Anti-CD27 Antibodies and Uses Thereof

Abstract

The present invention provides isolated antibodies that specifically bind to CD27 with high affinity.The present invention provides methods of treating a subject with cancer, comprising administering to the subject a therapeutically effective amount of an anti-CD27 antibody, either as monotherapy or in combination with a checkpoint inhibitor, such as an anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibody.

Description

[Technical field]

[0001] Throughout this application, various publications are cited by author name and date in parentheses, or by patent or patent publication number. Full citations for these publications are collected at the end of this specification, immediately preceding the claims. The disclosures of these publications are incorporated herein by reference in their entireties in order to more fully indicate the state of the art to those skilled in the art at the time the invention is described and claimed herein. However, these disclosures are incorporated by reference into this application only to the extent that there is no contradiction between the incorporated information and information provided by express disclosure in this specification. Furthermore, the citation of a cited reference herein should not be construed as an admission that such reference is prior art to the present invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 652,790, filed April 4, 2018, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence listings provided electronically via EFS-WEB The electronic sequence listing (name: 13107-WO-PCT_ST25.txt, size: 23,885 bytes; and creation date: April 1, 2019) provided in connection with this specification is incorporated by reference in its entirety.

[0004] FIELD OF THEINVENTION The present invention relates to monoclonal antibodies (mAbs) that specifically bind to CD27 and methods of treating cancer in a subject comprising administering to the subject the anti-CD27 antibodies (Abs) as monotherapy or in combination with anti-cancer agents, such as immune checkpoint inhibitors, and / or certain chemotherapy and radiation therapies. [Background technology]

[0005] 2. Background of the Invention Human cancers carry numerous genetic and epigenetic alterations and produce neoantigens that are potentially recognizable by the immune system (Chakravarthi et al., 2016). The adaptive immune system, consisting of T and B lymphocytes, has powerful anti-cancer capabilities and acquires specificity with a broad ability to respond to diverse tumor antigens. Furthermore, the immune system contains considerable flexibility and memory elements. Harnessing all of these attributes of the adaptive immune system makes immunotherapy unique among all cancer treatment modalities.

[0006] Over the past decade, the development of specific immune checkpoint pathway inhibitors for cancer treatment has been revealed (Chen and Mellman, 2013; Lesokhin et al., 2015), including Abs such as ipilimumab (Yervoy®), which binds to and inhibits cytotoxic T lymphocyte antigen-4 (CTLA-4) for the treatment of patients with advanced melanoma, and nivolumab (Opdivo®), pembrolizumab (Keytruda®), and cemiplimab-rwlc (LIBTAYO®), which specifically bind to the PD-1 receptor and block the inhibitory PD-1 / PD-L1 signaling pathway (Iwai et al., 2017). This pathway is also blocked by Abs including atezolizumab (Tecentriq®), durvalumab (Imfinzi®), and avelumab (Bavencio®), which specifically bind to PD-L1.

[0007] Nivolumab is a fully human immunoglobulin (Ig) G4 (S228P) mAb that selectively blocks the interaction with PD-1 ligands, PD-L1 and PD-L2 (US Patent 8,008,449; Wang et al., 2014), thereby blocking the downregulation of antigen-specific T cell responses to both foreign (including tumor) and self-antigens and enhancing immune responses to these antigens. Nivolumab has recently been approved for several cancers, including melanoma, lung cancer, renal cell carcinoma, classical Hodgkin lymphoma, head and neck cancer, urothelial carcinoma, MSI-H or dMMR metastatic colorectal cancer and hepatocellular carcinoma, and is currently being clinically evaluated as monotherapy or in combination with other anticancer agents for additional tumor types. However, only a small proportion of patients, generally less than about 25%, benefit from treatment with checkpoint inhibitors, and considerable efforts are currently being directed at the efficacy of immunotherapy in combination with checkpoint inhibitors and other anticancer agents or treatments. PD-1 / PD-L1 inhibitors have proven successful in treating a broad range of cancers, recognizing their potential as the backbone of various future drug combinations in immuno-oncology, and the race to develop the most effective combinations has begun (see, e.g., Mahoney et al., 2015; Ott et al., 2017).

[0008] CD27, a 55 kDa type I transmembrane protein of the tumor necrosis factor receptor (TNFR) family, costimulates T cell activation after binding to its ligand CD70. In humans, CD27 is constitutively and exclusively expressed on naive T cells and upregulated on activated T and B cells, whereas expression of CD70, a type II transmembrane protein, is highly regulated and occurs only transiently on activated T cells, B cells, and dendritic cells (DCs) (Wajant, 2016). CD27 plays a key role in naive T cell expansion, effector function expansion, and T cell immunity and the generation and long-term maintenance of responder T cell pools (Hendricks et al., 2000). CD27-deficient mice exhibit reduced T cell immunity, memory to influenza virus, and cytolytic activity of activated NK cells (Hendricks et al., 2000; De Colvenaer et al., 2011), whereas CD70 deficiency reduces the expression of effector CD8 + CD27 is expressed at higher levels in human peripheral blood than in human tumors, and Tregs are CD4 + They express higher levels of CD27 than T cells. CD27-driven costimulation is CD8 + It is believed to lower the T cell receptor activation threshold of T cells, allowing them to respond to low affinity antigens and expanding the functional T cell repertoire (van Gisbergen et al., 2011). Blockade of the CD27-CD70 interaction inhibits primary CD8 + It reduces T cell responses (Taraban et al., 2004; Sanchez et al., 2007) and inhibits CD8 + This results in a reduction in T cell memory (Dolfi et al., 2008). Importantly, CD27 costimulation blockade completely abrogates anti-CD40 therapeutic responses in a mouse lymphoma model (French et al., 2007).

[0009] CD27 has a role in the early generation of primary immune responses and is required for the generation and long-term maintenance of T cell immunity. CD27-CD70 binding leads to activation of the NF-κB and MAPK8 / JNK pathways. The adaptor proteins TRAF2 and TRAF5 have been shown to mediate signaling resulting from CD27 binding. Evidence for the clinical relevance of active CD27 in maintaining proper immune function is provided by fatal EBV-associated lymphoproliferative disorders or persistent EBV viremia in patients lacking functional CD27 (Salzer et al., 2013; van Montfrans et al., 2012).

[0010] Enhanced T cell stimulation by CD27 agonism is manifested by increased proliferation and secretion of IFN-γ (Ramakrishna et al., 2015). In ex vivo studies using peripheral blood mononuclear cells (PBMCs) from melanoma patients, CD27 agonist treatment enhanced CD8 T cell proliferation against melanoma antigens. + CD27 agonist-mediated costimulation increases T cell responses, along with IFN-γ production, in non-tumor-bearing mice (Bullock et al., 2014). +It has been shown to cooperate with the co-inhibitory checkpoint PD-L1 blockade by bringing T cells out of quiescence (Buchan et al., 2015). In preclinical studies, anti-CD27 agonist treatment has been shown to inhibit experimental lung metastasis (Roberts et al., 2010) and inhibit the growth of syngeneic tumors (French et al., 2007; Roberts et al., 2010; He et al., 2013; Sakanishi and Yagita, 2010). It has also been shown that CD27 agonism with PD-1 blockade increases antigen-specific CTL responses that are effective in eradicating TC-1 tumors (Ahrends et al., 2016), and the anti-human CD27 agonist monoclonal antibody (mAb), varlilumab, cooperates with PD-L1 blockade for protection against lymphoma in human-CD27 transgenic mice (Buchan et al., 2018). Varlilumab is in clinical trials in combination with the anti-PD-1 mAb, nivolumab, in a number of cancer patients and has been well tolerated to date, with initial evidence of clinical activity in patient subsets of tumor types that are generally resistant to PD-1 inhibitor monotherapy (Sanborn et al., 2017). Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention relates to the evaluation of non-ligand blocking CD27 agonist antibodies (Abs) in inducing anti-tumor immunity and maintaining CD70-mediated costimulation and agonism. The present invention also relates to the evaluation of the combination of non-ligand blocking CD27 agonist Abs with checkpoint blockade therapy for anti-tumor activity. There continues to be a need for high affinity, non-ligand blocking anti-CD27 Abs that exhibit potent agonist activity to enhance anti-tumor immune responses. [Means for solving the problem]

[0012] Summary of the Invention The present invention relates to a mAb or antigen-binding portion thereof that specifically binds to human CD27 (hCD27) and does not block its binding to CD70 ligand, as measured by surface plasmon resonance (SPR) or flow cytometry, wherein the Ab or fragment thereof (a) binds to hCD27 with a K of about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 1 nM to about 100 nM, about 10 nM to about 70 nM, about 10 nM to about 50 nM, or about 40 nM to about 45 nM. D and / or (b) an EC of about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, about 0.005 nM to about 0.5 nM, about 0.01 nM to about 0.4 nM, or about 0.02 nM to about 0.25 nM in naive human T cells stimulated with anti-CD3 Ab and anti-CD28 Ab. 50 The present invention relates to a mAb or antigen-binding portion thereof that induces NF-κB and MAPK signaling in a mammal.

[0013] The present invention relates to mAbs or antigen-binding portions thereof that specifically bind to an epitope located within a discontinuous region spanning approximately amino acid residues 21-41 and 52-57 of hCD27 (the sequence of which is SEQ ID NO:1), as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and / or fast photochemical oxidation of proteins (FPOP) epitope mapping.

[0014] The present invention relates to a heavy chain variable region (VCL) comprising: (a) a CDR1 comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:2, a CDR2 comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:3, and a CDR3 comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:4. H and (b) a light chain variable region (V) comprising a CDR1 comprising consecutively linked amino acids having the sequence set forth in SEQ ID NO:5, a CDR2 comprising consecutively linked amino acids having the sequence set forth in SEQ ID NO:6, and a CDR3 comprising consecutively linked amino acids having the sequence set forth in SEQ ID NO:7. L In one embodiment, the present invention relates to a mAb or antigen-binding portion thereof that specifically binds to hCD27, the mAb comprisingH comprises contiguous amino acids having the sequence set forth in SEQ ID NO: 8. In certain embodiments, V L comprises contiguous amino acids having the sequence set forth in SEQ ID NO: 9. In certain embodiments, the mAb comprises a heavy chain comprising contiguous amino acids having the sequence set forth in SEQ ID NO: 12 and a light chain comprising contiguous amino acids having the sequence set forth in SEQ ID NO: 13. In certain embodiments, the mAb is the mAb designated BMS-986215.

[0015] The present invention relates to any mAb of the above mAb or its antigen-binding portion, or a mAb or its antigen-binding portion that specifically binds to the same epitope of hCD27 that is substantially the same as them. The present invention further relates to a mAb or its antigen-binding portion that cross-competes with a control Ab or its control antigen-binding portion for binding to hCD27.

[0016] Any of the above mAbs or their antigen-binding portions are referred to as anti-CD27 mAbs or their antigen-binding portions in the following embodiments.

[0017] In certain embodiments, any of the anti-CD27 mAbs or antigen-binding portions thereof (a) do not inhibit CD70 binding to CD27 as measured by surface plasmon resonance (SPR) or flow cytometry; (b) do not specifically bind rat CD27 and / or mouse CD27 as measured by SPR or flow cytometry; (c) do not specifically bind one or more TNF receptor superfamily members selected from the group consisting of CD30, HVEM, DR5, 4-1BB, CD40, OX40, GITR, and any combination thereof; (d) do not specifically bind to one or more human tissues at a concentration of up to 10 μg / mL, wherein the human tissue is selected from the group consisting of thyroid, lung, skin, uterus, prostate, liver, kidney, pancreas, adrenal gland, pituitary, placenta, testis, cerebrum, cerebellum, heart, peripheral nerve, and any combination thereof; (e) do not specifically bind to one or more human tissues at a concentration of up to 10 μg / mL, wherein the human tissue is selected from the group consisting of thyroid, lung, skin, uterus, prostate, liver, kidney, pancreas, adrenal gland, pituitary, placenta, testis, cerebrum, cerebellum, heart, peripheral nerve, and any combination thereof; (f) can induce proliferation and / or IFN-γ secretion in Ab-stimulated naive and preactivated human T cells; (g) can induce CD4 + CD45RO + (h) can induce increased IL-2 release from Staphylococcal enterotoxin B (SEB)-stimulated human PBMCs; (i) can increase IL-2 release by at least two-fold when combined with anti-PD-1 Ab; (j) can inhibit monocyte-derived dendritic cells (MDDCs) and CD4+ cells co-cultured in the presence of soluble OKT3. + (k) reverse Treg-mediated suppression of responder T cells; and (k) enhance human T cell proliferation and induction of IFN-γ secretion by soluble CD70. The compound has one or more characteristics selected from the group consisting of:

[0018] In certain embodiments, the anti-CD27 mAb or any of its antigen-binding portions has a K for hCD27 of about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 1 nM to about 100 nM, about 10 nM to about 70 nM, about 10 nM to about 50 nM, or about 40 nM to about 45 nM. D Combine with.

[0019] In certain embodiments, either the anti-CD27 mAb or its antigen-binding portion binds to human T cells with an EC of about 0.1 nM or less, about 0.09 nM or less, about 0.08 nM or less, about 0.07 nM or less, about 0.06 nM or less, about 0.05 nM or less, about 0.04 nM or less, about 0.01 nM to about 0.1 nM, about 0.025 nM to about 0.075 nM, or about 0.03 nM to about 0.06 nM. 50 and binds.

[0020] In certain embodiments, either the anti-CD27 mAb or its antigen-binding portion binds to cynomolgus T cells with an EC of about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, about 0.2 nM or less, about 0.1 nM or less, about 0.01 nM to about 0.5 nM, about 0.025 nM to about 0.4 nM, about 0.04 to about 0.3 nM, or about 0.06 nM to about 0.2 nM. 50 and binds.

[0021] In certain embodiments, either the anti-CD27 mAb or its antigen-binding portion comprises a heavy chain constant region that is of the human IgG1, IgG2, IgG3, or IgG4 isotype. In certain preferred embodiments, the anti-CD27 mAb or its antigen-binding portion comprises a heavy chain constant region that is of the human IgG1 isotype. In other embodiments, the anti-CD27 mAb or its antigen-binding portion comprises a heavy chain constant region that is of the human IgG4 isotype.

[0022] In certain embodiments, any of the anti-CD27 mAbs is a full-length Ab of the IgG1, IgG2, IgG3, or IgG4 isotype.

[0023] In certain embodiments, any of the anti-CD27 antigen binding moieties is an Ab fragment or a single chain Ab. In certain embodiments, the Ab fragment is selected from the group consisting of Fab, F(ab')2, Fd and Fv fragments, single domain Abs, single chain variable fragments (scFv), bivalent scFvs (di-scFv) and bivalent scFvs (bi-scFv), bispecific antibodies, minibodies, CDRs and any combination thereof.

[0024] In certain embodiments, either the anti-CD27 mAb or an antigen-binding portion thereof is a human Ab or a fragment thereof.

[0025] In certain embodiments, the anti-CD27 mAb or any of its antigen-binding portions is a humanized Ab or fragment thereof.

[0026] In certain embodiments, the anti-CD27 mAb or any of its antigen-binding portions is a chimeric Ab or fragment thereof.

[0027] In certain embodiments, either the anti-CD27 mAb or an antigen-binding portion thereof is an hCD27 agonist.

[0028] In certain embodiments, either the anti-CD27 mAb or an antigen-binding portion thereof does not inhibit CD70 binding to CD27 as measured by SPR or flow cytometry.

[0029] In certain embodiments, either the anti-CD27 mAb or an antigen-binding portion thereof does not specifically bind to rat CD27 and / or mouse CD27 as measured by SPR or flow cytometry.

[0030] In certain embodiments, none of the anti-CD27 mAbs or antigen-binding portions thereof specifically binds to one or more TNF receptor superfamily members selected from the group consisting of CD30, HVEM, DR5, 4-1BB, CD40, OX40, GITR, and any combination thereof.

[0031] In certain embodiments, the anti-CD27 mAb, or any of its antigen-binding portions, does not specifically bind to one or more human tissues at a concentration of up to 10 μg / mL, wherein the human tissue is selected from the group consisting of thyroid, lung, skin, uterus, prostate, liver, kidney, pancreas, adrenal gland, pituitary gland, placenta, testis, cerebrum, cerebellum, heart, peripheral nerve, and any combination thereof.

[0032] In certain embodiments, either the anti-CD27 mAb or antigen-binding portion thereof can induce NF-κB and MAPK signaling in naive and pre-activated human T cells stimulated with anti-CD3 Ab and anti-CD28 Ab. In certain embodiments, the mAb or antigen-binding portion thereof has an EC of about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, about 0.005 nM to about 0.5 nM, about 0.01 nM to about 0.4 nM, or about 0.02 nM to about 0.25 nM. 50 can induce NF-κB and MAPK signaling.

[0033] In certain embodiments, the anti-CD27 mAb or any of its antigen-binding portions can induce proliferation and / or IFN-γ secretion in a CHO-svCD3-CD32A assay. In certain embodiments, the mAb or antigen-binding portion thereof has an EC of about 0.05 nM or less, about 0.04 nM or less, about 0.03 nM or less, about 0.0005 nM to about 0.05 nM, about 0.0005 nM to about 0.04 nM, about 0.0005 nM to about 0.03 nM, about 0.001 nM to about 0.05 nM, about 0.001 nM to about 0.04 nM, or about 0.001 nM to about 0.03 nM. 50 can induce proliferation and / or IFN-γ secretion.

[0034] In certain embodiments, the anti-CD27 mAb or any of its antigen-binding portions binds to CD4 + CD45RO +In certain embodiments, the mAb or antigen-binding portion thereof has an EC of about 0.01 nM or less, about 0.009 nM or less, about 0.008 nM or less, about 0.007 nM or less, about 0.006 nM or less, about 0.005 nM or less, about 0.001 nM to about 0.01 nM, about 0.002 nM to about 0.008 nM, about 0.003 nM to about 0.007 nM, or about 0.004 nM to about 0.006 nM. 50 can induce proliferation.

[0035] In certain embodiments, either the anti-CD27 mAb or antigen-binding portion thereof can increase IL-2 release from SEB-stimulated human PBMCs. In certain embodiments, the mAb or antigen-binding portion thereof can increase IL-2 release by more than about 50% in the presence of a crosslinker, for example, compared to a negative control IgG1 mAb.

[0036] In certain embodiments, either the anti-CD27 mAb, or an antigen-binding portion thereof, when combined with an anti-PD-1 Ab, can increase IL-2 release by at least about 2-fold.

[0037] In one embodiment, the anti-CD27 mAb or any of its antigen-binding portions is administered to MDDCs and soluble OKT3 in the presence of co-cultured CD4 + may reverse Treg-mediated suppression of responder T cells. In certain embodiments, the mAb or antigen-binding portion thereof may reverse Treg-mediated suppression by at least about 70%, for example, compared to a negative control IgG1 mAb.

[0038] In certain embodiments, the anti-CD27 mAb or any of its antigen-binding portions is capable of enhancing human T cell proliferation and induction of IFN-γ secretion by soluble CD70. In certain embodiments, the mAb or antigen-binding portion thereof has an EC of about 0.01 nM or less, about 0.009 nM or less, about 0.008 nM or less, about 0.007 nM or less, about 0.006 nM or less, about 0.005 nM or less, about 0.001 nM to about 0.01 nM, about 0.002 nM to about 0.008 nM, or about 0.003 nM to about 0.005 nM. 50In certain embodiments, the mAb or antigen-binding portion thereof has an EC of about 0.01 nM or less, about 0.009 nM or less, about 0.008 nM or less, about 0.007 nM or less, about 0.001 nM to about 0.01 nM, about 0.002 nM to about 0.008 nM, or about 0.005 nM to about 0.007 nM. 50 can enhance the induction of IFN-γ secretion.

[0039] The present invention relates to an immunoconjugate comprising either an anti-CD27 mAb or an antigen-binding portion thereof linked to a therapeutic agent. In certain embodiments, the therapeutic agent is a cytotoxin. In certain other embodiments, the therapeutic agent is a radioisotope.

[0040] The present invention relates to bispecific molecules comprising either an anti-CD27 mAb, or an antigen-binding portion thereof, linked to a binding domain that has a binding specificity different from the anti-CD27 mAb, or an antigen-binding portion thereof.

