for an intact antibody or its F(ab')2 fragment to bind to TNFR2 in an antiparallel conformation, the TNFR2 receptor will bind the antibody or fragment at the specified amino acid motif. The binding sites of the parallel dimers are too close to each other and therefore prevent antibody binding. Furthermore, the trimer TNFα-TNFR2 complex masks the epitopes bound by antagonistic TNFR2 antibodies because these residues are located inside the trimer structure. Figure 15C This is an image showing a known and publicly available structural model of the trimer of TNFR2 as a trimer containing TNF trimer. Shaded residues are also shown in this structure, representing amino acids within human TNFR2 bound by the latent antagonist antibodies A and B.

[0184] Figure 16A This is a graph showing the effect of TNFRAB1 on the proliferation of T-reg cells (gray shading) isolated from patients with ovarian cancer and T-reg cells (black shading) isolated from subjects who have never shown ovarian cancer. Figure 16B This is a graph showing the effect of TNFRAB2 on the proliferation of T-reg cells (gray shading) isolated from patients with ovarian cancer and T-reg cells (black shading) isolated from subjects who have never shown ovarian cancer.

[0185] Figure 17A This is a graph showing the effect of TNFRAB1 on the proliferation of isolated T-reg cells from subjects who have never shown ovarian cancer. Figure 17B This is a graph showing the effect of TNFRAB2 on the proliferation of isolated T-reg cells from subjects who have never shown ovarian cancer. Figure 17C This is a graph showing the effect of TNFRAB1 on the proliferation of T-reg cells isolated from subjects exhibiting ovarian cancer. Figure 17D This is a graph showing the effect of TNFRAB2 on the proliferation of T-reg cells isolated from subjects exhibiting ovarian cancer. Figure 17EThis is a graph showing the effect of TNFRAB1 on the proliferation of T-reg cells isolated from subjects who have never shown ovarian cancer (relative to...). Figure 17A (Duplicate datasets). Figure 17F This is a graph showing the effect of TNFRAB2 on the proliferation of T-reg cells isolated from subjects who have never shown ovarian cancer (relative to...). Figure 17B (Duplicate datasets). Figure 17G This is a graph showing the effect of TNFRAB1 on the proliferation of T-reg cells isolated from subjects exhibiting ovarian cancer (relative to...). Figure 17C (Duplicate datasets). Figure 17H This is a graph showing the effect of TNFRAB2 on the proliferation of T-reg cells isolated from subjects exhibiting ovarian cancer (relative to...). Figure 17D (Duplicate datasets).

[0186] Figure 18A This is a structural model of the TNFRS member, human death receptor 3 (DR3), as shown in an antiparallel dimeric conformation. The four cysteine-rich domains of DR3 are represented by Roman numerals I-IV. This structural model is reproduced from Tengchuan, Original Archival Copy of Thesis, Structural Characterization of TNF Receptors and Ligands, Chicago, IL, 2008, which is disclosed herein by reference in its entirety. Figure 18B This is a structural model of TNFR1 in an antiparallel dimeric conformation. In this model, the N-terminus of one TNFR1 monomer is located near the center of another TNFR1 monomer. Figure 18C This is a structural model of TNFR1 in an alternative antiparallel dimer conformation. In this model, the N-terminus and C-terminus of the TNFR1 monomer are close to each other. Figure 18B and 18C Retrieved from Naismith et al., Structure 4:1251-1262 (1996), the disclosure of which is incorporated herein by reference in its entirety. Detailed Implementation

[0187] The antagonistic TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) inhibit the activation of TNFR2 on cells expressing TNFR2 by binding to the receptor (e.g., on the outer surface of T-reg cells, TNFR2-expressing cancer cells, or myeloid-derived suppressor cells (MDSCs), and thus prevent the protein from recruiting its homologous ligand TNFα. TNFα enhances TNFR2 signaling by nucleating the trimer of the TNFR2 protein. It is this trimerization event that brings the individual TNFR2 proteins close together and initiates signaling via the MAPK / NFκB / TRAF2 / 3 pathway, ultimately leading to cell growth and escape from apoptosis. TNFR2-binding peptides (e.g., single-chain peptides, antibodies, and antibody fragments) can antagonize this interaction by binding to the receptor and preventing TNFα from inducing this structural change. For example, one possible mechanism is the formation of an antiparallel TNFR2 dimer, which is the inactive structural form of the receptor.

[0188] The peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) can be used to inhibit the activity of other members of the tumor necrosis factor receptor superfamily (TNFRS). For example, the antibodies and antigen-binding fragments of the present invention can bind TNFRS members, such as DR3 or TNFR1, in an antiparallel dimeric conformation. By binding to a TNFRS member in an antiparallel dimeric structure, the antibody or its antigen-binding fragment forms a complex with the TNFRS member, wherein receptor residues that bind homologous ligands (such as TNFα in the case of TNFR2) are chelated within the complex. Therefore, the antibodies or their antigen-binding fragments of the present invention can prevent ligand-mediated trimerization of TNFRS members and thereby prevent activation of TNFRS members by forming complexes with TNFRS members, thus spatially preventing endogenous ligands from entering their homologous binding sites within the receptor. Known exemplary TNFRS members employing an antiparallel dimeric conformation include: TNFR1, TNFR2, Fas, DCR3, DR3, TRAIL-R1 (DR4), TRAIL-R2 (DR5), TRAIL-R3, TRAIL-R4, DR6, EDAR, CD271, OPG, RANK, LTβR, TWEAK-R, HVEM, CD27, CD30, CD40, CD137, OX40, GITR, BCMA, TACI, BAFFR, EDAR2, TROY, and RELT, etc. Therefore, the peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) can be used to bind TNFRS members such as TNFR2 in an antiparallel dimeric conformation to inhibit the activity of TNFRS members in target cells (e.g., T-reg cells, TNFR2+ cancer cells, or myeloid-derived suppressor cells). For example, the antagonistic peptides of the present invention (e.g., single-chain peptides, antibodies, or antigen-binding fragments thereof) can bind DR3 (e.g., as shown in the original text) in an antiparallel dimeric conformation. Figure 18A (as shown) and / or bind TNFR1 in an antiparallel dimeric conformation (e.g., as shown) Figure 18B As shown). Figure 18A and 18B As shown, the antagonistic peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) can bind TNFRS members such as DR3 and / or TNFR1 (e.g., ...) in an antiparallel conformation. Figure 18A and 18B As shown), this makes the homologous ligand binding sites spatially inaccessible and weakens TNFRS member-mediated signal transduction. For example, the antagonistic DR3 antibody or its antigen-binding fragment of the present invention can bind to the DR3 amino acid sequence (gene library). TM (GENBANK TMEpitopes within residues 138-150 of accession number AAQ88676.1. The antagonistic TNFR1 antibody or its antigen-binding fragment of this invention can bind to the TNFR1 amino acid sequence (gene library). TM Epitopes within residues 185-197 of accession number NP_001056.

[0189] The peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) can be dominant antagonists (e.g., dominant TNFR2 antagonistic peptides, such as single-chain peptides, antibodies, or antigen-binding fragments thereof). Dominant TNFR2 antagonistic peptides are those that can bind TNFR2 (e.g., in an antiparallel dimer conformation) and inhibit TNFR2-mediated signal transduction even in the presence of agonists (such as TNFα or IL-2). For example, in the presence of a TNFR2 agonist, dominant antagonistic TNFR2 peptides can inhibit the proliferation of cell populations (such as T-reg cells, TNFR2-expressing cancer cells, or myeloid-derived suppressor cells) by, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, relative to such cell populations not treated with dominant antagonistic TNFR2 peptides. Conversely, recessive antagonistic TNFR2 peptides can bind TNFR2 and inhibit TNFR2-mediated signaling, but the ability of these peptides is weakened in the presence of a TNFR2 agonist. For example, the IC50 of a latent antagonistic TNFR2 peptide, measured in the presence of a TNFR2 agonist (such as TNFα or IL-2), in T-reg cell death assays, TNFR2+ cancer cell death assays, or myeloid-derived suppressor cell death assays. 50 It can be enhanced, for example, by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, relative to the IC50 of the dominant antagonistic TNFR2 peptide. 50 This document describes, for example, in Example 9 below, an example of a cell death assay that can be used to measure the antagonistic effect of TNFR2 antibodies. Therefore, the dominant antagonistic TNFR2 peptides of the present invention (such as single-chain peptides, antibodies, or antigen-binding fragments thereof) can be used to inhibit the proliferation of TNFR2-expressing cells (e.g., T-reg cells, TNFR2+ cancer cells (such as ovarian cancer cells), or myeloid-derived suppressor cells), even in the presence of growth-inducing signals (such as TNFα or IL-2).

[0190] The peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) can be used to attenuate the activity (e.g., proliferation) of T-reg cells, which are typically accompanied by T cell-mediated cytotoxicity against their own cells, such as T lymphocyte attack on tumor cells. Antagonistic TNFR2 peptides can be administered to mammalian subjects (e.g., humans) (e.g., via any of the various routes of administration described herein) to prolong the duration of adaptive immune responses (e.g., responses against cancer cells or pathogenic organisms). In this way, the antagonistic TNFR2 peptides of the present invention can synergistically enhance T lymphocyte-based therapies against cancer and infectious diseases in conjunction with existing techniques. For example, the TNFR2 antagonists of the present invention can be administered to inhibit T-reg cell activity, thereby enhancing the cytotoxic effects of tumor-reactive T cells. TNFR2 antagonists can also synergistically promote the survival of tumor-reactive T cells (e.g., lymphocyte depletion and growth factor therapy) in conjunction with existing strategies, thereby prolonging the duration of antitumor responsiveness in vivo. Antagonistic TNFR2 peptides (e.g., single-chain peptides, antibodies, and antigen-binding fragments) can also be used to treat various infectious diseases in mammalian subjects (e.g., humans) because inhibition of T-reg growth and proliferation promotes the activity of CD8+ T lymphocytes capable of attacking pathogens. Furthermore, the antagonistic TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments thereof) can be used to treat a variety of infectious diseases, such as Mycobacterium tuberculosis, in agricultural farm animals (e.g., bovine mammals, pigs, cattle, horses, sheep, goats, cats, dogs, rabbits, hamsters, guinea pigs, or other non-human mammals).

[0191] Antagonistic TNFRS member peptides

[0192] TNFRS member proteins exhibit conformational properties, exemplified by TNFR2. For example, these receptors are known to adopt an antiparallel dimer conformation and are activated upon trimerization induced by binding to homologous ligands. The amino acid sequences and three-dimensional structures of TNFRS member proteins are known in the art. For instance, structural studies of ligand-free TNFα receptors have revealed that these proteins adopt an antiparallel dimer conformation, as described, for example, in Naismith et al., J. Biol. Chem. [Journal of Biochemistry] 290:13303 (1995), which is disclosed herein by reference in its entirety. Furthermore, human death receptor 3 (DR3) has been shown to adopt an antiparallel dimer conformation (Tengchuan, Original Archival Copy of Thesis, Structural Characterization of TNF Receptors and Ligands, Chicago, IL, 2008 [Original Archival Copy of the Thesis, Structural Characterization of TNF Receptors and Ligands, Chicago, IL, 2008], which is disclosed herein by reference in its entirety). Similarly, the TRAIL receptor is known to form an antiparallel dimer (Shirley et al., Rec. Pat. Anticanc. Drug Disc. [Patent Records and Antitumor Drug Discovery] 6:311 (2011), the disclosure of which is incorporated herein by reference in its entirety). Additionally, TRAF6 has been shown to adopt this structural conformation (Marienfeld et al., Mol. Cell. Biol. [Molecular Cell Biology] 26:9209 (2006); Yin et al., Biochem. [Biochemistry] 48:10558 (2009); and Yin et al., Nat. Struct. Mol. Biol. [Nature Structural and Molecular Biology] 16:658 (2009), the disclosure of each of these references is incorporated herein by reference in its entirety). NGFR has been shown to adopt an antiparallel dimer conformation (Bibel et al., Genes Dev. [Genes and Development] 14:2919 (2000), the disclosure of which is incorporated herein by reference in its entirety). Furthermore, CD40 is known to exhibit this structural motif (Smulski et al., J. Biol. Chem. 288:10914 (2013), the disclosure of which is incorporated herein by reference in its entirety). Additionally, CD137 has been shown to exist in an antiparallel dimeric state (Vinay et al., CD137 Pathway: Immunology and Diseases, New York, NY, 2006, the disclosure of which is incorporated herein by reference in its entirety).In addition, FAS, CD40, OX40, and CD27 have been shown to adopt an antiparallel dimeric conformation (Tartaglia et al., J. Biol. Chem. 267:4304 (1992), the disclosure of which is incorporated herein by reference in its entirety). BAFF-R has also been found to exhibit this structural motif (Kim et al., Nat. Struct. Biol. 10:342 (2003), the disclosure of which is incorporated herein by reference in its entirety).

[0193] Peptides (e.g., single-chain peptides, antibodies, and antigen-binding fragments) that bind to amino acid sequences within TNFRS member proteins that share or are structurally homologous to one or more of the following motifs in human TNFR2: KCRPGFGV (SEQ ID NO:20), CKPCAPGTF (SEQ ID NO:21), CAPLRKCR (SEQ ID NO:11), DSTYTQL (SEQ ID NO:8), PECLSCGS (SEQ ID NO:9), or RICTCRPG (SEQ ID NO:10) can bind to and stabilize TNFRS member proteins in an antiparallel dimer conformation. For example, epitopes within TNFR member proteins that have at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) with one of the aforementioned amino acid sequences in human TNFR2 can be identified using conventional protein sequence alignment techniques known in the art. Antagonistic TNFRS member peptides that bind to one or more of these homologous epitopes can then be developed using various library screening and in vitro display techniques as described herein (e.g., see Example 15 below). Antagonistic TNFRS member peptides (e.g., single-chain peptides, antibodies, and antigen-binding fragments) developed in this manner can bind to TNFRS member proteins in an antiparallel dimer conformation, thereby making the homologous ligand binding site spatially inaccessible and thus preventing receptor trimerization and activation. Specifically, single-chain peptides, antibodies, or their antigen-binding fragments that specifically bind to TNFRS members and stabilize in an antiparallel dimer conformation exhibit binding specificity only to a single TNFRS member and do not cross-react with other TNFRS members.

[0194] Antagonistic TNFR2 peptide

[0195] The anti-TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) are capable of interacting with TNFR2 and inhibiting its activity. Therefore, the anti-TNFR2 antibodies of the present invention can selectively antagonize TNFα-TNFR2 interaction rather than promote TNFR2 signaling. This is particularly important for therapeutic applications such as cancer immunotherapy, because activation following TNFR2 association with TNFα leads to the propagation of the MAPK and TRAF2 / 3 signaling cascades and NF-κB-mediated activation of gene transcription involved in T-reg cell growth and escape from apoptosis (Faustman et al., Nat. Rev. Drug Disc. [Nature Reviews: Drug Discovery], 9:482-493, 2010). The TNFR2 peptides of the present invention can bind TNFR2 with high affinity and can spatially isolate the receptor from TNFα, rather than allowing TNFα to bind to TNFR2 to initiate TNFR2 signaling, for example by binding TNFR2 in an antiparallel dimeric conformation, where the TNFα binding site is spatially inaccessible. Therefore, the antibodies of the present invention can be used to inhibit the growth and proliferation of T-reg cells and can be administered to mammalian subjects (such as human patients with cell proliferation disorders or infectious diseases) to enhance the efficacy of immune responses (e.g., immune responses against cancer cells or pathogenic organisms) in patients.

[0196] The antagonistic TNFR2 single-chain peptides, antibodies, or antigen-binding fragments thereof of the present invention can additionally bind to and inactivate TNFR2 on the surface of cancer cells (such as tumor cells). For example, the antagonistic TNFR2 single-chain peptides, antibodies, and antigen-binding fragments thereof described herein can bind to TNFR2 on the surface of Hodgkin lymphoma cells or cutaneous non-Hodgkin lymphoma cells, T-cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, or renal cell carcinoma cells, etc. The ability of the antagonistic TNFR2 single-chain peptides, antibodies, and antigen-binding fragments thereof of the present invention to directly bind TNFR2 on cancer cells provides another pathway through which these molecules can weaken cancer cell survival and proliferation. For example, the antagonistic TNFR2 single-chain polypeptide, antibody, or antigen-binding fragment thereof of the present invention, such as an antibody or antigen-binding fragment containing one or more heavy chain and / or light chain CDRs of TNFRAB1 or TNFRAB2 (or a CDR having at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) of the heavy chain and / or light chain CDR of TNFRAB1 or TNFRAB2, can directly bind TNFR2 on the surface of cancer cells (e.g., cutaneous T-cell lymphoma cells, ovarian cancer cells, colon cancer cells, or multiple myeloma cells, such as ovarian cancer cells) to reduce cell proliferation.

[0197] The antagonistic TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) can demonstrate the ability to attenuate T-reg and / or cancer cell proliferation even in the presence of TNFR2 agonists (such as TNFα) or agonistic TNFR2 antibodies or growth-promoting molecules (such as IL-2). Not limited by mechanism, the antagonistic TNFR2 single-chain peptides, antibodies, or antigen-binding fragments of the present invention can exhibit properties attributable to the ability of these antibodies or antigen-binding fragments to bind TNFR2 and stabilize the antiparallel dimer conformation of this receptor. This structural configuration does not enhance NFκB signaling. By maintaining TNFR2 in an inactive structural state, the antagonistic TNFR2 single-chain peptides, antibodies, or antigen-binding fragments of the present invention can prevent TNFR2 agonists from restoring cell growth.

[0198] Another property that antagonistic TNFR2 peptides (e.g., single-chain peptides, antibodies, and antigen-binding fragments) can exhibit is the ability to reduce not only the proliferation of T-reg cells, TNFR2+ cancer cells, and / or MDSCs, but also the total amount of T-reg cells, TNFR2+ cancer cells, and / or MDSCs in a sample (e.g., in a patient such as a human patient). The antagonistic TNFR2 single-chain polypeptide, antibody, or antigen-binding fragment thereof of the present invention can reduce the total amount of T-reg cells, cancer cells (such as cutaneous T-cell lymphoma cells, ovarian cancer cells, colon cancer cells, renal cell carcinoma cells, or multiple myeloma cells, etc.), and / or MDSCs in samples treated with the antagonistic TNFR2 polypeptide (such as samples isolated from human patients undergoing treatment for cancer or infectious diseases as described herein) by, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 50%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, relative to samples not treated with the antagonistic TNFR2 antibody or antigen-binding fragment thereof.

[0199] The ability of the antagonistic TNFR2 peptides (e.g., single-chain peptides, antibodies, and antigen-binding fragments) of the present invention to attenuate the growth of T-reg cells and / or cancer cells may be due to the ability of these peptides to reduce the amount of soluble TNFR2 in a sample (e.g., a sample isolated from a human patient undergoing treatment for a cancer or infectious disease as described herein). Soluble TNFR2 can be secreted by, for example, T-reg cells and can interfere with the ability of TNFR2 antagonists to localize TNFR2 on the surface of T-reg cells, TNFR2+ cancer cells, or MDSCs by binding to and chelating these antagonists in the extracellular environment. By reducing TNFR2 secretion, the antagonistic TNFR2 single-chain peptides, antibodies, or antigen-binding fragments thereof of the present invention can make T-reg cells, TNFR2+ cancer cells, and / or MDSCs increasingly susceptible to therapeutic molecules (such as antagonistic TNFR2 antibodies or antigen-binding fragments thereof, and / or other anticancer agents described herein or known in the art that can be used in conjunction with the compositions and methods of the present invention).

[0200] The antagonistic TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) can inhibit the proliferation of or reduce the total number of T-reg cell populations in samples (e.g., samples isolated from human patients undergoing treatment for cancer or infectious diseases as described herein), and can selectively act on T-reg cells in an active dividing state. The antagonistic TNFR2 single-chain peptides, antibodies, or antigen-binding fragments of the present invention can selectively target the expression of CD25. 高 and CD45RA 低 Active T-reg cells, without targeting, for example, cells expressing CD25 中 and CD45RA 高 Resting T-reg cells. For example, the antagonistic TNFR2 peptide of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) can reduce CD25 expression. 高 and CD45RA 低 The proliferation of T-reg cell populations, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, was reduced relative to the absence of CD25 expression. 高 and CD45RA 低 T-reg cell populations expressing proteins, such as CD25 Med and CD45RA 高 This refers to the T-reg cell population of the protein.

[0201] The antagonistic TNFR2 antibody of the present invention can inhibit the growth of, for example, T-reg cells, cancer cells, and / or MDSCs with similar potency to that exhibited by antigen-binding fragments of such antibodies. For example, removing the Fc region of the antagonistic TNFR2 antibody of the present invention may not alter the molecule's ability to attenuate the proliferation of T-reg cells and / or cancer cells or reduce their total number in a sample (e.g., a sample isolated from a human patient who has undergone treatment for cancer or an infectious disease as described herein). The antagonistic TNFR2 antibody of the present invention and its antigen-binding fragment can act through a pathway different from antibody-dependent cytotoxicity (ADCC), where the Fc region is required to recruit effector proteins to induce cell death. Furthermore, the antagonistic TNFR2 antibody or its antigen-binding fragment is less susceptible to loss of inhibitory potency in the presence of a cross-linking agent. Therefore, the antagonistic TNFR2 antibody or its antigen-binding fragment of the present invention can exhibit therapeutic activity in various isotypes (such as IgG, IgA, IgM, IgD or IgE) or in various forms (such as monoclonal antibodies or their antigen-binding fragments, polyclonal antibodies or their antigen-binding fragments, humanized antibodies or their antigen-binding fragments, primate antibodies or their antigen-binding fragments, bispecific antibodies or their antigen-binding fragments, multispecific antibodies or their antigen-binding fragments, dual variable immunoglobulin domains, monovalent antibodies or their antigen-binding fragments, chimeric antibodies or their antigen-binding fragments, single-chain Fv molecules (scFv), biantibodies, triantibodies, nanobodies, antibody-like protein scaffolds, domain antibodies, Fv fragments, Fab fragments, F(ab')2 molecules, and tandem scFv (taFv)).

