Human 4-1BB agonist antibody and method of use thereof

Abstract

Providing a human 4-1BB agonist antibody and a method for using it. [Solution] Isolated monoclonal antibodies or recombinant monoclonal antibodies that bind to 4-1BB are provided. In some cases, the antibodies of the embodiments can be used for the detection, diagnosis and / or therapeutic treatment of human diseases such as cancer. Increasing evidence has shown that anti-4-1BB monoclonal antibodies have strong antitumor properties, which in itself is due to their potent CD8 + This results in T cell activation, IFN-γ production, and induction of cytolytic markers. However, the need for improved human 4-1BB antibodies remains unmet. This disclosure provides a means to address this problem.

Description

[Technical Field]

[0001] This application claims the interests of U.S. Provisional Patent Application No. 62 / 977,658, filed on 17 February 2020, which is incorporated herein by reference in its entirety.

[0002] Incorporation of sequence lists The sequence listing, created on February 16, 2021, and contained in a file named "UTFCP1467WO_ST25.txt", is 23KB (measured on Microsoft Windows®) and submitted electronically with this specification and is incorporated herein by reference. background 1. Field This invention generally relates to the field of molecular biology, and more specifically to tumor necrosis factor superfamily receptor (TNF-FSFR; 4-1BB; CD137) agonist antibodies. [Background technology]

[0003] 2. Explanation of related technologies 4-1BB (CD137), a member of the TNF receptor superfamily, is an activation-inducing T cell costimulatory molecule (Vinay and Kwon, 2012). Signaling via 4-1BB upregulates survival genes, enhances cell division, induces cytokine production, and prevents activation-induced cell death in T cells. The importance of the 4-1BB pathway is highlighted in many diseases, including cancer. Increasing evidence has shown that anti-4-1BB monoclonal antibodies possess strong antitumor properties, which in itself is due to their potent CD8 + This is a result of T cell activation, IFN-γ production, and the ability to induce cytolytic markers. Furthermore, combination therapy with anti-4-1BB and other anticancer therapies such as radiation has robust tumor regression ability against non-immunogenic or low-immunogenic tumors. In addition, ex vivo anti-4-1BB activated CD8 from previously tumor-treated animals. +Adoptive transfer of T cells efficiently inhibits tumor progression in recipient mice inoculated with fresh tumors. Furthermore, targeting tumors with a variant of 4-1BBL against 4-1BB also has a strong antitumor effect. However, the need for improved human 4-1BB antibodies remains unmet.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Means for Solving the Problems

[0005] Summary In a first embodiment, the present disclosure provides an isolated monoclonal antibody that specifically binds to 4-1BB and (a) the first V H CDR is identical to SEQ ID NO: 3, (b) the second V H CDR is identical to SEQ ID NO: 5, (c) the third V H CDR is identical to SEQ ID NO: 7, (d) the first V L CDR is identical to SEQ ID NO: 11, (e) the second V L CDR is identical to SEQ ID NO: 13, and (f) the third V L CDR is identical to SEQ ID NO: 15, or (a) the first V H CDR is identical to SEQ ID NO: 19, (b) the second V H CDR is identical to SEQ ID NO: 21, (c) the third V H CDR is identical to SEQ ID NO: 23, (d) the first V L CDR is identical to SEQ ID NO: 27, (e) the second V L CDR is identical to SEQ ID NO: 29, and (f) the third V L CDR is identical to SEQ ID NO: 31, or (a) the first V HCDR is identical to sequence number 35, and (b) the second V H CDR is identical to sequence number 37, and (c) the third V H CDR is identical to sequence number 39, and (d) the first V L CDR is identical to sequence number 43, and (e) the second V L CDR is identical to sequence number 45, and (f) the third V L CDR is identical to sequence number 47, or (a) the first V H CDR is identical to sequence number 51, and (b) the second V H CDR is identical to sequence number 53, and (c) the third V H CDR is identical to sequence number 55, and (d) the first V L CDR is identical to sequence number 59, and (e) the second V L CDR is identical to sequence number 61, and (f) the third V L The CDR is identical to sequence number 63.

[0006] In some embodiments, the antibody is clone 54 V H Domain (SEQ ID NO: 2) and V are at least approximately 80% identical, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. H Domain and clone 54 V L Domain (SEQ ID NO: 10) and V that are at least approximately 80% identical, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. L Includes the domain. In certain embodiments, the antibody is clone 54 of V H Domain (SEQ ID NO: 2) and the same V H Domain and clone 54 V L Domain (SEQ ID NO: 10) and the same V L Includes the domain.

[0007] In certain embodiments, the antibody is V of clone 135B. HDomain (SEQ ID NO: 18) and V are at least approximately 80% identical, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. H Domain and clone 135B's V L Domain (SEQ ID NO: 26) and V that are at least approximately 80% identical, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. L Includes the domain. In certain embodiments, the antibody is V of clone 135B. H Domain (SEQ ID NO: 18) and the same V H Domain and clone 135B's V L Domain (SEQ ID NO: 26) and the same V L Includes the domain.

[0008] In some embodiments, the antibody is V of clone 138. H Domain (SEQ ID NO: 34) and V that are at least approximately 80% identical, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. H Domain and clone 138 V L Domain (SEQ ID NO: 42) and V that are at least approximately 80% identical, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. L Includes the domain. In certain embodiments, the antibody is clone 138 V H Domain (SEQ ID NO: 34) and the same V H Domain and clone 138 V L Domain (SEQ ID NO: 42) and the same V L Includes the domain.

[0009] In some embodiments, the antibody is V of clone 49A. H Domain (SEQ ID NO: 50) and V that are at least approximately 80% identical, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. H Domain and the V of clone 49A LDomain (SEQ ID NO: 58) and a V that is at least approximately 80% identical, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. L Includes the domain. In certain embodiments, the antibody is V of clone 49A. H Domain (SEQ ID NO: 50) and the same V H Domain and the V of clone 49A L Domain (SEQ ID NO: 58) and the same V L Includes the domain.

[0010] In certain embodiments, the antibody is recombinant. In some embodiments, the antibody is IgG, IgM, IgA, or its antigen-binding fragment. In certain embodiments, the antibody is Fab', F(ab')2, F(ab')3, monovalent scFv, bivalent scFv, or a single-domain antibody. In certain embodiments, the antibody is human, humanized, or deimmunized. In further embodiments, the antibody is conjugated with an imaging agent, chemotherapeutic agent, toxin, or radionuclide.

[0011] Further embodiments provide compositions comprising the antibody of this embodiment in a pharmaceutically acceptable carrier. Isolated polynucleotide molecules comprising nucleic acid sequences encoding the antibody of this embodiment are also provided herein.

[0012] Another embodiment is clone 54 V H Domain CDR1-3 (SEQ ID NOs: 3, 5, and 7), clone 135B V H Domain CDR1-3 (SEQ ID NOs. 19, 21, and 23), clone 138 V H Domain CDR1-3 (SEQ ID NOs. 35, 37, and 39), or V of clone 49A H Antibody V containing domain CDR1-3 (SEQ ID NOs. 51, 53, and 55) H We provide recombinant polypeptides that include domains.

[0013] Further embodiments include V 54. L Domain CDR1-3 (SEQ ID NOs. 11, 13, and 15), V of 135BL Domain CDR1-3 (SEQ ID NOs. 27, 29, and 31), V of 138 L Domain CDR1-3 (SEQ ID NOs. 43, 45, and 47), or V of 49A L Antibody V containing domain CDR1-3 (SEQ ID NOs. 59, 61, and 63) L We provide recombinant polypeptides that include domains.

[0014] In a further embodiment, the V of clone 54 H Antibody V containing domain CDR1-3 (SEQ ID NOs: 3, 5, and 7) H Domain and 54 V L Antibody V containing domain CDR1-3 (SEQ ID NOs: 11, 13, and 15) L Domain; Clone 135B V H Antibody V containing domain CDR1-3 (SEQ ID NOs: 19, 21, and 23) H Domain and 135B V L Antibody V containing domain CDR1-3 (SEQ ID NOs: 27, 29, and 31) L Domain; Clone 138 V H Antibody V containing domain CDR1-3 (SEQ ID NOs: 35, 37, and 39) H Domain and 138 V L Antibody V containing domain CDR1-3 (SEQ ID NOs: 43, 45, and 47) L Domain; or clone V of 49A H Antibody V containing domain CDR1-3 (SEQ ID NOs. 51, 53, and 55) H Domain and 49A V L Antibody V containing domain CDR1-3 (SEQ ID NOs. 59, 61, and 63) L Recombinant polypeptides containing domains are provided.

[0015] Further embodiments provide isolated polynucleotide molecules comprising nucleic acid sequences encoding the polypeptides of this embodiment. Host cells comprising antibodies of this embodiment or one or more polynucleotide molecules encoding the recombinant polypeptides of this embodiment are also provided herein. In some embodiments, the host cells are mammalian cells, yeast cells, bacterial cells, ciliated cells, or insect cells.

[0016] Another embodiment is a method for producing an antibody, wherein (a) the V of the antibody of this embodiment L Chain and V H The present invention provides a method comprising the steps of (b) expressing one or more polynucleotide molecules encoding a chain in cells, and (b) purifying an antibody from the cells.

[0017] Further embodiments provide a method for treating a subject having cancer, comprising the step of administering an effective amount of the antibody of this embodiment to the subject.

[0018] In some embodiments, cancer is breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, skin cancer, brain tumor, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

[0019] In certain embodiments, the antibody is contained in a pharmaceutically acceptable composition. In some embodiments, the antibody is administered systemically. In some embodiments, the antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically.

