Human 4-1bb agonist antibodies and methods of use thereof

Abstract

Provided are isolated or recombinant monoclonal antibodies that bind to 4-1BB. 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.

Description

[0001] This application is a divisional application of the invention patent application filed on February 17, 2021, with application number 202180023415.9 and invention title "Human 4-1BB Agonist Antibody and Method of Using Thereof".

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 977,658, filed on February 17, 2020 (which is incorporated herein by reference in its entirety).

[0003] Merging of sequence lists

[0004] The sequence list contained in the file named “UTFCP1467WO_ST25.txt” (which is 23 KB (measured in Microsoft Windows®) and was created on February 16, 2021) is submitted electronically and incorporated into this document by reference. Background of the Invention

[0005] 1. Field

[0006] This invention generally relates to the field of molecular biology. More specifically, this invention relates to tumor necrosis factor superfamily receptor (TNFSFR; 4-1BB; CD137) agonist antibodies.

[0007] 2. Description of relevant technologies

[0008] 4-1BB (CD137) (a member of the TNF receptor superfamily) is an activation-induced T-cell costimulatory molecule (Vinay and Kwon, 2012). Through 4-1BB signaling, survival genes are upregulated, cell division is enhanced, cytokine production is induced, and activation-induced cell death in T cells is prevented. The importance of the 4-1BB pathway has been highlighted in many diseases, including cancer. Increasing evidence suggests that anti-4-1BB monoclonal antibodies possess potent antitumor properties, which are directly attributable to their strong activation of CD8. + The results demonstrate the ability of T cells to produce IFN-γ and induce cell lysis markers. Furthermore, combination therapy with anti-4-1BB and other anticancer agents (e.g., radiation) exhibits robust tumor regression against non-immunogenic or poorly immunogenic tumors. Further, in vitro anti-4-1BB-activated CD8+ from pre-tumor-treated animals... + Adoptive transfer of T cells effectively suppressed tumor progression in recipient mice that had been inoculated with fresh tumors. Furthermore, targeting tumors with a 4-1BBL variant against 4-1BB also exhibited a potent anti-tumor effect. However, the need for improved human 4-1BB antibodies remains unmet. Invention Overview

[0009] In a first embodiment, this disclosure provides an isolated monoclonal antibody, wherein the antibody specifically binds to 4-1BB and comprises: (a) a first V H CDR is the same as SEQ ID NO: 3; (b) the second V H CDR is the same as SEQ ID NO: 5; (c) the third V H CDR is the same as SEQ ID NO: 7; (d) the first V L CDR is the same as SEQ ID NO: 11; (e) the second V L CDR is the same as SEQ ID NO: 13; and (f) the third V L CDR is the same as SEQ ID NO:15; or (a) the first V H CDR is the same as SEQ ID NO: 19; (b) the second V H CDR is the same as SEQ ID NO: 21; (c) the third V H CDR is the same as SEQ ID NO: 23; (d) the first V L CDR is the same as SEQ ID NO: 27; (e) the second V L CDR is the same as SEQ ID NO: 29; and (f) the third V L CDR is the same as SEQ ID NO: 31; or (a) the first V H CDR is the same as SEQ ID NO: 35; (b) the second V H CDR is the same as SEQ ID NO: 37; (c) the third V H CDR is the same as SEQ ID NO: 39; (d) the first V L CDR is the same as SEQ ID NO: 43; (e) the second V L CDR is the same as SEQ ID NO: 45; and (f) the third V L CDR is the same as SEQ ID NO: 47; or (a) the first V H CDR is the same as SEQ ID NO: 51; (b) the second V H CDR is the same as SEQ ID NO: 53; (c) the third V H CDR is the same as SEQ ID NO: 55; (d) the first V LCDR is the same as SEQ ID NO: 59; (e) the second V L CDR is the same as SEQ ID NO: 61; and (f) the third V L CDR is the same as SEQ ID NO: 63.

[0010] In some respects, the antibody comprises V with clone 54. H The domain (SEQ ID NO: 2) is at least approximately 80% identical, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the V. H Domain, and V with clone 54 L The domain (SEQ ID NO: 10) is at least approximately 80% identical, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the V. L Domain. In a particular aspect, the antibody contains V with clone 54. H The structural domain (SEQ ID NO: 2) is the same as the V. H Domain, and V with clone 54 L The same V as the structural domain (SEQ ID NO: 10) L Structural domain.

[0011] In some respects, the antibody contains V similar to clone 135B. H The domain (SEQ ID NO: 18) is at least approximately 80% identical, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the V. H Domain, and V with clone 135B L The domain (SEQ ID NO: 26) is at least approximately 80% identical, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the V. L Structural domain. In a particular aspect, the antibody comprises a V domain similar to that of clone 135B. H The same V as the structural domain (SEQ ID NO: 18) H Domain, and V with clone 135B L The same V as the structural domain (SEQ ID NO: 26) L Structural domain.

[0012] In some respects, the antibody comprises V with clone 138. HThe domain (SEQ ID NO: 34) is at least approximately 80% identical, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the V. H Domain, and V with clone 138 L The domain (SEQ ID NO: 42) is at least approximately 80% identical, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the V. L Structural domain. In a particular aspect, the antibody contains a V domain similar to that of clone 138. H The structural domain (SEQ ID NO: 34) is the same as the V. H Domain, and V with clone 138 L The same V as the structural domain (SEQ ID NO: 42) L Structural domain.

[0013] In some respects, the antibody comprises V with clone 49A. H The domain (SEQ ID NO: 50) is at least approximately 80% identical, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the V. H Domain, and V with clone 49A L The domain (SEQ ID NO: 58) is at least approximately 80% identical, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the V. L Structural domain. In a particular aspect, the antibody contains a V domain similar to that of clone 49A. H The same V as the structural domain (SEQ ID NO: 50) H Domain, and V with clone 49A L The same V as the structural domain (SEQ ID NO: 58) L Structural domain.

[0014] In some respects, the antibody is recombinant. In some respects, the antibody is IgG, IgM, IgA, or an antigen-binding fragment thereof. In some respects, the antibody is Fab', F(ab')2, F(ab')3, monovalent scFv, bivalent scFv, or a single-domain antibody. In a particular respect, the antibody is a human antibody, a humanized antibody, or a de-immunized antibody. In a further respect, the antibody is conjugated to an imaging agent, a chemotherapeutic agent, a toxin, or a radionuclide.

[0015] A further embodiment provides a composition comprising an antibody of the embodiments of the present invention in a pharmaceutically acceptable carrier. An isolated polynucleotide molecule comprising a nucleic acid sequence encoding an antibody of the embodiments of the present invention is also provided herein.

[0016] Another embodiment provides a recombinant polypeptide comprising: antibody V H A structural domain containing clone 54 of V H CDRs 1-3 (SEQ ID NO: 3, 5 and 7) of the domain, and V of clone 135B H CDRs 1-3 of the domain (SEQ ID NO: 19, 21 and 23), V of clone 138 H CDRs 1-3 (SEQ ID NO: 35, 37 and 39) of the domain or V of clone 49A H CDRs 1-3 of the domain (SEQ ID NO: 51, 53 and 55).

[0017] A further embodiment provides a recombinant polypeptide comprising: antibody V L A structural domain containing 54 V L CDRs 1-3 (SEQ ID NO: 11, 13 and 15) of the structural domain, and V of 135B L CDRs 1-3 (SEQ ID NO: 27, 29 and 31) of the structural domain, and V of 138 L CDRs 1-3 (SEQ ID NO: 43, 45 and 47) of the structural domain or V of 49A L CDR1-3 of the structural domain (SEQ ID NO: 59, 61 and 63).

[0018] In a further embodiment, a recombinant polypeptide is provided, the recombinant polypeptide comprising: V comprising clone 54 H Antibody V containing CDRs 1-3 (SEQ ID NO: 3, 5 and 7) of the structural domain H The structural domain, and the V containing 54 L Antibody V containing CDR1-3 (SEQ ID NO: 11, 13 and 15) domains L Domain; V containing clone 135B H Antibody V containing CDR 1-3 (SEQ ID NO: 19, 21 and 23) of the structural domain H The structural domain, and the V containing 135B L Antibody V containing CDR 1-3 (SEQ ID NO: 27, 29 and 31) of the structural domain L Domain; V containing clone 138H Antibody V containing CDR 1-3 (SEQ ID NO: 35, 37 and 39) of the structural domain H The structural domain, and the V containing 138 L Antibody V containing CDR 1-3 (SEQ ID NO: 43, 45 and 47) of the structural domain L Domain; or V containing clone 49A H Antibody V containing CDR 1-3 (SEQ ID NO: 51, 53 and 55) of the structural domain H The structural domain, and V containing 49A L Antibody V containing CDR 1-3 (SEQ ID NO: 59, 61 and 63) of the structural domain L Structural domain.

[0019] Further embodiments provide isolated polynucleotide molecules comprising nucleic acid sequences encoding polypeptides of embodiments of the present invention. Host cells are also provided herein, comprising one or more polynucleotide molecules encoding antibodies or recombinant polypeptides of embodiments of the present invention. In some aspects, the host cells are mammalian cells, yeast cells, bacterial cells, ciliate cells, or insect cells.

[0020] Another embodiment provides a method for preparing an antibody, the method comprising: (a) expressing one or more V antibodies encoding the antibodies of the embodiments of the present invention in cells. L and V H (a) a chain of polynucleotide molecules; and (b) the antibody is purified from the cells.

[0021] A further embodiment provides a method for treating a subject with cancer, the method comprising administering an effective amount of an antibody according to an embodiment of the invention to the subject.

[0022] In some respects, the cancers referred to are breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, skin cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

[0023] In some respects, the antibody is in a pharmaceutically acceptable composition. In some respects, the antibody is administered systemically. In some respects, the antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally.