[0041] The present invention relates to a composition comprising: (a) any of the anti-CD27 mAbs, or antigen-binding portions thereof; (b) any of the immunoconjugates; or (c) any of the bispecific molecules; and a pharma- ceutically acceptable carrier. In certain embodiments, the composition further comprises an additional Ab, or antigen-binding portion thereof. In certain embodiments, the additional Ab, or antigen-binding portion thereof, is an anti-PD-1 Ab, an anti-programmed death-ligand-1 (PD-L1) Ab, an anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) Ab, an anti-lymphocyte activation gene-3 (LAG-3) Ab, an anti-B- and T-lymphocyte attenuator (BTLA) Ab, an anti-T-cell immunoglobulin and mucin domain-3 (TIM-3) Ab, an anti-killer immunoglobulin-like receptor (KIR) Ab, an anti-killer cell lectin-like receptor G1 (KLRG-1) Ab, an anti-adenosine A2a receptor (A2aR) Ab, an anti-natural killer cell receptor 2B4 (CD244) Ab, an anti-CD160 Ab, an anti-CD161 Ab, an anti-CD162 Ab, an anti-CD163 Ab, an anti-CD164 Ab, an anti-CD165 Ab, an anti-CD166 Ab, an anti-CD167 Ab, an anti-CD168 Ab, an anti-CD169 ... The additional Ab or antigen-binding portion thereof is selected from the group consisting of anti-induced T cell costimulatory molecule (ICOS) Ab, anti-CD137 (4-1BB) Ab, anti-CD134 (OX40) Ab, anti-glucocorticoid-induced TNFR-related protein (GITR) Ab, anti-herpes virus entry mediator (HVEM) Ab, and any combination thereof.

[0042] The present invention relates to isolated nucleic acids encoding either anti-CD27 mAbs or antigen-binding portions thereof.

[0043] The present invention relates to expression vectors comprising any of the isolated nucleic acids.

[0044] The present invention relates to host cells containing any of the expression vectors.

[0045] The present invention relates to a transgenic mouse comprising human immunoglobulin heavy and light chain transgenes, wherein the mouse expresses either an anti-CD27 mAb.

[0046] The present invention relates to hybridomas prepared from any of the transgenic mice, wherein the hybridomas produce anti-CD27 mAbs.

[0047] The present invention relates to a method of producing an anti-CD27 mAb or an antigen-binding portion thereof, comprising expressing an anti-CD27 mAb or an antigen-binding portion thereof from a host cell and isolating the anti-CD27 mAb or an antigen-binding portion thereof from the host cell.

[0048] The present invention relates to a method of treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of any of an anti-CD27 mAb or antigen-binding portion thereof, any of an immunoconjugate, any of a bispecific molecule, or any of a pharmaceutical composition such that the subject is treated.

[0049] The present invention relates to a method of inhibiting tumor cell proliferation in a subject, the method comprising administering to the subject a therapeutically effective amount of any of the anti-CD27 mAbs or antigen-binding portions thereof, any of the immunoconjugates, any of the bispecific molecules, or any of the pharmaceutical compositions such that tumor cell proliferation is inhibited.

[0050] In some embodiments, any of the methods for treating cancer or inhibiting tumor growth further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agent for treating cancer.In some embodiments, the additional therapeutic agent is a compound that reduces the inhibition or increases the stimulation of the immune system.In some embodiments, the additional therapeutic agent is a small molecule compound, a macrocyclic peptide, a fusion protein, or an Ab.In some embodiments, the additional therapeutic agent is an antagonist Ab or an antigen-binding portion thereof that specifically binds to PD-1, PD-L1, CTLA-4, LAG-3, BTLA, TIM-3, KIR, KLRG-1, A2aR, CD244, CD160, TIGIT, or VISTA.In some embodiments, the additional therapeutic agent is an agonist Ab or an antigen-binding portion thereof that specifically binds to ICOS, CD137(4-1BB), CD134(OX40), CD27, GITR, or HVEM. In some embodiments, the additional therapeutic agent is an antagonist Ab or antigen-binding portion thereof that specifically binds to PD-1 or PD-L1, disrupts the interaction between PD-1 and PD-L1, and inhibits PD-1 / PD-L1 signal transduction. In some embodiments, the additional therapeutic agent is an antagonist Ab or antigen-binding portion thereof that specifically binds to PD-1 or PD-L1, and is a chimeric, humanized, or human mAb or antigen-binding portion thereof. In some embodiments, the additional therapeutic agent is an antagonist Ab or antigen-binding portion thereof that specifically binds to PD-1 and cross-competes with nivolumab for binding to human PD-1. In some embodiments, the additional therapeutic agent is an antagonist Ab or antigen-binding portion thereof that specifically binds to PD-1, and is nivolumab, pembrolizumab, or cemiplimab. In certain embodiments, the additional therapeutic agent is an antagonist Ab or an antigen-binding portion thereof that specifically binds to PD-L1 and cross-competes with the Ab named BMS-936559 (WO2013 / 173223) for binding to human PD-L1. In certain embodiments, the additional therapeutic agent is an antagonist Ab or an antigen-binding portion thereof that specifically binds to PD-L1 and is atezolizumab, durvalumab, avelumab, or the Ab named BMS-936559 (WO2013 / 173223).

[0051] In some embodiments, the cancer in any of the methods described herein for treating cancer or inhibiting tumor growth is a solid tumor or the tumor cell is a cell of a solid tumor. In some embodiments, the solid tumor is colon carcinoma or fibrosarcoma. In a further embodiment, the solid tumor is squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous NSCLC, non-squamous NSCLC, head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN), breast cancer, triple negative breast cancer (TNBC), male breast cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, small intestine cancer, liver Cancer, hepatocellular carcinoma (HCC), pancreatic cancer (PAC), pancreatic ductal adenocarcinoma (PDAC), kidney cancer, renal cell carcinoma (RCC), bladder cancer, urethral cancer, ureteral cancer, colorectal cancer (CRC), rectal cancer, colon carcinoma, anal cancer, endometrial cancer, prostate cancer, metastatic castration-resistant prostate cancer (mCRPC), neuroblastoma, glioma, glioblastoma, glioblastoma multiforme (GBM) , germ cell tumors, pediatric sarcoma, sinonasal natural killer, melanoma, skin cancer, bone cancer, cervical cancer, uterine (endometrial) cancer, endometrial carcinoma, uterine sarcoma, fallopian tube carcinoma, ovarian cancer, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, testicular cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, penile cancer, renal pelvis carcinoma, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain cancer, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, pediatric solid tumors, environmentally induced cancer, virus-associated cancer, virus-origin cancer, advanced cancer, unresectable cancer, metastatic cancer, refractory cancer, recurrent cancer, and any combination thereof.

[0052] In some embodiments, the cancer in any of the disclosed methods for cancer treatment or tumor growth inhibition is a hematopoietic tumor or the tumor cell is a hematopoietic tumor cell.In some embodiments, the hematopoietic tumor is a T-cell lymphoma.In some embodiments, the hematopoietic tumor is selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), multiple myeloma, smoldering myeloma, monoclonal gammopathy of undetermined significance (MGUS), progressive, metastatic, refractory and / or recurrent hematopoietic tumor and any combination of said hematopoietic tumor.

[0053] In certain embodiments, the subject in any of the methods of treatment or inhibition is a human.

[0054] The present invention relates to a kit for treating a subject having cancer, comprising: (a) one or more dosages of any of an anti-CD27 mAb or antigen-binding portion thereof, any of an immunoconjugate, any of a bispecific molecule, or any of a pharmaceutical composition in the range of about 0.1 to about 20 mg / kg body weight; and (b) instructions for using the one or more dosages in any of a method for treating cancer or inhibiting tumor growth.

[0055] The present invention relates to a kit for treating a subject having cancer, the kit comprising: (a) one or more dosages of any of an anti-CD27 mAb or antigen-binding portion thereof, any of an immunoconjugate, any of a bispecific molecule, or any of a pharmaceutical composition, in the range of about 0.1 to about 20 mg / kg body weight; (b) one or more dosages of any of an Ab or antigen-binding portion thereof that specifically binds PD-1 or PD-L1, in the range of about 200 to about 1600 mg; and (c) instructions for administering said one or more dosages in any method of treatment or inhibition, the kit comprising a further therapeutic agent, wherein the further therapeutic agent is an antagonist Ab or antigen-binding portion thereof that specifically binds PD-1 or PD-L1.

[0056] Other features and advantages of the present invention will be apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all cited references, including scientific articles, GenBank entries, patents, and patent applications cited throughout this specification, are hereby expressly incorporated by reference. [Brief description of the drawings]

[0057] [Figure 1] Figure 1(A)-(B) show the binding affinity of Abs to human (A) and cynomolgus (B) T cells in flow cytometry analysis. The graphs show the mean fluorescence intensity (MFI) associated with nanomolar (nM) concentrations of BMS-986215 and 1F5 human anti-CD27 mAbs, along with a human IgG1 (hIgG1) control Ab in (A). "FL2" in (A) indicates measurements in fluorescence channel 2 (FL2). The graphs show that BMS-986215 has a higher affinity for human and cynomolgus CD27 than 1F5.

[0058] [Diagram 2]Figure 2(A)-(B) shows that BMS-986215 is a non-ligand blocking anti-CD27 mAb. (A) shows a description of a flow cytometry assay of human T cells pretreated with 10 μg / ml human CD70 (hCD70), a ligand for CD27, followed by treatment with ligand blocking (B) or non-blocking (NB) anti-CD27 Ab (aCD27). Ab binding in this assay was detected by a labeled anti-human IgG Ab (*, ahIgG). (B) shows a graph where the assay in (A) was performed with BMS-986215, ligand blocking 1F5 and human IgG1 (control) Abs. Binding of BMS-986215 was detected, indicating that CD70 binding to CD27 does not block BMS-986215 binding. Binding of ligand blocking 1F5 Ab was not detected. (C) shows a representation of a flow cytometry assay in which human T cells were exposed to the Abs described for (A) in the presence of 10 μg / ml soluble human CD70. CD70 is labeled anti-CD70 Ab (*, aCD70). (D) shows a graph of the assay of (C) performed with BMS-986215, ligand-blocking 1F5 and human IgG1 (control) Abs. CD70 remained detectable with increasing concentrations of BMS-986215, indicating that CD70 binding was not blocked by BMS-986215. CD70 binding was reduced with increasing concentrations of ligand-blocking 1F5 Ab. "MFI", "hIgG1" and "FL2" are as described for Figure 1. "FL1" in (B) indicates measurements in fluorescence channel 1 (FL1).

[0059] [Diagram 3]Figure 3 (A)-(C) show BMS-986215 epitope mapping. (A) shows sequence coverage of CD27 obtained by hydrogen / deuterium exchange mass spectrometry (HDX-MS) and fast photochemical oxidation of proteins (FPOP) epitope mapping methods. The sequence shown is the mature CD27 protein without the 20 amino acid signal peptide sequence of SEQ ID NO:1. Amino acids 1-172 in (A) correspond to amino acids 21-192 of SEQ ID NO:1. (B) shows HDX-MS data analysis showing that the N-terminal region of CD27 (corresponding to amino acids 21-41 of SEQ ID NO:1) showed significant protection by BMS-986215 binding. (C) shows the FPOP relative protection percentage of four peptides of CD27, with amino acids 32 to 37 of the mature protein (corresponding to amino acids 52 to 57 of SEQ ID NO:1) having the highest FPOP protection percentage (75%), indicating the binding epitope of CD27 through interaction with Fab.

[0060] [Figure 4] Figure 4(A)-(B) shows an illustration of the effect of ligand-blocking anti-CD27 Ab (A) and a non-ligand-blocking Ab, BMS-986215 (B), in terms of interaction with Fc gamma receptor (FcγR) and CD27 stimulation of T cells. CD27 costimulation by binding of T cells to its ligand CD70 occurred in the presence of BMS-986215 (B), but not in the presence of ligand-blocking Ab (A).

[0061] [Diagram 5] 5(A)-(C) show that BMS-986215 results in greater induction of NF-κB signaling compared to 1F5 in a primary T cell signaling assay. Graphs show NF-κB signal intensity based on 0, 15, 30 and 60 min treatment times with the indicated amounts (μg / ml) of BMS-986215 (A), 1F5 (B) and IgG1 control (C) Abs.

[0062] [Figure 6]Figure 6(A)-(B) shows that BMS-986215 results in greater induction of T cell proliferation and IFN-g secretion in co-cultures of CD4+ T cells and CHO-scCD3-CD32a cells (CHO cells expressing anti-CD3 OKT3 mAb and single chain anti-CD3 scFvAb derived from hCD32a-131His(FcγRIIa)) compared to 1F5. Co-cultures were treated with BMS-986215, 1F5 or hIgG1 (control) at the concentrations (nM) indicated on the graph. (A) shows T cell proliferation in counts per minute (CPM) and (B) shows the amount of IFN-gamma secreted by T cells after treatment in picograms per milliliter (pg / ml).

[0063] [Figure 7] Figure 7(A)-(D) shows that BMS-986215, compared with 1F5, induced greater T cell proliferation and IFN-γ secretion in cocultures of CHO-scCD3-CD32a cells with CD4+CD45RO+ memory T cells ((A)-(B)) or CD4+CD45RA+ naive T cells ((C)-(D)), with T cell proliferation, IFN-γ levels, and Abs as described in Figure 6.

[0064] [Figure 8] Figure 8 shows that BMS-986215 results in a mean fold induction of >2.5 in IFN-γ secretion (pg / ml) from CD4+CD25- T cells from different donors (donor 1 and donor 2) co-cultured with B cells and stimulated with various amounts (ng / ml) of Staphylococcal Enterotoxin B (SEB). Stimulated co-cultures were stimulated with BMS-986215 or anti-keyhole limpet hemocyanin Ab, g1F glycoform (KLH-g1f).

[0065] [Figure 9]Figures 9(A)-(E) show that interleukin-2 (IL-2) release was increased from SEB-stimulated human PBMCs in the presence of soluble cross-linked BMS-986215 compared to controls without BMS-986215. PBMCs from three donors (donors A, B and C) stimulated with SEB were treated with soluble anti-human IgG1 (hIgG1, control), anti-human OX40 (OX40.21), 1F5 or BMS-986215 Abs at the indicated concentrations (nM) in the presence (Figures 9(A) and (C)) or presence (Figures 9(B), (D) and (E)) of a cross-linker (anti-human Fcγ Ab). PBMCs without SEB or Ab treatment (cells only) and PBMCs treated with SEB but not Ab (SEB only) were controls.

[0066] [Figure 10] Figure 10 (A)-(F) shows that BMS-986215 acts synergistically with nivolumab and ipilimumab to improve IL-2 production upon T cell stimulation with SEB. PBMCs from two donors (donors A and B) treated with SEB were treated with soluble anti-human IgG1 (hIgG1, control), 1F5 or BMS-986215 Abs at the indicated concentrations (nM), alone (CD27) or together with nivolumab (Nivo combo) or ipilimumab (Ipi combo).

[0067] [Figure 11]Figure 11 (A)-(C) shows that BMS-986215 reverses regulatory T cell (Treg)-mediated suppression and induces expansion of responder T cells (Tresp). (A) shows the p-effect of BMS-986215 and IgG1 (control) Ab on Treg-mediated suppression of MDDC and CD4+ Tresp co-cultured in the presence of soluble anti-human CD3 mAb, OKT3. Treg:Tresp ratio is shown on the x-axis and percentage expansion of CD4+ Tresp is shown on the y-axis. BMS-986215 reversed the suppression by more than 70%. (B) and (C) show the number of Tresp cells (Foxp3- events in (B)) and the number of Foxp3+ Treg cells (Foxp3+ events in (C)) after culturing Tresp cells in wells coated with OKT3 and BMS-986215(.8), 1F5 or anti-KLH (KLH) Ab in the presence of soluble anti-CD28 for 7 days. (C) shows the number of cells in the CD45RA+ and CD45RO+ Treg populations.

[0068] [Figure 12] Figure 12b shows moderate induction of ADCC activity by BMS-986215. (A) and (B) show experiments in which BMS-986215, 1F5 or IgG1 isotype (control) Ab was added to co-cultures of primary natural killer (NK) cells as effector cells and activated T cells as target cells at an effector to target ratio of 10:1 (n=8).

[0069] [Figure 13]Figure 13 (A)-(D) show agonist activity by BMS-986215 in T cell activation in the absence of FcγR and enhancement by soluble human CD70 (shCD70). (A) shows illustration of the effect of ligand-blocking anti-CD27 Ab (1F5) and non-ligand blocking Ab (BMS-986215) in an assay in which human CD4+ T cells were co-cultured with CHO-OKT3 cells (CHO cells expressing single chain anti-CD3 scFvAb derived from anti-CD3 OKT3 mAb). (B)-(E) show graphs of the assays in (A) performed by treating co-cultures with BMS-986215, 1F5 or hIgG1 (control) Abs without ((B) and (C)) or with 10 μg / ml shCD70 ((D) and (E)) and assaying for T cell proliferation ((B) and (D)) or IFN-γ secretion ((C) and (E)). Figure 13(B) and (C) show that BMS-986215, but not 1F5, exhibits weak agonist activity by enhancing T cell proliferation (B) and IFN-γ secretion in the absence of FcγR and without shCD70. Figure 13(D) and (E) show that BMS-986215, but not 1F5, enhances human T cell activation (proliferation and IFN-γ secretion) by soluble CD70 protein.

[0070] [Figure 14]Figure 14 (A)-(F) shows that an anti-mouse CD27 mAb, clone 8H5, a surrogate for BMS-986215, enhances anti-PD-1 mAb activity in mouse tumor models. BALB / c mice subcutaneously implanted with 1x106 CT26 cells as a mouse colon cancer model were treated with 10mg / kg of a combination of control mouse IgG2a and control mouse IgG1-D265A isotype mAbs (A: control mIgG2a + control mIgG1, D265A); an anti-mouse CD27 (anti-mCD27) surrogate for BMS-986215, designated mAb 8H5 formulated as a mouse IgG1 isotype (B: anti-mCD27, 8H5, mIgG1); 8H5 formulated as a mouse IgG2a isotype (C: anti-mCD27, 8H5, mIgG2a); and an anti-mouse PD-1 mAb formulated as an Fc-inactive mouse IgG1-D265A isotype. Treatment was performed on days 6, 9, and 13 post-implantation with intraperitoneal injections of mAbs (D: anti-mPD-1, mIgG1, D265A); combination of anti-mPD-1, mIgG1, D265A and anti-mCD27, 8H5, mIgG1 (E); and combination of anti-mPD-1, mIgG1, D265A and anti-mCD27, 8H5, mIgG2a (F). CD27 agonism with anti-mCD27 mAbs in combination with PD-1 blockade improved tumor growth inhibition (TGI) and resulted in more tumor-free (TF) mice (Figure 14(E): 70% TGI, 3 / 10 TF, respectively) than with single agent treatment (Figure 14(B), anti-mCD27: 22% TGI, 0 / 10 TF; Figure 14(D), anti-mPD-1: 27% TGI, 2 / 10 TF). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0071] Detailed Description of the Invention The present invention relates to mAbs that specifically bind to CD27 and methods of treating cancer in a patient comprising administering to the patient an anti-CD27 Ab alone or in combination with an anti-cancer agent, such as an immune checkpoint inhibitor.

[0072] term In order that this disclosure may be more readily understood, certain terms are first defined. As used herein, unless otherwise expressly stated herein, each of the following terms has the meaning set forth below. Further definitions are set forth herein.

[0073] Where an embodiment is described herein using the term "comprising," other similar embodiments are also provided that are indicated using the terms "consisting of" and / or "consisting essentially of."

[0074] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those of ordinary skill in the art with a general dictionary of many of the terms used herein.

[0075] Units, prefixes, and symbols are in their International System of Units (SI) accepted form. Numerical ranges are inclusive of the numbers defining the range. The headings provided herein do not limit the disclosure of the various embodiments, which can be obtained by reference to this specification in its entirety. Thus, the terms defined immediately below are more fully defined by reference to this specification in its entirety.

[0076] "Administering" refers to the physical introduction of a therapeutic agent or a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. The preferred route of administration of therapeutic Abs, such as anti-CD27 Abs and anti-PD-1 Abs, is intravenous administration. Other routes of administration include, for example, intramuscular, subcutaneous, intraperitoneal or other parenteral routes of administration, such as by injection or infusion. The term "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration. Administration may also be performed, for example, once, multiple times and / or over one or more periods of time.