[0020] In further embodiments, the method further includes the step of administering at least a second anticancer therapy. In some embodiments, the second anticancer therapy is surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy. In certain embodiments, the second anticancer therapy includes adoptive T-cell therapy. In some embodiments, the second anticancer therapy includes immunotherapy. In certain embodiments, the immunotherapy is an immune checkpoint inhibitor such as an anti-CTLA-4 antibody, an anti-PD-L1 antibody, and / or an anti-PD1 antibody. In certain embodiments, Epidemic checkpoint inhibitors are human programmed cell death 1 (PD-1) conjugated antagonists, PD-1 conjugated antagonists, or PD-2 conjugated antagonists. In some embodiments, the PD-1 conjugated antagonist is a monoclonal antibody or its antigen-binding fragment. In certain embodiments, the PD-1 conjugated antagonist is nivolumab, pembrolizumab, CT-011, BMS 936559, MPDL328OA, or AMP-224.

[0021] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description, it should be understood that the detailed description and specific examples illustrating specific embodiments of the present invention are given only as illustrations. [Brief explanation of the drawing]

[0022] The following drawings form part of this specification and are included to further illustrate specific aspects of the invention. The invention may be better understood by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein.

[0023] [Figure 1A-1] Figures 1A-1B: Multiple high-affinity anti-human 4-1BB antibodies were generated. (Figure 1A) ELISA results of the first post-fusion antibody supernatant against recombinant human 4-1BB. (Figure 1B) Titration curve of the 4-1BB monoclonal antibody clone. [Figure 1A-2] Figures 1A-1B: Multiple high-affinity anti-human 4-1BB antibodies were generated. (Figure 1A) ELISA results of the first post-fusion antibody supernatant against recombinant human 4-1BB. (Figure 1B) Titration curve of the 4-1BB monoclonal antibody clone. [Figure 1B] Figures 1A-1B: Multiple high-affinity anti-human 4-1BB antibodies were generated. (Figure 1A) ELISA results of the first post-fusion antibody supernatant against recombinant human 4-1BB. (Figure 1B) Titration curve of the 4-1BB monoclonal antibody clone.

[0024] [Figure 2A] Figures 2A-2B: This clone 54 4-1BB antibody (referred to herein as Curranlumab) amplifies human TILs with high efficiency. Using two variants of clone 54 (Curranlumab), human tumor-infiltrating lymphocytes (TILs) were amplified from patient tumor masses, and the efficiency of each in generating pure, proliferated CD8+ T cell products was compared with that of urelumab (BMS-663513). [Figure 2B] Figures 2A-2B: This clone 54 4-1BB antibody (referred to herein as Curranlumab) amplifies human TILs with high efficiency. Using two variants of clone 54 (Curranlumab), human tumor-infiltrating lymphocytes (TILs) were amplified from patient tumor masses, and the efficiency of each in generating pure, proliferated CD8+ T cell products was compared with that of urelumab (BMS-663513).

[0025] [Figure 3] Figure 3: PCR using several combinations of Ig variable domain primers. Asterisks indicate the correct antibody transcripts for the VH and VL chains. The remaining PCR bands observed are aberrant transcripts confirmed by sequencing.

[0026] [Figure 4A] Figures 4A-4B: Glioblastoma and colorectal cancer tumors were isolated, and tumor-infiltrating T cells were amplified using the 41BB agonist antibody clone 54 as either human IgG1 or mouse IgG2a. The degree of amplification, CD8 percent, and CD4 percent are shown. Total TILs from tumors of glioblastoma and colorectal cancer patients were amplified with clone 54 IgG1 and clone 54 IgG1 (Figure 4A). Percentage of CD3+CD8+TILs or CD3+CD4+TILs generated by the amplification method (Figure 4B). [Figure 4B]Figures 4A-4B: Glioblastoma and colorectal cancer tumors were isolated, and tumor-infiltrating T cells were amplified using the 41BB agonist antibody clone 54 as either human IgG1 or mouse IgG2a. The degree of amplification, CD8 percent, and CD4 percent are shown. Total TILs from tumors of glioblastoma and colorectal cancer patients were amplified with clone 54 IgG1 and clone 54 IgG1 (Figure 4A). Percentage of CD3+CD8+TILs or CD3+CD4+TILs generated by the amplification method (Figure 4B). [Modes for carrying out the invention]

[0027] Description of Exemplary Embodiments The tumor necrosis factor superfamily receptor (TNFSFR), also known as 4-1BB and CD137, co-stimulates the activation of myeloid cells, including T cells, NK cells, and antigen-presenting dendritic cells. In CD8 T cells, 4-1BB activation increases their proliferation, cytotoxicity, cytokine production, mitochondrial mass, and viability. Therefore, in this study, we generated 4-1BB agonist antibodies. To generate antibodies targeting human 4-1BB, mice were alternately immunized with cell vaccines and recombinant protein-based vaccines. Among many lead candidates, we selected clone 54 4-1BB antibodies that were sequenced and expressed in various mouse and human isotypes. In this study, the IgG2a version of clone 54 4-1BB antibody was found to be highly efficient in tumor infiltration of CD8. We discovered that this process amplifies and matures T cells.

[0028] Accordingly, in certain embodiments, the present disclosure provides human 4-1BB agonist antibodies, particularly 4-1BB IgG2a agonist monoclonal antibodies. These antibodies may be manufactured under GMP-compliant conditions. These antibodies may be used in patients in monospecific or bispecific formats. In further embodiments, these antibodies may be administered to cancer patients alone as immunotherapy or in combination with additional immunotherapy such as immune checkpoint inhibitors (e.g., anti-PD1 antibodies or anti-CTLA4 antibodies).

[0029] I. Definition As used herein, “essentially not containing” with respect to a particular ingredient means that none of the particular ingredient is intentionally included in the composition and / or contaminants. This term is used to mean that the component is present only as a trace or in trace amounts. Therefore, the total amount of a particular component resulting from any unintended contamination of the composition is far below 0.05%, preferably below 0.01%. Compositions in which the amount of a particular component cannot be detected by standard analytical methods are most preferred.

[0030] As used herein, "a" or "an" may mean one or more. As used in the claims, when used in combination with the word "comprising," "a" or "an" may mean one or more.

[0031] The use of the term "or" in the claims means "and / or" unless it is explicitly indicated to refer only to the substitutes, or unless the substitutes are mutually exclusive. While used to describe taste, this disclosure supports the definitions of "and / or" referring only to substitutes. Where used herein, "another" may mean at least a second or third or more. The term "about" generally means plus or minus 5% of the stated value.

[0032] "Treatment" and "to treat" refer to the administration or application of a therapeutic agent to a subject, or the performance of a procedure or modality on a subject, for the purpose of obtaining therapeutic benefits from a disease or health-related symptom.

[0033] "Subject" and "Patient" refer to either human or non-human animals, such as primates, mammals, and vertebrates. In certain embodiments, the subject is human.

[0034] As used throughout this application, the terms “therapeutic benefit” or “therapeutic effect” refer to any medical treatment of the condition that promotes or enhances the well-being of the subject. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of the disease. For example, treatment of cancer may include, for instance, a reduction in tumor size, a reduction in tumor invasiveness, a decrease in the rate of cancer growth, or a prevention of metastasis. Treatment of cancer may also refer to an extension of the survival of the subject with cancer.

[0035] "Anticancer" drugs can negatively affect target cancer cells / tumors by, for example, promoting the death of cancer cells, inducing apoptosis in cancer cells, slowing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting an immune response against cancer cells or tumors, preventing or inhibiting the progression of cancer, or extending the lifespan of the subject with cancer.

[0036] In this specification, the term “antibody” is used in its broadest sense and specifically includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments insofar as they exhibit the desired biological activity.

[0037] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, where, for example, the individual antibodies constituting the population are identical except for expected mutations, such as spontaneous mutations that may exist in small amounts. Thus, the modifier “monoclonal” indicates that the antibody features are not a mixture of individual antibodies. In certain embodiments, such a monoclonal antibody typically comprises an antibody containing a target-binding polypeptide sequence, where the target-binding polypeptide sequence is obtained by a process comprising the selection of a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be the selection of a unique clone from a pool of clones, such as a hybridoma clone, a phage clone, or a recombinant DNA clone. It should be understood that the selected target-binding sequence may be further modified, for example, to improve affinity for a target, to humanize the target-binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to produce a multispecific antibody, and that an antibody containing a modified target-binding sequence is also a monoclonal antibody of the present invention. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are typically advantageous in that they are not contaminated by other immunoglobulins.

[0038] The terms “contacted” and “exposed” are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to or directly juxtaposed with target cells when applied to cells. To achieve cell death, for example, both agents are delivered to cells in a total amount effective in killing the cells or preventing them from dividing.

[0039] The term "immune checkpoint" refers to molecules, such as proteins, in the immune system that provide inhibitory signals to components of the immune response to maintain balance. Known immune checkpoint proteins include CTLA-4, PD1 and its ligands PD-L1 and PD-L2, as well as LAG-3, BTLA, B7H3, B7H4, TIM3, and KIR. It is recognized in the art that pathways involving LAG3, BTLA, B7H3, B7H4, TIM3, and KIR constitute an immune checkpoint pathway similar to the CTLA-4 and PD-1-dependent pathway (see, for example, Pardoll, 2012; Mellman et al., 2011).