[0024] In another aspect, the method further includes administering at least a second anticancer therapy to the subject. In some aspects, the second anticancer therapy is surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy. In some aspects, the second anticancer therapy includes adoptive T-cell therapy. In some aspects, the second anticancer therapy includes immunotherapy. In a particular aspect, 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 a particular aspect, the immune checkpoint inhibitor is a human programmed cell death 1 (PD-1) binding antagonist, a PDL1 binding antagonist, or a PDL2 binding antagonist. In some aspects, the PD-1 binding antagonist is a monoclonal antibody or its antigen-binding fragment. In some respects, the PD-1 binding antagonist is nivolumab, pembrolizumab, CT-011 (or pidilizumab), BMS 936559, MPDL328OA (or atezolizumab), or AMP-224.

[0025] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that while the detailed description and specific embodiments illustrate certain embodiments of the invention, they are given by way of example only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Brief description of the attached diagram

[0026] The accompanying drawings form part of this specification and are included to further illustrate certain aspects of the invention. A better understanding of the invention can be achieved by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein.

[0027] Figure 1A-1B A variety of high-affinity anti-human 4-1BB antibodies were generated. Figure 1A The results of an ELISA using the initial post-fusion antibody supernatant against recombinant human 4-1BB are shown. Figure 1B Titration curve of 4-1BB monoclonal antibody clone.

[0028] Figure 2A-2BThe present invention utilizes the clone 54 4-1BB antibody (referred to herein as Curranlumab) to efficiently amplify human TILs. Two variants of clone 54 (Curranlumab) were used to amplify human tumor-infiltrating lymphocytes (TILs) from patient tumor masses, and the efficiency of each variant in generating pure, expanded CD8+ T cell products was compared with that of urrelumab (BMS-663513).

[0029] Figure 3 PCR using several primer combinations with Ig variable domains. Asterisks indicate correct antibody transcripts for the VH and VL chains. The remaining PCR bands observed are abnormal transcripts confirmed by sequencing.

[0030] Figures 4A-4B Glioblastoma and colorectal cancer tumors were isolated, and tumor-infiltrating T cells were expanded using the 41BB agonist antibody clone 54 (as human IgG1 or mouse IgG2a). The extent of expansion, CD8 percentage, and CD4 percentage are shown. Total TILs from glioblastoma and colorectal cancer tumors amplified with clone 54 IgG1 and clone 54 IgG1 are also shown. Figure 4A The percentage of CD3+CD8+ TIL or CD3+CD4+ TIL generated by the extended method (). Figure 4B ).

[0031] Description of illustrative implementation schemes

[0032] Tumor necrosis factor superfamily receptors (TNFSFRs) (also known as 4-1BB and CD137) co-stimulate the activation of T cells, NK cells, and myeloid cells (including antigen-presenting dendritic cells). On CD8 T cells, 4-1BB activation increases their proliferation, cytotoxicity, cytokine production, mitochondrial quality, and viability. Therefore, a 4-1BB agonist antibody was generated in this study. Mice were immunized with alternating cell vaccines and recombinant protein-based vaccines to generate antibodies targeting human 4-1BB. Among many lead candidates, clone 54 4-1BB antibody was selected, sequenced, and expressed (in a wide variety of mouse and human isotypes). This study found that the IgG2a version of clone 54 4-1BB antibody very effectively expanded and matured tumor-infiltrating CD8 T cells.

[0033] Therefore, in some embodiments, this disclosure provides human 4-1BB agonist antibodies, particularly 4-1BB IgG2a agonist monoclonal antibodies. The antibodies of the present invention can be prepared under GMP-compliant conditions. The antibodies of the present invention can be used in patients in monospecific or bispecific form. In a further aspect, the antibodies of the present invention can be administered as immunotherapy to patients with cancer, alone or in combination with other immunotherapies such as immune checkpoint inhibitors (e.g., anti-PD1 or anti-CTLA4 antibodies).

[0034] I. Definition

[0035] As used herein, "substantially free" with respect to a particular component means that none of the particular component was intentionally formulated into the composition and / or is present only as a contaminant or in trace amounts. Therefore, the total amount of the particular component due to any unintentional contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferably, a composition is one in which the amount of the particular component is undetectable by standard analytical methods.

[0036] As used herein in the specification, "a" or "an" may mean one or more. As used herein in the claims, when combined with the word "comprising (including)," the word "a" or "an" may mean one or more.

[0037] The use of the term "or" in the claims is intended to mean "and / or" unless it is explicitly stated that it refers only to alternatives or that the alternatives are mutually exclusive, although this disclosure supports the definition of referring only to alternatives and "and / or". As used herein, "another" may mean at least a second or more. The term "about" generally means the stated value ±5%.

[0038] "Treatment" refers to the administration or application of a therapeutic agent to a subject or the performance of a procedure or program on a subject for the purpose of obtaining therapeutic benefits from a disease or health-related condition.

[0039] "Subject" and "patient" refer to humans or non-human animals, such as primates, mammals, and vertebrates. In a particular embodiment, the subject is a human.

[0040] The terms “treatment benefit” or “treatment-effective” as used throughout this application mean anything that promotes or enhances the well-being of a subject with respect to the medical treatment relating to the condition. This includes, but is not limited to, reducing the frequency or severity of signs or symptoms of the disease. For example, cancer treatment may involve, for instance, reducing the size of a tumor, reducing the invasiveness of a tumor, slowing the growth rate of cancer, or preventing metastasis. Cancer treatment may also refer to prolonging the survival of a subject with cancer.

[0041] "Anti-cancer" agents can negatively affect cancer cells / tumors in subjects, for example by promoting the killing of cancer cells, inducing apoptosis of cancer cells, reducing 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 immune responses against cancer cells or tumors, preventing or inhibiting cancer progression, or increasing the lifespan of subjects with cancer.

[0042] The term “antibody” is used in the broadest sense in this document and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity.

[0043] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, for example, the individual antibodies constituting the group are identical except for the possible presence of small amounts of mutations (e.g., naturally occurring mutations). Therefore, the modifier "monoclonal" indicates that the antibody is characterized as not being a mixture of discrete antibodies. In some embodiments, such monoclonal antibodies typically comprise antibodies containing a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a process comprising selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select a unique clone from a plurality of clones (e.g., a collection of hybridoma clones, phage clones, or recombinant DNA clones). It should be understood that the selected target-binding sequence can be further modified to, for example, improve affinity for the target, humanize the target-binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, generate multispecific antibodies, etc.; and antibodies containing such modified target-binding sequences are also monoclonal antibodies of the present invention. Unlike polyclonal antibody preparations, which typically consist of different antibodies targeting different determinants (epitopes), monoclonal antibody preparations contain a single monoclonal antibody targeting a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous because they are typically not contaminated by other immunoglobulins.

[0044] When applied to cells, the terms "contact" and "exposure" are used herein to describe processes by which a therapeutic construct and a chemotherapy or radiotherapy agent are delivered to or placed directly alongside target cells. For example, to achieve cell killing, the two agents are delivered to the cells in a combined amount effective for killing cells or preventing their division.

[0045] The term "immune checkpoint" refers to molecules, such as proteins, in the immune system that provide inhibitory signals to their components in order to balance the immune response. 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. Pathways involving LAG3, BTLA, B7H3, B7H4, TIM3, and KIR are considered in the art to constitute immune checkpoint pathways similar to those of CTLA-4 and PD-1 (see, for example, Pardoll, 2012; Mellman et al., 2011).

[0046] "Immune checkpoint inhibitors" refer to any compound that inhibits the function of immune checkpoint proteins. Inhibition includes both reduction and complete blockage of function. In particular, the immune checkpoint proteins are human immune checkpoint proteins. Therefore, the immune checkpoint protein inhibitors are specifically inhibitors of human immune checkpoint proteins.

[0047] II. Anti-41BB antibody

[0048] In some embodiments, an antibody or fragment thereof is considered that binds to at least a portion of 4-1BB and activates signal transduction (e.g., to stimulate an immune response). As used herein, the term "antibody" is intended to refer broadly to any immunobinding agent, 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.

[0049] In some embodiments, the anti-41BB antibody comprises CDRs 1-3 (GYSFTDYN (SEQ ID NO: 3), INPNYGTT (SEQ ID NO: 5), and ARSPVEDYFDY (SEQ ID NO: 7)) of the heavy chain of SEQ ID NO: 2 and CDRs 1-3 (SSVSSSY (SEQ ID NO: 11), STS (SEQ ID NO: 13), and QQYSGYPLIT (SEQ ID NO: 15)) of the light chain of SEQ ID NO: 10. The anti-41BB antibody may have at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1, 2, 9, or 10. The anti-41BB antibody may have at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 17, 18, 25, or 26. The anti-41BB antibody may have at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 33, 34, 41, or 42. The anti-41BB antibody may have at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 49, 50, 57, or 58. The heavy chain and light chain sequences are described below.