[0077] "Antibody" (Ab) refers to a glycoprotein immunoglobulin (Ig) or antigen-binding portion thereof that specifically binds an antigen and contains at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain contains a heavy chain variable region (herein referred to as V H The heavy chain constant region of an IgG Ab contains three constant domains, C H1 , C H2 and C H3 Each light chain comprises a light chain variable region (herein V L The light chain constant region of an IgG Ab contains one constant domain, C L Includes: V H and V L The regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L contains three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable regions of the heavy and light chains contain the binding domains that interact with antigens. Various methods have been used to delineate the CDR domains within Abs, including the Kabat, Chothia, AbM, contact and IMGT definitions. The constant regions of Abs may mediate the binding of Ig to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0078] Ig may be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art, including, but not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the Ab class or subclass (e.g., IgM, IgG1, or IgG4) encoded by the heavy chain constant region genes. The term "antibody" includes, by way of example, both naturally occurring and non-naturally occurring Abs, monoclonal and polyclonal Abs, chimeric and humanized Abs, human or non-human Abs, fully synthetic Abs, and single chain Abs. Non-human Abs may be partially or fully humanized by recombinant methods to reduce immunogenicity in humans. Unless otherwise specified and contrary to the context, the term "antibody" also includes any antigen-binding fragment or portion of said Ig, including monovalent and bivalent fragments or portions and single chain Abs.

[0079] An "isolated" Ab refers to an Ab that is substantially free of other Abs having different antigen specificities (e.g., an isolated Ab that specifically binds CD27 is substantially free of Abs that specifically bind antigens other than CD27). However, an isolated Ab that specifically binds hCD27 may have cross-reactivity with other antigens, such as CD27 polypeptides from different species, such as cynomolgus monkeys. Additionally, an isolated Ab may be purified to be substantially free of other cellular material and / or chemicals.

[0080] The term "monoclonal" Ab (mAb) refers to a non-naturally occurring preparation of Ab molecules of single molecular composition, i.e., Ab molecules that are essentially identical in primary sequence and display a single binding specificity and affinity for a particular epitope. A mAb is an example of an isolated Ab. mAbs may be produced by hybridoma, recombinant, transgenic, or other techniques known to those of skill in the art.

[0081] A "chimeric" Ab refers to an Ab in which the variable region is derived from one species and the constant region is derived from another species, such as an Ab in which the variable region is derived from a murine Ab and the constant region is derived from a human Ab.

[0082] "Human" mAb (HuMAb) refers to a mAb having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the Ab contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. Human Abs of the invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human" Ab is not intended to include Abs in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been grafted onto human framework sequences. The terms "human" Ab and "fully human" Ab are used interchangeably.

[0083] "Humanized" mAbs refer to mAbs in which some, most or all of the amino acids outside the CDR domains of a non-human mAb are replaced with the corresponding amino acids from a human immunoglobulin. In some embodiments of a humanized form of an Ab, some, most or all of the amino acids outside the CDR domains are replaced with amino acids from a human immunoglobulin, while some, most or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are tolerated as long as they do not abolish the Ab's ability to bind to a particular antigen. "Humanized" Abs retain antigen specificity similar to the original Ab.

[0084] An "anti-antigen" Ab refers to an Ab that specifically binds to the antigen. For example, an anti-CD27 Ab is an Ab that specifically binds to CD27, while an anti-PD-1 Ab is an Ab that specifically binds to PD-1. As used herein, an "anti-PD-1 / anti-PD-L1" Ab refers to an Ab used to interfere with the PD-1 / PD-L1 signaling pathway, which may be an anti-PD-1 Ab or an anti-PD-L1 Ab.

[0085] An "antigen-binding portion" (also called an "antigen-binding fragment") of an Ab refers to one or more fragments of an Ab that retain the ability to specifically bind to the antigen bound by the whole Ab.

[0086] "Cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells within the body. Uncontrolled cell division and proliferation leads to the formation of malignant tumors that can invade nearby tissues and even metastasize to distant sites in the body via the lymphatic system or bloodstream.

[0087] "CD27" refers to a receptor that is a member of the tumor necrosis factor receptor superfamily. CD27 is required for the generation and long-term maintenance of T cell immunity and binds to CD70. CD27 is constitutively expressed on most mature T cells, memory B cells and a portion of natural killer cells. The interaction of CD27 with its ligand CD70 plays an important role in the following processes: 1) CD27-mediated costimulation of T cells leads to activation, proliferation, survival and maturation of effector capacity and memory; 2) CD27-mediated costimulation of human B cells activates and promotes plasma cell production, proliferation and immunoglobulin production; and 3) CD27-mediated costimulation of natural killer cells induces cytolytic activity. As used herein, the term "CD27" includes hCD27, variants, isoforms and species homologs of hCD27 and analogs that share at least one common epitope with hCD27. The complete hCD27 sequence can be found under GenBank Accession No. AAH12160. CD27 expression is well documented in various types of lymphomas and leukemias, such as chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma, and marginal zone B-cell lymphoma.

[0088] "Cytotoxic T-lymphocyte antigen-4" (CTLA-4) refers to an immunoinhibitory receptor belonging to the CD28 family. CTLA-4 is expressed exclusively on T cells in vivo and binds two ligands, CD80 and CD86 (also called B7-1 and B7-2, respectively). As used herein, the term "CTLA-4" includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologs of hCTLA-4 and analogs that share at least one common epitope with hCTLA-4. The complete hCTLA-4 sequence can be found under GenBank Accession No. AAB59385.

[0089] The term "immunotherapy" refers to treating a subject having or at risk of having or relapsing from a disease by methods involving the induction, enhancement, suppression or other modification of the immune response.

[0090] "Treatment" or "treatment" of a subject refers to any type of intervention or process, including administration of an active agent, to a subject for the purpose of reversing, alleviating, ameliorating, arresting, slowing or preventing the onset, progression, development, severity or recurrence of a symptom, complication, condition or biochemical manifestation associated with a disease.

[0091] "Planned Death-1" (PD-1) refers to an immunoinhibitory receptor belonging to the CD28 family that is predominantly expressed on preactivated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1 and analogs that share at least one common epitope with hPD-1. The complete hPD-1 amino acid sequence can be found under GENBANK® Accession No. U64863.

[0092] "Planned death-ligand-1" (PD-L1) is one of two cell surface glycoprotein ligands for PD-1 (the other is PD-L2), which downregulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1 and analogs that share at least one epitope in common with hPD-L1. The complete hPD-L1 sequence can be found in GENBANK® Accession No. Q9NZQ7.

[0093] A "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates, such as non-human primates, sheep, dogs, and rodents, such as mice, rats, and guinea pigs. In a preferred embodiment, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0094] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of drug or agent that, when used alone or in combination with other therapeutic agents, protects a subject from disease onset or promotes disease regression, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention or reduction of functional or disability impairment due to disease morbidity. Furthermore, the terms "effective" and "efficacy" in relation to treatment include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote disease regression, e.g., cancer regression, in a patient. Physiological safety refers to an acceptable level of toxicity or other adverse physiological effects (adverse effects) at the cell, organ and / or organism level resulting from drug administration. The efficacy of a therapeutic agent can be evaluated using a variety of methods known to practitioners of ordinary skill, such as by assaying the activity of the drug in humans during clinical trials, in animal models predictive of efficacy in humans, or in in vitro assays.

[0095] As an example of tumor treatment, a therapeutically effective amount of an anticancer agent preferably inhibits cell proliferation or tumor growth by at least about 20%, preferably at least about 40%, more preferably at least about 60%, even more preferably at least about 80% and even more preferably at least about 100% relative to untreated subjects.In a preferred embodiment of the present invention, tumor regression can be observed and continued for a period of at least about 30 days, more preferably at least about 60 days or even more preferably at least about 6 months.Regardless of these final measurements of therapeutic effectiveness, the evaluation of immunotherapy drugs must subtract "immune-related" response patterns.

[0096] "Immune-related" response patterns refer to clinical response patterns frequently observed in cancer patients treated with immunotherapeutic agents that produce antitumor effects by inducing cancer-specific immune responses or modifying innate immune processes. This response pattern is characterized by an initial increase in tumor burden or the appearance of new lesions followed by a favorable therapeutic effect, which for traditional chemotherapeutic agents would be classified as disease progression and is synonymous with drug failure. Thus, proper evaluation of immunotherapeutic agents may require long-term monitoring of the effects of these agents on the target disease.

[0097] A therapeutically effective amount of a drug includes a "prophylactically effective amount," which is any amount of drug that, when administered alone or in combination with other therapeutic agents to a subject at risk of developing a disease (e.g., a subject with a pre-malignant condition at risk of developing cancer) or at risk of disease recurrence, prevents disease (e.g., cancer) onset or recurrence. In a preferred embodiment, a prophylactically effective amount completely prevents disease onset or recurrence. "Preventing" disease onset or recurrence means reducing the likelihood of disease onset or recurrence or completely preventing disease onset or recurrence.

[0098] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination of the alternatives. As used herein, the terms "a," "an," and "the" are understood to refer to "one or more" of any described or listed elements.

[0099] The term "and / or," as used herein, is to be construed as a specific disclosure of each of the two specified features or elements, with or without the other. Thus, the term "and / or" used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (single) and "B" (single). Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to include each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0100] The term "about" refers to a number, composition, or characteristic within an acceptable error range for a particular value, composition, or characteristic, as determined by one of ordinary skill in the art, which depends in part on how the value, composition, or characteristic is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation, as is customary in the art. Alternatively, it may mean within a range of ±20%, more commonly within a range of ±10%. When a particular value, composition, or characteristic is provided in the specification and claims, unless otherwise indicated, the meaning of "about" should be assumed as being within an acceptable error range for that particular value, composition, or characteristic. With respect to the frequency of administration of a drug administration regimen, the terms "about once a week," "about once every two weeks," or any other similar administration interval term, as used herein, refer to approximate numerical values. For example, "about once a week" may include every 7 days ±1 day, i.e., every 6 days to every 8 days. "About once every two weeks" includes every 14 days ±3 days, i.e., every 11 days to every 17 days. Similar approximations apply, for example, approximately once every three weeks, approximately once every four weeks, approximately once a month, or approximately once every three to six months or more.

[0101] The terms "substantially the same" or "essentially the same" refer to a sufficiently high degree of similarity between two or more values, compositions, or characteristics that one of skill in the art would consider the difference between those values, compositions, or characteristics to have little or no biological and / or statistical significance within the context of the properties being measured. The difference between the measured values ​​can be, for example, less than about 30%, preferably less than about 20%, and more preferably less than about 10%.

[0102] Any concentration range, percentage range, ratio range, or integer range described herein should be understood to include every integer value within the described range and, where appropriate, fractions thereof (e.g., 1 / 10 and 1 / 100 of an integer), unless otherwise specified.

[0103] Various aspects of the invention are described in further detail in the following subsections.

[0104] Anti-CD27 mAb In one aspect, the present invention relates to an isolated Ab, particularly a mAb or antigen-binding portion thereof, that specifically binds to hCD27. In one embodiment, the sequence of hCD27 is shown in SEQ ID NO:1.

[0105] Specific binding of anti-CD27 Ab to CD27 Anti-CD27 Abs, including the mAb of the present invention or antigen-binding portions thereof, specifically bind to CD27. Abs generally have a dissociation constant (K D ) specifically binds to its cognate antigen with high affinity as reflected by any K greater than about 100 μM. D As used herein, an IgG Ab that "specifically binds" to an antigen has a K of about 100 nM or less. D"Substantially identical" refers to an Ab that binds to an antigen with high affinity and substantially the same antigen, but does not bind to unrelated antigens with high affinity, meaning that it has a high degree of sequence identity with the antigen, for example, at least 80%, at least 90%, preferably at least 95%, more preferably at least 97% or even more preferably at least 99% sequence identity with the sequence of the antigen. In some embodiments, the Ab that specifically binds to hCD27 also cross-reacts with CD27 antigen from some primate species, such as cynomolgus monkeys. In some embodiments, the Ab that specifically binds to hCD27 does not cross-react with CD27 antigen or antigens other than CD27, such as Axl or PD-1 antigen, from rodent species, such as mouse and / or rat.

[0106] The term "K" used here D " is k off vs. k on Ratio (i.e., k off / k on ) and expressed in molar concentrations (e.g., nM). on " refers to the binding rate or "on-rate" of the binding of an Ab to its antigen interaction, while the term "k off " refers to the dissociation rate of the Ab-antigen complex. D Values ​​can be determined using methods well established in the art, such as surface plasmon resonance (SPR) or biolayer interferometry (BLI; ForteBio, Fremont, Calif.). K determined by different methods for one Ab D The values ​​can vary considerably, e.g., up to 1,000-fold. Therefore, the K D To compare these values, D It is important that values ​​are determined using the same methodology. Unless expressly stated and unless the context indicates otherwise, the K D Values ​​were determined by SPR using a BIACORE® biosensor system (GE Healthcare, Chicago, Ill.).

[0107] In certain embodiments of the presently disclosed invention, an anti-CD27 mAb, or antigen-binding portion thereof, is coupled to hCD27 at about 100 nM or less, about 95 nM or less, about 90 nM or less, about 85 nM or less, about 80 nM or less, about 75 nM or less, about 70 nM or less, about 65 nM or less, about 60 nM or less, about 55 nM or less, about 50 nM or less, about 45 nM or less, about 40 nM or less, about 1 nM to about 100 nM, about K of 1 nM to about 70 nM, about 1 nM to about 50 nM, about 1 nM to about 45 nM, about 5 nM to about 100 nM, about 5 nM to about 70 nM, about 5 nM to about 50 nM, about 5 nM to about 45 nM, about 10 nM to about 100 nM, about 10 nM to about 70 nM, about 10 nM to about 50 nM, about 10 nM to about 45 nM, or about 40 nM to about 45 nM. D In certain embodiments, the anti-CD27 mAb, or antigen-binding portion thereof, binds to hCD27 with a K of about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 1 nM to about 100 nM, about 10 nM to about 70 nM, about 10 nM to about 50 nM, or about 40 nM to about 45 nM. D In one embodiment, K D At about 37° C. and about 25° C., the K of the anti-CD27 mAb or antigen-binding portion thereof is about 10 nM to about 100 nM, about 10 nM to about 75 nM, about 10 nM to about 50 nM, or about 10 nM to about 45 nM at about 37° C. and about 25° C. D In certain embodiments, the anti-CD27 mAb, or antigen-binding portion thereof, binds to hCD27 at about 37° C. with a K of about 15 nM to about 100 nM, about 15 nM to about 75 nM, about 15 nM to about 50 nM, about 15 nM to about 45 nM, about 20 nM to about 100 nM, about 20 nM to about 75 nM, about 20 nM to about 50 nM, about 20 nM to about 45 nM, about 30 nM to about 100 nM, about 30 nM to about 75 nM, about 30 nM to about 50 nM, about 30 nM to about 45 nM, or about 40 nM to about 45 nM. DIn certain embodiments, the anti-CD27 mAb, or antigen-binding portion thereof, binds to hCD27 at about 25° C. with a K of about 45 nM or less, about 40 nM or less, about 35 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 1 nM to about 45 nM, about 1 nM to about 40 nM, about 1 nM to about 30 nM, about 1 nM to about 20 nM, about 5 nM to about 45 nM, about 5 nM to about 40 nM, about 5 nM to about 30 nM, about 5 nM to about 20 nM, about 10 nM to about 45 nM, about 10 nM to about 40 nM, about 10 nM to about 30 nM, or about 10 nM to about 20 nM. D In certain embodiments, the anti-CD27 mAb, or antigen-binding portion thereof, binds to hCD27 with a K of about 41 nM to about 44 nM at about 37° C. and / or about 13 nM to about 16 nM at about 25° C. D Combine with.

[0108] Binding of anti-CD27 Ab to specific epitopes A variety of methods can be used to probe the binding epitopes of Abs, including hydrogen / deuterium exchange mass spectrometry (HDX-MS) and fast photochemical oxidation of proteins (FPOP).

[0109] HDX-MS probes protein 3D structure and 3D dynamics in solution by monitoring the rate and extent of deuterium exchange of backbone amide hydrogen atoms (Huang and Chen, 2015; Wei et al., 2014). The level of HDX depends on the solvent affinity to backbone amide hydrogen atoms and protein hydrogen bonds. Protein mass gain due to HDX can be rigorously measured by MS. When this technique is coupled with enzymatic digestion, structural characteristics at the peptide level can be analyzed, allowing differentiation of surface-exposed peptides from the folded interior. Typically, deuterium labeling and subsequent quenching experiments are performed, followed by enzymatic digestion, peptide separation and MS analysis.

[0110] FPOP is a complementary protein footprinting technique for characterizing protein 3D structure by determining the level of oxidation of amino acid side chains induced by hydroxyl (OH) radicals in general (Yan et al., 2014; Jones et al., 2011). The degree of side chain oxidation depends on the solvent affinity of the amino acid side chains and the chemical properties of the exposed amino acids. FPOP is a novel method for photochemical labeling in flow systems using H 2 O 2 A laser irradiation of 1000 nm is used, and the radical lifetime is controlled to about 1 μs by the introduction of radical scavengers to avoid excessive labeling. The oxidized proteins are subsequently digested with enzymes and analyzed by MS. The change in peptide oxidation level under different conditions is used to obtain the changes in the local three-dimensional structure and to characterize the protein interface.

[0111] In one embodiment, an isolated Ab of the invention, comprising a mAb or an antigen-binding portion thereof, specifically binds to an epitope located within a discontinuous region spanning approximately amino acid residues 21-41 and 52-57 of hCD27 (the sequence of which is SEQ ID NO:1), as determined by HDX-MS and / or FPOP epitope mapping.

[0112] Structurally defined anti-CD27 Ab The present invention also relates to a V polypeptide that specifically binds to hCD27 and comprises consecutively linked amino acids having the sequence shown in SEQ ID NO:8. H and V comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:9. L Also provided is an isolated Ab, preferably a mAb or antigen-binding portion thereof, comprising CDR1, CDR2 and CDR3 domains, each of which is a CDR1, CDR2 and CDR3 domain.

[0113] Various methods have been developed to delineate the CDR domains within Abs. In addition to the widely used Kabat definition, others have been used, including the Chothia, AbNum, AbM, contact and IMGT definitions, which seek to address the shortcomings of the Kabat definition.

[0114] The efforts of Kabat and colleagues (Wu and Kabat, 1970; Kabat et al., 1983) were based on the assumption that the CDRs contain the most variable positions in Abs and could therefore be identified by alignment of the fairly limited number of Ab sequences available at the time. Based on this alignment, Kabat et al. introduced a numbering scheme for the residues in the hypervariable regions and determined which positions were the beginning and end of each CDR (http: / / bioinf.org.uk / abs / simkab.html).

[0115] The Chothia definition is based on the analysis of a small number of Ab structures to determine the correlation between the Ab sequence and the structural loop regions of its CDRs (Chothia et al., 1987; 1989; Al-Lazikani et al., 1997; http: / / bioinf.org.uk / abs / Chothia.html). The boundaries of FRs and CDRs were determined, and the latter were shown to adopt a restricted set of three-dimensional structures based on the presence of certain residues at key positions in the CDRs and flanking FRs. The resulting Chothia numbering scheme is nearly identical to the Kabat scheme, but structural considerations have led to the addition of a number of variants, V. L CDR1 and V HThe insertion in CDR1 has been placed at a different position. As more experimental data become available, the boundaries of the CDRs are being reanalyzed and redefined. Abhinandan and Martin (2008) analyzed Ab sequence alignments in a structural context and found that about 10% of the sequences in the manually assigned Kabat database contained errors or inconsistencies. They proposed a modified version of the Chothia scheme that was structurally corrected through the CDRs and frameworks, and developed a software tool (AbNum; available at http: / / www.bioinf.org.uk / abs / abnum / ) that applies Kabat, Chothia and modified Chothia numbering in an automated and robust manner. The other method, the AbM definition, represents a compromise between the Kabat and Chothia definitions and is used by the Oxford Molecular Group's AbM Ab modeling software (http: / / www.bioinf.org.uk / abs; Martin et al., 1989).

[0116] Contact definitions were based on analysis of Ab-antigen contacts in complex crystal structures available in the Protein Data Bank ( http: / / bioinf.org.uk / abs / ; MacCallum et al., 1996 ).

[0117] A more recent attempt to define CDRs is that of the IMGT database (Lefranc et al. (2003; http: / / www.imgt.org), which organizes nucleotide sequence information for Ig, T cell receptor (TcR) and major histocompatibility complex (MHC) molecules. Based on the alignment of over 5000 Ig and TcR variable region sequences, it proposes a uniform numbering system for Ig and TcR sequences.