[0202] Specific binding properties of antagonistic TNFR2 peptides

[0203] The specific binding of the single-chain polypeptide, antibody, or antibody fragment of the present invention to human TNFR2 can be determined by any of a variety of established methods. Affinity can be quantified by various measurements, including the antibody concentration (IC50) required to achieve half-maximal inhibition of TNFα-TNFR2 interaction in vitro. 50 The equilibrium constant (K) for the dissociation of the peptide-TNFR2 complex. D The equilibrium constant K describes the interaction between TNFR2 and the peptide of this invention. D It is the chemical equilibrium constant of the reaction in which the TNFR2-antibody complex dissociates into solvent-separated TNFR2 and non-interacting antibody molecules.

[0204] The polypeptides of the present invention (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) comprise a K+ concentration of less than 100 nM (e.g., 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM). D Those peptides that specifically bind to TNFR2. In some embodiments, the antibodies of the present invention are in doses less than 1 nM (e.g., 990 pM, 980 pM, 970 pM, 960 pM, 950 pM, 940 pM, 930 pM, 920 pM, 910 pM, 900 pM, 890 pM, 880 pM, 870 pM, 860 pM, 850 pM, 840 pM, 830 pM, 820 pM, 810 pM, 800 pM, 790 pM, 780 pM). M, 770pM, 760pM, 750pM, 740pM, 730pM, 720pM, 710pM, 700pM, 690pM, 680pM, 670pM, 660pM, 650 pM, 640pM, 630pM, 620pM, 610pM, 600pM, 590pM, 580pM, 570pM, 560pM, 550pM, 540pM, 530pM, 520 pM, 510pM, 500pM, 490pM, 480pM, 470pM, 460pM, 450pM, 440pM, 430pM, 420pM, 410pM, 400pM, 39 0pM, 380pM, 370pM, 360pM, 350pM, 340pM, 330pM, 320pM, 310pM, 300pM, 290pM, 280pM, 270pM, 26 K (0pM, 250pM, 240pM, 230pM, 220pM, 210pM, 200pM, 190pM, 180pM, 170pM, 160pM, 150pM, 140pM, 130pM, 120pM, 110pM, 100pM, 90pM, 80pM, 70pM, 60pM, 50pM, 40pM, 30pM, 20pM, 10pM, 5pM, or 1pM) D Those antibodies that specifically bind to TNFR2.

[0205] The peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) can also be characterized by a variety of in vitro binding assays. These assays can be used to determine the K-value of TNFR2 single-chain peptides, antibodies, or fragments thereof. D or IC 50Examples of experiments include, for example, surface plasmon resonance, isothermal titration calorimetry, fluorescence anisotropy, and ELISA-based assays. ELISA represents a particularly useful method for analyzing antibody activity because such assays typically require minimal antibody concentrations. A common signal analyzed in typical ELISA assays is luminescence, which is usually a result of the activity of a peroxidase conjugated to a second antibody that specifically binds to a first antibody (e.g., the TNFR2 antibody of the present invention). The peptides of the present invention are capable of binding to TNFR2 and its derived epitopes (such as those containing residues 142-146 (KCRPG) of one or more of SEQ ID NO:7 in human TNFR2, e.g.) Figure 2A and 2B The epitopes (shown) and isolated peptides derived from TNFR2 are structurally pre-organized with various residues in a manner that mimics the conformation of these amino acids in natural proteins. For example, the peptides of the present invention can bind to peptides containing any one of the amino acid sequences of SEQ ID NO: 11, 19, 20, and 34-117, or peptides containing about 10 to about 30 consecutive or discontinuous amino acids between positions 80 and 130 of SEQ ID NO: 7. In a direct ELISA assay, this binding can be quantified, for example by analyzing the luminescence that occurs when an antigen-antibody complex (e.g., 2,2'-azono-di-3-ethylbenzothiazoline sulfonate) is incubated with an HRP-conjugated secondary antibody. For example, when incubated with a surface-immobilized antigen and an HRP-conjugated secondary antibody in the presence of an HRP substrate (see, for example, Example 3), the peptides of the present invention can induce a luminescent response of about 400 absorbance units or more. In some embodiments, the observed emission may be from about 400 to about 900 absorbance units (e.g., 400-900 absorbance units, 500-800 absorbance units, or 600-700 absorbance units). In certain cases, the observed emission may be from about 600 to about 900 absorbance units (e.g., 600-900 absorbance units or 700-800 absorbance units).

[0206] Kinetic properties of antagonistic TNFR2 peptides

[0207] In addition to the thermodynamic parameters of TNFR2-peptide interactions, it is also possible to quantitatively characterize the kinetic association and dissociation of the single-chain peptides, antibodies, or antibody fragments of the present invention with TNFR2. This can be accomplished, for example, by monitoring the rate of antibody-antigen complex formation according to an established procedure. For instance, surface plasmon resonance (SPR) can be used to determine the formation (k) of the antibody-TNFR2 complex. on ) and dissociation (k offThe rate constant of the antibody-TNFR2 complex can also be calculated. These data also allow for the calculation of the equilibrium constant (K0) for the dissociation of the antibody-TNFR2 complex. D ), because the equilibrium constant for the dissociation of this unimolecular molecule can be expressed as k. off With k on The ratio of values. SPR is a technique particularly advantageous for determining the kinetic and thermodynamic parameters of receptor-antibody interactions because the experiment does not require modifying a component by attaching a chemical label. Instead, the receptor is typically immobilized on a solid metal surface pulsed with an increasing concentration of antibody solution. Antibody-receptor binding induces a distortion in the angle of reflection of incident light on the metal surface, and this change in refractive index over time as the antibody is introduced into the system can be fitted to an established regression model to calculate the association and dissociation rate constants of the antibody-receptor interaction.

[0208] The peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) exhibit high kJ / kJ levels when interacting with TNFR2. on and low k off This value is consistent with high-affinity receptor binding. For example, the peptides of the present invention can bind at values ​​greater than 10. 4 M -1 s -1 (For example, 1.0x10) 4 M -1 s -1 1.5x10 4 M -1 s -1 2.0x10 4 M -1 s -1 2.5x10 4 M -1 s -1 3.0x10 4 M -1 s -1 3.5x10 4 M - 1 s -1 4.0x10 4 M -1 s -1 4.5x10 4 M -1 s -1 5.0x10 4 M -1 s -1 5.5x10 4 M -1 s -1 6.0x10 4 M -1 s -1、6.5x10 4 M -1 s -1 、7.0x10 4 M -1 s -1 、7.5x10 4 M -1 s -1 、8.0x10 4 M -1 s -1 、8.5x10 4 M -1 s -1 、9.0x10 4 M -1 s -1 、9.5x10 4 M -1 s -1 、1.0x10 5 M -1 s -1 、1.5x10 5 M -1 s -1 、2.0x10 5 M -1 s -1 、2.5x10 5 M -1 s -1 、3.0x10 5 M -1 s -1 、3.5x10 5 M -1 s -1 、4.0x10 5 M -1 s -1 、4.5x10 5 M -1 s -1 、5.0x10 5 M -1 s -1 、5.5x10 5 M -1 s -1 、6.0x10 5 M -1 s -1 、6.5x10 5 M -1 s -1 、7.0x10 5 M -1 s -1 、7.5x10 5 M -1 s-1 8.0x10 5 M -1 s -1 8.5x10 5 M -1 s -1 9.0x10 5 M -1 s -1 9.5x10 5 M -1 s -1 Or 1.0x10 6 M -1 s -1 In the presence of TNFR2, it exhibits k on Value. When bound to TNFR2, the peptide of the present invention exhibits a low kJ value. off The value is due to the antibody's ability to interact with different TNFR2 epitopes with high affinity. Residues within these epitopes form strong intermolecular contacts with TNFR2, which helps to slow the dissociation of the peptide-TNFR2 complex. This high receptor affinity manifests as a low kJ / kJ value. off Value. For example, when conjugated with TNFR2, the antibody of the present invention can exhibit a value of less than 10. -3 s -1 (For example, 1.0x10) -3 s -1 9.5x10 -4 s -1 9.0x10 -4 s -1 8.5x10 -4 s -1 8.0x10 -4 s -1 7.5x10 -4 s -1 7.0x10 -4 s -1 6.5x10 -4 s -1 6.0x10 -4 s -1 5.5x10 -4 s -1 5.0x10 -4 s -1 4.5x10 -4 s -1 4.0x10 -4 s -1 3.5x10 -4 s -1 3.0x10 -4 s -1 2.5x10-4 s -1 2.0x10 -4 s -1 1.5x10 -4 s -1 1.0x10 -4 s -1 9.5x10 -5 s -1 9.0x10 -5 s -1 8.5x10 -5 s -1 8.0x10 -5 s -1 7.5x10 -5 s -1 7.0x10 -5 s -1 6.5x10 -5 s -1 6.0x10 -5 s -1 5.5x10 -5 s -1 5.0x10 -5 s -1 4.5x10 -5 s -1 4.0x10 -5 s -1 3.5x10 -5 s -1 3.0x10 -5 s -1 2.5x10 -5 s -1 2.0x10 -5 s -1 1.5x10 -5 s -1 Or 1.0x10 -5 s -1 ) of k off value.

[0209] Epitopes within TNFR2 bound by antagonistic TNFR2 peptides

[0210] The high affinity of the peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) for TNFR2, accompanying the rapid initiation of peptide-TNFR2 complex formation and the slow dissociation of these complexes, makes these peptides highly suitable for therapeutic applications as inhibitors of T-reg cell growth and proliferation. For example, high-kJ... onThe values ​​indicate that the peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) can localize to the surface of cells expressing TNFR2 (e.g., T-reg cells) and rapidly associate with TNFR2, thereby preventing receptor activation that might otherwise be induced by TNFα (e.g., by inhibiting TNFα-induced trimerization of TNFR2). Furthermore, the slow dissociation of the peptide-TNFR2 complex indicates a long half-life of the complex in vivo, leading to a stable and sustained downregulation of the growth of TNFR2-expressing cells (e.g., sustained downregulation of T-reg growth). These ideal thermodynamic and kinetic parameters of TNFR2 binding are consistent with the strong intermolecular contact established when the peptides of the present invention associate with TNFR2.

[0211] One of the difficulties in developing anti-TNFR2 peptides (e.g., single-chain peptides, antibodies, and antigen-binding fragments) capable of antagonizing TNFR2 has been elucidated by epitopes within TNFR2 that participate in the formation of antagonistic complexes rather than those that promote signal transduction. Various discrete peptide fragments found within the primary structure of TNFR2 bind to the antagonistic antibodies of the present invention by virtue of the spatial orientation of these residues in the native conformation of the receptor. Notably, many isolated linear TNFR2-derived peptides appear not to interact with antagonistic TNFR2 peptides (e.g., single-chain peptides, antibodies, and antibody fragments) due to the different conformations exhibited by these peptides when structurally pre-organized in the full-length protein and when isolated in solution, making these residues difficult to identify. Epitope mapping analysis using restricted cyclic and bicyclic peptides derived from different regions of TNFR2 shows that the antagonistic TNFR2 antibodies of the present invention bind epitopes from different regions of the TNFR2 amino acid sequence in a conformation-dependent manner. Particularly important epitopes that bind to the antagonistic TNFR2 peptide of the present invention and promote receptor antagonism are those containing one or more residues of the KCRPG motif (SEQ ID NO: 19) located at positions 142-146 of SEQ ID NO: 7 within human TNFR2. One or more of these residues belong to a larger epitope (e.g., residues 142-149 of SEQ ID NO: 7, shown in...). Figure 2A In, residues 137-144 of SEQ ID NO:7 are shown in Figure 2B(The text refers to a specific type of peptide, which can interact with the antagonistic TNFR2 antibody of the present invention.) Knowledge of those residues that selectively bind to antagonistic TNFR2 peptides can be used to identify and design a wide range of antagonistic TNFR2 antibodies and their antigen-binding fragments using library screening techniques (e.g., those described herein or known in the art). For example, structurally rigidified peptides (e.g., peptides having sequences of SEQ ID NO: 42, 50, 52-54, and 61-63) containing one or more residues within the KCRPG sequence can be used to screen and select peptides (e.g., single-chain peptides, antibodies, and antibody-like scaffolds) that bind to these epitopes with high affinity and selectivity.

[0212] Several different residues within TNFR2 bind to antagonistic TNFR2 peptides (such as the single-chain peptides, antibodies, and antibody fragments of the present invention) and establish strong intermolecular contacts with these antibodies. Notably, the functional antagonistic TNFR2 peptide of the present invention selectively binds to an epitope comprising one or more residues of amino acids 142-146 (KCRPG, SEQ ID NO:19) of SEQ ID NO:7 within human TNFR2. The spatial orientation of this epitope is shown in... Figure 4The antagonistic TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) are capable of selectively binding to TNFR2 epitopes containing one or more of these residues and do not exhibit specific binding to epitopes containing residues 56-60 (KCSPG, SEQ ID NO: 12) of SEQ ID NO: 7 within human TNFR2. For example, the peptides of the present invention (e.g., single-chain peptides, antibodies, and antibody fragments) do not exhibit specific binding to one or all of the epitopes containing residues 48-67 (QTAQMCCSKCSPGQHAKVFC, SEQ ID NO: 18) of SEQ ID NO: 7 within human TNFR2, nor do they exhibit specific binding to epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) of that sequence, nor do they exhibit specific binding to epitopes containing conserved amino acid substitutions relative to that sequence (provided the amino acid sequence KCSPG is present in the epitope). Peptides that have been shown to exhibit the following ability lack inhibitory (antagonistic) activity: binding to epitopes comprising one or more residues of amino acids 142-146 of SEQ ID NO:7 within human TNFR2 and epitopes comprising residues 56-60 of SEQ ID NO:7 within human TNFR2. In this way, the ability of TNFR2 antibodies to distinguish these epitopes and specifically interact with one or more of the epitopes comprising residues 142-146 of SEQ ID NO:7 within human TNFR2, and not to participate in the specific binding to epitopes comprising residues 56-60 of SEQ ID NO:7 within human TNFR2, characterizes the peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) that antagonize TNFR2 signaling.

[0213] An exemplary procedure that can be used to predict the inhibitory activity of the TNFR2 single-chain polypeptide, antibody, or antibody fragment of the present invention is to compare the affinity of the polypeptide for a peptide containing a KCRPG motif (e.g., a linear peptide having the sequence KQEGCRLCAPLRKCRPGFGV, SEQ ID NO:17) with the affinity of the same antibody or antibody fragment for a peptide containing a KCSPG sequence (e.g., a linear peptide having the sequence QTAQMCCSKCSPGQHAKVFC, SEQ ID NO:18). For example, the antagonistic TNFR2 antibody TNFRAB1 specifically binds to a peptide fragment defined by residues 130-149 (KQEGCRLCAPLRKCRPGFGV, SEQ ID NO:17) of SEQ ID NO:7 within human TNFR2 with an affinity 40 times greater than that defined by residues 48-67 (QTAQMCCSKCSPGQHAKVFC, SEQ ID NO:18) of SEQ ID NO:7 within human TNFR2. The antagonistic TNFR2 peptide of the present invention (e.g., single-chain peptide, antibody, and antigen-binding fragment) binds to an epitope containing one or more residues of the KCRPG sequence (SEQ ID NO:19) with an affinity at least 10 times greater than that of the same single-chain peptide, antibody, or antigen-binding fragment to a peptide containing the KCSPG sequence (SEQ ID NO:12) of human TNFR2. For example, the antagonistic TNFR2 peptide of the present invention (e.g., single-chain peptide, antibody, and antigen-binding fragment thereof) binds to one or more residues of the KCRPG sequence (SEQ ID NO:19) containing human TNFR2 with an affinity 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or greater than 1000 times greater than that of the same single-chain peptide, antibody, or antigen-binding fragment to a peptide containing the KCSPG sequence (SEQ ID NO:12) containing human TNFR2. Peptides (e.g., single-chain peptides, antibodies, or antigen-binding fragments thereof) that bind with epitopes containing one or more residues of the KCRPG sequence (amino acids 142-146 of SEQ ID NO:7 in human TNFR2) and epitopes containing the KCSPG motif (amino acids 56-60 of SEQ ID NO:7 in human TNFR2) with similar affinity (e.g., affinity less than 10-fold difference) are not considered antagonistic TNFR2 peptides of the present invention.

[0214] In addition to one or more residues of amino acids 142-146 of SEQ ID NO:7, the polypeptide of the present invention may also bind a larger epitope comprising at least five consecutive or discontinuous residues from positions 130-149 (KQEGCRLCAPLRKCRPGFGV, SEQ ID NO:17) of SEQ ID NO:7 in human TNFR2, or an epitope exhibiting at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) of the sequence, and one or more residues of an epitope containing a conserved amino acid substitution relative to the sequence. For example, the antagonistic TNFR2 antibody or antibody fragment of the present invention can specifically bind to an epitope containing residues 142-149 (KCRPGFGV, SEQ ID NO:20) of SEQ ID NO:7 in human TNFR2, or an epitope exhibiting at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) with that sequence (provided that one or more residues of the KCRPG sequence are present in that epitope). Alternatively or additionally, the antagonistic TNFR2 antibody of the present invention can specifically bind to an epitope containing residues 137-144 (CAPLRKCR, SEQ ID NO:11) of SEQ ID NO:7 in human TNFR2, or an epitope exhibiting at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) with that sequence (provided that one or more residues of the KCRPG sequence are present in that epitope).

[0215] The antagonistic TNFR2 single-chain polypeptide, antibody, and antibody fragment of the present invention may optionally bind a downstream epitope consisting of at least five consecutive or discontinuous residues at positions 150-190 (ARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI, SEQ ID NO:22) in human TNFR2, as well as an epitope exhibiting at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) with respect to the sequence, and an epitope containing a conserved amino acid substitution relative to the sequence. For example, in some embodiments, the antagonistic TNFR2 peptide of the present invention can specifically bind an epitope comprising residues from positions 161-169 (CKPCAPGTF, SEQ ID NO:21) of SEQ ID NO:7 in human TNFR2, as well as an epitope exhibiting at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) with respect to that sequence, and an epitope containing a conserved amino acid substitution relative to that sequence.

[0216] In addition to interacting with one or more residues of the KCRPG motif (SEQ ID NO:19), the antagonistic TNFR2 peptide of the present invention can also specifically bind to an epitope in human TNFR2 containing at least five consecutive or discontinuous residues from positions 75-128 of SEQ ID NO:7 (CDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCAL, SEQ ID NO:13), as well as epitopes exhibiting at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) with respect to the sequence, and epitopes containing conserved amino acid substitutions relative to the sequence. The anti-TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) can also specifically bind to epitopes in human TNFR2 containing at least five consecutive or discontinuous residues from positions 75-91 (CDSCEDSTYTQLWNWVP, SEQ ID NO:14) of SEQ ID NO:7, as well as epitopes exhibiting at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) with respect to the sequence, and epitopes containing conserved amino acid substitutions relative to the sequence. In some embodiments, the anti-TNFR2 peptide of the present invention (e.g., single-chain peptide, antibody, and antigen-binding fragment) can specifically bind to an epitope containing residues at positions 80-86 (DSTYTQL, SEQ ID NO:8) of SEQ ID NO:7 in human TNFR2, as well as epitopes exhibiting at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) with respect to that sequence, and epitopes containing conserved amino acid substitutions relative to that sequence. The antagonistic TNFR2 peptide of the present invention (e.g., single-chain peptide, antibody, and antigen-binding fragment) can also specifically bind to an epitope comprising at least five consecutive or discontinuous residues from position 86-103 (LWNWVPECLSCGSRCSSD, SEQ ID NO:15) of SEQ ID NO:7 in human TNFR2, as well as an epitope exhibiting at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) with respect to the sequence, and an epitope containing a conserved amino acid substitution relative to the sequence.In some embodiments, the polypeptides of the present invention (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) can specifically bind an epitope comprising residues at positions 91-98 (PECLSCGS, SEQ ID NO:9) of SEQ ID NO:7 from human TNFR2, and an epitope exhibiting at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) with respect to that sequence, as well as an epitope containing a conserved amino acid substitution relative to that sequence. The polypeptides of the present invention (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) can also specifically bind an epitope comprising at least five consecutive or discontinuous residues at positions 111-128 (TREQNRICTCRPGWYCAL, SEQ ID NO:16) of SEQ ID NO:7 from human TNFR2, and an epitope exhibiting at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) with respect to that sequence, as well as an epitope containing a conserved amino acid substitution relative to that sequence. In some embodiments, the polypeptides of the present invention (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) can specifically bind epitopes containing residues 116-123 (RICTCRPG, SEQ ID NO: 10) of SEQ ID NO: 7 in human TNFR2, as well as epitopes exhibiting at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) with respect to that sequence, and epitopes containing conserved amino acid substitutions relative to that sequence. For example, the antagonistic TNFR2 single-chain polypeptides, antibodies, and antibody fragments of the present invention can specifically bind epitopes containing residues 116-123 (RICTCRPG, SEQ ID NO: 10) of SEQ ID NO: 7 in human TNFR2 and residues 137-144 (CAPLRKCR, SEQ ID NO: 11) of SEQ ID NO: 7 in human TNFR2.

[0217] The antagonistic TNFR2 polypeptide of the present invention can bind to epitopes within amino acids 112-131 (REQNRICTCRPGWYCALSKQ) of SEQ ID NO:7, as well as epitopes exhibiting at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) with respect to that sequence, and epitopes containing conserved amino acid substitutions relative to that sequence. Alternatively or additionally, the antagonistic TNFR2 single-chain polypeptide, antibody, or antigen-binding fragment of the present invention can bind to epitopes within amino acids 120-139 (CRPGWYCALSKQEGCRLCAP) of SEQ ID NO:7, as well as epitopes exhibiting at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) with respect to that sequence, and epitopes containing conserved amino acid substitutions relative to that sequence. The antagonistic TNFR2 peptide of the present invention can bind to an epitope within amino acids 128-147 (LSKQEGCRLCAPLRKCRPGF) of SEQ ID NO:7, as well as an epitope exhibiting at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) with respect to that sequence, and an epitope containing a conserved amino acid substitution relative to that sequence. The antagonistic TNFR2 peptide of the present invention can optionally bind to an epitope within amino acids 136-155 (LCAPLRKCRPGFGVARPGTE) of SEQ ID NO:7, as well as an epitope exhibiting at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) with respect to that sequence, and an epitope containing a conserved amino acid substitution relative to that sequence.