[0050] Cloning 54 4-1BB, VH co-sequence starting from framework 1 (FR1) (positions 77-505), IgG2a

[0051] Nucleotide sequence:

[0052] GAGTTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGCGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGACTACAACATGAACTGGGTGAAGCAGAGCAATGGAAAGAGCCTTGAGTGGATTGGAGTAATTAATCCTAACTATGGTACTACTAGCTACAATCAGAAGTTCAAGGGCAAGGCCACATTTACTGTAGACCAATCTTCCAGCACAGCCTACATGCAGCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATCCCCGGTAGAGGACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGGTACAACTGGCTCCTCGGTGACTCTA (SEQ ID NO: 1)

[0053] Amino acid sequence:

[0054] EFQLQQSGPELVKPGASVKISCKASGYSFTDYNMNWVKQSNGKSLEWIGVINPNYGTTSYNQKFKGKATFTVDQSSSTAYMQLNSLTSEDSAVYYCARSPVEDYFDYWGQGTTLTVSSAKTTAPSVYPLAPVCGGTTGSSVTL (SEQ ID NO: 2)

[0055] CDR1:

[0056] GYSFTDYN (SEQ ID NO: 3)

[0057] GGTTACTCATTCACTGACTACAAC (SEQ ID NO: 4)

[0058] CDR2:

[0059] INPNYGTT (SEQ ID NO: 5)

[0060] ATTAATCCTAACTATGGTACTACT (SEQ ID NO: 6)

[0061] CDR3:

[0062] ARSPVEDYFDY (SEQ ID NO: 7)

[0063] GCAAGATCCCCGGTAGAGGACTACTTTGACTAC (SEQ ID NO: 8)

[0064] VL consensus sequence starting from framework 1 (FR1) (positions 91 - 470)

[0065] Nucleotide sequence:

[0066] GAAAATGTGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAAAGGTCACCATGACCTGCAGGGCCAGGTCAAGTGTAAGTTCCAGTTACTTGCACTGGTACCAGCAGAAGTCAGGTGCCTCCCCCAAACTCTGGATTTATAGCACATCCAACTTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGTGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTACAGTGGTTACCCACTCATCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTT (SEQ ID NO: 9)

[0067] Amino acid sequence:

[0068] ENVLTQSPAIMSASPGEKVTMTCRARSSVSSSYLHWYQQKSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLITFGAGTKLELKRADAAPTVSIFPPSSEQ (SEQ ID NO: 10)

[0069] CDR1:

[0070] SSVSSSY (SEQ ID NO: 11)

[0071] TCAAGTGTAAGTTCCAGTTAC (SEQ ID NO: 12)

[0072] CDR2:

[0073] STS (SEQ ID NO: 13)

[0074] AGCACATCC (SEQ ID NO: 14)

[0075] CDR3:

[0076] QQYSGYPLIT (SEQ ID NO: 15)

[0077] CAGCAGTACAGTGGTTACCCACTCATCACG (SEQ ID NO: 16)

[0078] Clone 135B 4-1BB, VH consensus sequence starting from framework 1 (FR1) (positions 45 - 407), IgG1

[0079] Nucleotide sequence:

[0080] AGGTGAAGCTGCAGCAGTCAGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATACACGTTCACTGACTACTACATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAATGATGGTACTACCTACTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATCCCTCTACGGTAGTAGCTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG (SEQ IDNO: 17)

[0081] Amino acid sequence:

[0082] VKLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDINPNNDGTTYYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARSLYGSSYYFDYWGQGTTLTVSS (SEQ ID NO: 18)

[0083] CDR1:

[0084] GYTFTDYY (SEQ ID NO: 19)

[0085] GGATACACGTTCACTGACTACTAC (SEQ ID NO: 20)

[0086] CDR2:

[0087] INPNNDGT (SEQ ID NO: 21)

[0088] ATTAATCCTAACAATGATGGTACT (SEQ ID NO: 22)

[0089] CDR3:

[0090] ARSLYGSSYYFDY (SEQ ID NO: 23)

[0091] GCAAGATCCCTCTACGGTAGTAGCTACTACTTTGACTAC (SEQ ID NO: 24)

[0092] VL common sequence starting from architecture 1 (FR1) (positions 46-379)

[0093] Nucleotide sequence:

[0094] GATATTGTGATGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCA ACCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAATGAGGACCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC (SEQ ID NO: 25)

[0095] amino acid sequence:

[0096] DIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIK (SEQ ID NO: 26)

[0097] CDR1:

[0098] KSVSTSGYSY (SEQ ID NO: 27)

[0099] AAAAGTGTCAGTACATCTGGCTATAGTTAT (SEQ ID NO: 28)

[0100] CDR2:

[0101] LVS (SEQ ID NO: 29)

[0102] CTTGTATCC (SEQ ID NO: 30)

[0103] CDR3:

[0104] QQSNEDPWT (SEQ ID NO: 31)

[0105] CAGCAAAGTAATGAGGACCCGTGGACG (SEQ ID NO: 32)

[0106] Cloner 138 4-1BB, VH co-sequence starting from frame 1 (FR1) (positions 56-409), IgG2b

[0107] Nucleotide sequence:

[0108] AGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTTGCATACATTAGTAGTGGCAGTAATTCCATCTACTATGCAGACACAGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCCTCGAATAATGGTTACTTCTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG (SEQ ID NO: 33)

[0109] Amino acid sequence:

[0110] VQLQESGGGLVKPGGSLKLSCAASGFTFSDYGMHWVRQAPEKGLEWVAYISSGSNSIYYADTVTGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCASNNGYFYFDYWGQGTTLTVSS (SEQ ID NO: 34)

[0111] CDR1:

[0112] GFTFSDYG (SEQ ID NO: 35)

[0113] GGATTCACTTTCAGTGACTATGGA (SEQ ID NO: 36)

[0114] CDR2:

[0115] ISSGSNSI (SEQ ID NO: 37)

[0116] ATTAGTAGTGGCAGTAATTCCATC (SEQ ID NO: 38)

[0117] CDR3:

[0118] ASNNGYFYFDY (SEQ ID NO: 39)

[0119] GCCTCGAATAATGGTTACTTCTACTTTGACTAC (SEQ ID NO: 40)

[0120] VL consensus sequence starting from framework 1 (FR1) (positions 63 - 378)

[0121] Nucleotide sequence:

[0122] ATTGTGATCACCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAGTCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTGACCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAC (SEQ ID NO: 41)

[0123] Amino acid sequence:

[0124] IVITQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTVSRVEAEDAATYYCQQWSSDPFTFGSGTKLEIK (SEQ ID NO: 42)

[0125] CDR1:

[0126] SSVSY (SEQ ID NO: 43)

[0127] TCAAGTGTAAGTTAC (SEQ ID NO: 44)

[0128] CDR2:

[0129] ATS (SEQ ID NO: 45)

[0130] GCCACATCC (SEQ ID NO: 46)

[0131] CDR3:

[0132] QQWSSDPFT (SEQ ID NO: 47)

[0133] CAGCAGTGGAGTAGTGACCCATTCACG (SEQ ID NO: 48)

[0134] Clone 49A 4-1BB, VH consensus sequence starting from framework 1 (FR1) (positions 65 - 427), IgG1

[0135] Nucleotide sequence:

[0136] AGGTGAAACTGCAGCAGTCAGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATACACGTTCACTGACTACTACATGAACTGGGTGAAGGAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAATGGTGGTTCTACCTACTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGAGAAGTCCTCCAGCACAGCCTTCATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATCCCTCTACGGTAGTACCTACTACTTTGACTACTGGGGCCAAGGCACCCCTCTCACAGTCTCCTCAG (SEQ IDNO: 49)

[0137] Amino acid sequence:

[0138] VKLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKESHGKSLEWIGDINPNNGGSTYYNQKFKGKATLTVEKSSSTAFMELRSLTSEDSAVYYCARSLYGSTYYFDYWGQGTPLTVSS (SEQ ID NO: 50)

[0139] CDR1:

[0140] GYTFTDYY (SEQ ID NO: 51)

[0141] GGATACACGTTCACTGACTACTAC (SEQ ID NO: 52)

[0142] CDR2:

[0143] INPNNGGS (SEQ ID NO: 53)

[0144] ATTAATCCTAACAATGGTGGTTCT (SEQ ID NO: 54)

[0145] CDR3:

[0146] ARSLYGSTYYFDY (SEQ ID NO: 55)

[0147] GCAAGATCCCTCTACGGTAGTACCTACTACTTTGACTAC (SEQ ID NO: 56)

[0148] VL consensus sequence starting from framework 1 (FR1) (positions 55 - 388)

[0149] Nucleotide sequence:

[0150] GATATTGTGCTGACCCAGTCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATATGAACTGGTACCAACAGAAGCCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGGAACCTATTACTGTCAGCAAAGTAATGACGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC (SEQ ID NO: 57)

[0151] Amino acid sequence:

[0152] DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAGTYYCQQSNDDPWTFGGGTKLEIK (SEQ ID NO: 58)

[0153] CDR1:

[0154] QSVDYDGDSY (SEQ ID NO: 59)

[0155] CAAAGTGTTGATTATGATGGTGATAGTTAT (SEQ ID NO: 60)

[0156] CDR2:

[0157] AAS (SEQ ID NO: 61)

[0158] GCTGCATCC (SEQ ID NO: 62)

[0159] CDR3:

[0160] QQSNDDPWT (SEQ ID NO: 63)

[0161] CAGCAAAGTAATGACGATCCGTGGACG (SEQ ID NO: 64)

[0162] Therefore, polyclonal or monoclonal antibodies, antibody fragments and binding domains and CDRs (including modified forms of any of the above), or conjugates of any of the above, specific for 4-1BB, one or more of their respective epitopes, can be generated by known methods and as described herein, regardless of whether such antigens or epitopes are isolated from natural sources or are synthetic derivatives or variants of natural compounds.

[0163] Examples of antibody fragments suitable for embodiments of the present invention include, but are not limited to: (i) Fab fragments, which are composed of V L V H C L and C H1 (ii) The “Fd” segment, which is composed of V H and C H1 (iii) The “Fv” fragment, which consists of a single antibody’s V domain. L and V H (iv) The “dAb” segment, which is composed of V H Domain composition; (v) Separate CDR regions; (vi) F(ab')2 fragments, which are bivalent fragments containing two connected Fab fragments; (vii) Single-chain Fv molecules (“scFv”), where V H Domain and V LThe domains are linked by allowing the two domains to combine to form a peptide linker that binds the domains; (viii) a bispecific single-chain Fv dimer (see U.S. Patent No. 5,091,513); and (ix) a diabody, which is a multivalent or multispecific fragment constructed by gene fusion (U.S. Patent Application Publication 20050214860). Fv, scFv, or diabody molecules can be linked by incorporation of V H and V L Stabilization is achieved by using disulfide bridges in the CH3 domain. Minibody antibodies containing scFv linked to the CH3 domain can also be prepared (Hu et al., 1996).