[0040] An "immune checkpoint inhibitor" refers to any compound that inhibits the function of an immune checkpoint protein. Inhibition includes reduced function and complete blockage. In particular, immune checkpoint proteins are human immune checkpoint proteins. Therefore, immune checkpoint protein inhibitors are specifically inhibitors of human immune checkpoint proteins.

[0041] II. Anti-41BB antibody In certain embodiments, an antibody or fragment thereof is intended to bind to at least a portion of 4-1BB and activate signaling, for example, to stimulate an immune response. As used herein, the term “antibody” is intended to broadly refer to any immunological conjugate, such as IgG, IgM, IgA, IgD, IgE, and genetically modified IgG, as well as polypeptides containing an antibody CDR domain that retains antigen-binding activity. The antibody may be selected from the group consisting of chimeric antibodies, affinity matured antibodies, polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, or antigen-binding antibody fragments, or natural or synthetic ligands. Preferably, the anti-41BB antibody is a monoclonal antibody or a humanized antibody.

[0042] In some embodiments, the anti-41BB antibody comprises the heavy chain CDR1-3 of SEQ ID NO: 2 (GYSFTDYN (SEQ ID NO: 3), INPNYGTT (SEQ ID NO: 5), and ARSPVEDYFDY (SEQ ID NO: 7)) and the light chain CDR1-3 of SEQ ID NO: 10 (SSVSSSY (SEQ ID NO: 11), STS (SEQ ID NO: 13), and QQYSGYPLIT (SEQ ID NO: 15)). The anti-41BB antibody may have at least 80%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 1, 2, 9, or 10. The anti-41BB antibody may have at least 80% sequence identity with SEQ ID NOs: 17, 18, 25, or 26. The anti-41BB antibody may have at least 80% sequence identity, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The anti-41BB antibody may have at least 80% sequence identity, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with SEQ ID NOs. 49%, 50, 57, or 58 may have at least 80% sequence identity, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The heavy chain and light chain sequences are shown below. Frame 1 (FR1) (positions 77-505) Clone 54 starting with IgG2a 4-1BB VH consensus sequence Nucleotide sequence: GAGTTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGCGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGACTACAACATGAACTGGGTGAAGCAGAGCAATGGAAAGAGCCTTGAGTGGATTGGAGTAATTAATCCTAACTATGGTACTACTAGCTACAATCAGAAGTTCAAGGGCAAGGCCACATTTACTGTAGA CCAATCTTCCAGCACAGCCTACATGCAGCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATCCCCGGTAGAGGACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCTGTTGTGGAGGTACAACTGGCTCCTCGGTGACTCTA (SEQ ID NO: 1) Amino acid sequence: EFQLQQSGPELVKPGASVKISCKASGYSFTDYNMNWVKQSNGKSLEWIGVINPNYGTTSYNQKFKGKATFTVDQSSSTAYMQLNSLTSEDSAVYYCARSPVEDYFDYWGQGTTLTVSSAKTTAPSVYPLAPVCGGTTGSSVTL(Sequence ID 2) CDR1: GYSFTDYN (Sequence ID 3) GGTTACTCATTCACTGACTACAAC(Sequence ID 4) CDR2: INPNYGTT (Sequence ID 5) ATTAATCCTAACTATGGTACTACT(Sequence ID 6) CDR3: ARSPVEDYFDY (Sequence ID 7) GCAAGATCCCCGGTAGAGGACTACTTTGACTAC (Sequence No. 8) VL consensus sequence starting from Frame 1 (FR1) (91-470th place) Nucleotide sequence: GAAAATGTGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAAAGGTCACCATGACCTGCAGGGCCAGTCAAGTGTAAGTTCCAGTTACTTGCACTGGTACCAGCAGAAGTCAGGTGCCTCCCCCAAACTCTGGATTTATAGCACATCCAACTTGGCTTCTGGAGTCCCTGCTCGCTTCAGTG GCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGTTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGTCAGTGGTTACCCACTCATCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTT (SEQ ID NO: 9) Amino acid sequence: ENVLTQSPAIMSASPGEKVTMTCRARSSVSSSYLHWYQQKSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLITFGAGTKLELKRADAAPTVSIFPPSSEQ(Sequence ID 10) CDR1: SSVSSSY (Sequence ID 11) TCAAGTGTAAGTTCCAGTTAC (Sequence No. 12) CDR2: STS (Sequence ID 13) AGCACATCC (Sequence ID 14) CDR3: QQYSGYPLIT(Sequence ID 15) CAGCAGTACAGTGGTTACCCACTCATCACG (Sequence ID 16) Frame 1 (FR1) (around positions 45-407) Clone 135B 4-1BB VH consensus sequence starting with IgG1 Nucleotide sequence: AGGTGAAGCTGCAGCAGTCAGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATACACGTTCACTGACTACTACATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAATGATGGTACTACCTACTACAAC CAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATCCCTCTACGGTAGTAGCTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG (SEQ ID NO: 17) Amino acid sequence: VKLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDINPNNDGTTYYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARSLYGSSYYFDYWGQGTTLTVSS(Sequence ID 18) CDR1: GYTFTDYY (Sequence ID 19) GGATACACGTTCACTGACTACTAC(Sequence ID 20) CDR2: INPNNDGT (Sequence ID 21) ATTAATCCTAACAATGATGGTACT(Sequence ID 22) CDR3: ARSLYGSSYYFDY (Sequence ID 23) GCAAGATCCCTCTACGGTAGTAGCTACTACTTTGACTAC (Sequence ID 24) VL consensus sequence starting from Frame 1 (FR1) (ranks 46-379) Nucleotide sequence: GATATTGTGATGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAAC CTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAATGAGGACCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC (SEQ ID NO: 25) Amino acid sequence: DIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIK (Sequence ID 26) CDR1: KSVSTSGYSY (Sequence ID 27) AAAAGTGTCAGTACATCTGGCTATAGTTAT(Sequence No. 28) CDR2: LVS (Sequence ID 29) CTTGTATCC (Sequence ID 30) CDR3: QQSNEDPWT (Sequence ID 31) CAGCAAAGTAATGAGGACCCGTGGACG (Sequence No. 32) Frame 1 (FR1) (positions 56-409) Clone 138 starting with IgG2b 4-1BB VH consensus sequence Nucleotide sequence: AGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTTGCATACATTAGTAGTGGCAGTAATTCCATCTACTATGC AGACACAGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCCTCGAATAATGGTTACTTCTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG (SEQ ID NO: 33) Amino acid sequence: VQLQESGGGLVKPGGSLKLSCAASGFTFSDYGMHWVRQAPEKGLEWVAYISSGSNSIYYADTVTGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCASNNGYFYFDYWGQGTTLTVSS(Sequence ID 34) CDR1: GFTFSDYG (Sequence ID 35) GGATTCACTTTCAGTGACTATGGA(Sequence No. 36) CDR2: ISSGSNSI (Sequence ID 37) ATTAGTAGTGGCAGTAATTCCATC (Sequence ID 38) CDR3: ASNNGYFYFDY (Sequence ID 39) GCCTCGAATAATGGTTACTTCTACTTTGACTAC (Sequence No. 40) VL consensus sequence starting from Frame 1 (FR1) (ranks 63-378) Nucleotide sequence: ATTGTGATCACCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCT GGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAGTCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTGACCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAC (SEQ ID NO: 41) Amino acid sequence: IVITQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTVSRVEAEDAATYYCQQWSSDPFTFGSGTKLEIK(Sequence ID 42) CDR1: SSVSY (Sequence ID 43) TCAAGTGTAAGTTAC (Sequence ID 44) CDR2: ATS (Sequence ID 45) GCCACATCC (Sequence ID 46) CDR3: QQWSSDPFT (Sequence ID 47) CAGCAGTGGAGTAGTGACCCATTCACG (Sequence No. 48) Frame 1 (FR1) (around 65-427th position) Clone 49A4-1BB starting with IgG1 VH Consensus Array Nucleotide sequence: AGGTGAAACTGCAGCAGTCAGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATACACGTTCACTGACTACTACATGAACTGGGTGAAGGAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAAACAATGGTGGTTCTACCTACTACAAC CAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGAGAAGTCCTCCAGCACAGCCTTCATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATCCCTCTACGGTAGTACCTACTACTTTGACTACTGGGGCCAAGGCACCCCTCTCACAGTCTCCTCAG (SEQ ID NO: 49) Amino acid sequence: VKLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKESHGKSLEWIGDINPNNGGSTYYNQKFKGKATLTVEKSSSTAFMELRSLTSEDSAVYYCARSLYGSTYYFDYWGQGTPLTVSS(Sequence ID 50) CDR1: GYTFTDYY (Sequence ID 51) GGATACACGTTCACTGACTACTAC(Sequence ID 52) CDR2: INPNNGGS (Sequence ID 53) ATTAATCCTAACAATGGTGGTTCT(Sequence ID 54) CDR3: ARSLYGSTYYFDY (Sequence ID 55) GCAAGATCCCTCTACGGTAGTACCTACTACTTTGACTAC (Sequence ID 56) VL consensus sequence starting from Frame 1 (FR1) (ranks 55-388) Nucleotide sequence: GATATTGTGCTGACCCAGTCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATATGAACTGGTACCAACAGAAGCCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAAT CTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGGAACCTATTACTGTCAGCAAAGTAATGACGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC (SEQ ID NO: 57) Amino acid sequence: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAGTYYCQQSNDDPWTFGGGTKLEIK (Sequence ID 58) CDR1: QSVDYDGDSY (Sequence ID 59) CAAAGTGTTGATTATGATGGTGATAGTTAT(Sequence ID 60) CDR2: AAS (Sequence ID 61) GCTGCATCC (Sequence ID 62) CDR3: QQSNDDPWT (Sequence ID 63) CAGCAAAGTAATGACGATCCGTGGACG (Sequence ID 64)

[0043] Thus, polyclonal or monoclonal antibodies, antibody fragments, and binding domains and CDRs (including any of the engineered forms described above) specific for 4-1BB, one or more of its respective epitopes, or any of the conjugates described above can be made by known means and as described herein, regardless of whether such antigen or epitope is isolated from a natural source or is a synthetic derivative or variant of a natural compound.