[0218] Antagonistic TNFR2 peptides that bind to TNFR2 from non-human animals

[0219] In addition to binding to epitopes containing the KCRPG motif within human TFNR2, the antagonistic TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) also include those that specifically bind to epitopes containing equivalent motifs within TNFR2 derived from non-human animals (such as non-human mammals, e.g., cattle, bison, mice, or rats). Table 2 below shows the positions of sequences equivalent to the human KCRPG motif in TNFR2 derived from exemplary non-human mammals:

[0220] Table 2. Sequence locations of KCRPG equivalents in TNFR2 from non-human mammals

[0221]

[0222] Epitopes derived from TNFR2 in non-human mammals discussed above that can be bound by the antagonistic TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) are summarized in the following sequence alignment. This sequence alignment shows partial sequences of TNFR2 derived from humans, cattle, bison, mice, and rats, as well as epitopes equivalent to the human KCRPG motif (highlighted in gray).

[0223]

[0224] antagonistic TNFR2 antibody TNFRAB1

[0225] Representative antagonistic TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, or antigen-binding fragments thereof) may be based on TNFRAB1, also referred herein as TNFR2 antagonist 1, which is a mouse antibody that antagonizes TNFRα-TNFR2 interaction and is capable of inhibiting TNFα-mediated T-reg cell proliferation (see, e.g.) Figure 5 ) Variable regions (e.g., heavy and light chain CDRs) and variants of TNFRAB1 that exhibit specific binding properties substantially similar to TNFRAB1 can be used to prepare the antagonistic TNFR2 antibody or its antigen-binding fragment of the present invention, for example by replacing the mouse constant region of TNFRAB1 with a non-natural constant region (e.g., a constant region derived from a human antibody) using methods known in the art or described herein.

[0226] The peptides of the present invention (e.g., single-chain peptides, antibodies, and antigen-binding fragments) can exhibit the same or similar binding properties as TNFRAB1. These properties are as follows: In the presence of TNFR2, TNFRAB1 exhibits a binding strength of 4.98 x 10⁻⁶. 6 M -1 s -1 high k on Value and 2.21x10 -4 s -1 low k off And approximately 44.4 pM of K when it combines with TNFR2 DThe high affinity of TNFRAB1 for TNFR2, accompanying the rapid formation and slow dissociation of the TNFRAB1-TNFR2 complex, is consistent with strong intermolecular contact based on this protein-protein interaction. TNFRAB1 binds to different epitopes within the primary structure of TNFR2, which are spatially arranged in the native conformation of the receptor. The KCRPGFGV motif (SEQ ID NO: 20), and particularly the KCRPG sequence (SEQ ID NO: 19), has been identified as a particularly important component of the functional epitope that establishes strong intermolecular contact with TNFRAB1, as determined by epitope mapping analysis (Figures 2 and 3). The interaction of these residues with the anti-TNFR2 peptide of the present invention selectively promotes antagonistic activity. It is noteworthy that the TNFR2 epitope containing amino acid residues 56-60 (KCSPG, SEQ ID NO:12) of SEQ ID NO:7 within human TNFR2 is clearly not part of the conformational epitopes specifically bound by TNFRAB1 or the antagonistic TNFR2 peptide of the present invention, as specific binding to either of these epitopes has been shown to result in loss or significant reduction of antagonistic activity. Therefore, TNFR2 peptides (e.g., single-chain peptides, antibodies, and antigen-binding fragments) that specifically bind to either of these epitopes (KCSPG and epitopes containing at least the KCR sequence, and more specifically, the KCRPG sequence of human TNFR2) are not considered the antagonistic TNFR2 peptide of the present invention.

[0227] In addition to binding to the epitope contained in the sequence KCRPGFGV (SEQ ID NO:20), TNFRAB1 also binds to a downstream epitope (CKPCAPGTF, SEQ ID NO:21) contained in the sequence defined by positions 161-169 of SEQ ID NO:7 within human TNFR2. The anti-TNFR2 single-chain peptide, antibody, and antibody fragment of the present invention can also bind to this epitope or a larger region within TNFR2 containing this epitope (e.g., a sequence containing at least five consecutive or discontinuous residues from positions 150-190 of SEQ ID NO:7 within human TNFR2 (ARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI, SEQ ID NO:22).

[0228] TNFRAB1 contains two heavy chains and two light chains, such as Figure 1A and 1B As shown in the image. The heavy chain of TNFRAB1 contains the following amino acid sequence (CDRs are shown in bold):

[0229] EVQLQESGGGLVKPGGSLKLSCAASGFTFSSYVMSWVRQTPEKRLEWVATISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARQRVDGYSSYWYFDVWGAGTAVTVSS(SEQ ID NO:2)

[0230] The sequence of the TNFRAB1 light chain is as follows (CDR is indicated in bold):

[0231] DIVLTQSPAIMSASPGEKVTITCSASSSVYYMYWFQQKPGTSPKLWIYSTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQRRNYPYTFGGGTKLEIKRA

[0232] (SEQ ID NO:4)

[0233] antagonistic TNFR2 antibody TNFRAB2

[0234] Representative antagonistic TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, or antigen-binding fragments thereof) may be based on TNFRAB2, also referred to herein as TNFR2 antagonist 2, an antibody that selectively binds to and inhibits TNFR2 by specifically binding to various epitopes within the receptor. For example, the antagonistic TNFR2 single-chain peptides, antibodies, or antigen-binding fragments thereof of the present invention may exhibit the same or similar binding properties as TNFRAB2. These properties are as follows: In the presence of TNFR2, TNFRAB2 exhibits a binding strength of 3.6099 x 10⁻⁶. 5 M -1 s -1 high k on Value and 2.24x10 -4 s -1 low k off And approximately 621 pM of K when it combines with TNFR2 D Epitopes containing residues 137-144 (CAPLRKCR, SEQ ID NO:11) of SEQ ID NO:7 in human TNFR2 have been identified as particularly important components of functional epitopes that establish strong intermolecular contacts with TNFRAB2, as determined by epitope mapping analysis (see, for example, Example 1 and...). Figure 2B and 3B This invention includes a TNFR2 antibody that specifically binds to the epitope and an antibody fragment.

[0235] In addition to binding to the epitope containing the residue CAPLRKCR (SEQ ID NO: 11), TNFRAB2 also binds to epitopes containing one or more residues at positions 80-86 (DSTYTQL, SEQ ID NO: 8) of SEQ ID NO: 7 in human TNFR2, positions 91-98 (PECLSCGS, SEQ ID NO: 9) of SEQ ID NO: 7 in human TNFR2, and positions 116-123 (RICTCRPG, SEQ ID NO: 10) of SEQ ID NO: 7 in human TNFR2. The TNFR2 single-chain peptides, antibodies, and antibody fragments of the present invention can also bind to one or more of these epitopes. Knowledge of the epitopes specifically bound by TNFRAB2 can be used to design and identify the peptides of the present invention. For example, any of the various in vitro peptide display techniques or combinatorial antibody library screenings described herein or known in the art can be used to screen for peptides (e.g., single-chain peptides, antibodies, and their antigen-binding fragments) capable of binding these epitopes with high affinity and selectivity.

[0236] Furthermore, the TNFR2 peptides of the present invention include peptides containing one or more heavy and light chain CDRs or heavy and light chain variable regions of TNFRAB2, as well as non-native constant regions of TNFRAB2 (e.g., constant regions derived from human antibodies). The heavy and light chain CDRs of TNFRAB2 are shown below:

[0237] TNFRAB2 CDR-H1:GYTFTDY(L / I)(SEQ ID NO:257)

[0238] TNFRAB2 CDR-H2:VDPEYGST(SEQ ID NO:258)

[0239] TNFRAB2 CDR-H3:ARDDGSYSPFDYWG(SEQ ID NO:259)

[0240] TNFRAB2 CDR-L1:QNINKY(SEQ ID NO:260)

[0241] TNFRAB2 CDR-L2: TYS or YTS

[0242] TNFRAB2 CDR-L3: CLQYVNL(L / I)T(SEQ ID NO:261)

[0243] As shown above, the CDR-H1 sequence of TNFRAB2 may contain a leucine or isoleucine residue at position 8 of this region. Similarly, TNFRAB2 CDR-L2 may include a TYS or YTS tripeptide, and TNFRAB2 CDR-L3 may contain a leucine or isoleucine residue at position 8 of this region. Notably, the CDR-L2 of TNFRAB2 is flanked by an N-terminal framework residue LLIR (SEQ ID NO:262) and a C-terminal framework residue TLE. Therefore, the antagonistic TNFR2 antibody or its antigen-binding fragment of the present invention comprises one or more of the above-described CDRs of TNFRAB2, as well as those flanking the CDR-L2 sequence of the antagonistic TNFR2 antibody or its antigen-binding fragment with N-terminal LLIR (SEQ ID NO:262) and C-terminal TLE residues.

[0244] Molecular determinants of TNFR2 affinity and antagonistic effects

[0245] Notably, there is significant sequence similarity between the CDRs of the antagonistic TNFR2 antibodies TNFRAB1 and TNFRAB2. Analysis of the common residues in the CDRs of these two antibodies provides a profound understanding of the molecular characterization of antibodies that bind to TNFR2 and exhibit antagonistic activity. Epitope mapping analysis shows that both TNFRAB1 and TNFRAB2 bind to TNFR2 in an antiparallel dimer configuration. These antibodies bind to epitopes containing residues 142-146 of SEQ ID NO:7 within TNFR2, but not to epitopes containing residues 56-60 of SEQ ID NO:7. The structural similarity between the corresponding CDR-H and CDR-L regions provides a basis for predicting residue substitutions that can preserve or enhance TNFR2 affinity and antagonistic activity. Examination of the heavy and light chain CDR sequences of these antibodies reveals that several residues and physicochemical features are preserved within the corresponding CDR sequences, while other positions within these CDRs can be significantly modified without loss of affinity and antagonistic function. For example, the CDR-H1 sequences of TNFRAB1 and TNFRAB2 are shown below:

[0246] GFTFSSY (TNFRAB1 CDR-H1, SEQ ID NO:23)

[0247] GYTFTDY(L / I) (TNFRAB2 CDR-H1, SEQ ID NO:257)

[0248] G-TF—-Y- (Common Sequence)

[0249] Sequence alignment revealed a common GXTFXXY motif, where "X" represents any amino acid. These CDR-H1 sequences are characterized by a conserved glycine residue at the first position and conserved threonine, phenylalanine, and tyrosine residues at the third, fourth, and seventh positions, respectively. Examination of these sequences indicates that substitution is permissible at the remaining positions within the CDR-H1 region. Side chains of varying polarities are permissible at the second position; for example, phenylalanine containing an unsubstituted and hydrophobic phenyl moiety and tyrosine containing a protonated hydroxyl substituent were both found at this position in the CDR-H1 regions of TNFRAB1 and TNFRAB2, respectively. Furthermore, although the fifth and sixth positions in the CDR-H1 of TNFRAB1 are occupied by polar serine residues, these positions are characterized by threonine containing an additional hydrophobic methyl substituent and aspartic acid, which is an anion in TNFRAB2 at physiological pH. This diversity suggests that these positions can be substituted with amino acids of different electrostatic properties without loss of TNFR2 affinity and antagonistic activity.

[0250] Sequence analysis of the CDR-H2 regions of TNFRAB1 and TNFRAB2 similarly revealed a set of conserved amino acids at different positions throughout these regions:

[0251] SSG—GSY (TNFRAB1 CDR-H2, SEQ ID NO:24)

[0252] VDPEYGST (TNFRAB2 CDR-H2, SEQ ID NO:258)

[0253] -----GS- (Shared Sequence)

[0254] The sequence alignment analysis shows that the CDR-H2 sequence exhibits a conserved GS motif at the C-terminus of the CDR-H2 region, with side chains allowing for variable molecular size, polarity, and electrostatic charge at the remaining positions. Furthermore, the corresponding CDR-H3 sequences of TNFRAB1 and TNFRAB2 are aligned as follows:

[0255] QRVDGYSSYWYFDV (TNFRAB1 CDR-H3, SEQ ID NO:25)

[0256] ARDDG-S-YSPFDYWG (TNFRAB2 CDR-H3, SEQ ID NO:259)

[0257] -R-DG-SY--FD--- (Common sequence)

[0258] Similar analyses of the CDR-H3 sequences of TNFRAB1 and TNFRAB2 revealed conserved arginine, aspartic, glycine, serine, tyrosine, and phenylalanine residues throughout the CDR. Notably, residues with different spatial and electrostatic properties are permitted at the remaining positions. For example, the first position of the CDR-H3 sequence allows amino acid residues of different sizes and hydrogen-bonding propensities, as the first position of the CDR-H3 in TNFRAB1 is characterized by a polar glutamine residue with a formamide side chain possessing both hydrogen bond donor and acceptor moieties, while the corresponding position in TNFRAB2 shows an alanine residue with an unfunctionalized methyl side chain. Furthermore, the third position in the aforementioned CDR-H3 sequences is characterized by a hydrophobic valine moiety in TNFRAB1 and an anionic aspartic moiety in the corresponding position in TNFRAB2.

[0259] Similar analyses revealed shared molecular features in the CDR-L sequences of TNFRAB1 and TNFRAB2. For example, the CDR-L1 sequences of TNFRAB1 and TNFRAB2 are shown below:

[0260] SASSSVYYMY (TNFRAB1 CDR-L1, SEQ ID NO:26)

[0261] QN--INK-Y (TNFRAB2 CDR-L1, SEQ ID NO:260)

[0262] Y (shared residues)

[0263] Examination of these sequences revealed that hydroxyl-containing tyrosine residues are characteristic at the final position of the CDR-L1, while residues with different physicochemical properties are permitted at the remaining positions. Similarly, analysis of the CDR-L2 regions of TNFRAB1 and TNFRAB2 revealed conserved amino acids at the final positions in these two regions:

[0264] STSNLAS (TNFRAB1 CDR-L2, SEQ ID NO:27)

[0265] TY----S or (TNFRAB2 CDR-L2)

[0266] YT----S

[0267] S (shared residues)

[0268] Analysis of the above sequence alignments shows that serine residues are characteristic at the third position of these CDR-L2 sequences, while substitution is widely permissible at the remaining residues. Similarly, the CDR-L3 sequences of TNRAB1 and TNFRAB2 are as follows:

[0269] Q-QRRNYPY------T (TNFRAB1 CDR-L3, SEQ ID NO:28)

[0270] CLQ---YVNL(L / I)T (TNFRAB2 CDR-L3, SEQ ID NO:261)

[0271] ------Y--------T (Common sequence)

[0272] Analysis of the CDR-L3 sequences of TNFRAB1 and TNFRAB2 revealed a preference for tyrosine and threonine residues at different positions within these regions, while allowing for amino acids with a wide range of physicochemical characteristics at other positions, including residues with cationic side chains (Arg), conformationally restricted side chains (Pro), and side chains of different polarities (e.g., Gln, Asn, Leu, and Val). The shared structural features of these CDR-H and CDR-L sequences provide a profound understanding of which residues are important for selectively binding one or more residues to the KCRPG epitope of TNFR2 in an antiparallel dimer configuration (positions 142-146 of SEQ ID NO:7, as shown in SEQ ID NO:19), and demonstrate that certain amino acids can vary while maintaining affinity and antagonistic activity.

[0273] Therefore, the antagonistic TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, or antigen-binding fragments thereof) can have heavy and light chain CDRs containing the above-mentioned common sequences. For example, the TNFR2 antagonist of the present invention can have an amino acid sequence Z 4 JZ 3 Z 5 (J)2Z 5 or Z 4 JZ 3 Z 5 (J)2Z 5 J's CDR-H1; possessing the amino acid sequence (J)3Z 4 Z 3 J or (J)5Z 4 Z 3 J's CDR-H2; possessing the amino acid sequence JZ 1 JZ 2 Z 4 JZ 3 JZ 5 (J)2Z 5 Z 2 Z 5 or JZ 1 JZ 2 Z 4 Z3 Z 5 (J)2Z 5 Z 2 Z 5 (J)2 CDR-H3; possessing the amino acid sequence (J)9Z 5 Or (J)5Z 5 CDR-L1; with the amino acid sequence (J)6Z 3 or (J)2Z 3 CDR-L2; and the amino acid sequence (J)5Z 5 (J)2Z 3 Or (J)3Z 5 (J)4Z 3 CDR-L3; where each J is independently a naturally occurring amino acid; each Z 1 Independently, naturally occurring amino acids containing cationic side chains at physiological pH; each Z 2 A naturally occurring amino acid that contains anionic side chains at physiological pH; each Z 3 Independently, these are naturally occurring amino acids containing polar, uncharged side chains at physiological pH; each Z 4 It is either glycine or alanine; and each Z is either glycine or alanine. 5 It is an amino acid that exists naturally and independently, containing a hydrophobic side chain.

[0274] In some embodiments, the antagonistic TNFR2 peptide of the present invention (e.g., a single-chain peptide, an antibody, or an antigen-binding fragment thereof) may have a CDR-H1 having the amino acid sequence GJTF(J)2Y (SEQ ID NO:276) or GJTF(J)2YJ (SEQ ID NO:277); a CDR-H2 having the amino acid sequence (J)3GSJ or (J)5GSJ; a CDR-H3 having the amino acid sequence JRJDGJSJY(J)2FDJ (SEQ ID NO:278) or JRJDGSY(J)2FD(J)3 (SEQ ID NO:279); a CDR-L1 having the amino acid sequence (J)9Y or (J)5Y; a CDR-L2 having the amino acid sequence (J)6S or (J)2S; and a CDR-L3 having the amino acid sequence (J)5Y(J)2T or (J)3Y(J)4T; wherein each J is independently a naturally occurring amino acid.

[0275] The antagonistic TNFR2 polypeptide of the present invention (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof) may have an amino acid sequence Z 4 FZ 3 Z 5 SSZ 5 or Z 4 YZ3 Z 5 TDZ 5 X's CDR-H1; possessing the amino acid sequence SSGZ 4 Z 3 Y (SEQ ID NO:263) or VDPEYZ 4 Z 3 T(SEQ ID NO:264) CDR-H2; with amino acid sequence QZ 1 VZ 2 Z 4 YZ 3 SZ 5 WYZ 5 Z 2 Z 5 (SEQ ID NO:265) or AZ 1 DZ 2 Z 4 Z 3 Z 5 SPZ 5 Z 2 Z 5 CDR-H3 of WG (SEQ ID NO:266); with the amino acid sequence SASSSVYYMZ 5 (SEQ ID NO:267) or QNINKZ 5 CDR-L1 (SEQ ID NO:268); STSNLAZ with amino acid sequence 3 (SEQ ID NO:269), TYZ 3 Or YTZ 3 CDR-L2; and QQRRNZ with the amino acid sequence 5 PYZ 3 (SEQ IDNO:270) or CLQZ 5 VNLXZ 3 CDR-L3 of (SEQ ID NO:271); where each Z 1 Independently, each Z contains an amino acid with a cationic side chain at physiological pH; 2 Independently, these are amino acids that contain anionic side chains at physiological pH; each Z 3 Independently, these are amino acids that contain polar, uncharged side chains at physiological pH; each Z 4 It is either glycine or alanine; each Z 5 Each X is an amino acid containing a hydrophobic side chain; and each X is either leucine or isoleucine.

[0276] In some embodiments, the antagonistic TNFR2 peptide of the present invention (e.g., a single-chain peptide, an antibody, or an antigen-binding fragment thereof) may have a CDR-H1 having the amino acid sequences GFTFSSY (SEQ ID NO:23), GYTFTDYX (SEQ ID NO:257), or an amino acid sequence having up to two amino acid substitutions relative to these sequences; a CDR-H2 having the amino acid sequences SSGGSY (SEQ ID NO:24), VDPEYGST (SEQ ID NO:258), or an amino acid sequence having up to two amino acid substitutions relative to these sequences; a CDR-H3 having the amino acid sequences QRVDGYSSYWYFDV (SEQ ID NO:25), ARDDGSYSPFDYWG (SEQ ID NO:259), or an amino acid sequence having up to two amino acid substitutions relative to these sequences; a CDR-L1 having the amino acid sequences SASSVYYMY (SEQ ID NO:26), QNINKY (SEQ ID NO:260), or an amino acid sequence having up to two amino acid substitutions relative to these sequences; or a CDR-L1 having the amino acid sequence STSNLAS (SEQ ID NO:25). CDR-L2 having amino acid sequences of SEQ ID NO:27), TYS, YTS or having up to two amino acid substitutions relative to SEQ ID NO:27; and CDR-L3 having amino acid sequences QQRRNYPYT (SEQ ID NO:28), CLQYVNLXT (SEQ ID NO:261) or having up to two amino acid substitutions relative to these sequences.

[0277] Humanized, primate, and chimeric antibodies derived from TNFRAB1 and / or TNFRAB2

[0278] The antibodies of the present invention comprise one or more CDRs containing TNFRAB1, or human humanized, primate, and chimeric antibodies containing a CDR that exhibits at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) with respect to any of these CDRs or sequences containing conserved mutations. The antibodies of the present invention comprise one or more CDRs containing TNFRAB2, or human humanized, primate, and chimeric antibodies containing a CDR that exhibits at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) with respect to any of these CDRs or sequences containing conserved mutations. For example, the antibodies of the present invention also include human humanized, primate, and chimeric antibodies containing one or more CDRs identical to the above CDRs (except for conserved amino acid substitutions). In some embodiments, the humanized, primate, or chimeric antibody may contain one or more CDRs of TNFRAB1 or CDRs that exhibit at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) with respect to any of the CDRs or sequences of TNFRAB1 containing a conserved mutation relative to these CDRs, and one or more CDRs of TNFRAB2 or CDRs that exhibit at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) with respect to any of the CDRs or sequences of TNFRAB2 containing a conserved mutation relative to these CDRs. For example, the antagonistic TNFR2 antibody of the present invention can be generated by incorporating any of the above CDRs into the framework regions (e.g., FW1, FW2, FW3, and FW4) of a human antibody. Exemplary framework regions that can be used to develop humanized anti-TNFR2 antibodies containing one or more of the above CDRs include, but are not limited to, those described in U.S. Patent Nos. 7,732,578, 8,093,068, and WO 2003 / 105782, which are incorporated herein by reference.