[0164] Antibody-like binding peptide mimics were also considered in the implementation plan. (Liu et al.) (2003) describes “antibody-like binding peptidomimetics” (ABiPs), which are peptides that can act as reduced antibodies and have the advantages of a longer serum half-life and a less cumbersome synthetic method.

[0165] Animals can be inoculated with antigens, such as the 4-1BB extracellular domain (ECD) protein, to generate antibodies specific to 4-1BB. Often, antigens are bound to or conjugated to another molecule to enhance the immune response. As used herein, a conjugate is any peptide, polypeptide, protein, or non-protein substance that binds to an antigen used to elicit an immune response in an animal. Antibodies generated in animals in response to antigen inoculation comprise a wide variety of dissimilar molecules (polyclonal antibodies), prepared from a wide variety of B lymphocytes that produce individual antibodies. Polyclonal antibodies are a mixed population of antibody classes, each capable of recognizing different epitopes on the same antigen. If the correct conditions are given for the production of polyclonal antibodies in an animal, the majority of antibodies in the animal's serum will recognize a collective epitope on the antigen compound to which the animal has been immunized. This specificity is further enhanced by affinity purification (to select only those antibodies that recognize the target antigen or epitope).

[0166] Monoclonal antibodies are a single class of antibodies in which each antibody molecule recognizes the same epitope because all antibody-producing cells originate from a single B-lymphocyte cell line. Methods for generating monoclonal antibodies (Mabs) generally begin along the same routes as those used to prepare polyclonal antibodies. In some embodiments, rodents such as mice and rats are used to generate monoclonal antibodies. In some embodiments, rabbit, sheep, or frog cells are used to generate monoclonal antibodies. The use of rats is well-known and can offer certain advantages. Mice (e.g., BALB / c mice) are routinely used, and they typically give a high percentage of stable fusions.

[0167] Hybridoma technology involves fusing a single B lymphocyte from a mouse previously immunized with the 4-1BB antigen with an uncontrolled proliferation of myeloma cells (typically mouse myeloma). This technology provides a method for propagating a single antibody-producing cell to an unlimited number of generations, thereby generating an unlimited quantity of structurally identical antibodies (monoclonal antibodies) with the same antigen or epitope specificity.

[0168] Plasma B cells (CD45) + CD5 - CD19 + The antibody-binding B cells can be isolated from freshly prepared rabbit peripheral blood mononuclear cells from immunized rabbits, and further selected for 4-1BB binding cells. After enriching antibody-producing B cells, total RNA can be isolated and cDNA synthesized. The DNA sequence from the antibody variable region of both the heavy and light chains can be amplified, constructed into a phage display Fab expression vector, and transformed into *E. coli*. E. coli The 4-1BB-specific binding Fab was selected through multiple rounds of enrichment panning and then sequenced. The selected 4-1BB binding targets were expressed as full-length IgG in rabbit and rabbit / human chimeric forms in human embryonic kidney (HEK293) cells (Invitrogen) using a mammalian expression vector system, and purified using a rapid protein liquid chromatography (FPLC) separation unit with G protein resin.

[0169] In one embodiment, the antibody is a chimeric antibody, such as an antibody comprising an antigen-binding sequence from a non-human donor grafted with a heterologous non-human, human, or humanized sequence (e.g., framework and / or constant domain sequence). Methods have been developed to replace the light and heavy chain constant domains of a monoclonal antibody with similar domains derived from humans, while keeping the variable region of the exogenous antibody intact. Alternatively, “fully human” monoclonal antibodies are generated in transgenic mice relating to human immunoglobulin genes. Methods have also been developed to convert the variable domains of a monoclonal antibody into a more human form by recombinantly constructing antibody variable domains having both rodent (e.g., mouse) and human amino acid sequences. In “humanized” monoclonal antibodies, only the hypervariable CDR is derived from a mouse monoclonal antibody, and the framework and constant regions are derived from human amino acid sequences (see U.S. Patent Nos. 5,091,513 and 6,881,557). It is believed that replacing the characteristic amino acid sequence in antibodies for rodents with the amino acid sequence found at the corresponding positions in human antibodies would reduce the likelihood of adverse immune responses during therapeutic use. Hybridomas or other antibody-producing cells may also undergo gene mutations or other changes that may or may not alter the binding specificity of antibodies produced by that hybridoma.

[0170] Methods for generating polyclonal antibodies in various animal species and for generating various types of monoclonal antibodies (including humanized, chimeric, and fully human) are well known in the art and highly predictable. For example, the following U.S. patents and patent applications provide enabling descriptions of such methods: U.S. Patent Application Nos. 2004 / 0126828 and 2002 / 0172677; and U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,196,265, 4,275,149, and 4,277,437. 4,366,241, 4,469,797, 4,472,509, 4,606,855, 4,703,003, 4,742,159, 4,767,720, 4,816,567, 4,867,973, 4,938,948, 4,946,778, 5,021,236, 5,164,296, 5,196 066、5,223,409、5,403,484、5,420,253、5,565,332、5,571,698、5,627,052、5,656,434、5,770,376、5,789,208、5,821,337、5,844,091、5,858,657、5,861,155、5,8 71,907, 5,969,108, 6,054,297, 6,165,464, 6,365,157, 6,406,867, 6,709,659, 6,709,873, 6,753,407, 6,814,965, 6,849,259, 6,861,572, 6,875,434, and 6,891,024. All patents, patent application publications, and other publications cited herein and therein are incorporated herein by reference.

[0171] Antibodies can be produced from any animal source, including birds and mammals. Preferably, the antibodies are from sheep, rodents (e.g., mice and rats), rabbits, goats, guinea pigs, camels, horses, or chickens. Additionally, newer techniques allow for the development and screening of human antibodies from human combinatorial antibody libraries. For example, phage antibody expression technology allows for the production of specific antibodies in the absence of animal immunization, as described in U.S. Patent No. 6,946,546 (which is incorporated herein by reference). These techniques are further described in: Marks (1992); Stemmer (1994); Gram et al. (1992); Barbas et al. (1994); and Schier et al. (1996).

[0172] It is fully anticipated that antibodies against 4-1BB will have the ability to neutralize or counteract the effects of 4-1BB, regardless of the animal species, monoclonal cell line, or other source of the antibody. Certain animal species may be less preferred for generating therapeutic antibodies because they may be more likely to induce an allergic response due to activation of the complement system via the antibody's "Fc" portion. However, intact antibodies can be enzymatically digested into an "Fc" (complement-binding) fragment, as well as an antibody fragment with a binding domain or CDR. Removal of the Fc portion reduces the likelihood of the antigen-antibody fragment triggering an undesirable immune response, and therefore antibodies without the Fc may be preferentially used for prophylactic or therapeutic treatment. As described above, antibodies can also be constructed as chimeric or partially or fully human to reduce or eliminate adverse immunological consequences resulting from the administration of antibodies produced in other species or having sequences from other species to animals.

[0173] Substitution variants typically involve the exchange of one amino acid for another at one or more sites within a protein and can be programmed to modulate one or more properties of the polypeptide, with or without loss of other functions or properties. Substitutions can be conservative, meaning one amino acid is replaced by an amino acid with a similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; 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, the substitution can be non-conservative, thereby affecting the function or activity of the polypeptide. Non-conservative changes typically involve substituting residues with chemically different residues, such as replacing a polar or charged amino acid with a nonpolar or uncharged amino acid, or vice versa.

[0174] The protein can be recombinant or synthesized in vitro. Alternatively, non-recombinant or recombinant proteins can be isolated from bacteria. It is also contemplated that bacteria containing such variants can be incorporated into the composition and method. Therefore, protein isolation is not required.

[0175] Consideration has been given to the presence of approximately 0.001 mg to approximately 10 mg of total polypeptides, peptides, and / or proteins per ml in the composition. Therefore, the protein concentration in the composition can 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 more (or any range derived therefrom). Among 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% can be antibodies that bind to 4-1BB.

[0176] Antibodies, or preferably the immunological portion of antibodies, may be chemically conjugated to other proteins or expressed as fusion proteins with other proteins. For the purposes of this specification and the appended claims, all such fused proteins are included in the definition of antibodies or the immunological portion of antibodies.

[0177] The implementation provides antibodies and antibody-like molecules, peptides, and polypeptides targeting 4-1BB, which are linked to at least one reagent to form antibody conjugates or payloads. To enhance the efficacy of antibody molecules as diagnostic or therapeutic agents, it is common practice to link, covalently bind, or conjugate at least one desired molecule or moiety. Such molecules or moiety can be, but are not limited to, at least one effector molecule or reporter molecule. Effector molecules include molecules having the desired activity (e.g., cytotoxic activity). Non-limiting examples of effector molecules attached to antibodies include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides, etc. Conversely, reporter molecules are defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to antibodies include enzymes, radiolabeled molecules, haptens, fluorescently labeled molecules, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands (e.g., biotin).

[0178] Several methods for attaching or conjugating antibodies to their conjugate portions are known in the art. Some attachment methods involve the use of metal chelate complexes, employing, for example, organic chelating agents such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3-6-diphenylglyuron-3, to attach the antibody. Monoclonal antibodies can also be reacted with enzymes in the presence of conjugating agents such as glutaraldehyde or periodate. Conjugates with fluorescein labels are prepared in the presence of these conjugating agents or by reaction with isothiocyanates.