[0118] The Kabat definition is the most commonly used method for predicting CDR domains, even though it was developed at a time when structural information of Abs was not available. Unless expressly stated and the context indicates otherwise, the CDRs disclosed herein have been identified using the Kabat definition (see Table 2). Accordingly, the present invention provides an isolated Ab, preferably a mAb or antigen-binding portion thereof, that specifically binds hCD27, comprising a set of six CDRs, at least one of which corresponds to a CDR sequence shown in Table 2. In one embodiment ... all three heavy chain variable regions (V H In one embodiment, the present invention provides an isolated Ab, preferably a mAb or antigen-binding portion thereof, that specifically binds hCD27, comprising all three light chain variable regions (V) shown in Table 2. L The present invention provides an isolated Ab, preferably a mAb or antigen-binding portion thereof, that specifically binds to hCD27, comprising the CDR sequences of

[0119] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: (a) a VD comprising a CDR1 having consecutively linked amino acids having the sequence shown in SEQ ID NO:2, a CDR2 having consecutively linked amino acids having the sequence shown in SEQ ID NO:3, and a CDR3 having consecutively linked amino acids having the sequence shown in SEQ ID NO:4; H ; and (b) a VDR1 comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:5, a CDR2 comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:6, and a CDR3 comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:7; L The present invention provides an isolated Ab, preferably a mAb or antigen-binding portion thereof, that specifically binds to hCD27, comprising:

[0120] In one embodiment, V H comprises consecutively linked amino acids having the sequence shown in SEQ ID NO:8.

[0121] In one embodiment, V Lcomprises consecutively linked amino acids having the sequence shown in SEQ ID NO:9.

[0122] In one embodiment, the Ab, preferably the mAb, comprises a heavy chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:12 and a light chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:13.

[0123] In certain embodiments, the Ab is the mAb designated BMS-986215.

[0124] V having an amino acid sequence highly similar or homologous to any of the amino acid sequences of the above anti-CD27 Abs. H and V L Anti-CD27 Abs that contain regions and retain the functional properties of these Abs are also suitable for use in the present methods. For example, suitable Abs include those comprising contiguously linked amino acids, each having a sequence that is at least 80% identical to the amino acid sequences set forth in SEQ ID NOs: 8 and / or 9, respectively. H and V L In further embodiments, the mAb includes a region, e.g., V H and / or V L The amino acid sequence exhibits at least 85%, preferably at least 90%, more preferably at least 95% or 99% identity with the sequence shown in SEQ ID NO: 8 and / or 9, respectively. As used herein, the percent sequence identity between two amino acid sequences is a function of the number of identical positions shared by these sequences over the length of the compared sequences (i.e., % identity = number of identical positions / total number of positions compared x 100), taking into account any number of gaps introduced to maximize the degree of sequence identity between the two sequences and the length of each such gap. Sequence comparison and percent identity determination between two sequences can be accomplished using mathematical algorithms well known to those skilled in the art.

[0125] In some embodiments, the isolated anti-CD27 Ab or antigen-binding portion thereof comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 isotype. In some preferred embodiments, the isolated anti-CD27 Ab or antigen-binding portion thereof is of human IgG1 isotype. In other embodiments, the isolated anti-CD27 Ab or antigen-binding portion thereof is of human IgG4 isotype. In some embodiments, the isolated anti-CD27 Ab is a full-length Ab of IgG1, IgG2, IgG3 or IgG4 isotype. In some preferred embodiments, the full-length Ab is of IgG1 isotype. In further embodiments, the full-length Ab is of IgG4 isotype.

[0126] Anti-CD27 mAb that cross-competes with a control Ab for binding to CD27 Also included within the scope of the present invention is an isolated Ab, preferably a mAb or an antigen-binding portion thereof, that specifically binds to CD27, e.g., hCD27 and / or cynomolgus CD27 (cCD27), and cross-competes with a control Ab or a control antigen-binding portion thereof for binding to hCD27. The ability of an Ab pair to "cross-compete" for binding to an antigen, e.g., CD27, indicates that a first Ab binds to substantially the same epitopic region of the antigen as a second Ab and sterically blocks the binding of the second Ab at that particular epitopic region, and conversely, that a second Ab binds to substantially the same epitopic region of the antigen as the first Ab and sterically blocks the binding of the first Ab at that particular epitopic region. Thus, the ability of a test Ab to competitively inhibit the binding of, e.g., mAb BMS-986215 to hCD27 indicates that the test Ab binds to substantially the same epitopic region of mAb BMS-986215 and hCD27.

[0127] A first Ab is considered to bind "substantially the same epitope" as a second Ab if the first Ab reduces the binding of the second Ab to the antigen by at least about 40%. Preferably, the first Ab reduces the binding of the second Ab to the antigen by more than about 50% (e.g., at least about 60% or at least about 70%). In a more preferred embodiment, the first Ab reduces the binding of the second Ab to the antigen by more than about 70% (e.g., at least about 80%, at least about 90% or about 100%). The order of the first and second Abs can be reversed, i.e., the "second" Ab can be bound to the surface first and the "first" can be contacted with the surface in the presence of the "second" Ab. The Abs are considered to "cross-compete" if a competitive reduction in binding to the antigen is observed regardless of the order in which the Abs are added to the immobilized antigen.

[0128] Cross-competing Abs bind to substantially the same epitope region of an antigen, such as the CD27 receptor, and are therefore predicted to have functional properties very similar to those of a control Ab. The higher the degree of cross-competition, the more similar the functional properties will be. For example, two cross-competing Abs are predicted to have essentially the same functional properties if each inhibits binding to the epitope of the other by at least about 80%. This similarity in function is predicted by the fact that cross-competing Abs have similar dissociation constants (K D ) are predicted to be closer if their affinity for binding to the epitope is similar.

[0129] Cross-competitive anti-antigen Abs can be easily identified based on their ability to detectably compete in standard antigen binding assays, including BIACORE® analysis, ELISA assays, or flow cytometry, using recombinant or cell surface expressed antigen molecules. As an example, a simple competition assay to identify whether a test Ab competes with BMS-986215 for binding to hCD27 can include (1) measuring the binding of BMS-986215 applied at saturating concentrations to a BIACORE® chip (or other suitable medium for SPR analysis) on which hCD27 is immobilized, and (2) measuring the binding of BMS-986215 to a hCD27-coated BIACORE® chip (or other suitable medium) on which the test Ab is pre-bound. The binding of BMS-986215 to the CD27-coated surface in the presence and absence of the test Ab is compared. A significant (e.g., greater than about 40%) decrease in binding of BMS-986215 in the presence of the test Ab indicates that both Abs recognize substantially the same epitope such that they compete for binding to the CD27 target. The percentage of binding of the first Ab to the antigen that is inhibited by the second Ab is calculated as [1-(detection binding value of the first Ab in the presence of the second Ab) / (detection binding value of the first Ab in the absence of the second Ab)] x 100. To determine whether multiple Abs cross-compete, the competitive binding assay is repeated, except for binding of the test Ab to the CD27-coated chip in the presence of BMS-986215.

[0130] In one embodiment, the present invention relates to isolated Abs, particularly mAbs or antigen-binding portions thereof, that specifically bind to the same epitope of hCD27 as any of the anti-CD27 Abs disclosed herein.

[0131] Characteristics of anti-CD27 mAb In certain embodiments, the isolated Ab of the invention, including a mAb or an antigen-binding portion thereof, has one or more characteristics described herein.

[0132] In certain embodiments, the isolated Ab of the invention, including the mAb or antigen-binding portion thereof, is an hCD27 agonist.

[0133] In certain embodiments, an isolated Ab of the invention, including a mAb or antigen-binding portion thereof, specifically binds to CD27, e.g., hCD27, and does not block its binding to a CD70 ligand (i.e., does not inhibit CD70 binding to CD27, as measured by surface plasmon resonance (SPR) or flow cytometry). Such mAbs or antigen-binding portions thereof may be referred to herein interchangeably as non-ligand blocking mAbs or antigen-binding portions thereof, non-ligand blocking mAbs or antigen-binding portions thereof, or ligand non-blockers.

[0134] In certain embodiments, the isolated Ab of the invention, including the mAb or antigen-binding portion thereof, does not specifically bind to rat CD27 and / or mouse CD27 as measured by SPR or flow cytometry.

[0135] In certain embodiments, the isolated Ab of the invention, including the mAb or antigen-binding portion thereof, does not specifically bind to one or more TNF receptor superfamily members selected from the group consisting of CD30, HVEM, DR5, 4-1BB, CD40, OX40, GITR, and any combination thereof.

[0136] In certain embodiments, the isolated Ab of the invention, including the mAb or antigen-binding portion thereof, does not specifically bind to one or more human tissues at a concentration of up to 10 μg / mL, wherein the human tissue is selected from the group consisting of thyroid, lung, skin, uterus, prostate, liver, kidney, pancreas, adrenal gland, pituitary gland, placenta, testis, cerebrum, cerebellum, heart, peripheral nerve, and any combination thereof.

[0137] In one embodiment, the isolated Ab of the present invention, including the mAb or antigen-binding portion thereof, induces NF-κB and MAPK signaling in naive and pre-activated human T cells stimulated with anti-CD3 Ab and anti-CD28 Ab. In one embodiment, the isolated Ab of the present invention, including the mAb or antigen-binding portion thereof, induces NF-κB and MAPK signaling in naive and pre-activated human T cells stimulated with anti-CD3 Ab and anti-CD28 Ab. Inhibits NF-κB and / or MAPK signaling in naive and pre-activated human T cells stimulated with Ab at about 0.5 nM or less, about 0.45 nM or less, about 0.4 nM or less, about 0.35 nM or less, about 0.3 nM or less, about 0.25 nM or less, about 0.2 nM or less, about 0.005 nM to about 0.5 nM, about 0.005 nM to about 0.4 nM, about 0.005 nM to about 0.3 nM, about 0.005 nM to about 0.25 nM, about 0.005 nM to about 0.2 nM, about 0.01 nM to about 0.5 ... M ~ about 0.4nM, about 0.01nM to about 0.3nM, about 0.01nM to about 0.25nM, about 0.01nM to about 0.2nM, about 0.015nM to about 0.5nM, about 0.015nM to about 0.4nM, about 0.015nM to about 0.3nM, about 0.01 EC of 5nM to about 0.25nM, about 0.015nM to about 0.2nM, about 0.02nM to about 0.5nM, about 0.02nM to about 0.4nM, about 0.02nM to about 0.3nM, about 0.02nM to about 0.25nM or about 0.02nM to about 0.23nM 50 In certain embodiments, the isolated Ab of the invention, including the mAb or antigen-binding portion thereof, induces NF-κB and MAPK signaling with an EC of about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, about 0.005 nM to about 0.5 nM, about 0.01 nM to about 0.4 nM, or about 0.015 to about 0.3 nM. 50 Guide with.

[0138] In certain embodiments, the isolated Ab of the present invention, comprising a mAb or an antigen-binding portion thereof, induces proliferation and / or IFN-γ secretion in a CHO-svCD3-CD32A assay. In certain embodiments, the isolated Ab of the present invention, comprising a mAb or an antigen-binding portion thereof, induces proliferation and / or IFN-γ secretion in a CHO-svCD3-CD32A assay at about 0.05 nM or less, about 0.045 nM or less, about 0.04 nM or less, about 0.035 nM or less, about 0.03 nM or less, about 0.025 nM or less, about 0.02 nM or less, about 0.015 nM or less, about 0.01 nM or less, about 0.009 nM or less, about 0.008 nM or less, about 0.0005 nM to about 0.05 nM, about 0.0005 nM to about EC of 0.04 nM, about 0.0005 nM to about 0.03 nM, about 0.0005 nM to about 0.02 nM, about 0.0005 nM to about 0.01 nM, about 0.0005 nM to about 0.009 nM, about 0.0005 nM to about 0.008 nM, about 0.001 nM to about 0.05 nM, about 0.001 nM to about 0.04 nM, about 0.001 nM to about 0.03 nM, about 0.001 nM to about 0.02 nM, about 0.001 nM to about 0.01 nM, about 0.001 nM to about 0.009 nM, or about 0.001 nM to about 0.008 nM 50 In certain embodiments, the isolated Ab of the invention comprising the mAb or antigen-binding portion thereof induces proliferation and / or IFN-γ secretion in a CHO-svCD3-CD32A assay with an EC of about 0.05 nM or less, about 0.04 nM or less, about 0.03 nM or less, about 0.025 nM or less, about 0.0005 nM to about 0.05 nM, about 0.0005 nM to about 0.04 nM, about 0.0005 nM to about 0.03 nM, about 0.001 nM to about 0.05 nM, about 0.001 nM to about 0.04 nM, or about 0.001 nM to about 0.03 nM. 50 In one embodiment, an isolated Ab of the invention, including a mAb or an antigen-binding portion thereof, induces proliferation in a CHO-svCD3-CD32A assay with an average EC 50 In one embodiment, the isolated Ab of the invention, including the mAb or antigen-binding portion thereof, induces IFN-γ secretion in a CHO-svCD3-CD32A assay with an average EC 50 Guide with.

[0139] In one embodiment, the isolated Ab of the present invention, comprising a mAb or an antigen-binding portion thereof, is a CD4 + CD45RO + In one embodiment, the isolated Ab of the present invention, including the mAb or antigen-binding portion thereof, inhibits the proliferation of memory T cells. + CD45RO + Inhibit memory T cell proliferation with an EC of about 0.01 nM or less, about 0.009 nM or less, about 0.008 nM or less, about 0.007 nM or less, about 0.006 nM or less, about 0.005 nM or less, about 0.001 nM to about 0.01 nM, about 0.002 nM to about 0.008 nM, about 0.003 nM to about 0.007 nM, or about 0.004 nM to about 0.006 nM. 50 Guide with.

[0140] In one embodiment, the isolated Ab of the present invention, comprising a mAb or an antigen-binding portion thereof, is a CD4 + CD45RO + In one embodiment, the isolated Ab of the present invention, including the mAb or antigen-binding portion thereof, induces IFN-γ secretion in memory T cells. + CD45RO + Reduce IFN-γ secretion in memory T cells to about 0.05 nM or less, about 0.04 nM or less, about 0.03 nM or less, about 0.02 nM or less, about 0.005 nM to about 0.05 nM, about 0.005 nM to about 0.04 nM, about 0.00 EC of 5nM to about 0.03nM, about 0.005nM to about 0.02nM, about 0.01nM to about 0.05nM, about 0.01nM to about 0.04nM, about 0.01nM to about 0.03nM or about 0.01nM to about 0.02nM 50 Guide with.

[0141] In certain embodiments, the isolated Ab of the present invention, including the mAb or antigen-binding portion thereof, increases IL-2 release from Staphylococcal Enterotoxin B (SEB)-stimulated human PBMCs. In certain embodiments, the isolated Ab of the present invention, including the mAb or antigen-binding portion thereof, increases IL-2 release from SEB-stimulated human PBMCs by more than about 50% in the presence of a crosslinker. In certain embodiments, the percentage increase in IL-2 release is compared to a negative control IgG1 mAb (e.g., a negative control hIgG1 mAb). In certain embodiments, the fold increase is at an Ab concentration of about 1 nM to about 10 nM.

[0142] In certain embodiments, an isolated Ab of the invention, comprising a mAb or antigen-binding portion thereof, when combined with an anti-PD-1 Ab, increases IL-2 release by at least about 2-fold, in certain embodiments, the fold increase is compared to the mAb or antigen-binding portion thereof in the absence of the anti-PD-1 Ab.

[0143] In one embodiment, the isolated Ab of the present invention, including the mAb or an antigen-binding portion thereof, is administered to MDDCs and to CD4 + In one embodiment, the isolated Ab of the present invention, including the mAb or an antigen-binding portion thereof, reverses Treg-mediated suppression of responder T cells. + reverses Treg-mediated suppression of responder T cells by at least about 70%. In certain embodiments, the percentage of reversal of Treg-mediated suppression is compared to a negative control IgG1 mAb (e.g., a negative control hIgG1 mAb).

[0144] In certain embodiments, the isolated Ab of the present invention, comprising a mAb or an antigen-binding portion thereof, enhances the induction of human T cell proliferation and IFN-γ secretion by soluble CD70. In certain embodiments, the isolated Ab of the present invention, comprising a mAb or an antigen-binding portion thereof, enhances the induction of human T cell proliferation and IFN-γ secretion by soluble CD70 with an EC of about 0.01 nM or less, about 0.009 nM or less, about 0.008 nM or less, about 0.007 nM or less, about 0.006 nM or less, about 0.005 nM or less, about 0.001 nM to about 0.01 nM, about 0.002 nM to about 0.008 nM, or about 0.003 nM to about 0.005 nM. 50 and / or enhance the induction of IFN-γ secretion with an EC of about 0.01 nM or less, about 0.009 nM or less, about 0.008 nM or less, about 0.007 nM or less, about 0.001 nM to about 0.01 nM, about 0.002 nM to about 0.008 nM, or about 0.005 nM to about 0.007 nM. 50 Enhance with.

[0145] In certain embodiments, the isolated Ab of the present invention, comprising a mAb or an antigen-binding portion thereof, has different characteristics compared to a negative control Ab and / or a different anti-CD27 Ab. In certain embodiments, the negative control Ab is a hIgG1 Ab. In certain embodiments, the different anti-CD27 Ab is 1F5, an anti-hCD27 having heavy and light chain sequences as shown by SEQ ID NOs: 20 and 21, respectively, as shown in Table 5. In certain embodiments, the isolated Ab of the present invention, comprising a mAb or an antigen-binding portion thereof, exhibits one or more significantly superior and / or therapeutically advantageous characteristics compared to a known anti-CD27 Ab. For example, in certain embodiments, the isolated Ab of the present invention, comprising a mAb or an antigen-binding portion thereof, exhibits one or more significantly superior and / or therapeutically advantageous characteristics compared to a negative control Ab and / or a different anti-CD27 Ab: D or EC 50are hCD27 and / or cCD27 agonists; are ligand non-blockers; do not specifically bind to rat CD27 and / or mouse CD27 as measured by SPR or flow cytometry; do not specifically bind to one or more TNF receptor superfamily members selected from the group consisting of CD30, HVEM, DR5, 4-1BB, CD40, OX40, GITR, and any combination thereof; do not specifically bind to one or more human tissues at a concentration of up to 10 μg / mL, wherein the human tissue is selected from the group consisting of thyroid, lung, skin, uterus, prostate, liver, kidney, pancreas, adrenal gland, pituitary, placenta, testis, cerebrum, cerebellum, heart, peripheral nerve, and any combination thereof; anti-CD3 Ab and anti-CD28 High induction of NF-κB and MAPK signaling in Ab-stimulated naive and preactivated human T cells; high induction of proliferation and / or IFN-γ secretion in CHO-svCD3-CD32A assays; CD4 + CD45RO + Provides high induction of memory T cell proliferation; CD4 + CD45RO + Produces a high induction of IFN-γ secretion in memory T cells; produces a high increase in IL-2 release from Staphylococcal enterotoxin B (SEB)-stimulated human PBMCs; co-cultured CD4 in the presence of MDDC and soluble OKT3 + providing high reversal of Treg-mediated suppression of responder T cells; enhancing or highly enhancing the induction of human T cell proliferation and IFN-γ secretion by soluble CD70; having high efficacy in inhibiting tumor cell and / or tumor growth in vitro and / or in vivo; and any combination thereof.

[0146] Binding of anti-CD27 Ab to T cells In certain embodiments, the isolated Ab of the invention comprising the mAb or antigen-binding portion thereof has an EC50 of about 0.1 nM or less, about 0.09 nM or less, about 0.08 nM or less, about 0.07 nM or less, about 0.06 nM or less, about 0.05 nM or less, about 0.04 nM or less, about 0.01 nM to about 0.1 nM, about 0.015 nM to about 0.09 nM, or about 0.02 nM to about 0.08 nM. 50 Combine with.

[0147] In certain embodiments, the isolated Ab of the invention comprising the mAb or antigen-binding portion thereof has an EC50 of about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, about 0.2 nM or less, about 0.1 nM or less, about 0.01 nM to about 0.5 nM, about 0.02 nM to about 0.4 nM, or about 0.03 to about 0.3 nM with cynomolgus T cells. 50 Combine with.