[0279] One strategy that can be used to design the humanized antibody of this invention is to align the heavy chain variable region and light chain variable region of an antagonistic TNFR2 antibody (such as TNFRAB1 or TNFRAB2) with the heavy chain variable region and light chain variable region of a co-human antibody. The heavy chain and light chain sequences of the co-human antibody are known in the art (see, for example, the “VBASE” human germline sequence database; also see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242, 1991; Tomlinson et al., J. Mol. Biol. 227:776-98, 1992; and Cox et al., Eur. J. Immunol. 24:827-836, 1994; all incorporated herein by reference). In this way, variable domain framework residues and CDRs can be identified by sequence alignment (see Kabat, ibid.). One or more CDRs of the heavy chain and / or light chain variable domains of a co-human antibody can be substituted with one or more corresponding CDRs of an antagonistic TNFR2 antibody (such as TNFRAB1 or TNFRAB2) to generate humanized TNFR2 antibodies. Exemplary variable domains of co-human antibodies include the following heavy chain variable domains:

[0280] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAVISENGSDTYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGGAVSYFDVWGQGTLVTVSS(SEQ ID NO:32)

[0281] And light chain variable structural domains:

[0282] DIQMTQSPSSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT(SEQ ID NO:33)

[0283] It is identified in U.S. Patent No. 6,054,297; which is incorporated herein by reference (CDR shown in bold and determined according to the method of Chothia et al., J. Mol. Biol [Journal of Molecular Biology], 196:901-917, 1987). These amino acid substitutions can be made, for example, by recombinant expression of polynucleotides encoding the heavy and light chains of humanized antibodies in host cells using methods known in the art or described herein.

[0284] Similarly, this strategy can also be used to generate primate-compatible anti-TNFR2 antibodies, since the CDRs of the heavy chain and / or light chain variable domains of the primate antibody concordant sequence can be replaced with one or more corresponding CDRs of TNFRAB1 and / or TNFRAB2. Concordant primate antibody sequences are known in the art (see, for example, U.S. Patent Nos. 5,658,570; 5,681,722; and 5,693,780; incorporated herein by reference).

[0285] In some embodiments, in addition to the CDR sequence from a TNFR2 antibody (such as TNFRAB1 or TNFRAB2), it may be desirable to introduce specific framework residues into the heavy and / or light chain variable domains of the human antibody. For example, U.S. Patent No. 6,054,297 identifies several instances where it may be advantageous to retain certain framework residues from the variable regions of the specific antibody heavy or light chains in the resulting humanized antibody. In some embodiments, framework residues may participate in non-covalent interactions with the antigen, thereby contributing to the antibody's affinity for the target antigen. In other cases, individual framework residues may modulate the conformation of the CDR, thereby indirectly affecting the antibody-antigen interaction. Alternatively, certain framework residues may form the interface between the VH and VL domains, and thus may contribute to the overall antibody structure. In still other cases, framework residues may constitute functional glycosylation sites (e.g., Asn-X-Ser / Thr), which may determine the antibody structure and antigen affinity after being linked to the carbohydrate moiety. In the cases described above, it may be advantageous to retain certain framework residues of the TNFR2 antibody (e.g., TNFRAB1 or TNFRAB2) in the antagonistic antibody and its antigen-binding fragment (e.g., humanized antibody) of the present invention, because various framework residues can promote high epitope affinity and improved biochemical activity of the antibody or its antigen-binding fragment.

[0286] The antibodies of the present invention also include antibody fragments, Fab domains, F(ab') molecules, F(ab')2 molecules, single-chain variable fragments (scFv), tandem scFv fragments, biantibodies, triantibodies, dual variable domain immunoglobulins, multispecific antibodies, bispecific antibodies, and xenogeneic specific antibodies containing one or more CDRs of TNFRAB1 or TNFRAB2 (e.g., CDRs containing the amino acid sequence of any one of SEQ ID NO: 23-28) or CDRs exhibiting at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) with any of these CDRs. The antibodies and antigen-binding fragments of the present invention include those that also contain CDRs with one to three amino acid substitutions (e.g., conserved or non-conserved substitutions) relative to the CDR sequence of TNFRAB1 or TNFRAB2. These molecules can be recombinantly expressed using techniques described herein or known in the art, for example by incorporating polynucleotides encoding these proteins into expression vectors for transfection in eukaryotic or prokaryotic cells, or by chemically synthesizing these molecules, for example by solid-phase peptide synthesis methods described herein or known in the art.

[0287] The antibodies of the present invention further include antibody-like scaffolds containing one or more CDRs of TNFRAB1 or TNFRAB2, or CDRs or sequences that exhibit at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) with one to three amino acid substitutions (e.g., conserved or non-conserved substitutions) relative to the CDR sequence of TNFRAB1 or TNFRAB2. Examples of antibody-like scaffolds include those containing a type III domain of fibronectin ten (… 10 The protein Fn3, whose tenth fibronectin type III domain contains BC, DE, and FG loops similar to those in the standard antibody, has been shown to contain these loops. 10 The tertiary structure of the Fn3 domain is similar to that of the variable region of the IgG heavy chain, and those skilled in the art can replace it with residues of TNFRAB1 and / or TNFRAB2CDR. 10 Residues in the BC, DE, and FG loops of Fn3 can be grafted onto a fibronectin scaffold with at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) of one or more CDRs, such as TNFRAB1 and / or TNFRAB2, or any of these CDRs or sequences containing conserved amino acid substitutions relative to these CDRs. This can be achieved by recombinantly expressing the modified CDRs in prokaryotic or eukaryotic cells. 10Fn3 domains are used to implement this (e.g., using the carriers and techniques described herein). WO 2000 / 034784, WO 2009 / 142773, WO 2012 / 088006, and U.S. Patent No. 8,278,419 report the use of... 10 The Fn3 domain serves as an example of an antibody-like scaffold for grafting CDRs from antibodies onto the BC, DE, and FG structural loops; it is incorporated herein by reference.

[0288] Antagonistic TNFR2 single-chain peptide

[0289] The TNFR2 antagonists of the present invention can be in the form of single-chain polypeptides, such as single-chain polypeptides containing one, two, or three heavy chain CDRs of a monoclonal TNFR2 antagonist antibody (e.g., TNFRAB1 or TNFRAB2) described herein, and / or one, two, or three light chain CDRs of a monoclonal TNFR2 antagonist antibody (e.g., TNFRAB1 or TNFRAB2). The single-chain polypeptides can be in the form of antibody fragments, such as antibody fragments described herein or known in the art, such as scFv fragments. Alternatively, the single-chain polypeptides can contain one or more CDRs described herein, which are covalently linked to each other using conventional bonding techniques known in the art (e.g., via amide bonds, thioether bonds, carbon-carbon bonds, or via linkers, such as peptide linkers described herein or known in the art, or multivalent electrophiles (e.g., bis(bromomethyl)aromatic derivatives, such as bis(bromomethyl)benzene or bis(bromomethyl)pyridine)).

[0290] For example, the antagonistic TNFR2 single-chain polypeptide of the present invention may have one or more heavy and light chain CDRs containing the aforementioned common sequence that promotes selective binding to TNFR2 epitopes (such as the KCRPG motif (SEQ ID NO: 19)) and induces TNFR2 antagonism. For example, the antagonistic TNFR2 single-chain polypeptide of the present invention may have an amino acid sequence Z 4 JZ 3 Z 5 (J)2Z 5 or Z 4 JZ 3 Z 5 (J)2Z 5 J's CDR-H1; possessing the amino acid sequence (J)3Z 4 Z 3 J or (J)5Z 4 Z 3 J's CDR-H2; possessing the amino acid sequence JZ 1 JZ 2 Z 4 JZ 3 JZ 5 (J)2Z5 Z 2 Z 5 or JZ 1 JZ 2 Z 4 Z 3 Z 5 (J)2Z 5 Z 2 Z 5 (J)2 CDR-H3; possessing the amino acid sequence (J)9Z 5 Or (J)5Z 5 CDR-L1; with the amino acid sequence (J)6Z 3 or (J)2Z 3 CDR-L2; and / or having the amino acid sequence (J)5Z 5 (J)2Z 3 Or (J)3Z 5 (J)4Z 3 CDR-L3; where each J is independently a naturally occurring amino acid; each Z 1 Independently, naturally occurring amino acids containing cationic side chains at physiological pH; each Z 2 A naturally occurring amino acid that contains anionic side chains at physiological pH; each Z 3 Independently, these are naturally occurring amino acids containing polar, uncharged side chains at physiological pH; each Z 4 It is either glycine or alanine; and each Z is either glycine or alanine. 5 It is an amino acid that exists naturally and independently, containing a hydrophobic side chain.

[0291] In some embodiments, the antagonistic TNFR2 single-chain polypeptide of the present invention may have a CDR-H1 having the amino acid sequence GJTF(J)2Y (SEQ ID NO:276) or GJTF(J)2YJ (SEQ ID NO:277); a CDR-H2 having the amino acid sequence (J)3GSJ or (J)5GSJ; a CDR-H3 having the amino acid sequence JRJDGJSJY(J)2FDJ (SEQ ID NO:278) or JRJDGSY(J)2FD(J)3 (SEQ ID NO:279); a CDR-L1 having the amino acid sequence (J)9Y or (J)5Y; a CDR-L2 having the amino acid sequence (J)6S or (J)2S; and / or a CDR-L3 having the amino acid sequence (J)5Y(J)2T or (J)3Y(J)4T; wherein each J is independently a naturally occurring amino acid.

[0292] The antagonistic TNFR2 single-chain polypeptide of the present invention can have an amino acid sequence Z 4 FZ 3Z 5 SSZ 5 or Z 4 YZ 3 Z 5 TDZ 5 X's CDR-H1; possessing the amino acid sequence SSGZ 4 Z 3 Y (SEQ ID NO:263) or VDPEYZ 4 Z 3 T(SEQ IDNO:264) CDR-H2; possessing the amino acid sequence QZ 1 VZ 2 Z 4 YZ 3 SZ 5 WYZ 5 Z 2 Z 5 (SEQ ID NO:265) or AZ 1 DZ 2 Z 4 Z 3 Z 5 SPZ 5 Z 2 Z 5 CDR-H3 of WG (SEQ ID NO:266); with the amino acid sequence SASSSVYYMZ 5 (SEQ ID NO:267) or QNINKZ 5 CDR-L1 (SEQ ID NO:268); STSNLAZ with amino acid sequence 3 (SEQ ID NO:269), TYZ 3 Or YTZ 3 CDR-L2; and / or having the amino acid sequence QQRRNZ 5 PYZ 3 (SEQ ID NO:270) or CLQZ 5 VNLXZ 3 CDR-L3 of (SEQ ID NO:271); where each Z 1 Independently, each Z contains an amino acid with a cationic side chain at physiological pH; 2 Independently, these are amino acids that contain anionic side chains at physiological pH; each Z 3 Independently, these are amino acids that contain polar, uncharged side chains at physiological pH; each Z 4 It is either glycine or alanine; each Z 5 Each X is an amino acid containing a hydrophobic side chain; and each X is either leucine or isoleucine.

[0293] In some embodiments, the antagonistic TNFR2 single-chain polypeptide of the present invention may have the following amino acid sequences: GFTFSSY (SEQ ID NO:23), GYTFTDYX (SEQ ID NO:257), or an amino acid sequence having up to two amino acid substitutions relative to these sequences; CDR-H1 having the amino acid sequences SSGGSY (SEQ ID NO:24), VDPEYGST (SEQ ID NO:258), or an amino acid sequence having up to two amino acid substitutions relative to these sequences; CDR-H3 having the amino acid sequences QRVDGYSSYWYFDV (SEQ ID NO:25), ARDDGSYSPFDYWG (SEQ ID NO:259), or an amino acid sequence having up to two amino acid substitutions relative to these sequences; CDR-L1 having the amino acid sequences SASSVYYMY (SEQ ID NO:26), QNINKY (SEQ ID NO:260), or an amino acid sequence having up to two amino acid substitutions relative to these sequences; or having the amino acid sequences STSNLAS (SEQ ID NO:27), TYS, YTS, or an amino acid sequence having up to two amino acid substitutions relative to these sequences. NO:27 CDR-L2 having an amino acid sequence with up to two amino acid substitutions; and / or CDR-L3 having the amino acid sequences QQRRNYPYT (SEQ ID NO:28), CLQYVNLXT (SEQ ID NO:261) or an amino acid sequence with up to two amino acid substitutions relative to these sequences.

[0294] Single-chain peptides can be generated by a variety of recombinant and synthetic techniques, such as recombinant gene expression or solid-phase peptide synthesis methods described herein or known in the art. For example, those skilled in the art can design polynucleotides encoding two or more of the CDRs described above, which are efficiently linked together within a frame to generate a continuous single-chain peptide containing these CDRs. Optionally, CDRs can be separated by spacers, such as by frame regions (e.g., frame sequences described herein or frame regions of germline common sequences of human antibodies) or flexible linkers (such as polyglycine or glycine / serine linkers described herein or known in the art). When generated by chemical synthesis methods, native chemical linking can optionally be used as a strategy for synthesizing long peptides (e.g., greater than 50 amino acids). Native chemical linking schemes are known in the art and have been described by Dawson et al. (Science, 266:776-779, 1994); incorporated herein by reference. Detailed descriptions of techniques for generating single-chain peptides, full-length antibodies, and antibody fragments are provided in the following sections.

[0295] Nucleic Acids and Expression Systems

[0296] The antagonistic TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, or antigen-binding fragments thereof) can be prepared using any of a variety of established techniques. For example, the antagonistic TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, or antigen-binding fragments thereof) can be prepared by recombinantly expressing one or more immunoglobulin light and heavy chain genes in host cells. For example, to express recombinant antibodies, host cells can be transfected with one or more expression vectors carrying DNA fragments encoding immunoglobulin antibody light and heavy chains, such that the light and heavy chains are expressed in the host cells and optionally secreted into a culture medium in which the host cells are cultured, from which the antibodies can be recovered. Standard recombinant DNA methods are used to obtain antibody heavy and light chain genes, integrate these genes into recombinant expression vectors, and introduce the vectors into host cells, as described in Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, NY, 1989); Current Protocols in Molecular Biology (Ausubel et al., eds., Greene Publishing Associates, 1989); and those described in U.S. Patent No. 4,816,397; which are incorporated herein by reference.

[0297] Vector for expressing antagonistic TNFR2 peptides

[0298] Viral genomes provide a rich source of vectors for the efficient delivery of exogenous genes into the genomes of cells (e.g., eukaryotic or prokaryotic cells). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically integrated into the genome of the target cell via general or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require the addition of proteins or reagents to induce gene integration. Examples of viral vectors include retroviruses, adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus), positive-strand RNA viruses (e.g., parvovirus and alpha virus), and double-stranded DNA viruses (including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, EB virus, cytomegalovirus), and poxviruses (e.g., vaccinia, modified vaccinia ankara (MVA), fowlpox, and canarypox)). Other viruses used to deliver polynucleotides encoding the antibody light and heavy chains or antibody fragments of the present invention include, for example, norovirus, capsular virus, flavivirus, reovirus, polypore virus, hepatotropic DNA virus, and hepatitis virus. Examples of retroviruses include: avian leukosis sarcoma, mammalian type C virus, type B virus, type D virus, HTLV-BLV group, lentivirus, foam virus (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Other examples include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses. Other examples of vectors are described, for example, by McVey et al. (US Patent No. 5,801,030); incorporated herein by reference.

[0299] Genome editing technology

[0300] In addition to viral vectors, various other methods have been developed for incorporating genes (e.g., those encoding antibody light and heavy chains, single-chain polypeptides, single-chain variable fragments (scFv), tandem scFv, Fab domains, F(ab')2 domains, biantibodies, and triantibodies, etc.) into the genome of target cells for polypeptide expression. One method for incorporating polynucleotides encoding anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) into prokaryotic or eukaryotic cells includes transposons. A transposon is a polynucleotide encoding a transposase and contains a polynucleotide sequence or gene of interest flanked by cleavage sites at the 5' and 3' positions. Once the transposon is delivered into the cell, expression of the transposase gene begins, and an active enzyme cleaving the gene of interest is produced from the transposon. This activity is mediated by site-specific recognition of the transposon cleavage sites by the transposase. In some embodiments, these cleavage sites may be terminal repeat sequences or inverted terminal repeat sequences. Once excised from the transposon, the gene of interest can be integrated into the genome of a prokaryotic or eukaryotic cell by catalyzing the cleavage of a similar excision site present in the nuclear genome using transposases. This allows genes encoding, for example, an anti-TNFR2 antibody or fragments or domains thereof to be inserted into the cleaved nuclear DNA at the excision site, and the incorporation process is completed by subsequently linking the gene of interest to the phosphodiester bonds of the DNA in the prokaryotic or eukaryotic cell genome. In some embodiments, the transposon may be a retrotransposon, such that the gene encoding the antibody is first transcribed into an RNA product and then retrotranscribed into DNA before incorporation into the prokaryotic or eukaryotic cell genome. Exemplary transposon systems include the piggyac transposon (described in detail in WO 2010 / 085699) and the sleeping beauty transposon (described in detail in US 20050112764); both are incorporated herein by reference.

[0301] Another useful method for integrating nucleic acid molecules encoding anti-TNFR2 peptides (e.g., single-chain peptides, antibodies, or their antigen-binding fragments) into the genome of prokaryotic or eukaryotic cells is the regularly spaced clustered short palindromic repeats (CRISPR) / Cas system, which originated as an adaptive defense mechanism against viral infection in bacteria and archaea. This CRISPR / Cas system consists of palindromic repeats within plasmid DNA and an associated Cas9 nuclease. This DNA and protein combination first directs site-specific DNA cleavage of the target sequence by incorporating exogenous DNA into the CRISPR locus. In turn, polynucleotides containing these exogenous sequences and repeat-spacer elements of the CRISPR locus are transcribed in the host cell to produce guide RNA, which can then anneal to the target sequence and localize the Cas9 nuclease to that site. In this way, highly site-specific Cas9-mediated DNA cleavage can be generated in the exogenous polynucleotide because the interaction that brings Cas9 close to the target DNA molecule is governed by RNA:DNA hybridization. Therefore, it is theoretically possible to design a CRISPR / Cas system to cleave any target DNA molecule of interest. This technology has been developed for editing eukaryotic genomes (Hwang et al., Nat. Biotech. [Nature Biotechnology], 31:227-229, 2013) and can be used as an efficient means of site-specific editing of eukaryotic or prokaryotic genomes to cleave DNA prior to incorporation of a polynucleotide encoding the anti-TNFR2 polypeptide of this invention. Regulation of gene expression using CRISPR / Cas has been described in U.S. Patent No. 8,697,359, which is incorporated herein by reference.

[0302] Alternative methods for site-specific cleavage of genomic DNA prior to incorporation of a polynucleotide encoding the TNFR2 antibody or antibody fragment of the present invention include the use of zinc finger nucleases and transcription activator-like effector nucleases (TALENs). Unlike the CRISPR / Cas system, these enzymes do not contain a guiding polynucleotide that targets a specific target sequence. Target specificity is controlled by a DNA-binding domain within these enzymes. Urnov et al. (Nat. Rev. Genet. [Nature Genetics Review], 11:636-646, 2010) and Joung et al. (Nat. Rev. Mol. Cell. Bio. [Nature Reviews Molecular Cell Biology], 14:49-55, 2013) describe zinc finger nucleases and TALENs for genome editing applications; these are incorporated herein by reference. Other genome editing techniques that can be used to incorporate polynucleotides encoding the antibodies of the present invention into the genome of prokaryotic or eukaryotic cells include the use of ARCUS TMBroad-spectrum nucleases can be rationally designed to cleave genomic DNA at specific sites. These enzymes are particularly advantageous for incorporating polynucleotides encoding the antagonistic TNFR2 polypeptide (e.g., single-chain polypeptide, antibody, or antibody fragment) of the present invention into the genome of prokaryotic or eukaryotic cells, given the structure-activity relationships already established for such enzymes. Therefore, single-chain broad-spectrum nucleases can be modified at certain amino acid positions to produce nucleases that selectively cleave DNA at desired sites. These single-chain nucleases have been extensively described, for example, in U.S. Patent Nos. 8,021,867 and 8,445,251; which are incorporated herein by reference.

[0303] Polynucleotide sequence elements

[0304] To express the antagonistic TNFR2 polypeptide of the present invention (e.g., a single-chain polypeptide, an antibody, or an antibody fragment thereof), for example, polynucleotides encoding the portion or full-length light and heavy chains as described above, or their CDRs, can be inserted into an expression vector, such that the gene is effectively linked to transcriptional and translational control sequences. The expression vector and expression control sequences are selected to be compatible with the host cells used for expression. The light and heavy chain polynucleotides encoding, for example, TNFR2 antibodies can be inserted into different vectors, or optionally, both polynucleotides can be incorporated into the same expression vector using techniques described herein or known in the art.

[0305] In addition to polynucleotides encoding the heavy and light chains of antibodies (or polynucleotides encoding single-chain polypeptides or antibody fragments such as scFv molecules), the recombinant expression vectors of the present invention may also carry regulatory sequences controlling the expression of antibody chain genes in host cells. The design of the expression vector (including the selection of regulatory sequences) can depend on factors such as the choice of host cells to be transformed or the desired protein expression level. For example, suitable regulatory sequences for expression in mammalian host cells include viral elements that guide high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (e.g., CMV promoters / enhancers), simian virus 40 (SV40) (e.g., SV40 promoters / enhancers), adenoviruses (e.g., the adenovirus major late promoter (AdMLP)), and polyomaviruses. For further description of viral regulatory elements and their sequences, see, for example, U.S. Patent Nos. 5,168,062, 4,510,245, and 4,968,615.