[0044] Examples of antibody fragments suitable for this embodiment include, but are not limited to, the following: (i) Fab fragments consisting of V L , V H , C L and C H1 domains; (ii) "Fd" fragments consisting of V H domains and C H1 domains; (iii) "Fv" fragments consisting of the V L domain and V H domain of a single antibody; (iv) "dAb" fragments consisting of V H domains; (v) isolated CDR regions; (vi) F(ab')2 fragments, bivalent fragments containing two linked Fab fragments; (vii) single-chain Fv molecules ("scFv") in which the V H domain and V L domain are linked by a peptide linker that allows the two domains to associate to form a binding domain; (viii) bispecific single-chain Fv dimers (see U.S. Patent No. 5,091,513); and (ix) diabodies, multivalent or multispecific fragments constructed by gene fusion (U.S. Patent Application Publication No. 20050214860). Fv, scFv, or diabody molecules can be stabilized by incorporation of disulfide bridges that link the V H and V L domains. Mini-bodies containing scFv linked to a CH3 domain can also be made (Hu et al., 1996).

[0045] Antibody-like peptide mimes are also intended in the embodiments. Liu et al. (2003) described “antibody-like peptide mimes” (ABiPs), which are peptides that act as paired-down antibodies and have the particular advantages of a longer serum half-life and a less complicated synthesis method.

[0046] To produce antibodies specific to 4-1BB, animals may be inoculated with antigens such as the 4-1BB extracellular domain (ECD) protein. Often, the antigen is conjugated to another molecule to enhance the immune response. As used herein, a conjugate is any peptide, polypeptide, protein, or non-proteinogenic substance conjugated to an antigen used to induce an immune response in an animal. Antibodies produced in an animal in response to antigen inoculation include a variety of non-identical molecules (polyclonal antibodies) made up of various individual antibodies that produce B lymphocytes. Polyclonal antibodies are a mixed population of antibody species, each capable of recognizing a different epitope on the same antigen. Given the correct conditions for polyclonal antibody production in an animal, most antibodies in the animal's serum will recognize a collective epitope on the antigen compound to which the animal is immunized. This specificity is further enhanced by affinity purification, which selects only the antibodies that recognize the antigen or epitope of interest.

[0047] Monoclonal antibodies are single-species antibodies in which all antibody molecules recognize the same epitope, because all antibody-producing cells originate from a single B lymphocyte cell line. Methods for producing monoclonal antibodies (MAbs) generally begin in the same manner as methods for preparing polyclonal antibodies. In some embodiments, rodents such as mice and rats are used in producing monoclonal antibodies. In some embodiments, rabbit, sheep, or frog cells are used for the production of monoclonal antibodies. The use of rats is well known and may offer certain advantages. Mice (e.g., BALB / c mice) are routinely used and generally give stable fusions at a high rate.

[0048] Hybridoma technology involves the fusion of a single B lymphocyte derived from a mouse pre-immunized with the 4-1BB antigen with immortalized myeloma cells (typically mouse myeloma). This technology provides a method for propagating a single antibody-producing cell over an unspecified number of generations so that an unlimited amount of structurally identical antibodies (monoclonal antibodies) with the same antigen or epitope specificity can be produced.

[0049] Plasma B cells (CD45 + CD5 - CD19 + ) can be isolated from newly prepared rabbit peripheral blood mononuclear cells of immunized rabbits, and further selection of 4-1BB-binding cells is possible. After enrichment of antibody-producing B cells, total RNA can be isolated and cDNA can be synthesized. The DNA sequences of the antibody variable region from both the heavy and light chains can be amplified, constructed into a phage display Fab expression vector, and transformed into E. coli. 4-1BB-specific binding Fab can be selected and sequenced by multiple enrichment pannings. The selected 4-1BB-binding hits can be expressed in full length in rabbit and rabbit / human chimeric morphologies using a mammalian expression vector system in human fetal kidney (HEK293) cells (Invitrogen). It can be expressed as IgG and purified using a protein G resin equipped with a high-performance protein liquid chromatography (FPLC) separation unit.

[0050] In one embodiment, the antibody is a chimeric antibody, for example, a non-human donor antibody transplanted onto a heterogeneous non-human, human, or humanized sequence (e.g., framework and / or constant domain sequence). These are antibodies containing antigen-binding sequences derived from a specific organism. Methods have been developed to replace the light chain constant domain and heavy chain constant domain of monoclonal antibodies with similar domains of human origin, while keeping the variable region of the exogenous antibody unchanged. Alternatively, "fully human" monoclonal antibodies are produced in mice into which human immunoglobulin genes have been introduced. Methods to make the variable domain of monoclonal antibodies more human-like have also been developed by recombining and constructing antibody variable domains that have amino acid sequences from both rodents, e.g., mice and humans. In "humanized" monoclonal antibodies, only the hypervariable CDR is derived from the mouse monoclonal antibody, while the framework and constant region are derived from human amino acid sequences (see U.S. Patents 5,091,513 and 6,881,557). By replacing the amino acid sequences in antibodies characteristic of rodents with amino acid sequences found at corresponding positions in human antibodies, the possibility of adverse immune reactions during therapeutic use is thought to be reduced. The antibody-producing hybridomas or other cells may also undergo gene mutations or other changes, which may or may not alter the binding specificity of the antibodies to which the hybridomas produce.

[0051] Methods for producing polyclonal antibodies in various animal species, as well as methods for producing various types of monoclonal antibodies, including humanized, chimeric, and fully human, are well known and highly predictable in the art. For example, the following U.S. patents and patent applications enable the description of such methods: U.S. Patent Applications Nos. 2004 / 0126828 and 2002 / 0172677; and U.S. Patents Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,196,265; 4,275,149; 4,277,437; 4,366,241; 4,469,797; and 4,4 No. 72,509; No. 4,606,855; No. 4,703,003; No. 4,742,159; No. 4,767,720; No. 4,816,567; No. 4,867,973 No. 4,938,948; No. 4,946,778; No. 5,021,236; No. 5,164,296; No. 5,196,066; No. 5,223,409; No. 5, No. 403,484; No. 5,420,253; No. 5,565,332; No. 5,571,698; No. 5,627,052; No. 5,656,434; No. 5,770,37 No. 6; No. 5,789,208; No. 5,821,337; No. 5,844,091; No. 5,858,657; No. 5,861,155; No. 5,871,907; No. 5 Patents No. 969,108; No. 6,054,297; No. 6,165,464; No. 6,365,157; ​​No. 6,406,867; No. 6,709,659; No. 6,709,873; No. 6,753,407; No. 6,814,965; No. 6,849,259; No. 6,861,572; No. 6,875,434; and No. 6,891,024. This specification and all patents, patent application publications, and other publications cited herein are incorporated by reference in this application.

[0052] Antibodies can be produced from any animal source, including birds and mammals. Preferably, the antibodies are from sheep, mice (e.g., rats and crickets), rabbits, goats, guinea pigs, camels, horses, or chickens. Furthermore, newer technologies enable the development and screening of human antibodies from human combinatorial antibody libraries. For example, bacteriophage antibody expression technology enables the production of specific antibodies in the absence of animal immunization, as described in U.S. Patent No. 6,946,546, incorporated herein by reference. These technologies are further described in Marks (1992); Stemmer (1994); Gram et al., (1992); Barbas et al., (1994); and Schier et al., (1996).

[0053] Antibodies against 4-1BB are expected to have the ability to neutralize or counteract the effects of 4-1BB, regardless of animal species, monoclonal cell lines, or other sources of antibodies. Certain animal species may be less desirable for producing therapeutic antibodies because they may be more likely to cause allergic reactions due to complement system activation by the "Fc" portion of the antibody. However, whole antibodies can be enzymatically digested into antibody fragments that have an "Fc" (complement-binding) fragment and a binding domain or CDR. Removal of the Fc portion reduces the likelihood that the antigen-antibody fragment will induce an undesirable immunological response, and therefore, antibodies without Fc may be preferred for prophylactic or therapeutic treatment. As described above, antibodies can also be constructed to be chimeric or partially or completely human in order to reduce or eliminate adverse immunological consequences resulting from administering antibodies produced in or containing sequences from other species to animals.

[0054] Substitutional variants typically involve replacing one amino acid with another at one or more sites within a protein and may be designed to modulate one or more properties of a polypeptide, with or without loss of other functions or properties. The substitution may be conservative, i.e., one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, changes from alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions may be non-conservative in such a way that the function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting a residue with a chemically different residue, such as a nonpolar or uncharged amino acid for a polar or charged amino acid, and vice versa.

[0055] The protein may be recombinant or synthesized in vitro. Alternatively, non-recombinant or recombinant proteins may be isolated from bacteria. Bacteria containing such variants are also intended to be realized in the composition and method. Therefore, it is not necessary to isolate the protein.

[0056] The composition is intended to contain a total polypeptide, peptide, and / or protein of approximately 0.001 mg to approximately 10 mg / ml. Therefore, the protein in the composition The concentration of the substance may be approximately, at least approximately, or at most approximately 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml, or greater (or any derivable range within that range). Of these, approximately, at least approximately, or at most approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% may be antibodies that bind to 4-1BB.