[0148] Functional antigen-binding portion of anti-CD27 Ab The anti-CD27 Abs provided by the present invention include antigen-binding fragments in addition to full-length Abs. It has been well-documented that the antigen-binding function of an Ab can be performed by a fragment of a full-length Ab. Examples of binding fragments encompassed within the term "antigen-binding portion" of an Ab include: (i) V L , V H , C L and C H1 (ii) a bivalent fragment, F(ab'), consisting of two Fab fragments linked by a disulfide bridge at the hinge region. 2 Fragment; (iii) V H and C H1 (iv) a single-arm V of an Ab; L and V H(v) single domain Abs (sdAbs) or nanobodies consisting of a single monomeric variable domain of an Ab. Conventional Abs, camelids such as camels, alpacas and llamas, and cartilaginous fish such as sharks and rays, contain a subset of heavy chain Abs (hcAbs) consisting of a heavy chain homodimer containing three CDRs and lacking light chains. The first sdAbs were originally derived from camelids (V H H fragment) or cartilaginous fish (V NAR They were engineered from hcAbs found in the IgG1Ab (HcAb fragments) but can also be produced by splitting the dimeric variable domains from conventional Abs. In addition to heavy chain variable domain derived sdAbs, light chain derived nanobodies have also been shown to selectively bind to specific antigens.

[0149] Ab fragments, initially obtained by proteolysis with enzymes such as papain and pepsin, have subsequently been engineered into monovalent and multivalent antigen-binding fragments. For example, the two domains of the Fv fragment, the V L and V H may be encoded by separate genes, but may be joined using recombinant methods with a synthetic linker peptide to form a monovalent molecule known as a single chain variable fragment (scFv). L and V H Bivalent or bivalent cFvs (di-scFv or bi-scFv) are formed by combining two V H Area and two V L ScFv dimers and higher order multimers can be produced using a linker peptide of less than 10 amino acids that is too short to allow the two variable regions to fold together, allowing the scFvs to dimerize and produce bispecific antibodies or form other multimers. Bispecific antibodies have been shown to bind their cognate antigens with higher affinity than the corresponding scFvs, and the K DThe dissociation constants are up to 40-fold lower than the values ​​of the α-linker. Ultrashort linkers (≦3 amino acids) result in the formation of trivalent triabodies or tetravalent tetrabodies, which exhibit even higher affinity for antigens than bispecific antibodies. Other variants include scFv-C H3 Dimeric minibodies and large scFv-Fc fragments (scFv-C H2 -C H3 dimers) and isolated CDRs may exhibit antigen-binding function. These Ab fragments are engineered using conventional recombinant techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact Abs. All of the above proteolytic and engineered fragments and related variants of Abs (see Hollinger and Hudson, 2005; Olafsen and Wu, 2010 for further details) are intended to be included within the term "antigen-binding portion" of an Ab.

[0150] In certain embodiments of the invention, the antigen-binding portion of an isolated anti-CD27 Ab is an Ab fragment or a single chain Ab. In certain embodiments, an Ab fragment is a Fab, F(ab') 2 , Fd and Fv fragments, sdAbs, single chain variable fragments (scFv), bivalent scFv (di-scFv) and bivalent cFv (bi-scFv), bispecific antibodies, minibodies and CDRs. In a preferred embodiment, the Ab fragment is selected from Fab, F(ab') 2 , Fd and Fv fragments and single chain variable fragments (scFv).

[0151] In one embodiment, the isolated anti-CD27 Ab or antigen-binding portion thereof is a human Ab or fragment thereof. In another embodiment, it is a humanized Ab or fragment thereof. In a further embodiment, it is a chimeric Ab or fragment thereof. In another embodiment, it is a mouse Ab or fragment thereof. For administration to a human subject, the Ab is preferably a chimeric Ab or, more preferably, a humanized or human Ab. Such chimeric, humanized, human or mouse mAbs can be produced and isolated by methods well known in the art.

[0152] Anti-CD27 immunoconjugates In another embodiment, the present invention relates to any of the isolated anti-CD27 Abs disclosed herein or antigen-binding portions thereof linked to a therapeutic agent such as a cytotoxin or a radioisotope. Such conjugates are referred to herein as "immunoconjugates." Cytotoxins can be conjugated to the Abs of the present invention using linker technology available in the art. Methods for producing radioimmunoconjugates are also established in the art.

[0153] bispecific molecule In another embodiment, the invention relates to a bispecific molecule comprising any of the isolated anti-CD27 Abs disclosed herein, or antigen-binding portions thereof, linked to a binding domain having a different binding specificity than the anti-CD27 mAb, or antigen-binding portion thereof. The binding domain can be a functional molecule, e.g., another Ab, an antigen-binding portion of an Ab, or a ligand for a receptor, such that the bispecific molecule produced binds to at least two different binding sites or target molecules.

[0154] Nucleic acids encoding anti-CD27 Abs and uses for Ab expression Another aspect of the invention relates to nucleic acids encoding isolated anti-CD27 Abs of the invention. The invention provides isolated nucleic acids encoding any of the CD27 Abs or antigen-binding portions thereof described herein. An "isolated" nucleic acid refers to a nucleic acid composition that is significantly different from naturally occurring nucleic acids, i.e., has a unique chemical identity, properties and utility. For example, isolated DNA, unlike naturally occurring DNA, is a free-standing piece of naturally occurring DNA and is not an essential part of the larger structural complexes, chromosomes, found in nature. Furthermore, isolated DNA, unlike naturally occurring DNA, can be used as a PCR primer or hybridization probe to, among other things, measure gene expression and measure and detect biomarker genes or mutations for disease diagnosis or treatment efficacy prediction. Isolated nucleic acids can also be purified to be substantially free of other cellular elements or other contaminants, e.g., other cellular nucleic acids or proteins, using standard techniques well known in the art.

[0155] Nucleic acids of the invention can be obtained using standard molecular biology techniques. For Ab expression by hybridomas (e.g., hybridomas prepared from transgenic mice carrying the human Ig genes described in Example 1), cDNAs encoding the light and heavy chains or variable regions of the Abs produced by the hybridomas can be obtained by standard PCR amplification techniques. H and V L Once the DNA fragments encoding the segments are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, for example, to convert the variable region DNA into full-length Ab chain genes, Fab fragment genes, or into scFv genes. For Abs obtained from an Ig gene library (e.g., using phage display techniques), nucleic acid encoding the Ab can be recovered from the library.

[0156] The nucleic acid of the invention can be, for example, RNA or DNA, such as cDNA or genomic DNA. In a preferred embodiment, the nucleic acid is cDNA.

[0157] The present invention also provides an expression vector comprising an isolated nucleic acid encoding an anti-CD27 Ab or an antigen-binding portion thereof. The present invention further provides a host cell comprising said expression vector. Eukaryotic cells, and most preferably mammalian host cells, are preferred as expression host cells because Abs expressed in such eukaryotic cells, and particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded, immunologically active Abs. Preferred mammalian host cells for expression of the recombinant Abs of the present invention include Chinese hamster ovary (CHO) cells (Kaufman and Sharp, 1982), NSO myeloma cells, COS cells, and SP2 cells.

[0158] The host cells can be used in a method for producing an anti-CD27 mAb or antigen-binding portion thereof, which method includes expressing the mAb or antigen-binding portion thereof in the host cell and isolating the mAb or antigen-binding portion thereof from the host cell. The host cells can be used ex vivo or in vivo. The DNA encoding the Ab heavy and light chains can be introduced into separate expression vectors, or more commonly, both are introduced into the same vector. The V of the Ab can be expressed by the following methods: H and V L Segment, V H The segment is C in the vector H operably coupled to the V segment; κ The segment is C in the vector L They can be used to produce full-length Abs of any isotype by inserting DNA encoding these variable regions into an expression vector that already encodes heavy and light chain constant regions of the desired isotype such that they are operably linked to the segments.

[0159] Another aspect of the invention relates to a transgenic mouse comprising human Ig heavy and light chain transgenes, wherein the mouse expresses any of the anti-CD27 HuMAbs described herein. The invention also encompasses hybridomas prepared from the mouse, wherein the hybridomas produce a HuMAb.

[0160] Anti-PD-1 / anti-PD-L1 Abs suitable for use in the therapeutic methods of the invention Anti-PD-1 Abs suitable for use in the cancer treatment methods, compositions or kits disclosed herein include isolated Abs, preferably mAbs or antigen-binding portions thereof, that bind to PD-1 with high specificity and affinity, block the binding of PD-L1 and / or PD-L2 to PD-1, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. Similarly, anti-PD-L1 suitable for use in these methods include isolated Abs, preferably mAbs or antigen-binding portions thereof, that bind to PD-L1 with high specificity and affinity, block the binding of PD-L1 to PD-1 and CD80 (B7-1), and inhibit the immunosuppressive effects of the PD-1 signaling pathway. In any of the therapeutic methods disclosed herein, the anti-PD-1 or anti-PD-L1 Abs include antigen-binding portions or fragments that bind to the PD-1 receptor or PD-L1 ligand, respectively, and exhibit similar functional properties for receptor-ligand binding to the whole Ab, reversing the inhibition of T cell activity, thereby upregulating the immune response.

[0161] Anti-PD-1 Ab mAbs that specifically bind PD-1 with high affinity are disclosed in U.S. Patent 8,008,449. Other anti-PD-1 mAbs are disclosed, for example, in U.S. Patents 7,488,802, 8,168,757, 8,354,509, and 9,205,148. The anti-PD-1 mAbs disclosed in U.S. Patent 8,008,449 have been shown to have some or all of the following characteristics: (a) a K of about 50 nM or less, as determined by an SPR (BIACORE®) biosensor system; D (b) does not substantially bind to human CD28, CTLA-4, or ICOS; (c) increases T cell proliferation, interferon-γ production, and IL-2 secretion in a mixed lymphocyte reaction (MLR) assay; (d) binds to human PD-1 and cynomolgus PD-1; (e) inhibits the binding of PD-L1 and PD-L2 to PD-1; (f) inhibits the binding of CD4 + CD25 -(g) relieve T cell proliferation and interferon-γ production from inhibition by Treg cells; (h) stimulate an antigen-specific memory response; and (i) inhibit tumor cell proliferation in vivo. Anti-PD-1 Abs useful in the treatment methods, compositions or kits of the invention include mAbs that specifically bind human PD-1 with high affinity and exhibit at least five, and preferably all, of the above characteristics. For example, anti-PD-1 Abs suitable for use in the therapeutic methods disclosed herein have (a) a K of about 10 nM to 0.1 nM as determined by SPR (BIACORE®). D (b) binds to human PD-1 in an MLR assay; (c) increases T cell proliferation, interferon-γ production, and IL-2 secretion; (d) inhibits the binding of PD-L1 and PD-L2 to PD-1; and (e) inhibits CD4 + CD25 - (e) reversing Treg-mediated inhibition of T cell proliferation and interferon-γ production; and (f) inhibiting tumor cell proliferation in vivo.

[0162] Other anti-PD-1 mAbs are described in, e.g., U.S. Patents 6,808,710, 7,488,802, 8,168,757, 8,354,509, and 9,987,500, U.S. Publication No. 2016 / 0272708, and PCT Publication Nos. WO2008 / 156712, WO2012 / 145493, WO2014 / 179664, WO2014 / 194302, WO2014 / 206107, WO2015 / 035606, WO2015 / 085847, WO2015 / 112900, WO2016 / 1 No. 06159, WO2016 / 197367, WO2017 / 020291, WO2017 / 020858, WO2017 / 024465, WO2017 / 024515, WO2017 / 025016, WO2017 / 025051, WO2017 / 040790, WO2017 / 106061, WO2017 / 123557, WO2017 / 132827, WO2017 / 133540, each of which is incorporated herein by reference in its entirety.

[0163] In certain embodiments, the anti-PD-1 mAb is selected from the group consisting of nivolumab (Opdivo®; former names 5C4, BMS-936558, MDX-1106 or ONO-4538), pembrolizumab (Keytruda®; former names lambrolizumab and MK-3475; see WO2008 / 156712A1), cemiplimab (LIBTAYO®; formerly known as REGN-2810; see WO2015 / 112800), PDR001 (see WO2015 / 112900), MEDI-0680 (former name AMP-514; see WO2012 / 145493), JS001 (see Liu and Wu, 2017), BGB-A317 (see WO2015 / 035606 and US2015 / 0079109), INCSHR1210 (SHR-1210; see WO2015 / 085847; see Liu and Wu, 2017), TSR-042 (ANB011; see WO2014 / 179664), GLS-010 (WBP3055; see Liu and Wu, 2017), AM-0001 (see WO2017 / 123557), STI-1110 (see WO2014 / 194302), AGEN2034 (see WO2017 / 040790) and MGD013 (see WO2017 / 106061).

[0164] In certain preferred embodiments of any of the therapeutic methods described herein that include administration of an anti-PD-1 Ab, the anti-PD-1 Ab is nivolumab (Opdivo®), which has already been approved by the U.S. Food and Drug Administration (FDA) for the treatment of several different cancers. Nivolumab is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor Ab that selectively blocks interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking downregulation of anti-tumor T cell function (described as mAb C5 in U.S. Patent 8,008,449; Wang et al., 2014). In other preferred embodiments, the anti-PD-1 Ab is pembrolizumab (Keytruda®; a humanized monoclonal IgG4 Ab against PD-1, described as h409A11 in U.S. Patent 8,354,509), which is also approved for several cancer indications.

[0165] Anti-PD-1 Abs useful in the methods, compositions, or kits of the invention specifically bind human PD-1 (hPD-1) and also include isolated Abs, preferably mAbs, that cross-compete with any of the anti-PD-1 Abs described herein, such as nivolumab (5C4; see, e.g., U.S. Patent No. 8,008,449; WO2013 / 173223) and pembrolizumab for binding to human PD-1. Control Abs, e.g., Abs that cross-compete with nivolumab or pembrolizumab for binding to an antigen, in this case human PD-1, can be readily identified by standard PD-1 binding assays, such as BIACORE® analysis, ELISA assays, or flow cytometry (see, e.g., WO2013 / 173223). In certain embodiments, the anti-PD-1 Abs bind to the same epitope as any of the anti-PD-1 Abs described herein, such as nivolumab or pembrolizumab.

[0166] Anti-PD-1 Abs useful in the methods of the invention include Fab, F(ab') 2, Fd or Fv fragments, sdAbs, scFv, di-scFv or bi-scFv, bispecific antibodies, minibodies or antigen-binding portions comprising isolated CDRs (see Hollinger and Hudson, 2005; Olafsen and Wu, 2010 for further details).

[0167] In some embodiments, the isolated anti-PD-1 Ab or antigen-binding portion thereof comprises a heavy chain constant region that is of a human IgG1, IgG2, IgG3 or IgG4 isotype. In some preferred embodiments, the anti-PD-1 Ab or antigen-binding portion thereof comprises a heavy chain constant region that is of a human IgG4 isotype. In other embodiments, the anti-PD-1 Ab or antigen-binding portion thereof is of a human IgG1 isotype. In some other embodiments, the IgG4 heavy chain constant region of the anti-PD-1 Ab or antigen-binding portion thereof comprises an S228P mutation (numbered according to the EU numbering system, Kabat et al., 1991; alternatively, S241P, numbered according to the Kabat system, Kabat et al., 1987), which replaces a serine residue in the hinge region with a proline residue normally found at the corresponding position in IgG1 isotype Abs. This mutation, present in nivolumab, prevents Fab arm exchange with endogenous IgG4 Abs while maintaining the low affinity for activating Fc receptors associated with wild-type IgG4 Abs (Wang et al., 2014). In yet other embodiments, the Ab comprises a light chain constant region which is a human kappa or lambda constant region.

[0168] In another embodiment of the method, the anti-PD-1 Ab or antigen-binding portion thereof is a mAb or antigen-binding portion thereof. For administration to a human subject, the anti-PD-1 Ab is preferably a chimeric Ab or, more preferably, a humanized or human Ab. Such chimeric, humanized or human mAbs can be produced and isolated by methods well known in the art, for example, as described in U.S. Patent No. 8,008,449.

[0169] Anti-PD-L1 Ab Because anti-PD-1 and anti-PD-L1 target the same signaling pathway and have been shown to exhibit comparable levels of efficacy in a variety of cancers in clinical trials (see, e.g., Brahmer et al., 2012; WO2013 / 173223), anti-PD-L1 Abs may be substituted for anti-PD-1 Abs in combination in the therapeutic methods disclosed herein.

[0170] Anti-PD-L1 Abs suitable for use in the methods, compositions or kits of the invention are isolated Abs that bind to PD-L1 with high specificity and affinity, block the binding of PD-L1 to PD-1 and CD80, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. mAbs that specifically bind to PD-L1 with high affinity are disclosed in U.S. Patent 7,943,743. Other anti-PD-L1 mAbs are disclosed, for example, in U.S. Patents 8,217,149, 8,779,108, 9,175,082, 9,624,298 and 9,938,345 and PCT Publication WO2012 / 145493. Anti-PD-1 HuMAbs described in PD-1 HuMAbs have been shown to exhibit one or more of the following characteristics: (a) a K of about 50 mM or less as determined by SPR (BIACORE®); D (b) bind to human PD-1 in an MLR assay; (c) stimulate Ab responses; (d) inhibit the binding of PD-L1 to PD-1; and (e) reverse the suppressive effect of Tregs on T cell effector cells and / or dendritic cells. Anti-PD-L1 Abs for use in the therapeutic methods disclosed herein include isolated Abs, preferably mAbs, that specifically bind to human PD-L1 with high affinity and exhibit at least one, and in certain embodiments at least three, and preferably all, of the above characteristics. For example, anti-PD-L1 Abs suitable for use in these methods include those that (a) have a K of about 50 mM to 0.1 mM as determined by SPR (BIACORE®), D(b) bind to human PD-1 in an MLR assay; (c) inhibit the binding of PD-L1 to PD-1 and CD80; and (d) reverse the suppressive effects of Tregs on T cell effector cells and / or dendritic cells.

[0171] A suitable anti-PD-L1 Ab for use in the present methods is BMS-936559 (formerly MDX-1105; named 12A4 in U.S. Patent 7,943,743). Another suitable anti-PD-L1 Ab is atezolizumab (Tecentriq®; formerly known as RG7446 and MPDL3280A; named YW243.55S70 in U.S. Patent 8,217,149; Herbst et al., see also WO 2014), durvalumab (Imfinzi®; formerly known as MEDI-4736; named 2.14H9OPT in U.S. Patent 8,779,108), avelumab (Bavencio®; formerly known as MSB-0010718C; named A09-246-2 in U.S. Patent 9,624,298), STI-A1014 (named H6 in U.S. Patent 9,175,082), CX-072 (see WO 2016 / 149201), KN035 (see Zhang et al., 2017), LY3300054 (see, e.g., WO 2017 / 034916), and CK-301 (see Gorelik et al., 2017).

[0172] Anti-PD-L1 Abs suitable for use in the methods, compositions, or kits of the invention also include isolated Abs that specifically bind human PD-L1 and cross-compete for binding to human PD-L1 with a control Ab, which can be any of the anti-PD-L1 Abs disclosed herein, for example, BMS-936559 (12A4; see, e.g., U.S. Patent 7,943,743; WO 2013 / 173223), atezolizumab, durvalumab, avelumab, or STI-A1014. The ability of an Ab to cross-compete with a control Ab for binding to human PD-L1 predicts that such an Ab will bind to the same epitope region of PD-L1 as the control Ab, and, due to its binding to substantially the same epitope region of PD-L1, will have functional properties that are highly similar to the control Ab. In one embodiment, the anti-PD-L1 Ab binds to the same epitope as any of the anti-PD-L1 Abs described herein, e.g., atezolizumab, durvalumab, avelumab, or STI-A1014. Cross-competing Abs can be readily identified by their ability to cross-compete with a control Ab, such as atezolizumab or avelumab, in standard PD-L1 binding assays, such as BIACORE® analysis, ELISA assays, or flow cytometry, well known to those of skill in the art (see, e.g., WO 2013 / 173223).

[0173] In certain preferred embodiments, the isolated anti-PD-L1 Abs used in the methods are mAbs. In other embodiments, particularly for administration to human subjects, these Abs are preferably chimeric Abs or, more preferably, humanized or human Abs. Chimeric, humanized and human Abs can be produced and isolated by methods well known in the art, for example, as described in U.S. Patent 7,943,743.

[0174] In certain embodiments, the anti-PD-L1 Ab, or antigen-binding portion thereof, comprises a heavy chain constant region that is of the human IgG1, IgG2, IgG3, or IgG4 isotype. In certain other embodiments, the anti-PD-L1 Ab, or antigen-binding portion thereof, is of the human IgG1 or IgG4 isotype. In a further embodiment, the sequence of the IgG4 heavy chain constant region of the anti-PD-L1 Ab, or antigen-binding portion thereof, comprises a S228P mutation. In other embodiments, the Ab comprises a light chain constant region that is a human kappa or lambda constant region.