[0179] II. Treatment of the disease

[0180] Certain aspects of embodiments of the present invention can be used to prevent or treat diseases or conditions associated with 4-1BB signaling. The 4-1BB antibody of the present invention can be used to treat diseases such as cancer, infectious diseases, inflammatory diseases, or autoimmune diseases. Further methods for administering the vaccine are provided. Suitable vaccines include, for example, tumor cell vaccines, DNA vaccines, GM-CSF-modified tumor cell vaccines, or dendritic cell vaccines loaded with antigens.

[0181] In some embodiments, the compositions and methods of the present invention involve antibodies or antibody fragments targeting 4-1BB to activate their activity in cancer cell proliferation, and a second or additional therapy in combination therewith.

[0182] Therefore, in some embodiments, methods for treating or delaying the progression of cancer in an individual are provided herein, the methods comprising administering an effective amount of anti-41BB antibody to the individual. Examples of cancers considered 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 in the lungs, colon cancer, melanoma, and bladder cancer.

[0183] In some embodiments, the individual has cancer that is resistant (has been proven resistant) to one or more anticancer therapies. In some embodiments, resistance to anticancer therapies includes cancer recurrence or refractory cancer. Recurrence can refer to the reappearance of cancer at the original site or a new site after treatment. In some embodiments, resistance to anticancer therapies includes cancer progression during treatment with said anticancer therapies. In some embodiments, the cancer is in an early or late stage.

[0184] A. Pharmaceutical preparations

[0185] When conducting clinical application of therapeutic compositions containing inhibitory antibodies, it is often advantageous to prepare pharmaceutical or therapeutic compositions suitable for the intended application. In some embodiments, the pharmaceutical composition may contain, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may, for example, comprise from about 2% to about 75% or from about 25% to about 60% by weight of the unit, and any range from thereto.

[0186] The therapeutic compositions of embodiments of the present invention are advantageously administered in the form of injectable compositions (as liquid solutions or suspensions); they can also be prepared in solid forms suitable for dissolving or suspending in a liquid prior to injection. These preparations can also be emulsified.

[0187] The phrase "pharmaceutically or pharmacologically acceptable" means a molecular entity or composition that does not produce an adverse, allergic, or other inappropriate reaction when administered to animals, such as humans (as the case may be). In view of this disclosure, those skilled in the art will understand the preparation of pharmaceutical compositions comprising antibodies or other active ingredients. Furthermore, for animal (e.g., human) administration, it will be understood that the preparation should meet the sterility, pyrogenicity, overall safety, and purity standards required by the FDA's Office of Biostandards.

[0188] As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcohol / aqueous solutions, salt solutions, parenteral carriers such as sodium chloride, Ringer's glucose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delay agents, salts, pharmaceuticals, pharmaceutical stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluids, and nutritional supplements, as well as similar materials and combinations thereof, as will be known to those skilled in the art. The pH and precise concentration of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0189] The term "unit dose" or "dose" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of the therapeutic composition calculated to produce the desired response discussed above in relation to its administration (i.e., appropriate route and treatment regimen). The amount to be administered depends on both the number of treatments and the unit dose, depending on the desired effect. The actual dose of the composition of the embodiments of the present invention administered to a patient or subject can be determined by physical and physiological factors such as the subject's weight, age, health, and sex; the type of disease being treated; the extent of disease penetration; previous or concurrent treatment interventions; the patient's idiopathic condition; the route of administration; and the potency, stability, and toxicity of the particular therapeutic substance. For example, the dose can also be from about 1 μg / kg / body weight to about 1000 mg / kg / body weight (this range includes interventional doses) or more per administration, and any range derived therefrom. In non-limiting examples of the ranges that can be derived from the figures listed herein, the dosage may range from about 5 μg / kg / body weight to about 100 mg / kg / body weight, about 5 μg / kg / body weight to about 500 mg / kg / body weight, etc. In any case, the physician administering the medication will determine the concentration of the active ingredient in the composition and the appropriate dose for the individual subject.

[0190] The active compounds 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 liquid solutions or suspensions; they can also be prepared in solid forms suitable for preparing solutions or suspensions after the addition of liquid prior to injection; and the preparations can also be emulsified.

[0191] Suitable pharmaceutical forms for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be a fluid such that it can be readily injected. It should also be stable under the conditions of preparation and storage and must be protected against contamination by microorganisms such as bacteria and fungi.

[0192] Protein compositions can be formulated in neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of proteins) and those formed with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). 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, procaine, etc.

[0193] Pharmaceutical compositions may include solvents or dispersion media comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Appropriate flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Prevention of microbial action can be achieved using various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, isotonic agents (e.g., sugars or sodium chloride) will be preferred. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0194] B. Combined treatment

[0195] In some embodiments, the compositions and methods of the present invention involve antibodies or antibody fragments targeting 4-1BB to activate their activity, and second or additional therapies in combination therewith. For example, the disease may be cancer.

[0196] The methods and compositions (including combination therapies) enhance therapeutic or protective effects and / or increase the therapeutic effect of another anticancer or antiproliferative therapy. Therapeutic and prophylactic methods and compositions may be provided in combined amounts effective for achieving the desired effect (e.g., killing cancer cells and / or inhibiting cell proliferation). The process may involve contacting cells with an antibody or antibody fragment and a second therapy. Tissues, tumors, or cells may be contacted with one or more compositions or pharmacological preparations comprising one or more of the aforementioned reagents (i.e., antibodies or antibody fragments or anticancer reagents), or by contacting tissues, tumors, and / or cells with two or more different compositions or preparations, wherein one composition provides 1) an antibody or antibody fragment, 2) an anticancer reagent, or 3) both an antibody or antibody fragment and an anticancer reagent. Furthermore, it is contemplated that such combination therapies may be used in conjunction with chemotherapy, radiotherapy, surgical therapy, or immunotherapy.

[0197] When applied to cells, the terms "contact" and "exposure" are used herein to describe processes by which a therapeutic construct and a chemotherapy or radiotherapy agent are delivered to or placed directly alongside target cells. For example, to achieve cell killing, the two agents are delivered to the cells in a combined amount effective for killing cells or preventing their division.

[0198] Inhibitory antibodies can be administered before, during, after, or in various combinations with anticancer treatment. The administration can be at intervals ranging from simultaneous to minutes to days to weeks. In embodiments where the antibody or antibody fragment is administered separately from the anticancer agent, it is generally ensured that no significant time intervals exist between each delivery, so that the two compounds will still be able to exert a beneficial combined effect on the patient. In this case, it is considered that the antibody therapy and the anticancer therapy can be administered to the patient approximately 12 to 24 or 72 hours apart, more particularly approximately 6 to 12 hours apart. In some cases, it may be desirable to significantly prolong the treatment period, with intervals between the respective administrations ranging from several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks).

[0199] In some implementations, the treatment course will last 1-90 days or longer (such range includes intermediate days). It is considered that one agent may be administered on any day from day 1 to day 90 (such range includes intermediate days) or any combination thereof, and another agent may be administered on any day from day 1 to day 90 (such range includes intermediate days) or any combination thereof. Within a single day (a 24-hour period), the patient may be given one or more doses of the stated agent. Furthermore, after the treatment course, it is considered that there may be a period of time without anticancer treatment. This period may last 1-7 days, and / or 1-5 weeks, and / or 1-12 months or longer (such range includes intermediate days), depending on the patient's condition, such as their prognosis, physical condition, health, etc. The treatment cycle is expected to be repeated as needed.

[0200] Various combinations can be used. In the example below, antibody therapy is "A", and anticancer therapy is "B":

[0201] A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B

[0202] B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A

[0203] B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A.

[0204] Given the potential toxicity of the reagents (if any), administering any compound or therapy according to embodiments of the present invention to a patient will follow the general protocol for administering such compounds. Therefore, in some embodiments, there is a step to monitor for toxicity attributable to the combination therapy.

[0205] 1. Chemotherapy

[0206] According to embodiments of the present invention, a wide variety of chemotherapeutic agents can be used. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapy agent" is used to refer to compounds or compositions administered in the treatment of cancer. These agents or drugs are classified according to their patterns of activity within cells, such as whether and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly crosslink DNA, embed themselves in DNA, or induce chromosomal and mitotic aberrations by influencing nucleic acid synthesis.