[0057] An antibody, or preferably an immunological portion of an antibody, can be chemically conjugated into a fusion protein with another protein, or can be expressed as a fusion protein with another protein. For the purposes of this specification and the appended claims, all such fusion proteins are included in the definition of an antibody or an immunological portion of an antibody.

[0058] Embodiments provide antibodies and antibody-like molecules against 4-1BB, polypeptides, and peptides that are conjugated to at least one drug to form an antibody conjugate or payload. Conventionally, to enhance the efficacy of antibody molecules as diagnostic or therapeutic agents, it is practiced to conjugate, covalently bond, or complex at least one desired molecule or part. Such molecules or parts may, but are not limited to, at least one effector molecule or reporter molecule. Effector molecules include molecules having desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules attached to antibodies include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides, etc. In contrast, a reporter molecule is defined as any part that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands, e.g., biotin.

[0059] Several methods for attaching or conjugating antibodies to their conjugate portion are known in the art. Some conjugation methods include, for example, diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3-6-diphenyl glycoluryl-3( This includes using metal chelate conjugates attached to antibodies using organic chelating agents such as tetrachloro-3-6-diphenylglycouril-3. Monoclonal antibodies can also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanates.

[0060] II. Treatment of Diseases Certain aspects of this embodiment can be used to prevent or treat diseases or disorders related to 4-1BB signaling. The 4-1BB antibody can be used to treat diseases such as cancer, infectious diseases, inflammatory diseases, or autoimmune diseases. Further methods for administering vaccines are provided. Suitable vaccines include, for example, tumor cell vaccines, DNA vaccines, GM-CSF modified tumor cell vaccines, or antigen-loaded dendritic cell vaccines.

[0061] In certain embodiments, the compositions and methods of this embodiment include an antibody or antibody fragment against 4-1BB for activating its activity in cancer cell proliferation, in combination with a second or additional treatment.

[0062] Accordingly, in some embodiments, methods for treating cancer or delaying the progression of cancer in an individual are provided herein, comprising the step of administering an effective amount of anti-41BB antibody to the individual. Examples of cancers intended for treatment include lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphoma, precancerous lesions of the lung, colon cancer, melanoma, and bladder cancer.

[0063] In some embodiments, an individual has cancer that is resistant (or has been demonstrated to be resistant) to one or more anticancer therapies. In some embodiments, resistance to anticancer therapy includes recurrence of cancer or refractory cancer. Recurrence may refer to the reappearance of cancer at the original site or a new site after treatment. In some embodiments, resistance to anticancer therapy includes progression of cancer during treatment with anticancer therapy. In some embodiments, the cancer is in an early or late stage.

[0064] A. Pharmaceutical preparations When a therapeutic composition containing an inhibitory antibody is to be clinically applied, it is generally beneficial to prepare a pharmaceutical or therapeutic composition that is appropriate for the intended application. In certain embodiments, the pharmaceutical composition may contain, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may contain about 2% to about 75% by unit weight, or, for example, about 25% to about 60%, and any derivable range thereof.

[0065] The therapeutic composition of this embodiment is advantageously administered in the form of an injectable composition, either as a liquid solution or a suspension. A solid form suitable for dissolving or suspending in a liquid before injection may also be prepared. These preparations may be emulsified.

[0066] The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that, when administered to animals such as humans as necessary, do not cause harmful, allergic, or other adverse reactions. The preparation of pharmaceutical compositions containing antibodies or further active ingredients will be known to those skilled in the art in light of this disclosure. Furthermore, it will be understood that, in the case of administration to animals (e.g., humans), the formulations should meet the standards of sterility, pyrogenicity, general safety, and purity required by the FDA Office of Biological Standards.

[0067] As used herein, “pharmaceutically acceptable carrier” means any and all aqueous solvents (e.g., water, alcohol / aqueous solution, physiological solvent) as is known to those skilled in the art. Examples include saline solutions, parenteral excipients (e.g., sodium chloride, glucose Ringer's solution), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters (e.g., ethyl oleate)), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption retarders, salts, drugs, drug stabilizers, gels, binders, pharmaceutical excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluids, and nutritional supplements, materials of this type, and combinations thereof. The pH and precise concentrations of various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0068] The terms “unit dose” or “dosage” refer to physically distinct units suitable for use in a subject, each unit containing a predetermined amount of the therapeutic composition calculated to produce the desired response described above in relation to its administration, i.e., the appropriate route and treatment regimen. The amount administered depends on the desired effect, depending on both the number of treatments and the unit dose. The actual dose of the composition of this embodiment administered to a patient or subject can be determined by physical and physiological factors such as body weight, the subject’s age, health status and sex, the type of disease being treated, the extent of disease penetration, previous or concurrent therapeutic interventions, the patient’s idiopathic disease, the route of administration, and the potency, stability and toxicity of the particular therapeutic substance. For example, doses may also include approximately 1 μg / kg / body weight to approximately 1000 mg / kg / body weight per administration (such ranges include intervening doses) or more, and any range derivable therein. In non-limiting examples derived from the numbers listed herein, doses can be administered in ranges such as approximately 5 μg / kg / body weight to approximately 100 mg / kg / body weight, or approximately 5 μg / kg / body weight to approximately 500 mg / kg / body weight. In any case, the physician administering the medication will determine the concentration of the active ingredient in the composition and the appropriate dose for each individual subject.

[0069] The active compound can be formulated for parenteral administration, for example, for injection via intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. Typically, such compositions can be prepared as either a liquid solution or a suspension. Solid forms suitable for use in preparing a solution or suspension by adding liquid before injection can also be prepared. The formulation can also be emulsified.

[0070] Suitable pharmaceutical forms for injection include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injection solutions or dispersions. In all cases, the form must be sterile and fluid enough to be easily injected. It must also be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi.

[0071] Protein compositions can be formulated in neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the protein), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, and procaine.

[0072] Pharmaceutical compositions may include, for example, a solvent or dispersion medium containing water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Sustained absorption of injectable compositions can be achieved by the use of absorption-delaying agents, such as aluminum monostearate and gelatin in the composition.

[0073] B. Concurrent treatment In certain embodiments, the compositions and methods of this embodiment include an antibody or antibody fragment against 4-1BB to activate its activity in combination with a second or additional treatment. For example, the disease may be cancer.

[0074] Methods and compositions including combination therapies enhance therapeutic or protective effects, and / Alternatively, it enhances the therapeutic effect of other anti-cancer therapies or anti-hypertrophy therapies. Therapeutic and prophylactic methods and compositions achieve desired effects such as the death of cancer cells and / or inhibition of cell hyperproliferation. It can be provided in a combined amount effective to achieve the effect. This process may involve contacting cells with both an antibody or antibody fragment and a second therapeutic agent. Tissues, tumors, or cells may be contacted with one or more compositions or pharmacological preparations containing one or more drugs (i.e., antibodies or antibody fragments or anticancer agents), or tissues, tumors, and / or cells with two or more This can be achieved by contacting different compositions or formulations, where one composition provides 1) an antibody or antibody fragment, 2) an anticancer agent, or 3) both an antibody or antibody fragment and an anticancer agent. Furthermore, such combination therapy is intended to be used in combination with chemotherapy, radiotherapy, surgical therapy, or immunotherapy.

[0075] The terms “contacted” and “exposed” are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to or directly juxtaposed with target cells when applied to cells. To achieve cell death, for example, both agents are delivered to cells in a total amount effective in killing the cells or preventing them from dividing.

[0076] Inhibitory antibodies can be administered before, during, or after anticancer treatment, or in various combinations with anticancer treatment. Dosage may occur at intervals ranging from simultaneous to minutes to days to weeks. In embodiments where the antibody or antibody fragment is delivered to the patient separately from the anticancer agent, it is generally ensured that no significant time elapses between each delivery so that the two compounds can still exert a beneficial combined effect on the patient. In such cases, it is intended that antibody therapy and anticancer therapy can be delivered to the patient within approximately 12–24 hours or 72 hours from each other, more specifically, within approximately 6–12 hours from each other. In some situations, if several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapse between each delivery, it may be desirable to significantly extend the treatment period.

[0077] In certain embodiments, a series of treatments may last from 1 to 90 days or longer (such a range includes intervening days). One drug may be administered on any day from day 1 to day 90 (such a range includes intervening days) or in any combination thereof, and another drug may be administered on any day from day 1 to day 90 (such a range includes intervening days) or in any combination thereof. Within one day (24 hours), the patient may be administered a single or multiple dose of the drug. Furthermore, it is intended that there will be a period after a series of treatments during which no anticancer treatment is administered. This period may last from 1 to 7 days and / or from 1 to 5 weeks and / or from 1 to 12 months or longer (such a range includes intervening days), depending on the patient's condition, such as prognosis, physical strength, and health status. The treatment cycle is expected to be repeated as needed.

[0078] Various combinations can be used. In the following example, antibody therapy is "A" and anti-cancer therapy is "B". [ka]

[0079] The administration of any compound or therapeutic agent of this embodiment to a patient follows a general protocol for the administration of such compounds, taking into consideration any potential toxicity of the drug. Therefore, in some embodiments, there is a step to monitor toxicity resulting from the combination therapy.