[0175] The anti-PD-L1 Abs of the present invention also bind to PD-L1 and exhibit similar functional properties to the whole Ab in terms of receptor binding inhibition and immune system upregulation. 2 Also included are antigen-binding portions of the above Abs, including Fd, Fv and scFv, di-scFv or bi-scFv and scFv-Fc fragments, nanobodies, diabodies, triabodies, tetrabodies and isolated CDRs.

[0176] Treatment method Cancer treatment with anti-CD27 Ab as monotherapy As described in Example 8, the anti-mCD27 mAb, 8H5, inhibited tumor growth in CT26 colon carcinoma and fibrosarcoma tumor models (see FIG. 14 and Table 7). Thus, the present invention provides a method of treating a subject having cancer comprising administering to the subject a therapeutically effective amount of any of the anti-CD27 Abs, immunoconjugates or bispecific molecules disclosed herein, or a pharmaceutical composition comprising any of the anti-CD27 Abs, immunoconjugates or bispecific molecules, such that the subject is treated.

[0177] The invention also provides a method of inhibiting tumor cell proliferation in a subject comprising administering to the subject a therapeutically effective amount of any of the anti-CD27 Abs, immunoconjugates or bispecific molecules disclosed herein, or a pharmaceutical composition comprising any of the anti-CD27 Abs, immunoconjugates or bispecific molecules, such that tumor cell proliferation in the subject is inhibited.

[0178] Cancer treatment with anti-CD27 Ab in combination with other anti-cancer drugs As described in Example 8, the anti-mCD27 mAb, 8H5, enhances anti-mPD-1 mAb activity in various cancer types, including CT26 colon carcinoma, SA1N fibrosarcoma, and EG7 lymphoma mouse models (see FIG. 14 and Table 7). Thus, anti-CD27 Abs are much more effective at inhibiting tumor growth when combined with checkpoint inhibitors, such as anti-PD-1 Abs. Thus, the present invention provides a method of treating a subject with cancer, comprising administering to the subject a therapeutically effective amount of: (a) any of the anti-CD27 Abs, immunoconjugates, or bispecific molecules disclosed herein, or a pharmaceutical composition comprising any of the anti-CD27 Abs, immunoconjugates, or bispecific molecules; and (b) an additional therapeutic agent for cancer treatment, such that the subject is treated.

[0179] The invention also provides a method of inhibiting tumor cell proliferation in a subject, the method comprising administering to the subject a therapeutically effective amount of: (a) any of the anti-CD27 Abs, immunoconjugates or bispecific molecules disclosed herein, or a pharmaceutical composition comprising any of the anti-CD27 Abs, immunoconjugates or bispecific molecules; and (b) an additional therapeutic agent for the treatment of cancer, such that tumor cell proliferation in the subject is inhibited.

[0180] In certain preferred embodiments of any of the methods, the subject is a human patient.

[0181] In some embodiments, the additional therapeutic agent is a compound that reduces inhibition or increases stimulation of the immune system.For example, the additional therapeutic agent can be a small molecule compound, a macrocyclic peptide, a fusion protein, or an Ab.In further embodiments, the additional therapeutic agent is an antagonist Ab that specifically binds to PD-1, PD-L1, CTLA-4, LAG-3, BTLA, TIM-3, KIR, KLRG-1, A2aR, TIGIT, VISTA receptor, CD244, or CD160.In other embodiments, the additional therapeutic agent is an agonist Ab that specifically binds to ICOS, CD137, CD134, CD27, GITR, or HVEM.In some embodiments, the additional therapeutic agent is an antagonist Ab or an antigen-binding portion thereof that specifically binds to PD-1 or PD-L1, disrupts the interaction between PD-1 and PD-L1, and inhibits PD-1 / PD-L1 signal transduction. In some embodiments, the Ab or antigen-binding portion thereof that specifically binds to PD-1 or PD-L1 is a chimeric, humanized or human mAb or antigen-binding portion thereof. In some embodiments, the additional therapeutic agent is an antagonist Ab or antigen-binding portion thereof that specifically binds to PD-1 and cross-competes with nivolumab for binding to human PD-1. In some embodiments, the additional therapeutic agent is an antagonist Ab or antigen-binding portion thereof that specifically binds to PD-1 and is nivolumab or pembrolizumab. In some embodiments, the additional therapeutic agent is an antagonist Ab or antigen-binding portion thereof that specifically binds to PD-L1 and cross-competes with Ab named BMS-936559 (WO2013 / 173223) for binding to human PD-L1. In certain embodiments, the additional therapeutic agent is an antagonist Ab or an antigen-binding portion thereof that specifically binds to PD-L1, and is the Ab named atezolizumab, durvalumab, avelumab, or BMS-936559 (WO2013 / 173223).

[0182] Cancers suitable for treatment by the methods of the present invention Tumor immunotherapy, based on harnessing the immune system's virtually limitless flexibility to attack and destroy cancer cells, is applicable to the treatment of a wide range of cancers (see, e.g., Yao et al., 2013; Callahan et al., 2016; Pianko et al., 2017; Farkona et al., 2016; Kamta et al., 2017).

[0183] In certain embodiments, the disclosed therapeutic methods may be used to treat cancers that are solid tumors.

[0184] In some embodiments, the solid tumor is colon cancer or fibrosarcoma.In some embodiments, the solid tumor is squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous cell NSCLC, non-squamous cell NSCLC, head and neck cancer, breast cancer, esophageal cancer, gastric cancer, digestive cancer, small intestine cancer, liver cancer, hepatocellular carcinoma (HCC), hepatocellular carcinoma, gallbladder and bile duct cancer, pancreatic cancer (PAC), pancreatic ductal adenocarcinoma (PDAC), kidney cancer, renal cell carcinoma (RCC), bladder cancer, urethral cancer, ureteral cancer, colorectal cancer (CRC), colon cancer, anal cancer, endometrial cancer, prostate cancer, neuroblastoma, glioma, glioblastoma, germ cell tumor, pediatric sarcoma, sinonasal natural killer, melanoma, skin cancer, Merkel cell carcinoma, basal cell carcinoma, bone cancer, cervical cancer, uterine cancer, endometrial carcinoma, The cancer is selected from the group consisting of fallopian tube carcinoma, ovarian cancer, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, testicular cancer, endocrine system cancer, thymic tumor, thymoma, thyroid cancer, oral cancer, oral cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, penile cancer, renal pelvis carcinoma, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain cancer, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, neuroblastoma, pituitary adenoma, epidermoid carcinoma, childhood solid tumor, childhood sarcoma, rhabdomyosarcoma, cancer of unknown primary origin, environmentally induced cancer, virus-associated cancer, AIDS-associated cancer, cancer of viral origin, advanced cancer, unresectable cancer, metastatic cancer, refractory cancer, recurrent cancer, and any combination of the above solid tumors.

[0185] In certain embodiments, the solid tumor is a cancer selected from small cell lung cancer (SCLC), squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, and triple-negative breast cancer (TNBC).

[0186] The anti-CD27 Ab of the present invention may be effective in the early phase of the disease, where chemotherapy and / or radiation therapy are important treatment modalities, but there is a need to promote further sustainable antitumor immunity. In one embodiment, the solid tumor is a cancer selected from esophageal cancer, gastric cancer, rectal cancer, non-small cell lung cancer (NSCLC) and squamous cell carcinoma of the head and neck (SCCHN).

[0187] In certain other embodiments, the solid tumor is selected from melanoma, renal cancer, NSCLC, colorectal cancer, gastric cancer, bladder cancer, and glioblastoma.

[0188] In some embodiments, the therapeutic method of the present invention can be used to treat cancer that is a hematological malignancy or cancer whose tumor cells are cells of hematological malignancies.Hematological malignancies include liquid tumors derived from either of the two major blood cell lineages, i.e., myeloid cell lineage (producing granulocytes, erythrocytes, platelets, macrophages and mast cells) or lymphoid cell lineage (producing B, T, NK and plasma cells), including all types of leukemia, lymphoma and myeloma. Hematological malignancies that may be treated using the present therapeutic methods include, for example, cancers selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), multiple myeloma, smoldering myeloma, monoclonal gammopathy of undetermined significance (MGUS), progressive, metastatic, refractory and / or recurrent hematological malignancies, and any combination of said hematological malignancies.

[0189] In other embodiments, the hematological malignancy is acute, chronic, lymphoblastic and / or myeloid leukemia, e.g., ALL, AML, CLL and CML; lymphomas, e.g., HL, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), mantle cell lymphoma, marginal zone B-cell lymphoma (mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B-cell lymphoma and splenic marginal zone B-cell lymphoma), Burkitt's lymphoma, lymphoplasmacytic lymphoma (LPL; also known as Waldenstrom's hypergammaglobulinemia (WM)), hairy cell lymphoma and primary central nervous system (CNS) lymphomas, including NHL, of which about 85% are B-cell lymphomas; NHL, T-cell lymphomas including precursor T-lymphoblastic lymphoma / leukemia, T-lymphoblastic lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphomas such as cutaneous T-cell lymphoma (CTLC, i.e., mycosis fungoides, Sézary syndrome, and others), adult T-cell lymphoma / leukemia, angioimmunoblastic T-cell lymphoma, extranodal natural killer / T-cell lymphoma nasal type, enteropathy-associated intestinal T-cell lymphoma (EATL), anaplastic large cell lymphoma (ALCL) and unspecified peripheral T-cell lymphoma, acute myeloid lymphoma, lymphoplasmacytic lymphoma, monocytoid B-cell lymphoma, The cancer is selected from the following: follicular lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary effusion lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma and precursor B-lymphoblastic lymphoma; myeloma, such as multiple myeloma, smoldering myeloma (also called low-grade myeloma), monoclonal gammopathy of undetermined significance (MGUS), isolated plasmacytoma, IgG myeloma, light chain myeloma, non-secretory myeloma and amyloidosis; and any combination of said hematological tumors. The method is also applicable to the treatment of progressive, metastatic, refractory and / or recurrent hematological tumors.

[0190] Regarding combination therapy, the anti-PD-1 Ab, nivolumab, has been shown to be effective in treating many types of cancer (see, e.g., Brahmer et al., 2015; Guo et al., 2017; Pianko et al., 2017; WO2013 / 173223) and is currently undergoing clinical trials in several solid and hematological cancers. Nivolumab has been approved for the treatment of advanced melanoma, advanced non-small cell lung cancer, metastatic renal cell carcinoma, classical Hodgkin lymphoma, advanced squamous cell carcinoma of the head and neck, urothelial carcinoma, MSI-H or dMMR metastatic colorectal cancer, and hepatocellular carcinoma and small cell lung cancer (Drugs.com - Opdivo Approval History: https: / / www.drugs.com / history / opdivo.html), and is in clinical trials in many other cancers. Similarly, other anti-PD-1 drugs such as pembrolizumab (KEYTRUDA®) and cemiplimab (LIBTAYO®) and anti-PD-L1 drugs such as atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®) and avelumab (BAVENCIO®) have gained approval for various indications. Thus, a wide variety of cancers can be treated using the combination of anti-CD27 Ab and anti-PD-1 / PD-L1 Ab disclosed herein. The high degree of efficacy shown for this therapeutic combination allows for focus on cancers that suffer from significant unmet medical need.

[0191] Medical uses of anti-CD27 Ab and anti-PD-1 / anti-PD-L1 Ab As described above, the present invention provides an isolated anti-CD27 Ab, preferably a mAb or an antigen-binding portion thereof, for use in a method of treating a subject with cancer. The present invention further provides an isolated anti-CD27 Ab, preferably a mAb or an antigen-binding portion thereof, and a checkpoint inhibitor, such as an isolated anti-PD-1 / anti-PD-L1 Ab, preferably a mAb or an antigen-binding portion thereof, for use in combination in a method of treating a subject with cancer. The anti-CD27 Ab may be used as a monotherapy or in combination with a checkpoint inhibitor, such as an anti-PD-1 / anti-PD-L1 Ab, to treat the full range of cancers disclosed herein.

[0192] Certain embodiments of the present invention include an isolated anti-CD27 Ab or antigen-binding portion thereof of the present invention for the manufacture of a medicament for the treatment of a subject with cancer. The anti-CD27 Ab may be used alone or in combination with a checkpoint inhibitor, such as an isolated anti-PD-1 / anti-PD-L1 Ab or antigen-binding portion thereof, for the manufacture of a medicament for the treatment of a cancer patient. All uses of the anti-CD27 Ab and anti-PD-1 / anti-PD-L1 Ab for the manufacture of a medicament are broadly applicable to the full range of cancers disclosed herein.

[0193] The invention also provides an anti-CD27 Ab, or antigen-binding portion thereof, in combination with a checkpoint inhibitor, such as an isolated anti-PD-1 / anti-PD-L1 Ab, or antigen-binding portion thereof, for use in a method of treatment of cancer corresponding to all of the embodiments of the method of treatment using this therapeutic combination described herein.

[0194] Pharmaceutical Compositions and Dosing Regimens The Abs used in any of the therapeutic methods disclosed herein may be included in a composition, e.g., a pharmaceutical composition comprising the Ab and a pharma- ceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. Preferably, the carrier of the Ab-containing composition is suitable for intravenous (IV), intramuscular, subcutaneous (SC), parenteral, spinal or epidermal administration (e.g., by injection or infusion).

[0195] Options for SC injection include Halozyme Therapeutics' ENHANZE® drug delivery technology, which involves a co-formulation of Ab with recombinant human hyaluronidase enzyme (rHuPH20), which removes previous limitations on the volume of biologics and drugs that can be delivered subcutaneously due to the extracellular matrix (U.S. Patent 7,767,429). It may be possible to co-formulate two Abs used in a combination therapy into one composition for SC administration.

[0196] Pharmaceutical compositions of the present invention may contain one or more pharma- ceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers, and / or adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispersing agents.

[0197] Dosing regimens are adjusted to provide the optimum desired response, e.g., maximum therapeutic response and / or minimum adverse effects. For administration of anti-CD27, anti-PD-1 or anti-PD-L1 Abs or antigen-binding portions thereof included in a combined use, the dosage may range from about 0.01 to about 20 mg / kg, preferably about 0.1 to about 10 mg / kg of subject body weight. For example, the dosage may be about 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 5 mg / kg or 10 mg / kg of body weight. Alternatively, instead of a dose based on body weight, a fixed or constant dose, e.g., about 50 to about 2000 mg of Abs or antigen-binding portions thereof, may be administered. For example, a fixed dose of about 50 mg, 100 mg, 200 mg, 400 mg, 500 mg, 800 mg, 1000 mg, 1200 mg, 1500 mg, 1600 mg, or 2000 mg of antibody may be administered. The administration schedule is generally based on the typical pharmacokinetic properties of the Ab and is designed to achieve an exposure that results in sustained receptor occupancy (RO). Exemplary treatment regimes include administration about once a week, about once every two weeks, about once every three weeks, about once every four weeks, about once a month, about once every three to six months, or at longer intervals. In certain preferred embodiments, the anti-CD27, anti-PD-1, or anti-PD-L1 Abs or antigen-binding portions thereof are administered to the subject about once every two weeks. In other preferred embodiments, the Abs or antigen-binding portions thereof are administered about once every three weeks. In other preferred embodiments, the Abs or antigen-binding portions thereof are administered about once every four weeks. Dosage and scheduling may vary during the course of treatment. In one preferred embodiment, a fixed or constant dose of about 240 mg of anti-PD-1 Ab or antigen-binding portion thereof is administered to a subject approximately once every two weeks. In another preferred embodiment, a fixed or constant dose of about 480 mg of anti-PD-1 Ab or antigen-binding portion thereof is administered to a subject approximately once every four weeks. In one embodiment, the anti-PD-1 Ab is nivolumab.

[0198] When used in combination, subtherapeutic doses of one or both Abs may be used, e.g., doses of anti-CD27, anti-PD-1 and / or anti-PD-L1 Abs or antigen-binding portions thereof lower than the typical or approved monotherapy doses. For example, a dose of nivolumab lower than one of the approved doses, 3 mg / kg every 2 weeks, e.g., 1.0 mg / kg or less every 2, 3 or 4 weeks, would be considered subtherapeutic. RO data from 15 subjects receiving 0.3 mg / kg to 10 mg / kg of nivolumab indicates that PD-1 occupancy appears to be dose-independent in this dose range. Across doses, the mean occupancy was 85% (range, 70%-97%), with a mean plateau occupancy of 72% (range, 59%-81%) (Brahmer et al., 2010). Therefore, a dose of 0.3 mg / kg may allow sufficient exposure to lead to significant bioactivity.

[0199] The synergistic interactions seen in mouse tumor models (Example 8) may allow for the administration of subtherapeutic doses of either anti-CD27 Ab and anti-PD-1 / anti-PD-L1 Ab, or antigen-binding portion thereof, to cancer patients. In certain embodiments of the combination therapy methods of the invention, anti-CD27 Ab, or antigen-binding portion thereof, is administered to cancer patients at a subtherapeutic dose. In other embodiments, anti-PD-1 / anti-PD-L1 Ab, or antigen-binding portion thereof, is administered to patients at a subtherapeutic dose. In further embodiments, anti-PD-1 / anti-PD-L1 Ab and anti-CD27 Ab, or antigen-binding portion thereof, are administered to patients at subtherapeutic doses of each.

[0200] Administration of sub-therapeutic doses of one or both Abs may reduce adverse events compared to the use of higher doses of the individual Abs in monotherapy. Thus, the success of the combination therapy of the present invention may be indicated not only by the improved efficacy of the combination of these Abs compared to the monotherapy of these Abs, but also by the improved safety, i.e., reduced incidence of adverse events, due to the use of lower doses of the drugs in combination compared to the monotherapy doses.

[0201] In any of the embodiments of the methods disclosed herein, the anti-CD27, anti-PD-1 and / or anti-PD-L1 Abs are formulated for intravenous (IV) administration or subcutaneous (SC) injection. In certain embodiments, the anti-CD27 Ab, or antigen-binding portion thereof, and the anti-PD-1 / anti-PD-L1 Ab, or antigen-binding portion thereof, are administered sequentially to the subject. By "sequential" administration, it is meant that one of the anti-CD27 Ab and the anti-PD-1 / anti-PD-L1 Ab is administered before the other. Either Ab may be administered first; i.e., in certain embodiments, the anti-PD-1 / anti-PD-L1 Ab is administered before the anti-CD27 Ab, while in other embodiments, the anti-CD27 Ab is administered before the anti-PD-1 / anti-PD-L1 Ab. In certain embodiments, each Ab is administered by IV infusion, e.g., an infusion over about 30 minutes or about 60 minutes. In other embodiments, at least one Ab is administered by SC injection.

[0202] In one embodiment of sequential IV administration, the anti-CD27 Ab and anti-PD-1 / anti-PD-L1 Ab or portions thereof are administered within 30 minutes of each other, as is convenient for the patient. Generally, when both anti-CD27 Ab and anti-PD-1 / anti-PD-L1 Ab are administered IV on the same day, separate infusion bags and filters are used for each infusion. Immediately following the infusion of the first Ab, a saline flush is used to clear the Ab from the line, and then an infusion of the second Ab is started. In other embodiments, the two Abs are administered within 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, or 48 hours of each other.

[0203] Administration of at least one Ab via SC reduces the time required for administration by healthcare professionals and shortens the time for drug administration. For example, the use of SC injection shortens the time required for IV administration, which is typically about 30-60 minutes, to about 5 minutes. In one embodiment of sequential SC administration, the anti-CD27 Ab and the anti-PD-1 / anti-PD-L1 Ab or portions thereof are administered within 10 minutes of each other.

[0204] Since checkpoint inhibitor Abs have been shown to generate extremely durable responses, in part due to memory elements of the immune system (see, e.g., WO2013 / 173223; Lipson et al., 2013; Wolchok et al., 2013), the activity of administered anti-PD-1 / anti-PD-L1 Abs may continue for weeks, months, or even years. In one embodiment, the combination therapy method including sequential administration includes administration of anti-CD27 Ab to a patient who has been previously treated with anti-PD-1 / anti-PD-L1 Ab. In a further embodiment, anti-CD27 Ab is administered to a patient who has been previously treated with anti-PD-1 / anti-PD-L1 Ab and has progressed. In another embodiment, the combination therapy method including sequential administration includes administration of anti-PD-1 / anti-PD-L1 Ab to a patient who has been previously treated with anti-CD27 Ab, optionally with a patient whose cancer has progressed after treatment with anti-CD27 Ab.