[0207] Examples of chemotherapeutic agents include: alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, inprossurfan, and piperazine; aziridines, such as benzodopa, carboquinone, meturedopa, and uredopa; azacyclopropanes and methylamelamines, including hexamethylmelamine, triethylmelamine, triethylphosphamide, triethylthiophosphamide, and trihydroxymethylmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analogue topotecan); lichen inhibitors... Callystatin; CC-1065 (including its synthetic analogues, callystatin, callystatin, and pyrazin); cryptophytin (especially cryptophytin 1 and cryptophytin 8); dolastatin; pyrazinamide (including synthetic analogues, KW-2189 and CB1-TM1); argentin; pancratistatin; sarcodictyin; spongiform septicemia; nitrogen mustards, such as chlorambucil, naphthylambucil, cyclophosphamide, estradiol, ifosfamide, nitrogen mustard, oxynitrogen mustard hydrochloride, melphalan, neonitrogen mustard, benzyl mustard cholesterol, prednimustine, trazophosphatamide, and uracil mustard; nitrosoureas, such as carmustine, chloramphenicol, formustine, lomustine, and nimosine. Sine and ramustine; antibiotics, such as enematic antibiotics (e.g., calcipomycin, especially calcipomycin γ1I and calcipomycin ωI1); enematic anthracycline antibiotics, including enematic anthracycline antibiotic A; bisphosphonates, such as clophosphonates; esporamycin; and new carcinogen chromophores and related chromogens, enematic antibiotic chromophores, aclarubicin, actinomycin, authramycin, diazoserine, bleomycin, actinomycin C, carabicin, erythromycin, carcinogen, chromophore, actinomycin D, daunorubicin, detoxin, 6-diazo-5-oxo-L-leucine, doxorubicin (including morpholine doxorubicin, cyanomorpholine doxorubicin, 2-pyrrolidone) Drugs containing: doxorubicin and deoxydoxorubicin, epirubicin, isorubicin, idarubicin, maceralomycin, mitomycin C, mycophenolate mofetil, nogamycin, olivomycin, pepromycin, purulentin, triamcinolone acetonide, rhodorubicin, streptozotocin, streptozotocin, tuberculin, ubenmex, fenestrated statin, and zolrubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as folate, pteroxate, and trimethoprim; purine analogs, such as fludarabine, 6-mercaptopurine, thiopurine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azouridine, carmoflurane, cytarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, and fluorouridine.Androgens, such as calotestosterone, drotalbuterol propionate, cyclothionol, meandrazan, and testrolide; antiadrenergic drugs, such as mitotane and tramostan; folic acid supplements, such as folinic acid; acetoglucan lactone; aldehyde phosphoramide glycoside; 5-aminolevulinic acid; enturacil; acridine; bestrabucil; bifenthrin; edatrazine; desphosphonamide; colchicine; desaccharin; elformithine; eletyl acetate; epoxigin; etoradin; gallium nitrate; hydroxyurea; lentinan; chlorhexidine Damage; methanotrophs, such as maytansine and anthraquinone; mitoxantrone; mitoxantrone; mopiperazine; nitroxacin; pentostatin; phenamet; pirarubicin; loxoantrone; podophyllotoxin; 2-ethylhydrazine; procarbazine; PSK polysaccharide complex; razorazine; lizoxin; cizonan; germanospiramine; Alternaria ketoacid; triaminoquinone; 2,2′,2″-trichlorotriethylamine; trichothecene compounds (especially T-2 toxin, verracurin). (in) A, Bacitracin A and Serpentin); Urethane; Vinpocetine; Dacarbazine; Mannomustine; Dibromomannitol; Dibromoeutherol; Piperobromide; Gacytosine; Arabin (“Ara-C”); Cyclophosphamide; Taxanes, such as paclitaxel and docetaxel; Gemcitabine; 6-Thioguanine; Mercaptopurine; Platinum coordination complexes, such as cisplatin, oxaliplatin and carboplatin; Vincristine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Long Vincristine; vinorelbine; novotoxin; teniposide; edaraxacum; donomycin; aminopterin; capecitabine; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin; procarbazine; purcamycin; gemcitabine; novotoxin; farnesyl-protein transferase inhibitors; antiplatinum; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0208] 2. Radiation therapy

[0209] Other factors that cause DNA damage and have been widely used include the targeted delivery of gamma rays, X-rays, and / or radioactive isotopes to tumor cells, commonly referred to as gamma rays, X-rays, and / or radioactive isotopes. Other forms of DNA damage have also been considered, such as microwaves, proton beam radiation (US Patents 5,760,395 and 4,870,287), and UV radiation. Most likely, all of these factors cause a wide range of damage to DNA, to DNA precursors, to DNA replication and repair, and to chromosome assembly and maintenance. Dosage ranges for X-rays vary widely, from daily doses of 50 to 200 roentgens over extended periods (3 to 4 weeks) to single doses of 2000 to 6000 roentgens. Dosage ranges for radioactive isotopes vary extensively and depend on the isotope's half-life, the intensity and type of radiation emitted, and the uptake by the proliferating cells.

[0210] 3. Immunotherapy

[0211] Those skilled in the art will understand that additional immunotherapies can be used in combination with or in conjunction with the methods of the described embodiments. In the context of cancer treatment, immunotherapeutic agents typically rely on the use of immune effector cells and molecules to target and destroy cancer cells. Immune effectors can be, for example, antibodies or chimeric antigen receptors (CARs) that are specific to certain markers on the surface of tumor cells. In a further aspect, the therapy may include the administration of T-cells or NK-cells that are targeted at specific cancer cells. Such cells may be engineered or simply selected for anti-cancer activity.

[0212] In some respects, antibodies alone can act as effectors of a therapy, or they can recruit other cells to actually influence cell killing. The antibodies can also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chains, cholera toxin, pertussis toxin, etc.) and act as targeting agents. Alternatively, the effector can be a lymphocyte carrying a surface molecule that interacts directly or indirectly with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0213] Antibody-drug conjugates have emerged as a breakthrough approach for developing cancer therapeutics. Cancer is one of the leading causes of death worldwide. Antibody-drug conjugates (ADCs) contain monoclonal antibodies (MAbs) covalently linked to a cytotoxic drug. This approach combines the high specificity of the MAb targeting its antigen with a highly potent cytotoxic drug, resulting in an "armed" MAb that delivers its payload (drug) to tumor cells with enriched levels of the antigen. Targeted drug delivery also minimizes its exposure in normal tissues, leading to reduced toxicity and an improved therapeutic index. This approach has been validated by the FDA approvals of two ADC drugs (ADCETRIS® (brentuximab vedotin), 2011; and KADCYLA® (trastuzumab-emtansine or T-DM1), 2013). Currently, there are over 30 ADC drug candidates in various phases of clinical trials for cancer treatment. As antibody engineering and linker-payload optimization become increasingly sophisticated, the discovery and development of new ADCs increasingly rely on the identification and validation of novel targets suitable for the methodology and the generation of targeted MAbs. Two criteria for ADC targets are upregulated / high expression levels in tumor cells and robust internalization.

[0214] In one aspect of immunotherapy, tumor cells must carry markers that are easily targeted, i.e., not present on most other cells. Many tumor markers exist, and any of these may be suitable for targeting in the context of the embodiments of the present invention. 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 aspect of immunotherapy is the combination of 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.

[0215] Examples of immunotherapies currently under investigation or in use include: immune adjuvants, such as Mycobacterium bovis (…). Mycobacterium bovis ), Plasmodium falciparum ( Plasmodium falciparum), dinitrochlorobenzene and aromatic compounds (US Patents 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapies, such as 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, such as TNF, IL-1, IL-2 and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; US Patents 5,830,880 and 5,846,945); and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2 and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; US Patent 5,824,311). The use of one or more anticancer therapies in conjunction with the antibody therapies described in this article has been considered.

[0216] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints are molecules in the immune system that either upregulate (e.g., co-stimulatory molecules) or downregulate signaling. 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 attenuator (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 T-cell activation V-domain Ig inhibitor (VISTA). In particular, the immune checkpoint inhibitor targets the PD-1 axis and / or CTLA-4.

[0217] The immune checkpoint inhibitors may be pharmaceuticals, such as small molecules, recombinant forms of ligands or receptors, or particularly antibodies, such as human antibodies (e.g., International Patent Publication WO2015016718; Pardoll, 2012; both incorporated herein by reference). Known inhibitors of immune checkpoint proteins or their analogues may be used, particularly chimeric, humanized, or human forms of antibodies. As those skilled in the art will appreciate, alternative and / or equivalent names may be used for certain antibodies mentioned in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this invention. For example, lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0218] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In one particular aspect, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In one particular aspect, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In one particular aspect, 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. US8735553, US8354509, and US8008449 (all incorporated herein by reference). Other PD-1 axis antagonists used in the methods presented herein are known in the art, for example, as described in U.S. Patent Applications US20140294898, US2014022021, and US20110008369 (all incorporated herein by reference).

[0219] In some embodiments, the PD-1 binding 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 binding antagonist is an immunoadhesive (e.g., an immunoadhesive comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab (also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO)® Pembrolizumab (also known as MK-3475, Merck 3475, Lanlolizumab, KEYTRUDA) is an anti-PD-1 antibody described in WO2006 / 121168. ® SCH-900475 is an anti-PD-1 antibody described in WO2009 / 114335. CT-011 (also known as hBAT or hBAT-1) is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224 (also known as B7-DCIg) is a PD-L2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0220] Another immune checkpoint that can be targeted using the methods presented in this paper is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession 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 expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T-cell costimulatory protein CD28, and both molecules bind to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and is likely important for their function. T cell activation via T cell receptors and CD28 leads to increased expression of CTLA-4 (an inhibitory receptor for the B7 molecule).

[0221] 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, an immunoadhesin, a fusion protein, or an oligopeptide.

[0222] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in this method can be generated using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. For example, the methods disclosed herein may use anti-CTLA-4 antibodies disclosed in the following publications: US8,119,129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly known as tesimumab), US Patent No. 6,207,156; Hurwitz et al., (1998) Proc Natl Acad Sci USA 95(17): 10067-10071; Camacho et al., (2004) J ClinOncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al., (1998) CancerRes 58:5301-5304. The teachings of each of the above-mentioned publications are incorporated herein by reference. Antibodies that compete with any of these well-known antibodies in the art for binding to CTLA-4 may also be used. For example, humanized CTLA-4 antibodies are described in International Patent Applications WO2001014424, WO2000037504 and U.S. Patent No. US8017114 (all incorporated herein by reference).

[0223] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or its antigen-binding fragments and variants (see, for example, WOO 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 with the aforementioned antibodies for binding to CTLA-4 and / or binds to the same epitopes on CTLA-4 as the aforementioned antibodies. In yet another embodiment, the antibody has at least approximately 90% variable region amino acid sequence identity with the aforementioned antibodies (e.g., at least approximately 90%, 95%, or 99% variable region identity with ipilimumab).

[0224] Other molecules used to modulate CTLA-4 include CTLA-4 ligands and receptors, which are described, for example, in U.S. Patent Nos. US5844905, US5885796 and International Patent Application Nos. WO1995001994 and WO1998042752 (all incorporated herein by reference); and immune adhesion, which is described, for example, in U.S. Patent No. US8329867 (incorporated herein by reference).

[0225] 4. Surgical procedures

[0226] Approximately 60% of people with cancer will undergo some type of surgery, including preventative, diagnostic or staging, curative, and palliative surgeries. Curative surgeries include resections in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and can be used in combination with other therapies, such as treatments according to embodiments of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgical procedures (Moss surgery).