[0080] 1.Chemotherapy A wide variety of chemotherapeutic agents can be used according to this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to include compounds or compositions administered in the treatment of cancer. These drugs or agents are classified by their mode of intracellular activity, such as whether and at what stage they affect the cell cycle. Alternatively, drugs may be characterized based on their ability to directly crosslink DNA, intercalate DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0081] Examples of chemotherapeutic agents include alkylating agents, e.g., thiotepa and cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan, and picosulfan; aziridines, e.g., benzodopa, carbocon, meturedopa, and uredopa; ethyleneimines and methylamelamines (altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine); acetogenins (especially bratacin and bratacinone); camptothecin (including its synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its synthetic analogs adzeresin, karzeresin, and bizeresin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); Dorastatin; Duocalmycin (including synthetic analogs, KW-2189 and CB1-TM1); Erytherobin; Pancratistatin; Sarcodicin; Spongestatin; Nitrogen mustards, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, novembichin, fenesterine, prednimustine, trophosphamide and uracil mustard; Nitroureas, e.g., carmustine, chlorozotosine, fotemustine, lomustine, nimustine and ranimustine; Antibiotics, e.g., engine antibiotics (e.g., calicheamicin, especially calicheamicin gammal I and calicheamicin omega I1); Dynemycin (including dynemycin A); Bisphosphonates, e.g., clodronate; Esperamycin;Similarly, neocardinostatin chromophore and related pigment protein enediin antibiotics chromophore, acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin, kactinomycin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detrevicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin) Including epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, mycophenolic acid, nogalamycin, olibomycin, peplomycin, potfiromycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin and zolbicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folate analogs, e.g., denopterin, pteropterin and tri Methrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine derivatives, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepithiostan, and testolactone; anti-adrenaline, e.g., mitotane and trilostane; folic acid supplements, e.g., folic acid; acegraton; aldofol Sphamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisanthren; edatraxate; defofamine; demecolsin; diazicon; elformitin; eriptinium acetate; epotilon; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidynin; meitansinoids, e.g., meitansin and ansamitosin; mitogwazone; mitoxantrone; mopidanmol; nitretrelin; pentostatin; fenamet; pirarubicin; rosoxantrone; podophyllic acid;2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex; Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triadicone; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, Beraclin A, Loridine A and Anguidin); Urethanes; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Taxoids, e.g. For example, paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum-coordinated complexes, e.g., cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids, e.g., retinoic acid; capecitabine; carboplatin, procarbazine, precomycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, trans-platinum, and any pharmaceutically acceptable salts, acids or derivatives of any of the above. ;

[0082] 2. Radiation therapy Other widely used factors that cause DNA damage include gamma rays, X-rays, and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damage factors are also considered, including microwaves, proton beam irradiation (US Patent Nos. 5,760,395 and 4,870,287), and UV irradiation. All of these factors are most likely to affect widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome construction and maintenance. The dose range for X-rays ranges from a daily dose of 50–200 roentgens over a long period (3–4 weeks) to a single dose of 2000–6000 roentgens. The dose range for radioisotopes varies widely and depends on the half-life of the isotope, the intensity and type of radiation emitted, and uptake by neoplastic cells.

[0083] 3. Immunotherapy Those skilled in the art will understand that additional immunotherapies may be used in combination with or in conjunction with the methods of the embodiments. In relation to cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Immune effectors may include, for example, antibodies or chimeric antigen receptors (CARs) specific to certain markers on the surface of tumor cells. In further embodiments, the treatment may include the administration of T cells or NK cells that target specific cancer cells. Such cells may be engineered for anti-cancer cell activity or simply selected.

[0084] In some embodiments, antibodies can function as therapeutic effectors on their own, or they can mobilize other cells to actually influence cell death. Antibodies can also be conjugated with drugs or toxins (such as chemotherapeutic agents, radionuclides, lysine A chain, cholera toxin, pertussis toxin, etc.) and function as targeting agents. Alternatively, the effector may be lymphocytes carrying surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

[0085] Antibody-drug conjugates have emerged as a groundbreaking approach to the development of cancer treatments. Cancer is one of the leading causes of death worldwide. Antibody-drug conjugates (ADCs) contain a monoclonal antibody (MAb) covalently bound to a cell-killing drug. This approach combines the high specificity of the MAb to an antigen target with a highly potent cytotoxic drug, resulting in an "armed" MAb that delivers the payload (drug) to tumor cells with concentrated levels of the antigen. Targeted drug delivery also minimizes its exposure to normal tissue, leading to reduced toxicity and improved therapeutic index. The approval of two ADC drugs by the FDA in 2011, ADCETRIS® (brentuximab vedotin), and in 2013, KADCYLA® (trastuzumab emtansine or T-DM1), demonstrated this approach. Currently, there are more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment. As antibody engineering and linker-payload optimization become increasingly mature, the discovery and development of new ADCs increasingly depend on this approach and the identification and validation of new targets suitable for the generation of targeted MAbs. Two criteria for ADC targets are upregulation of expression in tumor cells / It requires a high level of internalization and robust resilience.

[0086] In one embodiment of immunotherapy, tumor cells must possess some marker suitable for targeting, i.e., markers not present in most other cells. Many tumor markers exist, and any of them may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative embodiment of immunotherapy is to combine anticancer effects with immunostimulatory effects. Immunostimulatory molecules also exist, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, and γ-IFN, chemokines such as MIP-1, MCP-1, and IL-8, and growth factors such as FLT3 ligand.

[0087] Examples of immunotherapies currently under investigation or in use include: immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patent Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapies, e.g., interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapies, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward et al., 1998); and gene therapies, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward et al., 1998). Villaseca, 1998; U.S. Patent Nos. 5,830,880 and 5,846,945); monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). It is intended that one or more anticancer therapies may be used in conjunction with the antibody therapies described herein.

[0088] In some embodiments, immunotherapy may be immune checkpoint inhibitors. Immune checkpoints are molecules in the immune system that either initiate (e.g., co-stimulatory molecules) or deactivate signals. Inhibitory checkpoint molecules that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2aR), B7-H3 (also known as CD276), B and T lymphocyte attenuators (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene 3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and the V-domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0089] Immune checkpoint inhibitors may be drugs such as small molecules, ligands, or recombinant forms of receptors, or in particular antibodies such as human antibodies (e.g., International Publication No. 2015016718; Pardoll, 2012; both incorporated herein by reference). Known inhibitors of immune checkpoint proteins or their analogues may be used, in particular chimeric, humanized, or human-type antibodies may be used. As those skilled in the art will understand, for any specific antibodies referred to herein, alternative and / or equivalent names may be used. The name may be used. Such alternative and / or equivalent names may be used in the context of the present invention. They are interchangeable. For example, lambrolizumab is known to also be known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0090] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand-binding partner. In certain embodiments, the PD-1 ligand-binding partner is PDL1 and / or PDL2. In another embodiment, PDL1 binding An antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In certain embodiments, the PDL1 binding partner is PD-1 and / or B7-1. Another embodiment In form, a PDL2-binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In certain embodiments, the PDL2-binding partner is PD-1. The antagonist may be an antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509 and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, including those described in U.S. Patent Application Nos. 2014,029,4898, 2014,022,021 and 2011,000,8369, all of which are incorporated herein by reference.

[0091] In some embodiments, the PD-1 conjugated antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 conjugated antagonist is an immunoadhesin (e.g., an immunoadhesin containing an extracellular or PD-1 binding moiety of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 conjugated antagonist is AMP-224. Nivolumab is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, and is an anti-PD-1 antibody described in International Publication No. 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in International Publication No. 2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in International Publication No. 2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in International Publication Nos. 2010 / 027827 and International Publication Nos. 2011 / 066342.

[0092] Another immune checkpoint that can be targeted by the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank deposit number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when it binds to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is analogous to the T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2, respectively, on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulating signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. T cell activation by T cell receptors and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for the B7 molecule.

[0093] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide.

[0094] Anti-human CTLA-4 antibodies (or VH and / or VH derived therefrom) suitable for use in this method The VL domain can be generated using methods well known in the art. Alternatively, an anti-CTLA-4 antibody recognized in the art can be used. For example, the anti-CTLA-4 antibody disclosed in U.S. Patent No. 8,119,129, International Publication No. 01 / 14424, and International Publication No. 98 / 42752; International Publication No. 00 / 37504 (CP675,206, also known as tremelimumab; formerly tisilimmab), U.S. Patent No. 6,207,156; Hurwitz et al., (1998) Proc Natl Acad Sci USA 95(17):10067-10071; Camacho et al., (2004) J Clin Oncology 22(145):Abstract No.2505 (Antibody CP-675206); and Mokyr et al., (1998) Cancer Res 58:5301-5304 can be used in the manner disclosed herein. The teachings of each of the aforementioned publications are incorporated herein by reference. Antibodies that compete with any of the antibodies recognized in these arts for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application No. 2001014424, International Patent Application No. 2000037504 and U.S. Patent No. 8017114, all of which are incorporated herein by reference.

[0095] Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10 D1, MDX-010, MDX-101, and Yervoy®) or its antigen-binding fragments and variants (see, for example, International Publication No. 1 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for and / or binds to the same epitope on CTLA-4, similar to the antibody described above. In yet another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the antibody described above (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0096] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors, such as those described in U.S. Patent No. 5,844,905, U.S. Patent No. 5,885,796, and International Patent Applications WO1995,001,994, and WO1998,042,752 (all incorporated herein by reference), as well as immunoadhesion, such as that described in U.S. Patent No. 8,329,867 (incorporated herein by reference).