[0205] In certain other embodiments, the anti-PD-1 / anti-PD-L1 Abs and anti-CD27 Abs are co-administered, either mixed as one composition in a pharma- ceutically acceptable formulation for simultaneous administration or simultaneously as separate compositions in which each Ab is formulated with a pharma- ceutically acceptable composition.

[0206] kit Also within the scope of the present invention are kits comprising anti-CD27 Abs for therapeutic use. The kits generally include a label containing the intended use and instructions for use of the contents of the kit. The term label includes any writing or recording medium provided on or with the kit or which otherwise accompanies the kit. Thus, the present invention provides kits for treating a subject with cancer, comprising: (a) one or more doses of any of the anti-CD27 Abs or antigen-binding portions thereof, immunoconjugates or bispecific molecules disclosed herein, or pharmaceutical compositions comprising any of the anti-CD27 Abs or antigen-binding portions thereof, immunoconjugates or bispecific molecules, in a dosage range of about 0.1 to about 20 mg / kg body weight; and (b) instructions for use of the mAb in any of the therapeutic methods disclosed herein. The present invention further provides kits for treating a subject having cancer comprising: (a) one or more of any of the anti-CD27 Abs, or antigen-binding portions, immunoconjugates, or bispecific molecules disclosed herein, or pharmaceutical compositions comprising the anti-CD27 Abs, or antigen-binding portions, immunoconjugates, or bispecific molecules, in a dosage range of about 0.1 to about 20 mg / kg body weight; (b) one or more doses of any of the Abs, or antigen-binding portions thereof, that specifically bind PD-1 or PD-L1, in a dosage range of about 50 to about 2000 mg; and (c) instructions for using an anti-CD27 mAb and a checkpoint inhibitor, e.g., an anti-PD-1 / anti-PD-L1 mAb, in any of the combination therapy methods disclosed herein.

[0207] In some embodiments, the Abs are co-packaged in a unit dosage form. In some preferred embodiments for the treatment of human patients, the kit comprises an anti-human PD-1 Ab disclosed herein, e.g., nivolumab or pembrolizumab.

[0208] The present invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references cited throughout this specification are expressly incorporated herein by reference. EXAMPLES

[0209] Example 1 mAb production against CD27 Human anti-CD27 mAbs were generated by immunizing transgenic mice expressing human Ab genes with the human CD27 (hCD27) antigen to generate a repertoire of human Igs specific for CD27 in the mice. Rat anti-mouse CD27 mAbs were generated by immunizing rats with the mouse CD27 (mCD27) antigen.

[0210] Immunization of human immunoglobulin transgenic mice HuMAbs against hCD27 were generated by alternating immunization of human Ig transgenic mice (KM mice, line number 325432) with recombinant hCD27 containing hFc and histidine tagged protein (rhCD27-hFc-his) and CHO cells stably transfected with hCD27.

[0211] Generation of hybridomas producing mAbs against CD27 Mouse splenocytes were isolated from the above immunized mice that showed positive anti-CD27 IgG titers after immunization. Hybridomas were generated by fusion of splenocytes with the mouse myeloma SP2 / 0 fusion partner.

[0212] Immunization of rats A surrogate mAb against mCD27 was generated by immunizing rats with recombinant mouse CD27 containing an Fc tag protein (rmCD27-Fc).

[0213] Example 2 Screening and selection of human anti-human CD27 mAbs Screening for mAbs that selectively bind to human CD27 To generate HuMAbs that bind to hCD27, human Ig transgenic mice were immunized with hCD27 antigen as described in Example 1. Hybridoma supernatants were first screened for binding to hCD27 by enzyme-linked immunosorbent assay (ELISA). Antigen specificity was then confirmed by immunohistochemistry using hCD27 + Binding to T cells was confirmed, and the Abs were subsequently tested for their ability to block the interaction of CD27 with CD70 by flow cytometry and surface plasmon resonance (SPR).

[0214] Functional screening of agonistic anti-CD27 mAbs The agonistic activity of anti-CD27 Abs was determined in vitro using the CHO-svCD3-CD32a assay, in which anti-CD27 agonistic Abs cross-link and bind to human CD4 + Enhanced svCD3-mediated activation of T cells. The lead mAb, the nonligand blocker clone 16D9, was selected for its excellent binding affinity to h- and cCD27 and its excellent agonism of activated T cells. The lead mAb binds human CD3 + Using IL-2-activated human NK cells co-cultured with T cells, it was determined to have a relatively modest level of ADCC activity.

[0215] Optimization of anti-hCD27 HuMAb Clone 16D9 was then subjected to VH-A28T and VK-I20T-I22T-D43A framework reversions and S93N mutations in the light chain CDRs. The modified clone was engineered as a human IgG1 and named BMS-986215. BMS-986215 was engineered as a hIgG1 to allow for the Fc cross-linking required for CD27 agonism.

[0216] The complete hCD27 amino acid sequence is shown in Table 1, which can be found in GENBANK® Accession No. AAH12160. The underlined amino acids 1-20 in Table 1 represent the predicted signal peptide. See, e.g., CBS Prediction SignalP 4.1 (http: / / www.cbs.dtu.dk). [Table 1]

[0217] The amino acid sequences of the six CDR domains of HuMAb BMS-986215, defined using the Kabat method, are shown in Table 2. V of HuMAb BMS-986215 H , V L , C H , C L The amino acid sequences of the heavy and light chains are shown in Table 3. [Table 2]

[0218] [Table 3]

[0219] The Ab was recombinantly produced in CHO cells and purified by Protein A affinity chromatography. The Ab was characterized by SDS-PAGE, N-terminal sequencing and mass spectrometry analysis. The purity of the Ab was >98% as tested by size-exclusion chromatography (SEC). Specific aggregation propensity (SAP) modeling studies performed on the CDR regions of BMS-986215 showed a low SAP score, indicating a very low aggregation propensity. A single N-glycosylation site was identified at N297 of the heavy chain (underlined in Table 3), which showed a glycan profile including G0F, G1F and G2F structures, as expected for human IgG1 molecule expression in CHO cells. BMS-986215 showed high thermal and chemical stability. The thermal reversibility of this molecule at 78°C is 41%, which is higher than that typically seen for a typical human IgG1 molecule. The biophysical characteristics of BMS-986215 are summarized in Table 4.

[0220] Example 3 Characterization of BMS-986215 by binding kinetics, binding affinity, cross-reactivity, tissue specificity, and stability BMS-986215 specifically binds to human and cynomolgus CD27 Surface plasmon resonance (SPR) analysis confirmed the specific binding of BMS-986215 to hCD27. The apparent affinity (K) of the Ab to hCD27 was measured using the Fab fragment of BMS-986215 and hCD27-mouse Fc or hCD27-His tag protein. D ) was determined by SPR to be approximately 40 nM. Measured values ​​were 41-44 nM at 37 °C and 13-16 nM at 25 °C. The 3-fold loss in affinity from 25 °C to 37 °C was due to an increase in the dissociation rate, while the association rate remained unchanged. Kinetic analysis used a 1:1 fit binding model. K D was determined to be 0.045 nM by Scatchard analysis. EC binding of BMS-986215 to human T cells 50 The EC value was determined by flow cytometry to be 0.044 nM (range 0.022–0.076 nM, 0.044 ± 0.011 nM, n = 5). 50 The value was determined by flow cytometry to be 0.131 nM (range 0.03569–0.2657; 0.131 ± 0.069 nM, n = 3), indicating that BMS-986215 cross-reacts with cCD27.

[0221] [Table 4]

[0222] Flow cytometry analysis also demonstrated that BMS-986215 has higher affinity for h- and cCD27 compared to 1F5, a human anti-hCD27 control mAb. See Figures 1A and 1B, respectively. The heavy and light chain amino acid sequences of HuMAb 1F5 are shown in Table 5. Binding EC of BMS-986215 and 1F5 to human T cells in Figure 1A 50 The EC values ​​were 0.02210 nM and 1.088 nM, respectively. The EC values ​​determined by binding of BMS-986215 and 1F5 to cynomolgus T cells in Figure 1B 50The values ​​were 0.092 nM and 1.800 nM, respectively. [Table 5]

[0223] BMS-986215 is a non-ligand blocking mAb No inhibition of CD70 binding to CD27 or vice versa by BMS-986215 was detected by SPR or flow cytometry, consistent with this mAb being a non-ligand blocking Ab. See Figures 2B and 2D. For example, flow cytometry analysis was performed using human T cells pretreated with 10 μg / ml soluble human CD70, a ligand for CD27, followed by treatment with BMS-986215, 1F5 and human IgG1 (control) Abs. The presence or absence of Ab binding was detected using a labeled anti-human IgG Ab. Binding of BMS-986215 was detected, indicating that CD70 binding to CD27 does not block BMS-986215 binding. See Figure 2A, which shows the assay in schematic form, and Figure 2B, which shows a graph of the results. Binding of the ligand blocking 1F5 Ab was not detected. Id. Another flow cytometry analysis was performed in which human T cells were exposed to BMS-986215, 1F5 and human IgG1 (control) Abs in the presence of 10 μg / ml soluble human CD70. CD70 was detected with a labeled anti-CD70 Ab. CD70 remained detectable with increasing concentrations of BMS-986215, indicating that CD70 binding was not blocked by BMS-986215. See Figure 2C for a schematic of the assay and Figure 2D for a graph of the results. CD70 binding decreased with increasing concentrations of the ligand blocking Ab 1F5. Id.

[0224] tissue specificity In a preliminary tissue cross-reactivity evaluation, fluorescein isothiocyanate (FITC) conjugate BMS-986215 (research grade) was applied to frozen sections of 22 normal human tissues (1 or 2 donors each). As expected, specific staining was observed in mononuclear cell (MNC) subsets in the thymus, tonsils and spleen, lymphoid-rich tissues (colon, small intestine and stomach) and rarely in other tissues (thyroid, lung, skin, uterus, prostate, liver, kidney, pancreas, adrenal gland, pituitary, placenta and testis) (data not shown). In general, positive cells correlate with background inflammatory lesions. There were the greatest numbers of positively stained cells in the tonsils, thymus and small intestine. Positively stained lymphocytes were highly concentrated in T cell-rich areas (interfollicular areas) in the thymic medulla and tonsils and in the spleen (perifollicular-like / marginal zone and PALS). In the small intestine, positive cells were mainly distributed in the basal lamina propria and focal lymphoid aggregates / follicles. The cerebrum, cerebellum, heart, and peripheral nerves were negative.

[0225] Example 4 BMS-986215 epitope mapping Hydrogen / deuterium exchange mass spectrometry (HDX-MS) and fast photochemical oxidation of proteins (FPOP) methods were utilized to probe the region of CD27 to which BMS-986215 binds. The CD27 / BMS-986215 HDX-MS experiment provided 62% sequence coverage and included the N- and C-terminal regions of CD27. The FPOP measurement yielded 81% sequence coverage of CD27 and included the middle region of CD27. The total sequence coverage of CD27 was 98% in the combined HDX-MS and FPOP data set. See Figure 3(A). The sequence shown in Figure 3(A) is the mature CD27 protein without the signal peptide sequence of amino acids 1-20 of SEQ ID NO:1. Amino acids 1-172 in Figure 3(A) correspond to amino acids 21-192 of SEQ ID NO:1.

[0226] HDX-MS Prior to the epitope mapping experiments, a deuterated experiment was performed to generate a list of common peptides for recombinant full-length hCD27-His tag (10 μM) and the protein complex of CD27 and BMS-986215 (1:1 molar ratio). For HDX-MS experiments, 5 μL of each sample (CD27 or CD27 and BMS-986215) was added to a 20 μL D 2 O buffer (10mM phosphate buffer, D 2 Labeling reactions were initiated by diluting the solution in 10 mL of PBS (pH 7.0, pD 7.0). Reactions were run for various times: 20 s, 1 min, 10 min, and 240 min. At the end of each labeling reaction time, the reaction was stopped by adding quenching buffer (100 mM phosphate buffer with 4 M GdnCl and 0.4 M TCEP, pH 2.5, 1:1, v / v). 1 μL of pepsin / protease XIII (1:1 at 1 mg / mL, v / v) was added and the proteins were digested for 3 min on ice. The digestion solution was injected into a Waters HDX-MS system (Waters Corporation, Milford, MA) for analysis. Deuterium incorporation levels of common peptic peptides were monitored in the absence or presence of BMS-986215. HDX-MS data analysis of BMS-986215 on CD27 indicates that BMS-986215 binds to an epitope comprising the TPAPKSCPERHYWAQGKLCCQ (SEQ ID NO: 15, corresponding to amino acids 1-21 of the mature protein or amino acids 21-41 of SEQ ID NO: 1) region at the N-terminus of CD27.

[0227] FPOP FPOP experiments were performed with CD27 and the Fab complex of CD27 / BMS-986215 (1:1 molar ratio, 7.5 μM final concentration). 2 O 2 Hydroxyl groups were generated by photolysis, and the excitation wavelength was set to 248 nm. Just before labeling, 5 μL each of histidine and H 2 O 2 was added to a certain amount of protein. The final volume of the protein solution was 50 μL, and the histidine and H 2 O 2The final concentrations of were 500 μM and 15 mM, respectively. Laser energy was adjusted to 28 mJ / pulse (7.4 Hz). Both FPOP and no-laser control experiments were performed in duplicate. Each replicate was collected in a microcentrifuge tube containing 11 μL of quenching solution (800 nM catalase tetramer and 200 mM methionine). Samples were denatured, reduced, alkylated, and trypsin digested. Data acquisition was performed on a Thermo Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA) and a Waters Acquity UPLC system (Waters Corporation). Byonic TM A search engine (Proten Metrics, San Carlos, CA) was used to obtain sequencing coverage and identify oxidation sites. Relative oxidation levels of tryptic peptides were calculated manually for each repeat. Only peptides with statistically significant differences in relative oxidation between free and bound states (based on Student's T-test, p-value <0.05) were considered for further analysis. In the FPOP experiments, four CD27 peptides showed significant differences in oxidation levels: HYWAQGK (SEQ ID NO: 16, corresponding to amino acids 11-17 of the mature protein or amino acids 31-37 of SEQ ID NO: 1), LCCQMCEPGTFLVK (SEQ ID NO: 17, corresponding to amino acids 18-31 of the mature protein or amino acids 38-51 of SEQ ID NO: 1), DCDQHR (SEQ ID NO: 18, corresponding to amino acids 32-37 of the mature protein or amino acids 52-57 of SEQ ID NO: 1) and DCDQHR (SEQ ID NO: 19, corresponding to amino acids 88-93 of the mature protein or amino acids 108-113 of SEQ ID NO: 1). The FPOP protection percentage due to binding to Fab was calculated as (relative %FPOP difference in CD27-relative %FPOP difference in Fab) / (relative %FPOP difference in CD27)×100. Figure 3(C) shows the FPOP protection percentage of these four peptides. The DCDQHR peptide was shown to have the highest FPOP protection percentage (75%) and represents the binding epitope of CD27 upon interaction with BMS-986215 Fab. Based on the above HDX-MS and FPOP experiments, the CD27 epitope to which BMS-986215 binds is believed to contain two discontinuous binding regions, TPAPKSCPERHYWAQGKLCCQ (SEQ ID NO: 15) and DCDQHR (SEQ ID NO: 18), corresponding to amino acids 21-41 and 52-57 of SEQ ID NO: 1.

[0228] Example 5 CD27 agonism by BMS-986215 increases T cell activation Anti-CD27 mAb agonists require FcγR interaction (i.e., cross-linking) for activity. In the presence of a ligand-blocking mAb (e.g., 1F5), FcγR + Without the cells, there is no CD27 costimulation of T cells. See Figure 4(A). However, BMS-986215mAb is a non-ligand blocking mAb and biological assays were performed to determine its activity. Unlike ligand blocking mAbs such as 1F5, the data show that BMS-986215 was able to costimulate T cells in the presence of CD70 and without FcγR binding. See Figure 4(B).

[0229] BMS-986215 enhances NF-κB and MAPK signaling Primary T cell signaling assays were performed to observe the effect of BMS-986215 on NF-κB and MAPK signaling induction in naive and preactivated human T cells stimulated with anti-CD3 and anti-CD28 Abs. + T cells were stimulated with anti-CD3 and anti-CD28 Abs for 24 hours. Cells were seeded in 384-well plates at 10,000 cells / well and starved for 2 hours before Ab addition. Cells were then treated with BMS-986215, 1F5 or hIgG1 (control) Abs in combination with crosslinkers at a dose / time course and NF-κB and MAPK signaling were analyzed. BMS-986215 has an EC of 0.02 nM to 0.23 nM. 50 It was observed that BMS-986215 induced NF-κB and MAPK signaling. For example, Figure 5(A)-(C) shows the NF-κB signal intensity derived from Ab treatment, and BMS-986215 induced more NF-κB compared to 1F5.

[0230] BMS-986215 enhances T cell proliferation and IFN-γ secretion CD4 + Co-cultures of T cells and CHO-svCD3-CD32a cells were used to assay the effect of BMS-986215 on T cell proliferation and IFN-γ secretion. Briefly, human CD4 + T cells were co-cultured with CHO-svCD3-CD32a cells (CHO cells expressing anti-CD3 OKT3 mAb and single-chain anti-CD3 scFvAb derived from hCD32a-131His(FcγRIIa). Co-cultures were treated with BMS-986215, 1F5 or hIgG1 (control) Abs and T cell proliferation and IFN-γ secretion were analyzed. BMS-986215 was observed to costimulate T cells, with an EC of 0.001 nM to 0.028 nM. 50The mean EC 50 The values ​​were 0.009 nM and 0.0078 nM for proliferation and IFN-γ secretion, respectively. For example, Figures 6(A)-(B) show a much greater induction of proliferation and IFN-γ secretion by BMS-986215 compared to 1F5 in general. ECs associated with T cell proliferation in Figure 6(A) 50 The values ​​were 0.004503 nM and 0.03566 nM for BMS-986215 and 1F5, respectively. EC 50 The values ​​were 0.006235 nM and 0.04017 nM for BMS-986215 and 1F5, respectively. CD4 + CD45RO + Memory T cells or CD4 + CD45RA + Assays were also performed using co-cultures of naive T cells and CHO-svCD3-CD32a cells. The co-cultures were treated with BMS-986215, 1F5 or hIgG1 (control) Abs and analyzed for T cell proliferation and IFN-γ secretion. The results are shown in Figure 7, where BMS-986215 resulted in much higher levels of T cell proliferation and IFN-γ secretion than 1F5. For example, BMS-986215 suppressed CD4 + CD45RO + Memory T cell proliferation and IFN-γ production were measured using EC 50 Values ​​and EC comparison of 1F5 50 The values ​​were 0.073 nM and 0.089 nM, respectively. CD4 from 2 donors + CD25 - Further assays were performed in which T cells were separately co-cultured with B cells, stimulated with Staphylococcal Enterotoxin B (SEB), and treated with BMS-986215 or anti-Keyhole Limpet Hemocyanin Ab, g1F glycoform (KLH-g1f) (each Ab used at 10 μg / ml). The results are shown in Figure 8, where BMS-986215 resulted in a 2.5-fold greater induction of mean IFN-γ secretion than when 100 ng / ml of SEB was used.

[0231] BMS-986215 enhances IL-2 secretion T cells stimulated with SEB were assayed for interleukin-2 (IL-2) production in the presence or absence of cross-linker and soluble BMS-986215. Fresh PBMCs isolated from buffy coats of three donors (donors A–C) were seeded at 100,000 cells / well in tissue culture plates. SEB was added at 80 ng / mL. Cells were treated with soluble anti-human IgG1 (hIgG1), anti-human OX40 (OX40.21), control anti-hCD27 (1F5-IgG1) or BMS-986215 Abs. Abs were titrated 3-fold in the presence or absence of 2.5 μg / mL anti-human Fcγ Ab (Jackson ImmunoResearch, West Grove, PA) as a cross-linker. PBMCs without SEB or Ab treatment (cells only) and PBMCs treated with SEB but not Ab (SEB only) were used as controls. The results are shown in Figure 9. PMBCs from donors A and B were treated without (Figure 9(A) and (C)) or with (Figure 9(B) and (D)) soluble Ab cross-linking. PMBCs from donor C (Figure 9(E)) were treated with soluble Ab cross-linking only. The results show that IL-2 secretion is increased by more than 50% from SEB-stimulated human PBMCs in the presence of soluble cross-linker BMS-986215 compared to hIgG1. Further assays were performed to determine the effect of BMS-986215 on IL-2 production when combined with PD-1 blockade. Briefly, fresh PBMCs isolated from buffy coats of two donors (donors A and B) were seeded in tissue culture plates at 100,000 cells / well. SEB was added at 80 ng / mL. Cells were treated with 5 μg / ml of soluble anti-human IgG1 (hIgG1), 1F5 or BMS-986215 Abs alone or titrated in tandem with 0.1 μg / ml of nivolumab for PD-1 blockade or ipilimumab for CTLA-4 blockade. PBMCs without SEB or Ab treatment (cells only) were used as controls. The results are shown in Figure 10. IL-2 release by BMS-986215 was increased 2-fold when combined with PD-1 blockade compared to without PD-1 blockade.