[0306] In addition to the antibody chain or CDR gene and regulatory sequences, the recombinant expression vector of the present invention may also carry additional sequences, such as sequences that regulate vector replication in host cells (e.g., origin of replication) and selective marker genes. Selective marker genes facilitate selection in host cells in which the vector has been introduced (see, for example, U.S. Patents 4,399,216; 4,634,665; and 5,179,017). For example, typically, selective marker genes confer resistance to cytotoxic drugs (such as G418, puromycin, blastomycin, hygromycin, or methotrexate) in host cells in which the vector has been introduced. Suitable selective marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR host cells selected / amplified with methotrexate) and the neo gene (for G418 selection). To express the light and heavy chains of a TNFR2 antibody or a TNFR2 antibody fragment, an expression vector containing polynucleotides encoding the heavy and light chains can be transfected into host cells using standard techniques.

[0307] Polynucleotides encoding modified antagonistic TNFR2 peptides

[0308] In some embodiments, the antagonistic TNFR2 peptide (e.g., the single-chain peptide, antibody, or antibody fragment of the present invention) may be similar to TNFRAB1 or TNFRAB2, but exhibit different characteristics in the sequence of one or more CDRs. In other cases, the peptide of the present invention may be similar to TNFRAB1 or TNFRAB2, but exhibit different characteristics in one or more frame regions. For example, one or more frame regions of TNFRAB1 or TNFRAB2 may be replaced by frame regions of human antibodies. Exemplary frame regions include, for example, the human frame region described in US 7,829,086 and the primate frame region described in EP 1945668; which are incorporated herein by reference. Alternatively, the peptide of the present invention (e.g., a single-chain peptide, antibody, or antigen-binding fragment thereof) may be similar to TNFRAB1 or TNFRAB2, but exhibit differences in the sequence of one or more CDRs and in one or more frame regions. To generate nucleic acids encoding such TNFR2 antagonist peptides, DNA fragments encoding one or more CDRs, such as light chain variable regions and heavy chain variable regions, can be produced by chemical synthesis (e.g., via solid-phase polynucleotide synthesis), in vitro gene amplification (e.g., via polymerase chain reaction), or by replication of polynucleotides in a host organism. For example, nucleic acids encoding the anti-TNFR2 peptides of the present invention can be obtained by amplifying and modifying germline DNA or cDNA encoding light and heavy chain variable sequences to incorporate CDRs of TNFRAB1 or TNFRAB2 into the framework residues of a co-antibody. In some embodiments, the humanized antagonistic TNFR2 antibody may comprise one or more CDRs of TFNRAB1 or variants thereof having at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) with any of these CDRs or sequences containing one to three amino acid substitutions (e.g., conserved or non-conserved substitutions) relative to the CDR sequence of TNFRAB1, and one or more CDRs of FNRAB2 or variants thereof having at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) with any of these CDRs or sequences containing one to three amino acid substitutions (e.g., conserved or non-conserved substitutions) relative to the CDR sequence of TNFRAB2. This can be achieved, for example, by site-directed mutagenesis of germline DNA or cDNA and amplification of the resulting polynucleotides using polymerase chain reaction (PCR) according to an established procedure.Germline DNA sequences of human heavy and light chain variable region genes are known in the art (see, for example, the “VBASE” human germline sequence database; also see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991; Tomlinson et al., J. Mol. Biol. 227:776-798, 1992; and Cox et al., Eur. J. Immunol. 24:827-836, 1994; all incorporated herein by reference). Chimeric nucleic acid constructs encoding human heavy and light chain variable regions containing one or more CDRs of TNFRAB1 and TNFRAB2 can be generated, for example, using established cloning techniques known in the art. In addition, polynucleotides encoding the heavy or light chain variable regions of TNFRAB1 or TFNRAB2 can be synthesized and used as templates for mutagenesis to produce variants as described herein using conventional mutagenesis techniques. Alternatively, DNA fragments encoding the variant can be synthesized directly (e.g., via established solid-phase nucleic acid chemical synthesis procedures).

[0309] Once DNA fragments encoding CDR or VH and VL regions of TNFRAB1, TNFRAB2, TNFRAB1-associated, or TFNRAB2-associated regions are obtained, these fragments can be further manipulated using standard recombinant DNA techniques, such as to convert variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VL or VH is effectively linked to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker.

[0310] This can be achieved, for example, by encoding V H The DNA is effectively linked to another DNA molecule encoding the heavy chain constant region domains (CH1, CH2, CH3 and optionally, CH4) to encode the V-type anti-TNFR2 antibody of the present invention. HThe isolated DNA from the region is converted into a full-length heavy chain gene (and a Fab heavy chain gene). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991), and DNA fragments covering these regions can be obtained by standard PCR amplification. Heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions, and in some embodiments, the IgG1 constant region. For Fab fragment heavy chain genes, the DNA encoding VH can be efficiently ligated to another DNA molecule encoding only the heavy chain CH1 domain.

[0311] The isolated DNA encoding the VL region of the present invention (e.g., a single-chain polypeptide, antibody, or antigen-binding fragment thereof) can be converted into a full-length light chain gene (and a Fab light chain gene) by efficiently linking the DNA encoding VL to another DNA molecule encoding the light chain constant region CL. Sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition (US Department of Health and Human Services, NIH Publication No. 91-3242, 1991)), and DNA fragments covering these regions can be obtained, for example, by amplification in prokaryotic or eukaryotic cells encoding polynucleotides, by PCR amplification, or by chemical polynucleotide synthesis. The light chain constant region can be a κ or λ constant region, but in some embodiments it is a κ constant region. To generate the scFv gene, the DNA segment encoding VH and VL is efficiently linked to another segment encoding a flexible linker, such as a polynucleotide encoding a flexible hydrophilic amino acid sequence (e.g., the amino acid sequence (Gly4Ser)3), allowing V... H and V L The sequence can be expressed as a continuous single-chain protein, where V L and V HThe regions are connected via connectors (see, for example, Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; McCafferty et al., Nature 348:552-554, 1990).

[0312] Recombinant DNA technology can also be used to remove some or all of the DNA encoding one or both of the light or heavy chains, which are not essential for binding TNFR2. The polypeptides of the present invention also comprise molecules expressed from such truncated DNA molecules. Furthermore, bifunctional polypeptides (e.g., bifunctional antibodies) can be generated, wherein one heavy chain and one light chain are derived from TNFRAB1 and / or TNFRAB2, and the other heavy and light chains are specific to antigens other than TNFR2. Such antibodies can be generated, for example, by crosslinking the heavy and light chains of TNFRAB1 and / or TNFRAB2 with the heavy and light chains of a second antibody using standard chemical crosslinking methods (e.g., by forming disulfide bonds). Bifunctional antibodies can also be prepared by expressing nucleic acid molecules engineered to encode bifunctional antibodies in prokaryotic or eukaryotic cells.

[0313] Bispecific antibodies can be generated by mutating amino acid residues in the light chain and / or heavy chain CDRs, i.e., antibodies that bind to TNFR2 and different antigens using the same binding site. In some embodiments, bispecific antibodies binding to two antigens (such as TNFR2 and a second cell surface receptor) can be generated by mutating amino acid residues surrounding the antigen binding site (Bostrom et al., Science 323:1610-1614, 2009). Bifunctional antibodies can be prepared by expressing polynucleotides engineered to encode bispecific antibodies.

[0314] The modified antagonistic TNFR2 antibodies and antibody fragments of the present invention can also be produced by chemical synthesis (e.g., by Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, 111; incorporated herein by reference). Variant antibodies can also be produced using cell-free synthetic platforms (see, e.g., Chu et al., Biochemia No. 2, 2001 (Roche Molecular Biologicals); incorporated herein by reference).

[0315] Host cells used to express antagonistic TNFR2 peptides

[0316] It is possible to express the polypeptides of the present invention (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) in prokaryotic or eukaryotic host cells. In some embodiments, the expression of the polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) is carried out in eukaryotic cells (e.g., mammalian host cells) for optimizing the secretion of properly folded and immunologically active antibodies. Exemplary mammalian host cells for expressing the recombinant polypeptides of the present invention (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) include Chinese hamster ovary (CHO) cells (including DHFR CHO cells described by Urlaub and Chasin (1980, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 77:4216-4220), used with, for example, DHFR selective markers described by Kaufman and Sharp (1982, Mol. Biol. [Molecular Biology] 159:601-621), NSO myeloma cells, COS cells, 293 cells, and SP2 / 0 cells. Other cell types that can be used to express single-chain polypeptides, antibodies, and their fragments include bacterial cells, such as BL-21(DE3) *Escherichia coli* cells, which can be transformed using vectors containing exogenous DNA according to established protocols. Other eukaryotic cells that can be used to express polypeptides include yeast cells, such as auxotrophic strains of *Saccharomyces cerevisiae*, which can be transformed and selectively grown in incomplete media according to established procedures known in the art. When a recombinant expression vector encoding an antibody gene (e.g., a gene encoding one or more CDRs, antibody heavy chains, or antibody light chains) is introduced into mammalian host cells, antibodies are produced by culturing the host cells for a period sufficient to allow expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown.

[0317] Peptides (e.g., single-chain peptides, antibodies, and their antigen-binding fragments) can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to generate portions of the complete antibody, such as Fab fragments or scFv molecules. The invention also includes methods in which the above procedures are modified according to established protocols known in the art. For example, it is desirable to transfect host cells with DNA encoding the light or heavy chain (but not both) of the anti-TNFR2 antibody of the present invention to generate the antigen-binding fragment of the antibody.

[0318] Once the anti-TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, or antigen-binding fragments thereof) are generated through recombinant expression, they can be purified by any method known in the art, such as methods used for purifying immunoglobulin molecules, for example by chromatography (e.g., ion exchange, affinity (particularly affinity for TNFR2 after selection of protein A or protein G) and size fractionation column chromatography), centrifugation, differential solubility, or by any other standard technique used for purifying proteins. Furthermore, the anti-TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, or antigen-binding fragments thereof) can be fused with heterologous peptide sequences described herein or otherwise known in the art to facilitate purification or generate therapeutic conjugates (see “antibody conjugates” below).

[0319] Once separated, the anti-TNFR2 antibody or its antigen-binding fragment can be further purified, for example by high-performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques in Biochemistry and Molecular Biology (Work and Burdon, eds., Elsevier, 1980) [Laboratory Techniques in Biochemistry and Molecular Biology (Work and Burdon eds., Elsevier, 1980)]; cited herein), or by gel filtration chromatography, such as in Superdex. TM The study was conducted on a 75 column (Pharmacia Biotech Ab, Uppsala, Sweden).

[0320] Platform for the generation and affinity maturation of antagonistic anti-TNFR2 peptides

[0321] Epitope mapping of TNFR2 that promotes receptor antagonism

[0322] The antagonistic TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, and their antigen-binding fragments) can be generated by screening a library of functionally functional peptides (e.g., single-chain peptides, antibodies, and their antigen-binding fragments) that can bind to epitopes within TNFR2 that selectively promote receptor antagonism rather than receptor activation. The linear peptides isolated from the TNFR2 protein may not adopt the same three-dimensional conformation as those peptide sequences located within the protein. TNFR2 provides a structurally fixed framework that deviates from the conformation of the individual peptide fragments and enhances these spatial orientations by establishing intramolecular contacts (e.g., hydrogen bonds, dipole-dipole interactions, salt bridges) and by differentially localizing various regions exposed to solvents (depending on the relative hydrophilicity and lipophilicity of these regions) (Mukai et al., Sci. Signal, 3: ra83-ra83, 2010). Conformational constraint of peptide fragments within TNFR2 can be achieved by incorporating amino acid residues of TNFR2 epitopes (e.g., epitopes that promote receptor antagonism) into structurally pre-organized peptide scaffolds (such as cyclic, bicyclic, tricyclic, or tetracyclic peptides). Cyclic and polycyclic peptides, such as these, confine peptide fragments to different three-dimensional conformations. This can be achieved by synthesizing peptide epitopes isolated from TNFR2 using established chemical synthesis methods (e.g., using solid-phase peptide synthesis as described herein) and incorporating cysteine ​​residues into sequences at N-terminal and C-terminal positions or various internal positions within the peptide chain. Incorporation of cysteine ​​residues chemically protected with protecting groups at the thiol moiety can be advantageous, and these protecting groups can be removed under conditions different from those used for removing other protecting groups within the peptide being synthesized and different from those used for assembling the peptide chain. Exemplary orthogonal protecting groups of cysteine ​​thiols include 4-methyltriphenylmethyl and 4-methoxytriphenylmethyl groups, each of which can be removed using diluted trifluoroacetic acid (examples are described, for example, in Isidro-Llobet et al., ChemRev. [Chemical Review], 109:2455-2504, 2009).

[0323] After introducing cysteine ​​residues into a synthetic peptide fragment derived from an epitope within TNFR2, the peptide can be cyclized by treating it with a polyvalent electrophilic agent such as a bis(bromomethyl) or tri(bromomethyl) aromatic derivative. Alternative polyvalent thiol reactive electrophilic agents can be used, such as 1,5-difluoro-2,4-dinitrobenzene, acyclic dibromoalkanes, and others (see, e.g., Jo et al., J. Am. Chem. Soc., 134:17704-17713, 2012; incorporated herein by reference). In some embodiments, it may be advantageous to prevent cysteine ​​residues in the synthetic peptide fragment from participating in the cyclization reaction. For example, it may be desirable to synthesize a polycyclic peptide containing multiple cysteine ​​residues such that only selected cysteine ​​thiols participate in the intramolecular crosslinking process. To prevent the unwanted involvement of these additional Cys thiol groups in the coupling reaction, a simple approach is, for example, to use Fmoc-Cys(Acm) (Fmoc-acetaminomethyl-L-cysteine) during peptide synthesis to introduce the protected Cys residue. Alternatively, Fmoc-Cys(StBu)-OH and / or the corresponding tert-butoxycarbonyl (Boc)-protected amino acid can be used. The Acm or StBu group is not removed during the normal TFA deprotection-cleavage reaction process, but requires oxidation (e.g., iodine I2) in the case of the Acm group or reduction (e.g., β-mercaptoethanol (excess) or 1,4-dithiothreitol (excess)) in the case of the StBu group to obtain the reduced thiol form of the peptide, which can be used directly or subsequently oxidized to the corresponding cysteine ​​peptide. In one embodiment, a peptide containing at least one Cys derivative (such as Cys(Acm) or Cys(StBu)) is used to allow selective deprotection of the Cys-thiol group. The selective deprotection of the Cys-thiol group allows the Cys-thiol group to be used for reactions at desired times, such as after peptide chain assembly is complete and before other residues in the deprotected peptide (see, for example, WO 2008 / 013454; incorporated herein by reference).

[0324] As an example, libraries of cyclic and polycyclic peptides containing combinations of individual fragments isolated from TNFR2 and fragments from different regions of TNFR2 can be synthesized using techniques described above to incorporate cysteine ​​residues at different sites within the peptide scaffold, using different electrophilic crosslinking agents (see, for example, Example 1 and Figure 3, SEQ ID NO: 34-117). These peptides can be immobilized on a solid surface and used with an established procedure using an ELISA-based screening platform to screen for molecules that bind antagonistic TNFR2 peptides (e.g., single-chain peptides, antibodies, or their antigen-binding fragments, such as TNFRAB1 or TNFRAB2). Using this assay, peptides that specifically bind to TNFRAB1 and / or TNFRAB2 with high affinity, for example, thus contain residues within the TNFR2 epitopes that preferentially bind TNFRAB1 and / or TNFRAB2, and these residues can be structurally pre-organized such that they resemble the conformation of the corresponding peptides in natural proteins. The resulting cyclic and polycyclic peptides (e.g., peptides having the sequence of any one of SEQ ID NO: 11, 19, 20, and 34-117, or peptides containing about 10 to about 30 consecutive or discontinuous amino acids between positions 80 and 130 of SEQ ID NO: 7) can be used to screen libraries of antibodies and their antigen-binding fragments to identify the anti-TNFR2 peptides of the present invention. Furthermore, since these bound peptides act as substitutes for epitopes within TNFR2 that promote receptor antagonism, peptides generated using this screening technique (e.g., single-chain peptides, antibodies, and their antigen-binding fragments) can bind to the corresponding epitopes in TNFR2 and are expected to antagonize receptor activity.

[0325] Library for screening antagonistic TNFR2 peptides

[0326] Methods for high-throughput screening of libraries of peptides (e.g., single-chain peptides, antibodies, or antibody fragments) capable of binding to epitopes within TNFR2 (e.g., epitopes presented by peptides having sequences of any one of SEQ ID NO: 11, 19, 20, and 34-117, or epitopes presented by peptides containing about 10 to about 30 consecutive or discontinuous amino acids between positions 80 and 130 of SEQ ID NO: 7) include, but are not limited to, display techniques, including phage display, bacterial display, yeast display, mammalian display, ribosome display, mRNA display, and cDNA display. The use of phage display to isolate ligands that bind to biologically relevant molecules has been reviewed, for example, in Felici et al. (Biotechnol. Annual Rev. 1:149-183, 1995), Katz (Annual Rev. Biophys. Biomol. Struct. 26:27-45, 1997), and Hoogenboom et al. (Immunotechnology 4:1-20, 1998). Several randomized combinatorial peptide libraries have been constructed to select peptides that bind to different targets (e.g., cell surface receptors or DNA) (reviewed by Kay (Perspect. Drug Discovery Des. 2,251-268, 1995); Kay et al. (Mol. Divers. 1:139-140, 1996)). Proteins and multimeric proteins have been successfully displayed as functional molecules by bacteriophages (see EP 0349578 A, EP 4527839 A, EP 0589877 A; Chiswell and McCafferty (Trends Biotechnol. 10, 80-84, 1992). Furthermore, functional antibody fragments (e.g., Fab, single-chain Fv [scFv]) have been expressed (McCafferty et al. (Nature 348:552-554, 1990), Barbas et al. (Proc. Natl. Acad Sci. USA 88:7978-7982, 1991), Clackson et al. (Nature 352:624-628, 1991)). These references are incorporated herein by reference in their entirety.

[0327] (i) Phage display technology

[0328] As an example, phage display technology can be used to screen libraries of peptides (such as single-chain peptides, antibodies, and their antigen-binding fragments) that can bind to functional molecules containing epitopes within TNFR2, such peptides that promote receptor antagonism (e.g., peptides having sequences of any one of SEQ ID NO: 11, 19, 20, and 34-117, and particularly those containing KCRPG motifs such as those in SEQ ID NO: 42, 50, 52-54, and 61-63). For example, polynucleotide libraries encoding single-chain antibody fragments (such as scFv fragments) containing randomized hypervariable regions can be obtained using established procedures (e.g., solid-phase polynucleotide synthesis or error-prone PCR techniques, see McCullum et al. (Meth. Mol. Biol. [Molecular Biology Methods] 634:103-109, 2010; incorporated herein by reference). These randomized polynucleotides can then be incorporated into the viral genome, such that the randomized antibody chains encoded by these genes are expressed on the surface of filamentous phages, for example, through covalent bonds between the antibody chains and capsid proteins (e.g., pIII capsid protein on the surface of M13 phages). This provides a physical link between the genotype and phenotype of the antibody chains. In this way, phage libraries displaying different antibody chains containing random mutations in the hypervariable regions (e.g., peptides having sequences of any one of SEQ ID NO: 11, 19, 20, and 34-117, or SEQ ID NO: 11, 19, 20, and 34-117) can be screened based on the ability of the external antibody chains to bind to TNFR2 epitopes. NO:7 contains a peptide with approximately 10 to approximately 30 consecutive or discontinuous amino acids between positions 80 and 130, and these epitopes have been immobilized to the surface using established procedures. For example, cyclic peptides containing KCRPG motifs (such as those from SEQ ID NO:7) can be immobilized by forming a covalent bond between the peptide and an epitope tag (e.g., biotin) and incubating the peptide in the wells of a microtiter plate. (Those indicated by NO: 42, 50, 52-54, and 61-63) are physically bound to the surface of a microtiter plate previously coated with a complementary tag (e.g., avidin) that binds to a peptide-linked tag with high affinity. Suitable epitope tags include, but are not limited to, maltose-binding proteins, glutathione S-transferases, polyhistidine tags, FLAG-tags, myc-tags, human influenza hemagglutinin (HA) tags, biotin, and streptavidin. Peptides containing epitopes presented by these molecules can be immobilized on surfaces containing such complementary molecules (e.g., maltose, glutathione, nickel-containing complexes, anti-FLAG antibodies, anti-myc antibodies, anti-HA antibodies, streptavidin, or biotin, respectively).In this way, phages can be incubated with a surface containing a fixed TNFR2-derived peptide for a suitable time to allow antibodies to bind to the bound peptide in the presence of an appropriate buffering system (e.g., a system containing physiological salt concentration, ionic strength, and maintained at physiological pH by a buffer). The surface can then be washed (e.g., with phosphate buffer containing 0.1% Tween-20) to remove phages that do not present antibody chains that interact with the TNFR2-derived peptide with an affinity greater than a certain threshold.