[0097] 4.Surgery Approximately 60% of people with cancer will undergo some form of surgery, including prophylactic surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery includes excision, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed. The treatment of this embodiment may be used in conjunction with other therapies such as chemotherapy, radiotherapy, hormone therapy, gene therapy, immunotherapy and / or alternative therapies. Tumor resection is performed when the tumor is small Both refer to partial physical removal. In addition to tumor resection, surgical procedures include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs procedure).

[0098] When cancerous cells, tissue, or part or all of a tumor are removed, a cavity may form in the body. Treatment can be achieved by perfusion, direct injection, or local application of additional anticancer therapy to the area. Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may also vary in dosage.

[0099] 5. Other medications To improve the therapeutic efficacy of the treatment, other agents may be used in combination with specific embodiments of this embodiment. These additional agents may include agents that affect the upregulation of cell surface receptors and GAP junctions, cell proliferation inhibitors and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferating cells to apoptosis-inducing factors, or other biological agents. Increasing the number of GAP junctions would increase intercellular signaling, thereby increasing the anti-hyperproliferative effect on adjacent hyperproliferating cell populations. In other embodiments, cell proliferation inhibitors or differentiation agents may be used in combination with specific embodiments of this embodiment to improve the anti-hyperproliferative effect of the treatment. Cell adhesion inhibitors are intended to improve the efficacy of this embodiment. Examples of cell adhesion inhibitors include focal adhesion kinase (FAK) inhibitors and lovastatin. It is further intended that other agents that increase the sensitivity of hyperproliferating cells to apoptosis, such as the antibody c225, may be used in combination with specific embodiments of this embodiment to improve the efficacy of the treatment.

[0100] III. Kits and Diagnostics Various embodiments of the model include therapeutic agents and / or other therapeutic agents and delivery agents. A kit is envisioned. In some embodiments, this embodiment envisions a kit for preparing and / or administering the therapeutic agent of the embodiment. The kit is for the pharmaceutical composition of this embodiment The kit may include one or more sealed vials containing any of the following: the kit may include, for example, at least one 4-1BB antibody, as well as the components of the embodiment, which may be prepared, formulated, and / or The kit may include reagents for administration or reagents for carrying out one or more steps of the method according to the present invention. In some embodiments, the kit may also include a suitable container that does not react with the components of the kit, such as Eppendorf tubes, assay plates, syringes, bottles, or tubes. The container may be made from a sterile material such as plastic or glass.

[0101] The kit may further include instructions outlining the procedures of the methods described herein, which are substantially the same as those described herein or are known to those skilled in the art. The instructional information may be in computer-readable media containing machine-readable instructions that, when executed using a computer, display actual or hypothetical procedures for delivering a pharmaceutically effective amount of therapeutic agent. [Examples]

[0102] IV. Examples The following embodiments are included to demonstrate preferred embodiments of the present invention. Those skilled in the art should understand that the techniques disclosed in the following embodiments represent techniques that the inventors have found to work well in carrying out the present invention and therefore may constitute a preferred form for its implementation. However, those skilled in the art should understand that, in light of this disclosure, many modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention, and similar or comparable results can still be obtained.

[0103] Example 1 - Production and characterization of anti-41BB antibody Mice were alternately immunized with cell vaccines (retrovirally engineered macrophages or dendritic cells expressing 4-1BB) and recombinant protein-based vaccines (recombinant human 4-1BB extracellular region with Fc or HIS tags) to generate antibodies targeting human 4-1BB, including clones 54 (IgG2a), 49A (IgG1), 138 (IgG2b), 151 (IgG3), and 111A (IgG1) (Figure 1). EC50 values ​​were determined by titration curves of the clones using ELISA (Figure 1B).

[0104] RNA Extraction: Hybridoma cell pellets were provided by Long Vien (Monoclonal Antibody Core Facility MDACC). Total mRNA was extracted from the hybridoma cell pellets. Total RNA was extracted from the hybridoma clone 271-54 41-BB cell pellet using the RNA extraction protocol (Zymo Research).

[0105] RT-PCR: cDNA was generated from RNA by reverse transcription using random primers (RT Superscrit III, Life Technologies). PCR reaction was performed to amplify both the VH and VL regions of monoclonal antibody DNA using variable domain primers, yielding the bands shown in Figure 3. The VH and VL products were gel-purified, cloned into the sequencing vector pCR®2.1-TOPO®, and transformed into DH5a cells. Next, positive transformants were screened by PCR using M16 forward and reverse primers (mFVIj forward primer (positions 59-76) sequence: ACTGCAGGTGTCCTCTCT (SEQ ID NO: 65); mRevIgG2a reverse primer (positions 526-506) sequence: TAACCCTTGACCAGGCATCC (SEQ ID NO: 66); mFVK4 / 5a forward primer (positions 47-67) sequence: TCAGCTTCYTGCTAATCAGTG (SEQ ID NO: 67); 1mRK reverse primer (positions 491-471) sequence: ACTGAGGCACCTCCAGATGTT (SEQ ID NO: 68)). Colonies selected for miniprep plasmid purification were analyzed by DNA sequencing (MDACC core facility).

[0106] Among numerous lead candidates, clone 54 4-1BB antibodies sequenced and expressed in various mouse and human isotypes were selected. Patient tumor masses were cultured in tumor-infiltrating lymphocyte medium to support their survival and rapid proliferation in cell culture. This study found that the mouse IgG2a version of clone 54 4-1BB antibody amplified and matured human tumor-infiltrating CD8 T cells and CD4 T cells as efficiently as the anti-human 4-1BB antibody urelumab (Figure 2B). Furthermore, clone 54 efficiently amplified tumor-infiltrating CD8 T cells and CD4 T cells from tumors of patients with glioblastoma or colorectal cancer (Figure 4).