[0227] After the removal of some or all cancerous cells, tissue, or tumors, a cavity may form in the body. Treatment can be accomplished by perfusion, direct injection, or local application of additional anticancer therapies to the area. Such treatments 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 have varying dosages.

[0228] 5. Other reagents

[0229] Other agents can be used in combination with certain aspects of embodiments of the present invention to improve therapeutic efficacy. These additional agents include: agents that affect the upregulation of cell surface receptors and GAP linkages, cell inhibition and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of highly proliferating cells to apoptosis-inducing agents, or other biological agents. Increased intercellular signaling resulting from increased GAP linkages will increase the anti-hyperproliferative effect on adjacent highly proliferating cell populations. In other embodiments, cell inhibition or differentiation agents can be used in combination with certain aspects of embodiments of the present invention to improve the anti-hyperproliferative efficacy of the treatment. Inhibitors of cell adhesion are considered for use in improving the efficacy of embodiments of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. Further consideration is given to using other agents that increase the sensitivity of highly proliferating cells to apoptosis, such as antibody c225, in combination with certain aspects of embodiments of the present invention to improve therapeutic efficacy.

[0230] III. Kits and Diagnostics

[0231] In various aspects of the embodiments described, kits are contemplated that comprise therapeutic agents and / or other therapeutic and delivery agents. In some embodiments, the present invention contemplates kits for preparing and / or administering the therapies of the embodiments described. The kit may comprise one or more sealed vials containing any of the pharmaceutical compositions of the embodiments of the present invention. The kit may include, for example, at least one 4-1BB antibody, and reagents for preparing, formulating, and / or administering the components of the embodiments or for performing one or more steps of the methods of the present invention. In some embodiments, the kit may also comprise suitable containers that do not react with the components of the kit, such as Eppendorf tubes, assay plates, syringes, bottles, or tubes. The containers may be made of sterilizable materials (e.g., plastic or glass).

[0232] The kit may also include instructions that outline the procedural steps of the methods set forth herein and that will be followed in accordance with those substantially the same as those described herein or known to those skilled in the art. The guidance information may be in a computer-readable medium containing machine-readable instructions that, when executed using a computer, demonstrate a real or virtual procedure for delivering a pharmaceutically effective amount of the therapeutic agent.

[0233] IV. Examples

[0234] The following embodiments are included to illustrate preferred embodiments of the invention. Those skilled in the art will recognize that the techniques disclosed in the following embodiments represent those that the inventors have found to function well in the practice of the invention, and therefore can be considered to constitute preferred modes of practice with respect to the invention. However, based on this disclosure, those skilled in the art will recognize that many variations can be made in the particular embodiments disclosed, and that they still yield the same or similar results without departing from the spirit and scope of the invention.

[0235] Example 1 – Generation and Characterization of Anti-41BB Antibody

[0236] Mice were immunized with alternating cell vaccines (retrovirally modified macrophages or dendritic cells expressing 4-1BB) and recombinant protein-based vaccines (recombinant human 4-1BB extracellular regions with Fc or HIS tags) to generate antibodies targeting human 4-1BB (Figure 1), including clone 54 (IgG2a), clone 49A (IgG1), clone 138 (IgG2b), clone 151 (IgG3), and clone 111A (IgG1). Titration curves of the clones were obtained by ELISA, and EC50 values ​​were determined. Figure 1B ).

[0237] RNA extraction The hybridoma cell granular pellet was provided by Long Vien (Monoclonal Antibody CoreFacility MDACC). Total mRNA was extracted from the hybridoma cell granular pellet. Total RNA was extracted from the granular pellet of hybridoma clone 271-54 41-BB cells using the RNA extraction assay protocol (Zymo Research).

[0238] RT-PCR cDNA was generated from RNA via reverse transcription using random primers (RT Superscrit III, Life Technologies). PCR amplification of the VH and VL regions of the monoclonal antibody DNA using variable domain primers yielded the results. Figure 3The bands in the image were observed. The VH and VL products were purified by gel electrophoresis and cloned into the sequencing vector pCR™2.1-TOPO®, which was then transformed into DH5a cells. Next, positive transformants were screened by PCR using the M16 forward and reverse primers (mFVIj forward primer (positions 59-76): ACTGCAGGTGTCCTCTCT (SEQ ID NO: 65); mRevIgG2a reverse primer (positions 526-506): TAACCCTTGACCAGGCATCC (SEQ ID NO: 66); mFVK4 / 5a forward primer (positions 47-67): TCAGCTTCYTGCTAATCAGTG (SEQ ID NO: 67); 1mRK reverse primer (positions 491-471): ACTGAGGCACCTCCAGATGTT (SEQ ID NO: 68)). Selected colonies were chosen for small-scale plasmid purification and analyzed by DNA sequencing (MDACC core facility).

[0239] Among numerous lead candidates, clone 54 4-1BB antibody was selected, sequenced, and expressed (in a variety of mouse and human isotypes). Patient tumor masses were cultured in tumor-infiltrating lymphocyte medium to support their survival and rapid proliferation in cell cultures. This study found that the mouse IgG2a version of clone 54 4-1BB antibody was as effective as the anti-human 4-1BB antibody urerucumab in expanding and maturing human tumor-infiltrating CD8 T cells and CD4 T cells. Figure 2B In addition, clone 54 effectively expanded tumor-infiltrating CD8 T cells and CD4 T cells from tumors of patients with glioblastoma or colorectal cancer (Figure 4).

[0240]

[0241] All methods disclosed and claimed herein can be made and performed based on this disclosure without excessive experimentation. While the compositions and methods of the invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations can be made to the methods described herein and to the steps or sequence of steps thereof without departing from the concept, spirit, and scope of the invention. More particularly, it will be apparent that certain chemically and physiologically relevant agents can be substituted for the agents described herein with the same or similar results. All such similar substitutions or modifications that will be apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention, as defined by the appended claims.

[0242] References

[0243] The following references are specifically incorporated herein by reference to provide exemplary procedures or other details that are supplementary to those set forth herein.

[0244] Austin-Ward and Villaseca Revista Medica de Chile , 126(7):838-845,1998.

[0245] Bukowski et al. Clinical Cancer Research ., 4(10):2337-2347, 1998.

[0246] Christodoulides et al. Microbiology , 144(Pt 11):3027-3037, 1998.

[0247] Davidson et al. J. Immunother ., 21(5):389-398, 1998.

[0248] Hanibuchi et al. Int. J. Cancer , 78(4):480-485, 1998.

[0249] Hellstrand et al. Acta Oncologica , 37(4):347-353, 1998.

[0250] Hollander, Front. Immun. , 3:3, 2012.

[0251] Hui and Hashimoto, Infection Immun., 66(11):5329-5336, 1998.

[0252] International Patent Publication No. WO2009 / 101611

[0253] International Patent Publication No. WO2010 / 027827

[0254] International Patent Publication No. WO2011 / 066342

[0255] International Patent Publication No. WO2015016718

[0256] Pardoll, Nature Rev Cancer, 12:252-264, 2012.

[0257] Qin et al. Proc. Natl. Acad. Sci. USA , 95(24):14411-14416, 1998.

[0258] US Patent No. 4,870,287

[0259] US Patent No. 5,091,513

[0260] US Patent No. 5,739,169

[0261] US Patent No. 5,760,395

[0262] US Patent No. 5,801,005

[0263] US Patent No. 5,824,311

[0264] US Patent No. 5,830,880

[0265] US Patent No. 5,846,945

[0266] US Patent No. 6,881,557

[0267] US Patent No. 8,008,449

[0268] US Patent No. 8,354,509

[0269] US Patent No. 8,735,553

[0270] US Patent Publication No. US20050214860

[0271] US Patent Publication No. US20110008369

[0272] US Patent Publication No. US2014022021

[0273] US Patent Publication No. US20140294898

[0274] Vinay and Kwon, Mol Cancer Ther, 11(5): 1062-70, 2012.

Claims

1. An isolated monoclonal antibody, wherein the antibody specifically binds to 4-1BB and comprises: (a) The first V H The CDR is the same as SEQ ID NO: 3; (b) The second V H The CDR is the same as SEQ ID NO: 5; (c) The third V H The CDR is the same as SEQ ID NO: 7; (d) The first V L The CDR is the same as SEQ ID NO: 11; (e) The second V L CDR is the same as SEQ ID NO: 13; and (f) The third V L The CDR is the same as SEQ ID NO: 15; or (a) The first V H The CDR is the same as SEQ ID NO: 19; (b) The second V H CDR is the same as SEQ ID NO: 21; (c) The third V H The CDR is the same as SEQ ID NO: 23; (d) The first V L CDR is the same as SEQ ID NO: 27; (e) The second V L CDR is the same as SEQ ID NO: 29; and (f) The third V L CDR is the same as SEQ ID NO: 31; or (a) The first V H The CDR is the same as SEQ ID NO: 35; (b) The second V H CDR is the same as SEQ ID NO: 37; (c) The third V H The CDR is the same as SEQ ID NO: 39; (d) The first V L The CDR is the same as SEQ ID NO: 43; (e) The second V L CDR is the same as SEQ ID NO: 45; and (f) The third V L The CDR is the same as SEQ ID NO: 47; or (a) The first V H The CDR is the same as SEQ ID NO: 51; (b) The second V H The CDR is the same as SEQ ID NO: 53; (c) The third V H The CDR is the same as SEQ ID NO: 55; (d) The first V L The CDR is the same as SEQ ID NO: 59; (e) The second V L CDR is the same as SEQ ID NO: 61; and (f) The third V L CDR is the same as SEQ ID NO:

63.