[0329] The affinity of the peptides remaining after the initial panning (i.e., screening) step can be adjusted by modifying the conditions of the washing step (e.g., by including a weakly acidic or alkaline component, or by including low concentrations of other TNFR2-derived peptides to compete with the immobilized peptide for antigen binding sites). In this way, the phage population remaining bound to the surface of the microtiter plate after the washing step is enriched with phages that bind TNFR2-derived peptide epitopes that promote receptor antagonism. The remaining phages can then be amplified by eluting the phages from the surface containing these peptides (e.g., by altering the ambient pH, ionic strength, or temperature) to reduce the strength of protein-protein interactions. The isolated phages can then be amplified, for example, by infecting bacterial cells, and the resulting phages can optionally be panned with additional repeated screening to further enrich those phage populations carrying anti-TNFR2 peptides with higher affinity. After these panning stages, phages exhibiting high-affinity antibodies or their antigen-binding fragments can then be isolated, and the genomes of these phages can be sequenced to identify the polynucleotide and polypeptide sequences encoding the antibodies. Phage display techniques such as these can be used to generate antibody chains, such as scFv fragments, tandem scFv fragments, and other antigen-binding fragments of the present invention, which can be used as TNFR2 antagonists. Exemplary phage display schemes for identifying antibody chains and their antigen-binding fragments that bind to specific antigens with high affinity are well established and described, for example, in U.S. Patent Nos. 7,846,892; WO 1997 / 002342; U.S. Patent Nos. 8,846,867 and WO 2007 / 132917; which are incorporated herein by reference. Similar phage display techniques can be used to generate antibody-like scaffolds of the present invention that bind epitopes within TNFR2 that promote receptor antagonism (e.g., 10 (The Fn3 domain) (e.g., an epitope presented by peptides having the sequence of any one of SEQ ID NO: 11, 19, 20, and 34-117, and particularly those containing the KCRPG motif as in SEQ ID NO: 42, 50, 52-54, and 61-63). Exemplary phage display schemes for identifying antibody-like scaffold proteins are described, for example, in WO 2009 / 086116; which is incorporated herein by reference.

[0330] (ii) Cell-based display technology

[0331] Other in vitro display techniques that utilize the linkage between the genotype and phenotype of solvent-exposed peptides (e.g., single-chain peptides, antibodies, or antigen-binding fragments thereof) include yeast and bacterial display. Yeast display techniques are established in the art and are generally advantageous because a large number of antibodies (typically up to 30,000) can be presented on the surface of a single yeast cell (see, for example, Boder et al. (Nat Biotechno. [Nature Biotechnology] 15:553, 1997); incorporated herein by reference). Yeast cells, which are larger than filamentous phages, enable additional screening strategies because flow cytometry can be used to analyze and sort yeast libraries. For example, established procedures can be used to generate libraries of bacterial or yeast cells expressing peptides containing randomized hypervariable regions (such as single-chain peptides, antibodies, or antibody fragments) (see, for example, U.S. Patent Nos. 7,749,501 and 2013 / 0085072; the respective teachings of which are incorporated herein by reference). For example, large yeast cell libraries expressing polynucleotides encoding initial scFv fragments can be prepared using established procedures (de Bruin et al., Nat Biotechnol [Nature Biotechnology] 17:397, 1999; cited herein). Yeast cells expressing these polynucleotides can then be incubated during a panning step with two different fluorescent molecules: one dye that binds to conserved residues within the antibody and thus reflects the amount of antibody displayed; and another dye that fluoresces at a different wavelength and binds to the antigen and thus indicates the amount of antigen bound. In these cases, it is useful to use cyclic or polycyclic peptides containing sequences of any one of SEQ ID NO: 11, 19, 20, and 34-117 (and particularly those containing KCRPG motifs such as those in SEQ ID NO: 42, 50, 52-54, and 61-63), which have optionally been conjugated to epitope tags (e.g., biotin) at residues not intended to interfere with antibody-antigen binding. This allows for the localization of antibody-antigen complexes using fluorescent dyes labeled with complementary tags (e.g., avidin). This provides great flexibility and immediate feedback for selection progress. Antibodies with higher affinity, rather than higher expression levels, can be easily selected by normalizing antibody display levels compared to phage display. In fact, it is possible to distinguish and sort antibodies whose affinity differs by only a factor of two (Van Antwerp and Wittrup (Biotechnol Prog. 16:31, 2000)).

[0332] (iii) Nucleotide display technology

[0333] The visualization technique for in vitro translation of randomized polynucleotide libraries also provides a powerful method for generating the anti-TNFR2 antibody of the present invention. For example, randomized DNA libraries encoding single-chain polypeptides, antibodies, or antigen-binding fragments thereof, containing mutations in designated hypervariable regions, can be obtained, for example, using established PCR-based mutagenesis techniques as described herein. The polynucleotides in these libraries may contain transcriptional regulatory sequences (such as promoters and transcription termination sequences) and may additionally encode sequences that increase the translation rate of the resulting mRNA construct (e.g., IRES sequences, 5' and 3' UTRs, poly-adenylated segments, etc.). These polynucleotide libraries can be incubated in a solution containing RNA polymerase and an appropriate buffer of RNA nucleoside triphosphates (NTPs) to enable the DNA sequences to be transcribed into competent mRNA molecules, which can then be translated by large and small ribosomal subunits, aminoacyl-tRNA molecules, and translation initiation and elongation factors present in solution (e.g., using...). In vitro protein synthesis kit, New England Designed mRNA modifications can enable antibody products to maintain covalent binding to the mRNA template via chemical binding with puromycin (e.g., see Keefe (Curr. Protoc. Mol. Biol. [Contemporary Molecular Biology Protocols], Chapter 24, Unit 24.5, 2001); incorporated herein by reference). Thus, this genotype-phenotype linkage can be used to select antibodies that bind to TNFR2-derived peptides (e.g., peptides having the sequence of any one of SEQ ID NO: 11, 19, 20, and 34-117, and particularly those containing the KCRPG motif as in SEQ ID NO: 42, 50, 52-54, and 61-63), by incubating the mRNA:antibody fusion construct with the peptide immobilized on the surface and panning in a manner similar to phage display technology (see, e.g., WO 2006 / 072773; incorporated herein by reference).

[0334] Optionally, similar techniques can be used to generate the polypeptides of the present invention (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof), except that the polypeptide product can be non-covalently bound to the ribosome-mRNA complex instead of being covalently bound via a puromycin linker. This platform, known as ribosome display, has been described, for example, in U.S. Patent No. 7,074,557; which is incorporated herein by reference. Alternatively, cDNA display can be used to generate antibodies, which is a technique similar to mRNA display, except that cDNA, rather than mRNA, covalently binds the antibody product via a puromycin linker. cDNA display technology offers the advantage of being able to perform screening steps under increasingly stringent conditions, such as adjusting salt concentration, ionic strength, pH, and / or ambient temperature to screen for antibodies with particularly high affinity for TNFR2-derived peptides. This is due to the higher natural stability of double-stranded cDNA compared to single-stranded mRNA. cDNA display screening techniques are described, for example, in Ueno et al. (Methods Mol. Biol. [Molecular Biology Methods], 805:113-135, 2012); which is incorporated herein by reference.

[0335] In addition to generating the anti-TNFR2 peptides of the present invention, in vitro display techniques (e.g., those described herein and those known in the art) also provide methods for improving the affinity of the anti-TNFR2 peptides of the present invention. For example, instead of screening libraries containing single-chain peptides, antibodies, and fragments thereof with completely randomized hypervariable regions, narrower libraries can be screened that are characterized by targeted mutations at specific sites within the hypervariable regions. This can be achieved, for example, by assembling a library of polynucleotides encoding antibodies or their antigen-binding fragments, wherein the polynucleotides encode random mutations only at specific sites within the hypervariable regions. These polynucleotides can then be expressed using techniques such as ribosome display, mRNA display, or cDNA display in organisms such as filamentous phages, bacterial cells, yeast cells, mammalian cells, or in vitro to screen for peptides (such as single-chain peptides, antibodies, or their antigen-binding fragments) that specifically bind to TNFR2 epitopes with improved binding affinity (e.g., peptides containing sequences of any one of SEQ ID NO: 11, 19, 20, and 34-117, and particularly those containing KCRPG motifs such as those in SEQ ID NO: 42, 50, 52-54, and 61-63). Yeast display is particularly suitable for affinity maturation and has previously been used to increase the affinity of single-chain antibodies to 48 fM K. D (Boder et al. (Proc NatlAcad Sci USA [Proceedings of the National Academy of Sciences of the United States of America] 97:10701, 2000)).

[0336] Additional in vitro techniques that can be used for the generation and affinity maturation of the antagonistic TNFR2 antibodies of the present invention include combinatorial libraries of peptides (such as single-chain peptides, antibodies, or antigen-binding fragments thereof) that screen for functional molecules capable of specifically binding to TNFR2-derived peptides (e.g., peptides having the amino acid sequence of any one of SEQ ID NO: 11, 19, 20, and 34-117, such as SEQ ID NO: 42, 50, 52-54, and 61-63). Combinatorial peptide libraries, such as antibody or antibody fragment libraries, can be obtained, for example, by expressing polynucleotides encoding randomized hypervariable regions of antibodies or antigen-binding fragments thereof in eukaryotic or prokaryotic cells. This can be achieved, for example, using gene expression techniques described herein or known in the art. Heterogeneous mixtures of antibodies can be purified, for example, by protein A or protein G selection, size fractionation column chromatography, centrifugation, differential solubility, and / or by any other standard techniques used for protein purification. Libraries of such heterogeneous mixtures of antibodies can be screened, for example, by incubating them with a TNFR2-derived peptide immobilized on a surface (e.g., a peptide immobilized on the surface of a solid-phase resin or in the wells of a microtiter plate) for a time sufficient to allow antibody-antigen binding. Unbound antibodies or fragments thereof can be removed by washing the surface with a suitable buffer (e.g., a solution buffered at physiological pH (about 7.4) and containing physiological salt concentration and ionic strength, and optionally containing a detergent such as TWEEN-20). The remaining bound antibodies can then be detected, for example, using an ELISA-based assay (see, e.g., U.S. Patent No. 4,661,445; incorporated herein by reference).

[0337] Further techniques for screening combinatorial libraries of peptides that specifically bind to TNFR2-derived peptides (e.g., peptides containing the amino acid sequences of any one of SEQ ID NO: 11, 19, 20, and 34-117, such as SEQ ID NO: 42, 50, 52-54, and 61-63) include bead-to-compound libraries for screening single-chain peptides or antibody fragments. The single-chain peptides and antibody fragments can be chemically synthesized on solid beads (e.g., using established separation-and-polymerization solid-phase peptide synthesis protocols) composed of hydrophilic, water-swellable materials, such that each bead displays a single antibody fragment. The heterogeneous bead mixture can then be incubated with a TNFR2-derived peptide, optionally labeled with a detectable moiety (e.g., a fluorescent dye) or conjugated to an epitope tag (e.g., biotin, avidin, a FLAG tag, or a HA tag), which can later be detected by treatment with complementary tags (e.g., avidin, biotin, an anti-FLAG antibody, or an anti-HA antibody, respectively). Beads containing antibody fragments that specifically bind TNFR2-derived peptides (e.g., peptides containing the amino acid sequences of any one of SEQ ID NO: 11, 19, 20, and 34-117, such as SEQ ID NO: 42, 50, 52-54, and 61-63) can be identified by analyzing the fluorescence properties of the beads followed by incubation with a fluorescently labeled antigen or complementary tag (e.g., by confocal fluorescence microscopy or by fluorescence-activated bead sorting; see, for example, Muller et al. (J. Biol. Chem., 16500-16505, 1996); incorporated herein by reference). Therefore, beads containing antibody fragments that specifically bind TNFR2-derived peptides can be separated from beads that do not contain high-affinity antibody fragments. The sequence of antibody fragments that specifically bind to TNFR2-derived peptides can be determined using techniques known in the art, including, for example, Edman degradation, tandem mass spectrometry, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), nuclear magnetic resonance (NMR), and 2D gel electrophoresis (see, for example, WO 2004 / 062553; incorporated herein by reference).

[0338] Negative screening of peptides

[0339] In addition to the methods described above for screening single-chain polypeptides, antibodies, or antibody fragments containing KCR or KCRPG motifs that specifically bind to epitopes derived from human TNFR2, negative screening can also be performed to eliminate single-chain polypeptides, antibodies, or antibody fragments that may also bind to epitopes containing KCSPG sequences. For example, a mixture of single-chain polypeptides, antibodies, or antibody fragments isolated by any of the above screening techniques can be screened for single-chain polypeptides, antibodies, or antibody fragments that also specifically bind to peptides containing KCSPG motifs derived from human TNFR2 (such as peptides containing residues 48-67 of SEQ ID NO:7 (QTAQMCCSKCSPGQHAKVFC, SEQ ID NO:18)). This can be achieved using any of the above methods or variations thereof, for example, by making the screened single-chain polypeptides, antibodies, or antibody fragments those previously identified as capable of specifically binding to one or more residues containing a KCRPG sequence (e.g., at least a KCR sequence). Exemplary techniques for negative screening include those described above or known in the art, such as phage display, yeast display, bacterial display, ribosome display, mRNA display, cDNA display, or surface-based combinatorial library screening (e.g., in ELISA format). Such screening techniques represent a useful strategy for identifying antagonistic TNFR2 single-chain peptides, antibodies, and antibody fragments, as peptides capable of binding to TNFR2 epitopes containing one or more residues of the KCSPG and KCRPG sequences have been shown to be lacking or have significantly reduced antagonistic activity.

[0340] Immunity in non-human mammals

[0341] Another strategy that can be used to generate the antagonistic TNFR2 antibody or antibody fragment of the present invention includes immunization of non-human mammals. Examples of non-human mammals that can be immunized to generate the antagonistic TNFR2 antibody and fragment of the present invention include rabbits, mice, rats, goats, guinea pigs, hamsters, horses, and sheep, as well as non-human primates. For example, established procedures for immunizing primates are known in the art (see, for example, WO 1986 / 6004782; incorporated herein by reference). Immunization represents a robust method for generating monoclonal antibodies by utilizing the antigen specificity of B lymphocytes. For example, monoclonal antibodies can be prepared using the Kohler-Millstein procedure (e.g., described in EP 0110716; incorporated herein by reference), in which spleen cells from non-human animals (e.g., primates) are immunized with peptides that present TNFR2-derived antigens that promote receptor antagonism (e.g., peptides containing the amino acid sequence of any one of SEQ ID NO: 11, 19, 20, and 34-117, such as SEQ ID NO: 42, 50, 52-54, and 61-63). Clonal expansions of B lymphocytes generated by immunization can be isolated from the animal's serum and subsequently fused with myeloma cells to form hybridomas. Hybridomas are particularly useful agents for antibody production because these immortalized cells can provide a persistent supply of antigen-specific antibodies. Antibodies from such hybridomas can then be isolated using techniques known in the art, for example, by affinity chromatography using reagents such as protein A or protein G to purify the antibodies from the cell culture medium.

[0342] Antagonistic TNFR2 peptide conjugates

[0343] Before administering the antagonistic TNFR2 peptide of the present invention to a mammalian subject (e.g., a human), it is desirable to conjugate the peptide (e.g., a single-chain peptide, an antibody, or an antigen-binding fragment thereof) to a second molecule, for example, to modulate the activity of the peptide in vivo. Any of a variety of established conjugation strategies well known in the art can be used to conjugate the antagonistic TNFR2 single-chain peptide, antibody, or fragment thereof to another molecule at the N-terminus or C-terminus of the light or heavy chain of the peptide. Examples of reactive functional group pairs that can be used to covalently tether the antagonistic TNFR2 single-chain peptide, antibody, or fragment thereof to another molecule include, but are not limited to, thiol pairs, carboxylic acid and amino groups, ketone and amino groups, aldehyde and amino groups, thiols and α,β-unsaturated moieties (such as maleimide or dehydroalanine), thiols and α-haloamides, carboxylic acids and hydrazides, aldehydes and hydrazides, and ketones and hydrazides.

[0344] Antagonistic TNFR2 single-chain peptides, antibodies, and fragments thereof can be directly covalently attached to another molecule via chemical conjugation as described herein. Alternatively, fusion proteins containing antagonistic TNFR2 single-chain peptides, antibodies, and fragments thereof can be recombinantly expressed from cells (e.g., eukaryotic or prokaryotic cells). This can be achieved, for example, by incorporating a polynucleotide encoding the fusion protein into the cell's nuclear genome (e.g., using techniques described herein or known in the art). Optionally, the single-chain peptides, antibodies, and fragments thereof of the present invention can be linked to a second molecule by forming a covalent bond between the antibody and a linker. The linker can then be subsequently conjugated to another molecule, or the linker can be conjugated to another molecule prior to being linked to the anti-TNFR2 single-chain peptide, antibody, or fragment thereof. Examples of linkers that can be used to form conjugates include peptide linkers, such as those containing naturally occurring or non-naturally occurring amino acids. In some embodiments, it is desirable to include D-amino acids in the linker because these residues are not present in naturally occurring proteins and are therefore more resistant to degradation by endogenous proteases. Fusion proteins containing peptide linkers can be prepared using chemical synthesis techniques (such as those described herein) or by recombinant expression of the polynucleotide encoding the fusion protein in cells (e.g., prokaryotic or eukaryotic cells). Linkers can be prepared using a variety of strategies well known in the art and depending on the reactive components of the linker, and can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under alkaline conditions, oxidation, disulfide bond reduction, nucleophilic cleavage, or organometallic cleavage (Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012).

[0345] Drug-peptide conjugate

[0346] The antagonistic TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, or antigen-binding fragments thereof) can be additionally conjugated, mixed, or administered separately with therapeutic agents (such as cytotoxic molecules). The conjugates of the present invention are suitable for the treatment or prevention of diseases associated with abnormal cell proliferation, such as cancers described herein. Exemplary cytotoxic agents that can be conjugated, mixed, or administered separately with antagonistic TNFR2 peptides include, but are not limited to, antitumor agents such as: acivitine; ararubicin; acodazole hydrochloride; acroline; adoron; doxorubicin; interleukin; hexamethylmelamine; ambroxol; amphetamine acetate; aminoglutethimide; acridine; anastrozole; atrazolyl; asparaginase; triamcinolone; azacitidine; azatiprine; azomycin; palmastat; benzoxetine; bicalutamide; bisamyl hydrochloride; bisamyl dimethylsulfate; bisamylpyridinium sulfate; buquina sodium; brompirimidine; busulfan; actinomycin C; capprotestone; camptothecin; carvacrol; carbetin; carboplatin; carmustine; carrarubicin hydrochloride; carzeolacin; sildenafil; Chloramic acid mustard; silromycin; cisplatin; cladribine; cobustatin α-4; cristatin stomate; cyclophosphamide; cytarabine; dacarbazine; daca(n-[2-(dimethyl-amino)ethyl]acridin-4-carboxamide); dextrin; daunorubicin hydrochloride; donomycin; decitabine; dextromethorphan; dezaguanine; dezaguanine mesylate; diaciconone; docetaxel; dolalastatin; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; drotaloferrin propionate; dazomycin; edaraxacum; efornithine hydrochloride; roserine; etharucin; enloplatin; enprofen; epipiperidine; epirubicin hydrochloride; irbuprofen; isopycin hydrochloride; estradiol; estradiol sodium phosphate; estanidazole; ethyl iodized oil 131; Etoposide; Etoposide phosphate; Chlorpheniramine; Fazodazole hydrochloride; Fazalabin; Feniveryl Aamine; Fluorouracil; Fludarabine phosphate; Fluorouracil; 5-fdump; Flucitabine; Phosphorione; Fostracin sodium; Gemcitabine; Gemcitabine hydrochloride; 198; Camptothecin; Hydroxyurea; Idarubicin hydrochloride; Ifosfamide; Imofocin; Interferon α-2a; Interferon α-2b; Interferon α-nl; Interferon α-n3; Interferon β-1a; Interferon γ-1b; Isopropylplatin; Irinotecan hydrochloride, Lanreotide acetate; Letrozole; Leuprorelin acetate; Riazol hydrochloride; Lometroxodium sodium; Lomustine; Loxoanthraquinone hydrochloride; Masrophenol; Metansine; Nitrogen mustard hydrochloride; Medroxyprogesterone acetate ; Methionyl acetate; Melphalan; Minoril; Mercaptopurine; Methotrexate; Methotrexate sodium; Chlorpheniramine; Metotepazole; Mitolidomide; Mitoxicin; Mitoxazole; Mitomarcin; Mitosperidone; Mitosperidone; Mitosperidone hydrochloride; Mycophenolic acid; Nocodazole; Nopramine; Omaplatin; Oxysulex; Paclitaxel; Peasparin; Pelibrycin; Pendimethicone; Peploycinsulfate; Pephosphatamide;Piperabromide; Piperabromide; Pirroanthraquinone Hydrochloride; Procamycin; Promethene; Porphyrom sodium; Methylmitromycin; Prenimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurane; Rhizomycin; Rhizomycin D; Lipoadenosine; Rogulam; Safungo; Safungo; Semustine; Citric acid; Spofos sodium; Sparmycin; Germanium spiroamine Hydrochloride; Spiromustine; Spiroplatin; Streptomycin; Streptozotocin; Strontium chloride (Sr) 89; sulfonamide; tamethasone; taxane; taxane diterpenes; tecogallan sodium; tegafur; teloanthraquinone hydrochloride; temopofen; teniposide; tiroxicam; testrolide; thioimidazoline; thioguanine; thiotepa; thymitaq; thiazolinone; tilazamine; raltitrexed; top53; topotecan hydrochloride, toremifene citrate; triptorene acetate; tricerebroside phosphate; trimethotraxa; trimethotraxa glucuronide; triptorelin; tobaccochloride hydrochloride; uramoxicillin Ting; Uretipatide; Vaportide; Vertepofen; Vincristine; Vincristine Sulfate; Vincristine Sulfate; Vincristine Sulfate; Vincristine Sulfate; Vinpicidin Sulfate; Vincristyl Sulfate; Vinrosine Sulfate; Vinorelbine Tartrate; Vinrodine Sulfate; Vinorelbine Sulfate; Vorticillin Sulfate; Zorbazoline; Zorbazoline; Netostatin; Zorbazoline Hydrochloride; 2-Chlorodeoxyadenosine; 2'-Deoxymyomycin; 9-Aminocamptothecin; Raltitrexed; N-Prophylyn-5,8-Didezofolate; 2-Chloro- 2'-Arabinose-Fluoro-2'-Deoxyadenosine; 2-Chloro-2'-Deoxyadenosine; Anisin; Trichostatin A; hPRL-G129R; CEP-751; Linolamine; Thiamine Mustard Gas; Nitrogen Mustard Gas (Dichloromethyldiethylamine); Cyclophosphamide; Melphalan; Chlorobutyrate Mustard; Ifosfamide; Busulfan; N-Methyl-N-nitrosourea (MNU); N,N'-Bis(2-chloroethyl)-N-nitrosourea (BCNU); N-(2-chloroethyl)-N' Cyclohexyl-N-nitrosourea (CCNU); N-(2-chloroethyl)-N'-trans-4-methylcyclohexyl-N-nitrosourea (MeCCNU); N-(2-chloroethyl)-N'-(diethyl)ethylphosphonate-N-nitrosourea (formustine); streptozotocin; dacarbazine (DTIC); mitozopranzamide; temozolomide; thiotepa; mitomycin C; AZQ; adolaxin; cisplatin; carboplatin; omaliplatin; oxaliplatin; C1-973; DWA 2114R; JM216; JM335; Bis(Platinum); Raltitrexed; Azacitidine; Cytarabine; Gemcitabine; 6-Mercaptopurine; 6-Thioguanine; Hypoxanthine; Teniposide; 9-Aminocamptothecin; Topotecan; CPT-11; Doxorubicin; Doxorubicin; Epirubicin; Idarubicin; Mitoxantrone; Loxoantrone; Actinomycin D; Acridine; Pyrazoline Acridine; All-trans Retinol; 14-Hydroxy-trans-Retinol; All-trans Retinic Acid; N-(4-Hydroxyphenyl)retinoin; 13-cis-Retinic Acid; 3-MethylTTNEB;9-cis-retinoic acid; fludarabine (2-F-ara-AMP); or 2-chlorodeoxyadenosine (2-Cda).