[0107] All methods disclosed and claimed herein can be carried out and performed without undue experimentation in light of this disclosure. While the compositions and methods of the present invention have been described in relation to preferred embodiments, it will be apparent to those skilled in the art that modifications can be applied to the methods and steps or order of steps of the methods herein without departing from the concept, spirit and scope of the invention. More specifically, certain chemically and physiologically relevant agents may be substituted with the agents described herein, and it will be apparent that the same or similar results will be achieved. All such similar substitutions and modifications, which will be apparent to those skilled in the art, are considered to fall within the spirit, scope and concept of the invention as defined by the appended claims. References The following references are incorporated herein by reference insofar as they provide exemplary procedures or other details that supplement those described herein. Austin-Ward and Villaseca, Revista Medica de Chile, 126(7):838-845, 1998. Bukowski et al., Clinical Cancer Res., 4(10):2337-2347, 1998. Christodoulides et al., Microbiology, 144(Pt 11):3027-3037, 1998. Davidson et al., J. Immunother., 21(5):389-398, 1998. Hanibuchi et al., Int. J. Cancer, 78(4):480-485, 1998. Hellstrand et al., Acta Oncologica, 37(4):347-353, 1998. Hollander, Front. Immun., 3:3, 2012. Hui and Hashimoto, Infection Immun., 66(11):5329-5336, 1998. International Patent Publication WO2009 / 101611 International Patent Publication WO2010 / 027827 International Patent Publication WO2011 / 066342 International Patent Publication No. WO2015016718 Pardoll, Nature Rev Cancer, 12:252-264, 2012. Qin et al., Proc. Natl. Acad. Sci. USA, 95(24):14411-14416, 1998. U.S. Patent No. 4,870,287 U.S. Patent No. 5,091,513 U.S. Patent No. 5,739,169 U.S. Patent No. 5,760,395 U.S. Patent No. 5,801,005 U.S. Patent No. 5,824,311 U.S. Patent No. 5,830,880 U.S. Patent No. 5,846,945 U.S. Patent No. 6,881,557 U.S. Patent No. 8,008,449 U.S. Patent No. 8,354,509 U.S. Patent No. 8,735,553 U.S. Patent Publication US20050214860 U.S. Patent Publication US20110008369 U.S. Patent Publication US2014022021 U.S. Patent Publication US20140294898 Vinay and Kwon, Mol Cancer Ther, 11(5): 1062-70, 2012. In certain embodiments, for example, the following are provided: (Item 1) An isolated monoclonal antibody, wherein the antibody specifically binds to 4-1BB, (a) The first V H CDR is identical to sequence number 3, (b) Second V H The CDR is identical to sequence number 5. (c) The third V H The CDR is identical to sequence number 7. (d) The first V L CDR is identical to sequence number 11, (e) Second V L CDR is identical to sequence number 13, and (f) Third V L Is the CDR identical to sequence number 15, or (a) The first V H The CDR is identical to sequence number 19. (b) Second V H The CDR is identical to sequence number 21. (c) The third V H The CDR is identical to sequence number 23. (d) The first V L The CDR is identical to sequence number 27. (e) Second V L The CDR is identical to sequence number 29, and (f) Third V L Is the CDR identical to sequence number 31, or (a) The first V H The CDR is identical to sequence number 35. (b) Second V H The CDR is identical to sequence number 37. (c) The third V H The CDR is identical to sequence number 39. (d) The first V L CDR is identical to sequence number 43, (e) Second V L CDR is identical to sequence number 45, and (f) Third V L Is the CDR identical to sequence number 47, or (a) The first V H The CDR is identical to sequence number 51. (b) Second V H The CDR is identical to sequence number 53. (c) The third V H The CDR is identical to sequence number 55. (d) The first V LThe CDR is identical to sequence number 59. (e) Second V L CDR is identical to sequence number 61, and (f) Third V L The CDR is identical to sequence number 63. Isolated monoclonal antibodies, including the above. (Item 2) The aforementioned antibody (a) The first V H CDR is identical to sequence number 3, (b) Second V H The CDR is identical to sequence number 5. (c) The third V H The CDR is identical to sequence number 7. (d) The first V L CDR is identical to sequence number 11, (e) Second V L CDR is identical to sequence number 13, and (f) Third V L The CDR is identical to sequence number 15. The antibodies listed in item 1, including the one described above. (Item 3) Clone 54 V H Domain (SEQ ID NO: 2) and V are at least approximately 80% identical. H Domain and clone 54 V L Domain (SEQ ID NO: 10) and V are at least approximately 80% identical. L The antibody described in item 2, including the domain. (Item 4) Clone 54 V H Domain (SEQ ID NO: 2) and V are at least approximately 90% identical. H Domain and clone 54 V L Domain (SEQ ID NO: 10) and V are at least approximately 90% identical. L The antibody described in item 2, including the domain. (Item 5) Clone 54 V H Domain (SEQ ID NO: 2) and the same V H Domain and clone 54 V L Domain (SEQ ID NO: 10) and the same VL The antibody described in item 2, including the domain. (Item 6) The aforementioned antibody (a) The first V H The CDR is identical to sequence number 19. (b) Second V H The CDR is identical to sequence number 21. (c) The third V H The CDR is identical to sequence number 23. (d) The first V L The CDR is identical to sequence number 27. (e) Second V L The CDR is identical to sequence number 29, and (f) Third V L The CDR is identical to sequence number 31. The antibodies listed in item 1, including the one described above. (Item 7) V of clone 135B H Domain (SEQ ID NO: 18) and V are at least approximately 80% identical. H Domain and clone 135B's V L Domain (SEQ ID NO: 26) and V are at least approximately 80% identical. L The antibody described in item 6, including the domain. (Item 8) V of clone 135B H Domain (SEQ ID NO: 18) and V are at least approximately 90% identical. H Domain and clone 135B's V L Domain (SEQ ID NO: 26) and V are at least approximately 90% identical. L The antibody described in item 6, including the domain. (Item 9) V of clone 135B H Domain (SEQ ID NO: 18) and the same V H Domain and clone 135B's V L Domain (SEQ ID NO: 26) and the same V L The antibody described in item 6, including the domain. (Item 10) The aforementioned antibody (a) The first V HThe CDR is identical to sequence number 35. (b) Second V H The CDR is identical to sequence number 37. (c) The third V H The CDR is identical to sequence number 39. (d) The first V L CDR is identical to sequence number 43, (e) Second V L CDR is identical to sequence number 45, and (f) Third V L The CDR is identical to sequence number 47. The antibodies listed in item 1, including the one described above. (Item 11) V of Clone 138 H Domain (SEQ ID NO: 34) and V are at least approximately 80% identical. H Domain and clone 138 V L Domain (SEQ ID NO: 42) and V are at least approximately 80% identical. L The antibody described in item 10, including the domain. (Item 12) V of Clone 138 H Domain (SEQ ID NO: 34) and V are at least approximately 90% identical. H Domain and clone 138 V L Domain (SEQ ID NO: 42) and V are at least approximately 90% identical. L The antibody described in item 10, including the domain. (Item 13) V of Clone 138 H Domain (SEQ ID NO: 34) and the same V H Domain and clone 138 V L Domain (SEQ ID NO: 42) and the same V L The antibody described in item 10, including the domain. (Item 14) The aforementioned antibody (a) The first V H The CDR is identical to sequence number 51. (b) Second V H The CDR is identical to sequence number 53. (c) The third V HThe CDR is identical to SEQ ID NO: 55, (d) the first V L The CDR is identical to SEQ ID NO: 59, (e) the second V L The CDR is identical to SEQ ID NO: 61 and (f) the third V L The CDR is identical to SEQ ID NO: 63 The antibody according to item 1, comprising this. (Item 15) The V of clone 49A H domain (SEQ ID NO: 50) and at least about 80% identical V H domain, and the V of clone 49A L domain (SEQ ID NO: 58) and at least about 80% identical V L domain, and the antibody according to item 14. (Item 16) The V of clone 49A H domain (SEQ ID NO: 50) and at least about 90% identical V H dome in, and the V of clone 49A L domain (SEQ ID NO: 58) and at least about 90% identical V L domain, and the antibody according to item 14. (Item 17) The V of clone 49A H domain (SEQ ID NO: 50) and identical V H domain, and the V of clone 49A L domain (SEQ ID NO: 58) and identical V L domain, and the antibody according to item 14. (Item 18) The antibody according to any one of items 1 to 5, wherein the antibody is recombinant. (Item 19) The antibody according to item 1, wherein the antibody is IgG, IgM, IgA or an antigen-binding fragment thereof. (Item 20) The antibody according to any one of items 1 to 19, wherein the antibody is Fab’, F(ab’)2, F(ab’)3, monovalent scFv, bivalent scFv or a single-domain antibody. (Item 21) The antibody described in any one of item 1 is a human antibody, a humanized antibody, or a deimmunized antibody. (Item 22) The antibody described in any one of items 1 to 21, wherein the antibody is conjugated with an imaging agent, a chemotherapeutic agent, a toxin, or a radionuclide. (Item 23) A composition comprising an antibody described in any one of items 1 to 22 in a pharmaceutically acceptable carrier. (Item 24) An isolated polynucleotide molecule containing a nucleic acid sequence encoding an antibody as described in any one of items 1 through 21. (Item 25) Clone 54 V H Domain CDR1-3 (SEQ ID NOs: 3, 5, and 7), clone 135B V H Domain CDR1-3 (SEQ ID NOs. 19, 21, and 23), clone 138 V H Domain CDR1-3 (SEQ ID NOs. 35, 37, and 39), or V of clone 49A H Antibody V containing domain CDR1-3 (SEQ ID NOs. 51, 53, and 55) H Recombinant polypeptides containing domains. (Item 26) 54 V L Domain CDR1-3 (SEQ ID NOs. 11, 13, and 15), V of 135B L Domain CDR1-3 (SEQ ID NOs. 27, 29, and 31), V of 138 L Domain CDR1-3 (SEQ ID NOs. 43, 45, and 47), or V of 49A L Antibody V containing domain CDR1-3 (SEQ ID NOs. 59, 61, and 63) L Recombinant polypeptides containing domains. (Item 27) Clone 54 V H Antibody V containing domain CDR1-3 (SEQ ID NOs: 3, 5, and 7) H Domain and 54 V L Antibody V containing domain CDR1-3 (SEQ ID NOs: 11, 13, and 15)L Domain; Clone 135B V H Antibody V containing domain CDR1-3 (SEQ ID NOs: 19, 21, and 23) H Domain and 135B V L Antibody V containing domain CDR1-3 (SEQ ID NOs: 27, 29, and 31) L Domain; Clone 138 V H Antibody V containing domain CDR1-3 (SEQ ID NOs: 35, 37, and 39) H Domain and 138 V L Antibody V containing domain CDR1-3 (SEQ ID NOs: 43, 45, and 47) L Domain; or clone V of 49A H Antibody V containing domain CDR1-3 (SEQ ID NOs. 51, 53, and 55) H Domain and 49A V L Antibody V containing domain CDR1-3 (SEQ ID NOs. 59, 61, and 63) L Recombinant polypeptides containing domains. (Item 28) An isolated polynucleotide molecule containing a nucleic acid sequence encoding a polypeptide as described in any one of items 25 to 27. (Item 29) A host cell containing one or more polynucleotide molecules encoding an antibody as described in any one of items 1 through 21 or a recombinant polypeptide as described in any one of items 25 through 27. (Item 30) The host cell according to item 29, wherein the host cell is a mammalian cell, yeast cell, bacterial cell, ciliated cell, or insect cell. (Item 31) A method for producing antibodies, (a) The V of the antibody described in any one of items 1 to 21 L Chain and V H A step of expressing one or more polynucleotide molecules encoding a chain in a cell, (b) A step of purifying the antibody from the cells. Methods that include... (Item 32) A method for treating a subject having cancer, comprising the step of administering to the subject an effective amount of an antibody described in any one of items 1 to 21. (Item 33) The method according to item 32, wherein the cancer is breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, skin cancer, brain tumor, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. (Item 34) The method according to item 32, wherein the antibody is present in a pharmaceutically acceptable composition. (Item 35) The method described in item 32, wherein the aforementioned antibody is administered systemically. (Item 36) The method according to item 32, wherein the antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally. (Item 37) The method according to item 32, further comprising the step of administering at least a second anticancer therapy to the subject. (Item 38) The method according to item 37, wherein the second anticancer therapy is surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy. (Item 39) The method according to item 37, wherein the second anticancer therapy includes adoptive T-cell therapy. (Item 40) The method according to item 37, wherein the second anti-cancer therapy includes immunotherapy. (Item 41) The method according to item 40, wherein the immunotherapy is an immune checkpoint inhibitor. (Item 42) The method according to item 41, wherein the at least one immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-L1 antibody, and / or an anti-PD1 antibody. (Item 43) The method according to item 28, wherein the immune checkpoint inhibitor is a human programmed cell death 1 (PD-1) binding antagonist, a PDL1 binding antagonist, or a PDL2 binding antagonist. (Item 44) The PD-1 conjugated antagonist is a monoclonal antibody or its antigen-binding fragment The method described in item 43. (Item 45) The method according to item 43, wherein the PD-1 binding antagonist is nivolumab, pembrolizumab, CT-011 (pizilizumab), BMS-936559 (MDX-1105), MPDL328OA (atezolizumab), or AMP-224 (PD-L2 Fc fusion protein).

Claims

[Claim 1] The invention described in the specification.

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