2. The antibody of claim 1, wherein the antibody comprises: (a) The first V H The CDR is the same as SEQ ID NO: 3; (b) The second V H The CDR is the same as SEQ ID NO: 5; (c) The third V H The CDR is the same as SEQ ID NO: 7; (d) The first V L The CDR is the same as SEQ ID NO: 11; (e) The second V L CDR is the same as SEQ ID NO: 13; and (f) The third V L CDR is the same as SEQ ID NO:

15.

3. The antibody of claim 2, wherein the antibody comprises V with clone 54 H The structural domain (SEQ ID NO: 2) is at least approximately 80% identical to the V. H Domain, and V with clone 54 L The domain (SEQ ID NO: 10) is at least approximately 80% identical to the V. L Structural domain.

4. The antibody of claim 2, wherein the antibody comprises V with clone 54 H The domain (SEQ ID NO: 2) is at least approximately 90% identical to the V. H Domain, and V with clone 54 L The domain (SEQ ID NO: 10) is at least approximately 90% identical to the V. L Structural domain.

5. The antibody of claim 2, wherein the antibody comprises V with clone 54 H The structural domain (SEQ ID NO: 2) is the same as the V. H Domain, and V with clone 54 L The same V as the structural domain (SEQ ID NO: 10) L Structural domain.

6. The antibody of claim 1, wherein the antibody comprises: (a) The first V H The CDR is the same as SEQ ID NO: 19; (b) The second V H CDR is the same as SEQ ID NO: 21; (c) The third V H The CDR is the same as SEQ ID NO: 23; (d) The first V L CDR is the same as SEQ ID NO: 27; (e) The second V L CDR is the same as SEQ ID NO: 29; and (f) The third V L CDR is the same as SEQ ID NO:

31.

7. The antibody of claim 6, wherein the antibody comprises V with clone 135B. H The domain (SEQ ID NO: 18) is at least approximately 80% identical to the V. H Domain, and V with clone 135B L The domain (SEQ ID NO: 26) is at least approximately 80% identical to the V. L Structural domain.

8. The antibody of claim 6, wherein the antibody comprises V with clone 135B. H The domain (SEQ ID NO: 18) is at least approximately 90% identical to the V. H Domain, and V with clone 135B L The domain (SEQ ID NO: 26) is at least approximately 90% identical to the V. L Structural domain.

9. The antibody of claim 6, wherein the antibody comprises V with clone 135B. H The same V as the structural domain (SEQ ID NO: 18) H Domain, and V with clone 135B L The same V as the structural domain (SEQ ID NO: 26) L Structural domain.

10. The antibody of claim 1, wherein the antibody comprises: (a) The first V H The CDR is the same as SEQ ID NO: 35; (b) The second V H CDR is the same as SEQ ID NO: 37; (c) The third V H The CDR is the same as SEQ ID NO: 39; (d) The first V L The CDR is the same as SEQ ID NO: 43; (e) The second V L CDR is the same as SEQ ID NO: 45; and (f) The third V L CDR is the same as SEQ ID NO:

47.

11. The antibody of claim 10, wherein the antibody comprises V with clone 138 H The domain (SEQ ID NO: 34) is at least approximately 80% identical to the V. H Domain, and V with clone 138 L The domain (SEQ ID NO: 42) is at least approximately 80% identical to the V. L Structural domain.

12. The antibody of claim 10, wherein the antibody comprises V with clone 138. H The domain (SEQ ID NO: 34) is at least approximately 90% identical to the V. H Domain, and V with clone 138 L The domain (SEQ ID NO: 42) is at least approximately 90% identical to the V. L Structural domain.

13. The antibody of claim 10, wherein the antibody comprises V with clone 138. H The structural domain (SEQ ID NO: 34) is the same as the V. H Domain, and V with clone 138 L The same V as the structural domain (SEQ ID NO: 42) L Structural domain.

14. The antibody of claim 1, wherein the antibody comprises: (a) The first V H The CDR is the same as SEQ ID NO: 51; (b) The second V H The CDR is the same as SEQ ID NO: 53; (c) The third V H The CDR is the same as SEQ ID NO: 55; (d) The first V L The CDR is the same as SEQ ID NO: 59; (e) The second V L CDR is the same as SEQ ID NO: 61; and (f) The third V L CDR is the same as SEQ ID NO:

63.

15. The antibody of claim 14, wherein the antibody comprises V with clone 49A H The structural domain (SEQ ID NO: 50) is at least approximately 80% identical to the V. H Domain, and V with clone 49A L The domain (SEQ ID NO: 58) is at least approximately 80% identical to the V. L Structural domain.

16. The antibody of claim 14, wherein the antibody comprises V with clone 49A H The domain (SEQ ID NO: 50) is at least approximately 90% identical to the V. H Domain, and V with clone 49A L The domain (SEQ ID NO: 58) is at least approximately 90% identical to the V. L Structural domain.

17. The antibody of claim 14, wherein the antibody comprises V with clone 49A H The same V as the structural domain (SEQ ID NO: 50) H Domain, and V with clone 49A L The same V as the structural domain (SEQ ID NO: 58) L Structural domain.

18. The antibody of any one of claims 1-5, wherein the antibody is recombinant.

19. The antibody of claim 1, wherein the antibody is IgG, IgM, IgA or an antigen-binding fragment thereof.

20. The antibody of any one of claims 1-19, wherein the antibody is Fab', F(ab')2, F(ab')3, monovalent scFv, bivalent scFv or single-domain antibody.

21. The antibody of any one of claims 1, wherein the antibody is a human antibody, a humanized antibody, or a deimmunized antibody.

22. The antibody of any one of claims 1-21, wherein the antibody is conjugated to an imaging agent, a chemotherapeutic agent, a toxin, or a radionuclide.

23. A composition comprising an antibody of any one of claims 1-22 in a pharmaceutically acceptable carrier.

24. An isolated polynucleotide molecule comprising a nucleic acid sequence encoding an antibody of any one of claims 1-21.

25. A recombinant polypeptide, said recombinant polypeptide comprising: antibody V H A structural domain containing clone 54 of V H CDRs 1-3 (SEQ ID NO: 3, 5 and 7) of the domain, and V of clone 135B H CDRs 1-3 of the domain (SEQ ID NO: 19, 21 and 23), V of clone 138 H CDRs 1-3 (SEQ ID NO: 35, 37 and 39) of the domain or V of clone 49A H CDRs 1-3 of the structural domain (SEQ IDNO: 51, 53 and 55).

26. A recombinant polypeptide, said recombinant polypeptide comprising: antibody V L A structural domain containing 54 V L CDRs 1-3 (SEQ ID NO: 11, 13 and 15) of the structural domain, and V of 135B L CDRs 1-3 (SEQ ID NO: 27, 29 and 31) of the structural domain, and V of 138 L CDRs 1-3 (SEQ ID NO: 43, 45 and 47) of the structural domain or V of 49A L CDRs 1-3 of the domain (SEQ ID NO: 59, 61 and 63).

27. A recombinant polypeptide, wherein the recombinant polypeptide comprises: V containing clone 54 H Antibody V containing CDRs 1-3 (SEQ ID NO: 3, 5 and 7) of the structural domain H The structural domain, and the V containing 54 L Antibody V containing CDR 1-3 (SEQ ID NO: 11, 13 and 15) of the structural domain L structural domain; V containing clone 135B H Antibody V containing CDR 1-3 (SEQ ID NO: 19, 21 and 23) of the structural domain H The structural domain, and the V containing 135B L Antibody V containing CDR 1-3 (SEQ ID NO: 27, 29 and 31) of the structural domain L structural domain; V containing clone 138 H Antibody V containing CDR 1-3 (SEQ ID NO: 35, 37 and 39) of the structural domain H The structural domain, and the V containing 138 L Antibody V containing CDR 1-3 (SEQ ID NO: 43, 45 and 47) of the structural domain L domain; or V containing clone 49A H Antibody V containing CDR 1-3 (SEQ ID NO: 51, 53 and 55) of the structural domain H The structural domain, and V containing 49A L Antibody V containing CDR 1-3 (SEQ ID NO: 59, 61 and 63) of the structural domain L Structural domain.

28. An isolated polynucleotide molecule comprising a nucleic acid sequence encoding a polypeptide of any one of claims 25-27.

29. A host cell comprising one or more polynucleotide molecules encoding an antibody of any one of claims 1-21 or a recombinant polypeptide of any one of claims 25-27.

30. The host cell of claim 29, wherein the host cell is a mammalian cell, yeast cell, bacterial cell, ciliate cell or insect cell.

31. A method for preparing antibodies, the method comprising: (a) Expressing one or more antibodies encoding any one of claims 1-21 in cells L and V H Chain-like polynucleotide molecules; and (b) Purify the antibody from the cells.

32. A method for treating a subject with cancer, the method comprising administering to the subject an effective amount of an antibody of any one of claims 1-21.

33. The method of claim 32, wherein the cancer is breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, skin cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

34. The method of claim 32, wherein the antibody is in a pharmaceutically acceptable composition.

35. The method of claim 32, wherein the antibody is administered systemically.

36. The method of claim 32, wherein the antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally.

37. The method of claim 32, further comprising administering at least a second anticancer therapy to the subject.

38. The method of claim 37, wherein the second anticancer therapy is surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

39. The method of claim 37, wherein the second anticancer therapy comprises adoptive T-cell therapy.

40. The method of claim 37, wherein the second anticancer therapy comprises immunotherapy.

41. The method of claim 40, wherein the immunotherapy is an immune checkpoint inhibitor.

42. The method of claim 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.

43. The method of claim 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.

44. The method of claim 43, wherein the PD-1 binding antagonist is a monoclonal antibody or an antigen-binding fragment thereof.

45. The method of claim 43, wherein the PD-1 binding antagonist is nivolumab, pembrolizumab, CT-011 (pildizumab), BMS-936559 (MDX-1105), MPDL328OA (atezilizumab), or AMP-224 (PD-L2 Fc fusion protein).

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