[0347] Other therapeutic compounds that can be conjugated, mixed, or administered separately with the antagonistic TNFR2 single-chain polypeptide, antibody, or antigen-binding fragment thereof of the present invention for the treatment, prevention, or investigation of the progression of diseases associated with abnormal cell proliferation include, but are not limited to, cytotoxic agents such as 20-pi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; acylfuvone; adenocyclopentol; adorexin; aldehyde interleukin; ALL-TK antagonists; hexamethylmelamine; ammustine; amidox; and ammonia Fostin; Aminolevulinic acid; Amrubicin; Acridine; Anagrelide; Anastrozole; Andrographolide; Angiogenesis inhibitor; Antagonist D; Antagonist G; Amrexol; Anti-dorsal morphogenetic protein-1; Anti-androgen, prostate cancer; Anti-estrogen; Antitumor; Antisense oligonucleotide; Afenidemycin glycine; Apoptosis gene regulator; Apoptosis regulator; Depurine nucleic acid; ara-CDP-DL-PTBA; Arginine deaminase; Asulacrine; Atamitan Amustine; Axinastatin 1; Axinastatin 2; Axinastatin 3; Azasetron; Azatoxin; Diazotyrosine; Baccatin III derivative; Balanol; Balastatin; BCR / ABL antagonist; Benzodihydroporphyrin; Benzoylstaurosporine; β-lactam derivative; β-alethine; Subaclavin B; betulinic acid; bFGF inhibitor; bicalutamide; bisulfanilamide; diazinon; bistratene A; bizole; breflate; bleomycin A2; bleomycin B2; bromipridine; budotitanol; butylcysteine ​​sulfinylimide; calcipotriol; carbostatin C; camptothecin derivatives (e.g., 10-hydroxycamptothecin); canarypox IL-2; capecitabine; formamide-amino-triazole; carboxy-amino-triazole; CaRest M3; CARN 700; chondroderma-derived inhibitors; cartilage-derived inhibitors; casein kinase inhibitors (ICOS); strychnine; cetrorexone; dihydroporphyrin; chloroquine sulfonamide; cisporin; cladribine; clomiphene analogs; clotrimazole; collinsin A; collinsin B; compressoritine A4; compressoritine analogs; conagenin; crambescidin 816; cristatin; cyclosporine cyclic peptide 8; cyclosporine cyclic peptide A derivative; curazin A; cyclopentafenone; cycloplatam; cypemycin; cytarabine octadecyl phosphate; cytolysin; hexestrol phosphate; dacizumab; decitabine; dehydrometasine B; 2'-deoxymyotrophic acid (DCF); diloxacin;Dextromethorphan; Dextrorazone; Dextrovertamycin; Dexaquinone; Membranosin B; Didox; Diethylnorspermide; Dihydro-5-azacytidine; Dihydropaclitaxel; 9-dioxazolidin; Bifenoxustin; Spongolactone; Diococcal; Dolasetron; Deoxyfluorouridine; Droxithiophene; Drocannabinol; Docalciferol SA; Ibuprofen; Icomustine; Edifosine; Ezocurumab; Elonisecide; Elimexene; Ethiributyrol; Epirubicin; Epothilone (A, R=H); B, R=Me); epithilone; iridarone; estradiol; estrogen agonists; estrogen antagonists; estradiol; etoposide; etoposide 4'-phosphate; exemestane; faldazole; fazalabin; fenivel-Amin; filgrastim; finasteride; flavonoid pyridoxine; flucalostemma; fluasterone; fludarabine; fluorodaunorunicin hydrochloride hydrochloride; phentermine; formestan; fostracin; formustin; gadotisarine; gallium nitrate; gallotaline; ganirilac; gelatinase inhibitor; gemcitabine; glutathione inhibitor; hepsulfam; thiamin; hexamethylenediacetamide; homoharringtonine (HHT); hypericin; ibandronic acid; idarubicin; edoxifene; ixorphine; imofoxine; ilomastal; imidazopiclone; imiquimod. Special; Immunostimulatory peptides; Insulin-like growth factor-1 receptor inhibitors; Interferon agonists; Interferon; Interleukin; Iodobenzylguanidine; Iodomycin; Embomiol; Irinotecan; Iloprap; Isopridine; Isobengazole; Isogamous sponge B; Itasetron; Jasplakinolide; Kahalalide F; Laminin N; Lanreitide; Lenamicin ycin); Levofloxacin; Lentinan sulfate; Leptolstatin; Letrozole; Leukemia inhibitory factor; Leukocyte alpha interferon; Leuprorelin + estrogen + progesterone; Leuprorelin; Levamisole; Lirozole; Linear polyamine analogs; Lipophilic disaccharide peptides; Lipophilic platinum compounds; Lissoclinamide 7; Lobaplatin; Earthworm phospholipids; Lometroxone; Clonidamine; Loxoanthraquinone; Lovastatin Loxoribine; Letotecan; Tisalinlutriol; Lisotheophylline; Cleavage peptide; Metansine; N-acetylmonase A; Marimasta; Masoroxyfen; Mammary serine; Matrix dissolving factor inhibitor; Matrix metalloproteinase inhibitor; Minoliqui; Meparon; Metetrelin; Methionin; Metoclopramide; MIF inhibitor; Mifepristone; Mitefocin; Mililastine; Mismatched double-stranded RNA; Glucosamine; Mitoguanidine hydrazone; Dibromoceroxyl; Mitomycin analogue; Mitonaphthylamine;Mitox toxin fibroblast growth factor-saponin; Mitoxantrone; Mofarotin; Moraxetin; Monoclonal antibody, Human chorionic gonadotropin; Monophosphoryl lipid A+ Mycobacterium cell wall SK; Moparidarol; Multidrug resistance gene inhibitor; Therapeutic multi-tumor inhibitor 1; Mustard anticancer agent; Indian Ocean sponge (mycaperoxide) B; Mycobacterium cell wall extract; Myriaporone; N-acetyldinalin; N-substituted benzamide; Nafarelin; Naretepen; Naloxone + Pentazocine; Napavin; Nafo Naphterpin; Natostin; Nedaplatin; Nemorubicin; Neridonic acid; Neutral endopeptidase; Nilumet; Nisamycin; Nitric oxide regulator; Nitrogen oxide antioxidant; Nitrullyn; O6-benzylguanine; Octreotide; Oicenone; Oligonucleotides; Ondansetron; Ondansetron; Oracin; Oral cytokine inducers; Omaplatin; Oxaliplatin; Oxarubicin; Oxamycin; Paclitaxel analogs Paclitaxel derivatives; Palauamine; Palmitoylrhizoxin; Pamidronitrol; Panomiphen; Paracoccin; Pazorptin; Pegaspargase; Pedesine; Sodium lignosulfonate; Pentostatin; Pentrozole; Perfluorobromoethane; Pephosphatamide; Perfrole; Benzobenzamide; Phenylacetylacetate; Phosphatase inhibitors; Sapelelin; Pilocarpine hydrochloride; Pirarubicin; Pyrithioxine; Placetin A; Placetin B; Plasminogen activator inhibitors Formulations; Platinum complexes; Platinum compounds; Platinum-triamine complexes; Podophyllotoxin; Porphyrin sodium; Methylmitomycin; Propylbis-Acridinone; Prostaglandin J2; Proteasome inhibitors; Protein A-based immunomodulators; Protein kinase C inhibitors; Protein kinase C inhibitors, microalgae; Protein tyrosine phosphatase inhibitors; Purine nucleoside phosphorylase inhibitors; Hydroxyalkaloid; Pyrazoline acridine; Pyridoxal-modified hemoglobin polyoxyethylene conjugates; RAF antagonists; Raltitrexed; Ramosetron; RAS farnesyltransferase; RAS inhibitors; RAS-GAP inhibitors; Desmethylretepritine; Rhenium Re 186-hydroxyethylphosphonic acid; radicin; ribonuclease; retinamide (RII); roguimine; roxithromycin; romotide; roquimetac; rubiginone B1; ruboxyl; safungo; saintopin; SarCNU; muscle chlorophyll A; saxaglastine; Sdi 1 mimic; semustine; senescence-derived inhibitor 1; positive oligonucleotide; signal transduction inhibitor; signal transduction regulator; single-chain antigen-binding protein;Cizonan; Sobuzosen; Borocarb sodium; Sodium phenylacetate; Solvorol; Somatostatin-binding protein; Sonamamine; Phosphoaspartic acid; Spiramycin D; Spiromustin; Senna; Spongin 1; Squalamine; Stem cell inhibitor; Stem cell division inhibitor; Stipipamide; Mesenchymal lysin inhibitor; Sulphinosine; Potent vasoactive intestinal peptide antagonist; Suradista; Suramin; Sorghum extract; Synthetic glycosaminoglycans; Tamoxifen methyl iodide; Taurolimustine; Tazarotene; Tecogallan sodium; Tegafur; Tellurapyrylium; Telomerase inhibitor; Temopofen; Temozolomide; Teniposide; Tetrachlorodecoxide; Tetrazomine; Pyruvate; Thalidomide; Ticcolamine; Thrombopoietin; Thrombopoietin mimics; Thymofasin; Thymopoietin receptor agonists; Thymotrehnem; Thyroid-stimulating hormone; Ethylcholinesterone (Tyrosine) Ethyl etiopurpurin; Tirazamine; Dicrenecrolein dichloride; Topotecan; Topsentin; Toremifene; Totipotent stem cell factor; Translation inhibitors; Retinoic acid; Triacetyluridine; Tricerebroside; Trimethoprim; Triptorelin; Tropanetron; Torotorandil; Tyrosine kinase inhibitors; Tyrosine phosphorylation inhibitors; UBC inhibitors; Ubenimex; Urogenital sinus-derived growth inhibitors; Urokinase receptor antagonists; Vaportide; Variolin B; Vector systems, Red blood cell gene therapy; Veraresole; Veratramine; Verdins; Vertepofen; Vinorelbine; Vinxaltine; Vitaxin; Voclonolone; Zanoteron; Zonipridine; Subbenzylidene Vitamin C; and Nettostatin ester.

[0348] Labeled anti-TNFR2 peptide

[0349] In some embodiments, for purification or detection purposes, a single-chain polypeptide, antibody, or antigen-binding fragment of an antagonistic TNFR2 may be conjugated to another molecule (e.g., an epitope tag). Examples of such molecules that can be used in protein purification include those that present structural epitopes that can be recognized by a second molecule. This is a common strategy for protein purification via affinity chromatography, in which molecules are immobilized on a solid support and exposed to a heterogeneous mixture containing a target protein conjugated to a molecule capable of binding to the immobilized compound. Examples of epitope-tagged molecules that can be conjugated to an antagonistic TNFR2 single-chain polypeptide, antibody, or fragment thereof for molecular recognition purposes include, but are not limited to, maltose-binding proteins, glutathione S-transferases, polyhistidine tags, FLAG tags, myc-tags, human influenza hemagglutinin (HA) tags, biotin, and streptavidin. Conjugates containing epitopes presented by these molecules can be recognized by complementary molecules such as maltose, glutathione, nickel-containing complexes, anti-FLAG antibodies, anti-myc antibodies, anti-HA antibodies, streptavidin, or biotin, respectively. For example, the antagonistic TNFR2 single-chain polypeptide, antibody, or fragment thereof of the present invention, which has been conjugated with epitope tags from a complex mixture of other proteins and biomolecules (e.g., DNA, RNA, carbohydrates, phospholipids, etc.), can be purified by treating the mixture with a solid-phase resin containing complementary molecules that can selectively recognize and bind to epitope tags of the antagonistic anti-TNFR2 antibody or fragment thereof. Examples of solid-phase resins include agarose beads, which are compatible with the purified product in aqueous solution.

[0350] The antagonistic TNFR2 peptide of the present invention can also be covalently attached to fluorescent molecules, for example, to detect antibodies or their antigen-binding fragments by fluorescence assays and / or by direct visualization using a fluorescence microscope. Exemplary fluorescent molecules that can be conjugated to the peptides of the present invention include green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, red fluorescent protein, phycoerythrin, allophycocyanin, Hurst, 4',6-diamidinyl-2-phenylindole (DAPI), propidium iodide, fluorescein, coumarin, rhodamine, tetramethylrhodamine, and cyanine. Further examples of fluorescent molecules suitable for conjugation to the peptides of the present invention are well known in the art and have been described in detail, for example, in U.S. Patent Nos. 7,417,131 and 7,413,874, each of which is incorporated herein by reference.

[0351] Antagonistic TNFR2 peptides containing fluorescent molecules are particularly useful for monitoring the cell surface localization characteristics of peptides of the present invention, such as single-chain peptides, antibodies, and fragments thereof. For example, cultured mammalian cells (e.g., T-reg cells) can be exposed to antagonistic TNFR2 single-chain peptides, antibodies, or fragments thereof of the present invention covalently bound to fluorescent molecules, and these cells can then be analyzed using conventional fluorescence microscopy techniques known in the art. Confocal fluorescence microscopy is a particularly powerful method for determining the cell surface localization of those antagonistic anti-TNFR2 single-chain peptides, antibodies, or fragments thereof bound to the outer surface of the cell membrane, because individual planes of the cell can be analyzed to distinguish antibodies or fragments thereof that have been internalized into the cell, for example, through receptor-mediated endocytosis. Furthermore, cells can be treated with antagonistic TNFR2 antibodies conjugated to fluorescent molecules emitting visible light at specific wavelengths (e.g., fluorescein fluorescein fluoresce at about 535 nm) and other fluorescent molecules known to be localized to specific sites on the surface of T-reg cells and fluoresce at different wavelengths (e.g., molecules localized to CD25 and fluoresce at about 599 nm). The resulting emission patterns can be visualized using confocal fluorescence microscopy, and images from both wavelengths can be combined to reveal information about the location of antagonistic TNFR2 single-chain peptides, antibodies, or their antigen-binding fragments on the surface of T-reg cells relative to other receptors.

[0352] For the purpose of detecting and visualizing antagonistic anti-TNFR2 peptides, bioluminescent proteins can also be incorporated into fusion proteins. Bioluminescent proteins (such as luciferase and jellyfish luminescent proteins) emit light as part of a chemical reaction with a substrate (e.g., luciferin and coelenterate). Exemplary bioluminescent proteins suitable for use as diagnostic sequences and methods of their use are described, for example, in U.S. Patent Nos. 5,292,658; 5,670,356; 6,171,809 and 7,183,092, each of which is incorporated herein by reference. Antagonistic TNFR2 single-chain peptides, antibodies, or fragments thereof labeled with bioluminescent proteins are useful tools for detecting the antibodies of the present invention after in vitro assays. For example, the presence of an antagonistic TNFR2 antibody already conjugated with a bioluminescent protein in a complex mixture of other proteins can be detected by separating the components of a mixture using gel electrophoresis methods known in the art (e.g., natural gel analysis) and then transferring the separated proteins onto a membrane for protein blotting. The detection of antagonistic TNFR2 antibodies in mixtures of other proteins can be achieved by treating the membrane with an appropriate luciferase substrate and then visualizing the mixture of proteins on the membrane using an established protocol.

[0353] The polypeptides of the present invention (e.g., single-chain polypeptides, antibodies, and fragments thereof) can also be conjugated to molecules containing a radioactive nucleus, allowing detection of the antibodies or fragments thereof by analyzing the radioactive emission pattern of the nucleus. Alternatively, antagonistic TNFR2 antibodies or fragments thereof can be directly modified by incorporating a radioactive nucleus into the antibody during protein preparation. Methionine (… 35 S), nitrogen ( 15 N) or carbon ( 13 C) Radioactive isotopes are incorporated into the antibodies or fragments thereof of the present invention. Optionally, tyrosine derivatives containing radioactive halogens can be incorporated into antagonistic TNFR2 antibodies or fragments thereof by culturing bacterial cells, for example, in a medium supplemented with radiolabeled tyrosine. It has been shown that radioactively halogenated tyrosine at the C2 position of a phenol system can be rapidly incorporated into elongated polypeptide chains using in vivo endogenous translation enzymes (US Patent No. 4,925,651; incorporated herein by reference). The halogens include fluorine, chlorine, bromine, iodine, and astatine. Furthermore, antagonistic TNFR2 antibodies or fragments thereof can be modified by functionalizing the antibodies or fragments thereof of the present invention with radioactive isotopes after isolation and purification from cell cultures. Halogens represent a class of isotopes that can be readily incorporated into purified proteins by aromatic substitution at tyrosine or tryptophan, for example via reaction of one or more of these residues with an electrophilic halogen substance. Examples of radioactive halogen isotopes include 18 F, 75 Br、 77 Br、 122 I, 123 I, 124 I, 125 I, 129 I, 131 I or 211 At.

[0354] Another alternative strategy for incorporating radioisotopes is to covalently link a chelating group to an antagonistic anti-TNFR2 peptide (e.g., a single-chain peptide, an antibody, or a fragment thereof). The chelating group can be covalently attached to the antagonistic TNFR2 peptide by linking it to a reactive functional group (such as a thiol, amino group, alcohol, or carboxylic acid). The chelating group can then be modified to contain any of a variety of metal radioisotopes, including but not limited to radionuclides such as… 125 I, 67 Ga、 111 In、 99 Tc, 169 Yb、 186 Re、 123 I, 124 I,125 I, 131 I, 99m Tc, 111 In、 64 Cu、 67 Cu、 186 Re、 188 Re、 177 Lu、 90 Y、 77 As、 72 As、 86 Y、 89 Zr、 211 At、 212 Bi、 213 Bior 225 Ac.

[0355] In some embodiments, it is desirable to combine the polypeptides of the present invention (e.g., single-chain polypeptides, antibodies, or fragments thereof) with metal ions (such as Gd) capable of binding from heavy elements or rare earth ions. 3+ Fe 3+ Mn 3+ or Cr 2+ The chelating group is covalently conjugated to the ion. Conjugates containing chelating groups coordinated with such paramagnetic metals are useful in MRI imaging applications. Paramagnetic metals include, but are not limited to, chromium (III), manganese (II), iron (II), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III), and ytterbium (III). In this way, antagonistic TNFR2 antibodies can be detected by MRI spectroscopy. For example, an antagonistic TNFR2 antibody or a fragment thereof conjugated with a chelating group bound to a paramagnetic ion can be given to a mammalian subject (e.g., a human patient) to monitor the distribution of the antibody after administration. This can be achieved by administering the antibody to the patient via any of the routes of administration described herein (e.g., intravenous) and subsequently recording the location of the administered antibody on MRI according to an established protocol.

[0356] To improve the solubility and stability of proteins in aqueous solutions, antagonistic TNFR2 peptides (e.g., single-chain peptides, antibodies, or fragments thereof) can be additionally conjugated to other molecules. Examples of such molecules include PEG, PSA, bovine serum albumin (BSA), and human serum albumin (HSA), etc. For example, antagonistic TNFR2 antibodies or fragments thereof can be conjugated to carbohydrate portions to evade detection by the immune system of a treated patient. This hyperglycosylation process reduces the immunogenicity of the therapeutic protein by sterically inhibiting the interaction of the protein with circulating B-cell receptors. Alternatively, antagonistic TNFR2 antibodies or fragments thereof can be conjugated to molecules that inhibit clearance from human serum and improve the pharmacokinetic characteristics of the antibodies of the present invention. Exemplary molecules that can be conjugated or inserted into the anti-TNFR2 antibodies or fragments thereof of the present invention to attenuate clearance and improve the pharmacokinetic characteristics of these antibodies and fragments include salvage receptor-binding epitopes. These epitopes are found in the Fc region of IgG immunoglobulins and have been shown to bind to Fc receptors and prolong the half-life of antibodies in human serum. The salvage receptor-binding epitope insertion into an anti-TNFR2 antibody or a fragment thereof can be achieved, for example, as described in U.S. Patent No. 5,739,277; which is incorporated herein by reference.

[0357] Modified antagonistic TFNR2 peptide

[0358] In addition to conjugation with other therapeutic agents and markers used for identification or visualization, the anti-TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, or fragments thereof) can also be modified to improve their pharmacokinetic profiles, biophysical stability, or inhibitory activity. For example, any cysteine ​​residue that does not participate in maintaining the proper conformation of the anti-TNFR2 antibody or its fragment may be substituted with isochoric or isoelectronic amino acids (e.g., serine) to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, one or more cysteine ​​bonds may be added to the antibody or its fragment to improve its stability (particularly when the antibody is an antibody fragment, such as an Fv fragment). This can be accomplished, for example, by altering the polynucleotide encoding the antibody heavy and light chains or the polynucleotide encoding the antibody fragment to encode an additional pair or more pairs of cysteine ​​residues that can form disulfide bonds under oxidative conditions to enhance the antibody's tertiary structure (see, for example, U.S. Patent No. 7,422,899; incorporated herein by reference).

[0359] Another useful modification that can be made to the anti-TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, or fragments thereof) includes altering the glycosylation properties of these antibodies and fragments. This can be achieved, for example, by substituting, inserting, or deleting amino acids in antagonistic TNFR2 antibodies to insert or remove glycosylation sites. Antibody glycosylation typically occurs in an N-linked or O-linked manner. N-linked glycosylation is the process by which the linking of the carbohydrate moiety to the antibody occurs at the side chain of an asparagine residue. Common amino acid sequences for N-linked glycosylation include the tripeptide sequences asparagine-X-serine (NXS) and asparagine-X-threonine (NXT), where X is any amino acid other than proline. Insertion of either of these tripeptide sequences into a peptide (e.g., an anti-TNFR2 antibody) creates a potential glycosylation site. O-linked glycosylation refers to the linking of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxy amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine are also competent substrates for glycoside formation. Therefore, glycosylation sites can be added to anti-TNFR2 antibodies by altering the amino acid sequence of the antibody (e.g., using recombinant expression techniques as described herein) to include one or more of the aforementioned tripeptide sequences that promote N-linked glycosylation or one or more serine or threonine residues of the original antibody sequence that produces O-linked glycosylation (see, for example, U.S. Patent No. 7,422,899; incorporated herein by reference).

[0360] Alternatively, it is desirable to modify the antibodies or fragments thereof of the present invention with respect to effector function, for example, to enhance the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions into the Fc region of the antibody. For example, cysteine ​​residues can be introduced into the Fc region of an anti-TNFR2 antibody or fragment thereof (e.g., by recombinant expression techniques as described herein) to promote the formation of additional interchain disulfide bonds in that region. The resulting homodimeric antibody can have increased conformational constraints, which can promote improved internalization capacity and / or increased complement-mediated cell killing and antibody-dependent cytotoxicity (ADCC). Homodimeric antibodies with enhanced antitumor activity can also be prepared using heterobifunctional crosslinkers as described, for example, by Wolff et al. (Canc. Res. [Cancer Research], 53:2560-2565, 1993); which are incorporated herein by reference. Alternatively, antibodies with dual Fc regions can be engineered, thereby potentially possessing enhanced complement lysis and ADCC capabilities (see Stevenson et al. (Anti-Canc. Drug Des. [Design of Anticancer Drugs], 3:219-230, 1989); incorporated herein by reference).

[0361] In some embodiments, the serum half-life of the anti-TNFR2 peptides (e.g., single-chain peptides, antibodies, or fragments thereof) of the present invention can be improved by incorporating one or more amino acid modifications, such as by altering the CH1 or CL region of the Fab domain to introduce a salvage receptor motif found, for example, in the two loops of the CH2 domain of the Fc region of IgG. Such modifications are described, for example, in U.S. Patent Nos. 5,869,046 and 6,121,022, which are incorporated herein by reference. Further framework modifications can also be made to reduce the immunogenicity of the antibody or fragment thereof or to reduce or remove T-cell epitopes present therein, as described, for example, in US 2003 / 0153043, which is incorporated herein by reference.

[0362] Treatment

[0363] Methods for treating cell proliferation disorders

[0364] The antagonistic TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, or fragments thereof) are useful therapeutic agents for treating a wide range of cancers and cell proliferation disorders. Antagonistic TNFR2 peptides (e.g., single-chain peptides, antibodies, or fragments thereof) can be administered to mammalian subjects (e.g., humans) with cell proliferation disorders (such as cancer) to enhance the effectiveness of adaptive immune responses against target cancer cells. Specifically, the antagonistic TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, or fragments thereof) can be administered to mammalian subjects (e.g., humans) to reduce or inhibit T-reg cell growth and activation (which allows tumor-infiltrating T lymphocytes to localize to cells presenting tumor-associated antigens and promote cytotoxicity). Furthermore, the peptides of the present invention can synergize with existing adoptive T-cell therapy platforms, as one limitation of the effectiveness of this strategy is the difficulty in prolonging the cytotoxicity of tumor-reactive T cells after infusion into mammalian subjects (e.g., humans). The peptides of the present invention can also promote the activity of allogeneic T lymphocytes that may express foreign MHC proteins and are increasingly susceptible to inactivation by the host immune system. For example, the antibodies and antigen-binding fragments of the present invention can alleviate the depletion of T-reg-mediated tumor-reactive T cells by inhibiting the growth and proliferation of T-reg cells that typically accompany T cell infusion. For example, the peptides of the present invention (e.g., single-chain peptides, antibodies, or fragments thereof) are capable of reducing the growth of T-reg cell populations by about 50% to about 200% (e.g., 50%, 75%, 100%, 125%, 150%, 175%, or 200%) relative to untreated cells. This reduction in cell growth even occurs in the presence of TNFα. In some embodiments, the peptides of the present invention (e.g., single-chain peptides, antibodies, or fragments thereof) are capable of limiting the growth of T-reg cell populations in the presence of TNFα by 90% to 150% (e.g., 90%, 100%, 110%, 20%, 130%, 140%, or 150%, as described, for example, in Example 4) relative to untreated cells. The antagonistic TNFR2 peptide of the present invention (e.g., a single-chain peptide, antibody, or fragment thereof) is also capable of limiting the proliferation of T-reg cell populations to less than 70% (e.g., 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1%) of the proliferation of untreated T-reg cell populations. The antagonistic TNFR2 peptide of the present invention (e.g., a single-chain peptide, antibody, or fragment thereof) is also capable of reducing the survival of T-reg cell populations by about 10% (e.g., about 20%, 30%, 40%, or 50% or more) relative to untreated T-reg cell populations.

[0365] The antagonistic TNFR2 peptide of the present invention can be administered to mammalian subjects (e.g., humans) with cancer to improve the patient's condition by promoting an immune response against cancer cells and tumorigenic material. The antibodies of the present invention can be administered to subjects, for example, via any of the routes of administration described herein. The peptides of the present invention can also be formulated with excipients, biologically acceptable carriers, and can optionally be conjugated, mixed, or co-administered separately (e.g., sequentially) with other therapeutic agents (e.g., anticancer agents). Cancers that can be treated by administration of the peptides of the present invention (e.g., single-chain peptides, antibodies, or fragments thereof) include cancers such as leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, gastric cardia cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip cancer and oral cancer, eye cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, and pharyngeal cancer.Specific cancers that can be treated by administering the antibodies of the present invention or their antigen-binding fragments include, but are not limited to, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer, Ewing sarcoma family, osteosarcoma and malignant fibrous histiocytoma, embryonal tumors of the central nervous system, and central nervous system tumors. Systemic germ cell tumors, craniopharyngioma, ependymoma, bronchial tumors, Burkitt lymphoma, carcinoid tumors, primary lymphoma, chordoma, chronic myeloproliferative neoplasms, colon cancer, extrahepatic bile duct carcinoma, ductal carcinoma in situ (DCIS), endometrial cancer, ependymoma, esophageal cancer, nasal glioma, extracranial germ cell tumors, gonadal germ cell tumors, fallopian tube cancer, osteofibrous histiocytoma, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), testicular germ cell tumors, gestational trophoblastic disease, glioma, childhood brainstem glioma, hairy cell leukemia, hepatocellular carcinoma, Langerhans cell tumor. Schwann cell histiocytosis, Hodgkin's lymphoma, hypopharyngeal carcinoma, islet cell tumor, pancreatic neuroendocrine tumor, nephroblastoma and other pediatric kidney tumors, Langerhans cell histiocytosis, small cell lung cancer, cutaneous T-cell lymphoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck carcinoma, midline carcinoma, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell tumor, myelodysplastic syndrome, nasal cavity and sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell carcinoma, etc. Ovarian cancer, low-potency ovarian cancer, pancreatic neuroendocrine tumors, papilloma-like hyperplasia, paraganglioma, sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary adenoma, pleural pulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi's sarcoma, rhabdomyosarcoma, Cezare syndrome, small bowel cancer, soft tissue sarcoma, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenström macroglobulinemia.

[0366] For example, the antagonistic TNFR2 peptides of the present invention (e.g., single-chain peptides, antibodies, or fragments thereof) and fragments thereof (e.g., Fab fragments), such as variants of TNFRAB1 and TNFRAB2 having non-natural constant regions, such as humanized TNFRAB1 and TNFRAB2 antibodies, can be administered to patients (e.g., mammalian patients, such as human patients) to treat Hodgkin's lymphoma or cutaneous non-Hodgkin's lymphoma, T-cell lymphoma, ovarian cancer, colon cancer, multiple myeloma, or renal cell carcinoma.

[0367] The anti-TNFR2 peptide of the present invention (e.g., a single-chain peptide, an antibody, or an antigen-binding fragment thereof) can also be co-administered with therapeutic antibodies that exhibit responsiveness to cancer cells. In this way, the antagonistic TNFR2 peptide of the present invention can not only synergize with T lymphocyte tumor reactivity and adaptive immune responses, for example, but also with other inhibitors of tumor cell growth. Examples of other therapeutic antibodies that can be used to treat cancer and other cell proliferation disorders include those that exhibit responsiveness to tumor antigens or cell surface proteins overexpressed on the surface of cancer cells. Exemplary antibodies that can be mixed, co-administered, or sequentially administered with the antagonistic TNFR2 peptide of the present invention include, but are not limited to, trastuzumab. bevacizumab cetuximab Parmumab Ipilimumab Rituximab ( and alenzab Ophamumab Geltuzumab Ozomicin Brutuximab 90 Y-teimozumab and 131 I-Tosimomab They are described in detail in Scott et al. (Cancer Immun., 12:14-21, 2012); incorporated herein by reference.

[0368] Physicians with ordinary skill in the art can readily determine the effective amount of the antagonistic TNFR2 peptide to administer to mammalian subjects (e.g., humans) in need. For example, a physician may begin by prescribing a dose of the peptide of the invention at a level below that required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. Alternatively, a physician may begin a treatment regimen by administering a high dose of the antagonistic TNFR2 antibody or antibody fragment, followed by gradually decreasing doses until a therapeutic effect is achieved (e.g., reduction of the volume of one or more tumors, reduction of T-reg cell populations, or relief of cell proliferation disorders). Typically, the appropriate daily dose of the antibody or its antigen-binding fragment of the invention will be the lowest dose of antibody that effectively produces a therapeutic effect. The single-chain peptide, antibody, or its antigen-binding fragment of the invention can be administered by injection, e.g., via intravenous, intramuscular, intraperitoneal, or subcutaneous injection, optionally near the site of the target tissue (e.g., tumor). The daily dose of the therapeutic composition of the antibody or its antigen-binding fragment of the present invention can be administered as a single dose throughout the day, week, month, or year, or as two, three, four, five, six, or more doses administered at appropriate intervals, optionally in unit dose form. Although it is possible to administer the antibody or its fragment of the present invention alone, it can also be administered as a pharmaceutical formulation in combination with excipients, carriers, and optionally additional therapeutic agents.

[0369] For the peptides of the present invention (e.g., single-chain peptides, antibodies, or fragments thereof), their ability to attenuate the progression of cell-proliferating diseases (such as cancer) can be monitored by any of a variety of methods known in the art. For example, a physician can monitor the response of a mammalian subject (e.g., a human) to treatment with the antibody, antibody fragment, or single-chain peptide of the present invention by analyzing the volume of one or more tumors in a patient. For example, the peptides of the prese...

Claims

1. Use of a combination of a tumor necrosis factor receptor 2 (TNFR2) antagonistic antibody or an antigen-binding fragment thereof and an anticancer agent in the preparation of a medicament for treating cancer in a subject of need, wherein said antibody or antigen-binding fragment thereof (i) does not bind to a peptide comprising amino acids 56-60 (KCSPG) of SEQ ID NO: 7, (ii) specifically binds to a peptide comprising amino acids 142-146 (KCRPG) of SEQ ID NO: 7 or an epitope within amino acids 150-190 of SEQ ID NO: 7, and (iii) comprises a complementarity-determining region (CDR): (a) CDR-H1 consisting of the amino acid sequence GYTFTDYX (SEQ ID NO: 257); (b) CDR-H2 consisting of the amino acid sequence VDPEYGST (SEQ ID NO: 258); (c) CDR-H3 consisting of the amino acid sequence ARDDGSYSPFDYWG (SEQ ID NO: 259); (d) CDR-L1 composed of the amino acid sequence QNINKY (SEQ ID NO: 260); (e) CDR-L2 composed of the amino acid sequence TYS or YTS; and (f) CDR-L3 consisting of the amino acid sequence CLQYVNLXT (SEQ ID NO: 261); Each X is either leucine or isoleucine.

2. The use according to claim 1, wherein the anticancer agent is a therapeutic antibody.

3. The use according to claim 1 or 2, characterized in that the antibody or its antigen-binding fragment and the anticancer agent are administered separately to the subject.

4. The use according to claim 3, wherein the antibody or its antigen-binding fragment is administered prior to the anticancer agent.

5. The use according to claim 3, wherein the antibody or its antigen-binding fragment is administered after the anticancer agent.

6. The use according to claim 1 or 2, characterized in that the antibody or its antigen-binding fragment and the anticancer agent are administered simultaneously to the subject.

7. The use according to claim 1 or 2, characterized in that the drug is administered together with a cytotoxic agent.

8. The use according to claim 7, wherein the drug and the cytotoxic agent are administered separately to the subject.

9. The use according to claim 8, wherein the drug is administered prior to the cytotoxic agent.

10. The use according to claim 8, wherein the drug is administered after the cytotoxic agent.

11. The use according to claim 7, wherein the drug and the cytotoxic agent are administered simultaneously to the subject.

12. The use as claimed in claim 1 or 2, characterized in that the drug is formulated for administration to the subject for a period of 1 day to 6 months.

13. The use according to claim 1 or 2, wherein the cancer is selected from leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, cardia cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip cancer and oral cancer, eye cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer and pharyngeal cancer.

14. The use according to claim 1 or 2, wherein the cancer is selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct carcinoma, extrahepatic carcinoma, Ewing sarcoma family, osteosarcoma and malignant fibrous histiocytoma, embryonal tumors of the central nervous system, and other related diseases. Germ cell tumors, craniopharyngioma, ependymoma, bronchial tumors, Burkitt lymphoma, carcinoid tumors, primary lymphoma, chordoma, chronic myeloproliferative neoplasms, colon cancer, extrahepatic bile duct carcinoma, ductal carcinoma in situ (DCIS), endometrial cancer, esophageal cancer, nasal glioma, extracranial germ cell tumor, gonadal extragerminal tumor, fallopian tube cancer, osteofibrous histiocytoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), testicular germ cell tumor, gestational trophoblastic disease, glioma, childhood brainstem glioma, hairy cell leukemia, liver cell tumors. Cellular carcinoma, Langerhans cell histiocytosis, Hodgkin's lymphoma, hypopharyngeal carcinoma, islet cell tumor, pancreatic neuroendocrine tumor, nephroblastoma and other pediatric kidney tumors, small cell lung cancer, cutaneous T-cell lymphoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck carcinoma, midline carcinoma, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell tumor, myelodysplastic syndrome, nasal cavity and sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer Germ cell ovarian cancer, low-potency ovarian cancer, papilloma-like hyperplasia, paraganglioma, sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary adenoma, pleural pulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi's sarcoma, Cezale syndrome, small bowel cancer, soft tissue sarcoma, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenström macroglobulinemia.

15. The use according to claim 1 or 2, wherein the medicament further comprises a pharmaceutically acceptable carrier, excipient, or stabilizer.

16. The use according to claim 1 or 2, wherein the drug is administered orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intraocularly, intratumorally, parenterally, topically, intrathecally, or intraventricularly.

17. The use according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is selected from monoclonal antibodies or antigen-binding fragments thereof, humanized antibodies or antigen-binding fragments thereof, primate antibodies or antigen-binding fragments thereof, bispecific antibodies or antigen-binding fragments thereof, multispecific antibodies or antigen-binding fragments thereof, bivariate immunoglobulin domains, monovalent antibodies or antigen-binding fragments thereof, chimeric antibodies or antigen-binding fragments thereof, single-chain Fv molecules (scFv), biantibodies, triantibodies, antibody-like protein scaffolds, Fv fragments, Fab fragments, F(ab')2 molecules, and tandem scFv (taFv).

18. The use as claimed in claim 1 or 2, wherein the subject is a mammal.

19. The use as claimed in claim 18, wherein the mammal is a human.

20. A combination comprising (a) a pharmaceutical composition comprising a TNFR2 antagonistic antibody or an antigen-binding fragment thereof and (b) an anticancer agent for treating cancer in a subject of need, wherein said antibody or antigen-binding fragment thereof (i) does not bind to a peptide comprising amino acids 56-60 (KCSPG) of SEQ ID NO: 7, (ii) specifically binds to a peptide comprising amino acids 142-146 (KCRPG) of SEQ ID NO: 7 or an epitope within amino acids 150-190 of SEQ ID NO: 7, and (iii) comprises a complementarity-determining region (CDR): (a) CDR-H1 consisting of the amino acid sequence GYTFTDYX (SEQ ID NO: 257); (b) CDR-H2 consisting of the amino acid sequence VDPEYGST (SEQ ID NO: 258); (c) CDR-H3 consisting of the amino acid sequence ARDDGSYSPFDYWG (SEQ ID NO: 259); (d) CDR-L1 composed of the amino acid sequence QNINKY (SEQ ID NO: 260); (e) CDR-L2 composed of the amino acid sequence TYS or YTS; and (f) CDR-L3 consisting of the amino acid sequence CLQYVNLXT (SEQ ID NO: 261); Each X is either leucine or isoleucine.

21. The combination of claim 20, wherein the anticancer agent is a therapeutic antibody.

22. The combination of claim 20 or 21, characterized in that the antibody or its antigen-binding fragment and the anticancer agent are administered separately to the subject.

23. The combination of claim 22, wherein the antibody or its antigen-binding fragment is administered prior to the anticancer agent.

24. The combination of claim 22, wherein the antibody or its antigen-binding fragment is administered after the anticancer agent.

25. The combination of claim 20 or 21, characterized in that the antibody or its antigen-binding fragment and the anticancer agent are administered simultaneously to the subject.

26. The combination of claim 20 or 21, characterized in that the pharmaceutical composition is administered together with a cytotoxic agent.

27. The combination of claim 26, wherein the pharmaceutical composition and the cytotoxic agent are administered separately to the subject.

28. The combination of claim 27, wherein the pharmaceutical composition is administered prior to the cytotoxic agent.

29. The combination of claim 27, wherein the pharmaceutical composition is administered after the cytotoxic agent.

30. The combination of claim 26, wherein the pharmaceutical composition and the cytotoxic agent are administered simultaneously to the subject.

31. The combination of claim 20 or 21, characterized in that the pharmaceutical composition is formulated for administration to the subject for a period of 1 day to 6 months.

32. The combination of claim 20 or 21, wherein the cancer is selected from leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, cardia cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip cancer and oral cancer, eye cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer and pharyngeal cancer.

33. The combination of claim 20 or 21, wherein the cancer is selected from ALL, AML, CLL, CML, adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, extrahepatic carcinoma, Ewing sarcoma family, osteosarcoma and malignant fibrous histiocytoma, embryonal tumors of the central nervous system, germ cell tumors of the central nervous system, craniopharyngioma, ependymoma, and bronchial tumors. Burkitt lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasm, colon cancer, extrahepatic bile duct cancer, DCIS, endometrial cancer, esophageal cancer, nasal glioma, extracranial germ cell tumor, gonadal germ cell tumor, fallopian tube cancer, osteofibrous histiocytoma, gastrointestinal carcinoid tumor, GIST, testicular germ cell tumor, gestational trophoblastic disease, glioma, childhood brainstem glioma, hairy cell leukemia, hepatocellular carcinoma, Langerhans cell histiocytosis, Hodgkin's disease. Golden lymphoma, hypopharyngeal carcinoma, islet cell tumor, pancreatic neuroendocrine tumor, nephroblastoma and other pediatric kidney tumors, small cell lung cancer, cutaneous T-cell lymphoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck carcinoma, midline carcinoma, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell tumor, myelodysplastic syndrome, nasal cavity and sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, NHL, NSCLC, epithelial ovarian cancer, germ cell ovarian cancer, low-potency ovarian cancer. Papilloma-like hyperplasia, paraganglioma, sinus and nasal cavity carcinoma, parathyroid carcinoma, penile cancer, pharyngeal carcinoma, pheochromocytoma, pituitary adenoma, pleural pulmonary blastoma, primary peritoneal carcinoma, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, Kaposi's sarcoma, Cezale syndrome, small bowel cancer, soft tissue sarcoma, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenström macroglobulinemia.

34. The combination of claim 20 or 21, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, or stabilizer.

35. The combination of claim 20 or 21, wherein the pharmaceutical composition is administered orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intraocularly, intratumorally, parenterally, topically, intrathecally, or intraventricularly.

36. The combination of claim 20 or 21, wherein the antibody or antigen-binding fragment thereof is selected from monoclonal antibodies or antigen-binding fragments thereof, humanized antibodies or antigen-binding fragments thereof, primate antibodies or antigen-binding fragments thereof, bispecific antibodies or antigen-binding fragments thereof, multispecific antibodies or antigen-binding fragments thereof, dual variable immunoglobulin domains, monovalent antibodies or antigen-binding fragments thereof, chimeric antibodies or antigen-binding fragments thereof, scFv, biantibodies, triantibodies, antibody-like protein scaffolds, Fv fragments, Fab fragments, F(ab')2 molecules, and taFv.

37. The combination of claim 20 or 21, wherein the subject is a mammal.

38. The combination of claim 37, wherein the mammal is a human.

Images