[0232] BMS-986215 reduces the suppression of responder T cells by regulatory T cells Forkhead box P3 (Foxp3), also known as regulatory T cells or Tregs + CD4 + T cells suppress immune responses and allow tumors to evade immune surveillance. Generally, Tregs express CD45RA or CD45RO. Tregs can suppress responder T cells (Tresp). The effect of BMS-986215 on Treg-mediated suppression was examined. In the first assay, CD45RA + Tregs were seeded in tissue culture plates at 50,000 cells / well and diluted 1:2 with 50,000 labeled Tresp / well. Next, 5,000 monocyte derived dendritic cells (MDDC) were added per well along with 0.3 μg / mL soluble muromonab-CD3 (OKT3), an anti-human CD3 mAb. Soluble BMS-986215 or control IgG1 Ab was then added at 10 μg / mL. Cells were incubated for 96 hours at 37°C to determine Treg:Tresp ratios and CD4 + The percentage proliferation of Tresp was determined by fluorescence-activated cell sorting (FACS). The results are shown in Figure 11(A). Co-culture of CD4+ cells in the presence of MDDC and soluble OKT3. + Treg-mediated suppression of Tresp was largely reversed by >70% by BMS-986215 in two experiments compared with control IgG1 Ab. In the second assay, 96-well U-bottom plates were coated with 3 μg / mL OKT3 and 10 μg / mL BMS-986215, 1F5 or anti-KLH Ab. Then, 20,000 Tresp per well were added. No additional IL-2 was added. Then, 1 μg / mL soluble anti-CD28 Ab was added. Cells were cultured for 7 days to detect Tresp and Foxp3. +The number of Treg cells was determined. The results are shown in Figure 11 (B) and (C). BMS-986215 inhibited Foxp3 + This resulted in a greater expansion of Tresp than Treg.

[0233] BMS-986215 induces moderate ADCC activity and low levels of Fc effector function BMS-986215, 1F5 or IgG1 (control) Ab was added to co-cultures of primary natural killer (NK) cells as effector cells and activated T cells or Ramos cells as target cells at a 10:1 ratio of effector to target (n=8). The results are shown in Figure 12 for T cells. BMS-986215 induced 20-44% lysis of T cells or Ramos cells, indicating a moderate induction of ADCC activity. Fc effector function assays demonstrated low levels of ADCP, CDC and C1q binding activity (data not shown). BMS-986215, in combination with or without anti-PD-1 mAb, did not mediate spontaneous cytokine secretion in fresh whole blood samples from eight healthy human donors (data not shown).

[0234] Agonistic activity of BMS-986215 on human T cells is enhanced by CD70 CD4 + Co-cultures of T cells and CHO-OKT3 cells were used to assay the effect of BMS-986215 on T cell proliferation and IFN-γ secretion in the absence of FcγR. + T cells were co-cultured with CHO-OKT3 cells (CHO cells expressing single chain anti-CD3 scFvAb derived from anti-CD3 OKT3 mAb). Co-cultures were treated with BMS-986215, 1F5 or hIgG1 (control) Ab with or without 10 μg / ml soluble human CD70 (shCD70) and T cell proliferation and IFN-γ secretion were analyzed. The assay is shown diagrammatically in Figure 13(A). The results are depicted in Figure 13, which shows that BMS-986215, but not 1F5, exhibited weak agonist activity by enhancing T cell proliferation (B) and IFN-γ secretion (C) in the absence of FcγR and without combination with shCD70. BMS-986215, but not 1F5, enhanced human T cell activation as measured by induction of T cell proliferation (D) and IFN-γ secretion (E) by soluble CD70 protein.

[0235] Example 6 Preclinical pharmacokinetics, receptor occupancy prediction and toxicity studies Pharmacokinetics and toxicity of BMS-986215 in cynomolgus monkeys Table 6 summarizes the pharmacokinetic (PK) parameters of BMS-986215 following single intravenous administration of 0.2 mg / kg and 4 mg / kg to cynomolgus monkeys. C was observed approximately 6 hours after the 0.2 mg / kg dose and approximately 24 hours after the 4 mg / kg dose. max With a 20-fold increase in the dose tested, the total body clearance (CLT) decreased from 2.2 mL / h / kg to 0.39 mL / h / kg. As a result, the half-life (t 1 / 2 The EC of BMS-986215 for binding to cynomolgus T cells was increased from 2.2 to 14 days. In addition, anti-drug Abs (ADAs) were detected in serum samples at 0.2 mg / kg. This suggests target-mediated drug deposition (TMDD), and ADAs may have led to the nonlinear PK observed with BMS-986215 in monkeys. 50 is approximately 5 ng / mL. At each dose level, BMS-986215 was administered intravenously at 1.6 mL / kg to three monkeys; two monkeys received vehicle for pharmacodynamic comparisons. Evaluation criteria included clinical observations, blood tests, PK, CD27 receptor occupancy (RO), circulating CD27 levels, lymphocyte subset analysis, cytokine levels, and ADA determination. Blood samples were obtained pre-dose and at various intervals up to 42 days post-dose. Overall, there were no adverse findings, including no adverse clinical observations and no changes in blood test parameters. There was a time- and dose-dependent increase in circulating CD27 levels. CD4 + and CD8+ High BMS-986215CD27 RO on T cells (>90%) was detected at 0.2 mg / kg for approximately 7 days and at 4 mg / kg for the duration of the experiment (42 days). + , Treg (Foxp3 + / CD4 + ), CD8 + T, CD20 + There were no significant changes in circulating lymphocyte subsets, including B and NK cells, or in circulating cytokine levels. [Table 6] PK parameters were calculated using non-compartmental methods. Values ​​are means ± SD. (N=3)

[0236] Pharmacokinetic / Pharmacodynamic Modeling of Whole Blood CD27 Receptor Occupancy of BMS-986215 in Monkeys CD27 RO was determined from whole blood in monkey PK / RO studies and was evaluated using the following E max Models were used to link with serum BMS-986215 concentrations.

number

[0237] Human pharmacokinetics and receptor occupancy prediction Human PK parameters for BMS-986215 were estimated from the monkey data. Due to non-linear PK, human clearance is predicted taking into account both non-target and target-mediated elimination processes as follows:

number

number

[0238] Example 7 CD27 and CD70 expression in human cancers Immunohistochemical analysis of CD27 expression in multiple human cancers To identify potential disease indications, the expression profile and distribution of CD27 in multiple cancer types was examined by immunohistochemistry (IHC) on regular full-size formalin-fixed paraffin-embedded (FFPE) sections using a commercially available anti-CD27 mAb, clone 137B4. Six tumor types and 22–55 samples from each tumor type were subjected to IHC staining, namely breast adenocarcinoma (BrC, n=35), cervical carcinoma (CC, n=23), colorectal adenocarcinoma (CRC, n=22), head and neck squamous cell carcinoma (HNSCC, n=46), hepatocellular carcinoma (HCC, n=55), and ovarian adenocarcinoma (OvC, n=27). Additionally, in many cases (n=13–37 samples), CD70 expression was assessed on adjacent sections using a commercially available anti-CD70 mAb. CD27 + Staining was observed in subsets of tumor infiltrating lymphocytes (TILs) in all tumor types tested. In general, CD27 + TILs are CD3 + The pattern was very similar to that of TIL. + TILs are CD3 + The abundance of CD27 in HNSCC and CC was significantly higher than that in TILs, suggesting that CD27 expression is seen in B cells in addition to T cells. + TILs had the highest levels, followed by CRC, BrC and HCC, while OvC showed the lowest levels. CD70 + Staining was observed in a small fraction of TILs and was CD27 + Among the six tumor types, CC, HCC, and CRC expressed higher levels of CD70 than HNSCC, BrC, and OvC. + In addition to detecting CD70 expression in TILs, CD70 + Labeling was identified in tumor cells in approximately 30% of CC and 15% of OvC cases. + In most tumor cells, expression was extremely low. The cutoff was 10% tumor cells. + Using this method, approximately 15% of CC cases were considered positive.

[0239] TCGA analysis of CD27 and CD70 expression As observed with other co-stimulatory and checkpoint receptors, The Cancer Genome Atlas (TCGA) database shows that CD27 is expressed at the RNA level in most tumors in a manner that correlates with other T cell signatures. Lymphomas are the exception, where CD27 is expressed by malignant B cells. CD27 expression itself does not correlate significantly with survival, with the exception of cervical and head and neck cancers, where its high expression correlates with improved survival (p=0.00001 and 0.01, respectively). After B lymphomas, the tumors that express CD27 the most are thyroid, renal cell, lung and head and neck. In the cervical cancer TCGA dataset, high CD70 expressers had better survival than low CD70 expressers in the high CD27 expressing population, suggesting that increased CD27 agonism by BMS-986215 conferred benefit.

[0240] Soluble CD27 It has been suggested that soluble CD27 (sCD27) may be a marker of immune cell activation that could be tracked as a pharmacodynamic (PD) marker in clinical trials (Huang et al., 2013). Specifically, sCD27 levels were tested in serum from patients with stage I-IV melanoma, CRC, RCC, and lung cancer, and compared to non-cancer controls from normal healthy volunteer (NHV) serum samples. A subset of cancer patients had 2-3 times higher serum sCD27 levels (2000-15,000 pg / mL) compared to NHV (2000-7000 pg / mL). This is different from and higher than in the literature, where sCD70 was reported as lower in cancer patients (150 pg / mL) versus NHV (500 pg / mL). We found that sCD27 levels increased in anti-CD27 mAb-treated cynomolgus monkeys, even though assays for detecting peripheral T cell activation did not show increased activation or proliferation. Therefore, the increase in sCD27 seen after anti-CD27 treatment is related to FcRn-mediated circulating T cells. 1 / 2 This could be the result of exceeding the yen.

[0241] Example 8 Mouse tumor assay To demonstrate that non-ligand blocking CD27 mAbs have anti-tumor activity in mouse tumor models, rat anti-mouse CD27 mAbs were generated as described in Example 1, and clone 8H5 was selected as a non-ligand blocking agonist Ab to serve as a surrogate for BMS-986215. This surrogate anti-CD27 Ab binds mCD27 with 0.25 nM affinity, does not block mouse CD70 binding, shows agonist activity in mouse splenic T cells, and was engineered with mIgG1 and mIgG2a Fc variants. As such, 8H5 is a suitable surrogate for BMS-986215. Clone 8H5 showed antitumor activity in both solid and hematological mouse tumor models, especially in combination with PD-1 checkpoint blockade. See, for example, FIG. 14 showing the results in the CT26 mouse colon carcinoma model and Table 7 summarizing the results obtained in the SA1N fibrosarcoma, CT26 and EGT T-cell lymphoma mouse models. The mIgG1 isotype of 8H5 showed only modest antitumor activity in the CT26 model with 21% tumor growth inhibition (TGI; FIG. 14(B)) compared to the mIgG2a and mIgG1, D265A controls (FIG. 14(A)). The mIgG2a isotype of 8H5 showed slightly higher, but still moderate, antitumor activity (FIG. 14(C)), and neither the mIgG1 nor the IgG2a isotypes resulted in any tumor-free (TF) mice in the 10 mice treated. The anti-tumor activity of 4H2, an anti-mPD-1 Ab with mIgG1-D265A isotype, was also tested. 4H2 is a chimeric rat-mouse anti-mPD-1 Ab constructed from a rat IgG2a anti-mouse PD-1 Ab in which the Fc portion was replaced with the Fc portion of a different mouse IgG isotype (WO2006 / 121168). In the CT26 tumor model, 4H2 showed moderate anti-tumor activity, resulting in 27% TGI and 2 out of 10 TF mice treated (Figure 14(D)). This anti-PD-1 activity was enhanced by 8H5, as shown by the combination of 8H5, mIgG1 resulting in 70% TGI and 3 / 10 TF mice (Figure 14(E); Table 7) and the combination of 8H5, mIgG2a resulting in 76% TGI and 4 / 10 TF mice (Figure 14(F); Table 7). This indicates that anti-CD27 interacts synergistically with checkpoint inhibitors such as anti-PD-1 to reduce cancer cell proliferation in vivo. Combinations of these Abs are synergistic if the antitumor effect of the combination is greater than the sum of the levels of inhibition exhibited by each individual Ab. Varying the isotype of Abs specific for T cell surface receptors (both costimulatory and coinhibitory) may potentially alter the antitumor activity potential of these Abs. Potent antitumor activity was observed in the SA1N fibrosarcoma model when tumor-bearing mice were treated with single agent 8H5 expressed as mIgG1 and mIgG2a (activating FcγR binder) mAb. The mIgG1 isotype of 8H5 demonstrated potent antitumor activity, resulting in 52% TGI and 8 TF mice out of 12 treated (Table 7). The mIgG2a isotype of 8H5 was even more potent, resulting in 98% TGI and 11 TF mice out of 12 treated (Table 7). Monotherapy activity was driven by CD8 expression at the tumor site. + Correlated with T cell expansion and Treg depletion. Recent preclinical studies have shown that agonistic CD27 Abs, when combined with PD-1 blockade, inhibit antigen-specific CD8 +It has been shown to increase T cell activity, which is effective in eradicating tumors (Ahrends et al., 2016). Anti-mouse CD27 isotype in combination with anti-mouse PD-1 showed similar levels of anti-tumor activity (70% and 76% TGI, respectively) and total tumor-free (TF) mice (3 / 10 and 4 / 10 TF, respectively) as measured by TGI on day 25 in the CT26 model. See Table 7. In addition to partial Treg depletion, CD4 + and CD8 + T cell numbers were decreased in the periphery (e.g., blood and spleen) compared with the tumor site. + T cell proliferation was observed with mIgG1, whereas mIgG2a appeared to be more potent in Treg depletion. These data suggest that anti-CD27 isotype variants are primarily responsible for Treg depletion (mIgG2a) and / or CD8 + This suggests that T cell expansion (mIgG1) results in anti-tumor activity. These studies demonstrate that CD27 agonism and PD-1 blockade result in reduced tumor burden and increased survival in syngeneic tumor models. See Table 7.

[0242] Neither the anti-mCD27 mAb 8H5, mIgG1 isotype, nor the mIgG2a isotype showed antitumor activity in murine EG7 T cell lymphoma cells, whereas the anti-PD1 mAb 4H2 showed minimal activity (Table 7). Nevertheless, 8H5 mIgG1 in combination with 4H2 synergistically resulted in 44% TGI and 1 / 10 TF mice in this lymphoma model (Table 7). [Table 7] a Data from AJ mice b Data from BALB / c mice c Data from C57BL / 6 mice Abbreviations: TGI = tumor growth inhibition 21-28 days after inoculation; TF = tumor-free at the end of the study.

[0243] References Abhinandan KR, Martin AC (2008) Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains. Mol Immunol 45:3832-9. Ahrends T, Babala N, Xiao Y et al. (2016) CD27 agonism plus PD-1 blockade recapitulates CD4+ T cell help in therapeutic anti-cancer vaccination. Cancer Res 76(10):2921-31. Al-Lazikani, Lesk AM, Chothia C (1997) Standard conformations for the canonical structures of immunoglobulins. J Mol Biol 273(4):927-48. Baitsch L, Legat A, Barba L, Fuertes Marraco SA, Rivals JP et al. (2012) Extended co-expression of inhibitory receptors by human CD8 T-cells depending on differentiation, antigen-specificity and anatomical localization. PloS One 7(2): e30852. Brahmer JR, Drake CG, Wollner I, Powderly JD, Picus J et al. (2010) Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates. J Clin Oncol 28:3167-75. Brahmer JR, Hammers H, Lipson EJ (2015) Nivolumab: targeting PD-1 to bolster antitumor immunity. Future Oncol 11(9):1307-26. Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL et al. (2012) Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med 366:2455-65. Buchan SL, Manzo T, Flutter B et al. (2015) OX40- and CD27-mediated costimulation synergizes with anti-PD-L1 blockade by forcing exhausted CD8+ T cells to exit quiescence. J Immunol 194:125-33. Buchan SL, Fallatah M, Thirdborough SM, Taraban VY, Rogel A et al. (2018) PD-1 blockade and CD27 stimulation activate distinct transcriptional programs that synergize for CD8+ T-cell driven anti-tumor immunity. Clin Cancer Res 24(10):2383-94. Bullock T, McClintic H, Jeong S et al. (2014) Immune correlates of varlilumab (CDX-1127) treated cancer patients are consistent with CD27 costimulatory activity. Society for Immunotherapy of Cancer (SITC) 29th Annual Meeting, November 6-9, 2014, National Habor, MD; Celldex Poster P115. Callahan M, Postow MA, Wolchok JD (2016) Targeting T cell co-receptors for cancer therapy. Immunity 44(5):1069-78. Chakravarthi BVSK, Nepal S, Varambally S (2016) Genomic and epigenomic alterations in cancer. Am J Pathol 186(7): 1724-35. Chen DS, Mellman I (2013) Oncology meets immunology: the cancer-immunity cycle. Immunity 39(1),1-10. Chothia C, Lesk AM (1987) Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol 196:901-17. Chothia C, Lesk AM, Tramontano A, Levitt M, Smith-Gill JS et al. (1989) Conformations of immunoglobulin hypervariable regions. Nature 342:877-83. De Colvenaer V, Taveirne S, Delforche M et al. (2011) CD27-deficient mice show normal NK-cell differentiation but impaired function upon stimulation. Immunol Cell Biol 89:803-11. Dolfi DV, Boesteanu AC, Petrovas C et al. (2008) Late signals from CD27 prevent Fas-dependent apoptosis of primary CD8+T cells. J. Immunol 180:2912-21. Drugs.com - Opdivo Approval History: https: / / www.drugs.com / history / opdivo.html, last accessed April 1, 2019. Farkona et al. (2016) Cancer immunotherapy: the beginning of the end of cancer? BMC Medicine 14:73. French RR, Taraban VY, Crowther GR et al. (2007) Eradication of lymphoma by CD8 T cells following anti-CD40 monoclonal antibody therapy is critically dependent on CD27 costimulation. Blood 109:4810-5. Gorelik L, Avgerinos G, Kunes Y, Marasco WA (2017) Preclinical characterization of a novel fully human IgG1 anti-PD-L1 mAb CK-301. In: Proceedings of the American Association for Cancer Research (AACR) Annual Meeting, Apr 1-5, 2017, Cancer Res 77(13 Suppl): Abstract No. 4606. Guo L, Zhang H, Chen B (2017) Nivolumab as Programmed Death-1 (PD-1) inhibitor for targeted immunotherapy in tumor. J Cancer 8(3):410-6. He LZ, Prostak N, Thomas LJ et al. (2013) Agonist anti-human CD27 monoclonal antibody induces T cell activation and tumor immunity in human CD27-transgenic mice. J Immunol 191:4174-83. Hendriks J, Gravestein LA, Tesselaar K et al. (2000) CD27 is required for generation and long-term maintenance of T cell immunity. Nat Immunol 1:433-40. Herbst RS, Soria JC, Kowanetz M, Fine GD, Hamid O et al. (2014) Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature 515: 563-7. Hollinger P, Hudson PJ (2005) Engineered antibody fragments and the rise of single domains. Nature Biotech 23(9):1126-36. Huang J, Jochems C, Anderson AM et al. (2013) Soluble CD27-pool in humans may contribute to T cell activation and tumor immunity. J. Immunol. 190:6250-8. Huang R, Chen G (2014) Higher order structure characterization of protein therapeutics by hydrogen / deuterium exchange mass spectrometry. Analytical and Bioanalytical Chemistry 406:6541-58. Iwai Y, Hamanishi J, Chamoto K, Honjo T (2017) Cancer immunotherapies targeting the PD-1 signaling pathway. J Biomed Sci 24(1):26. Jones LM, Sperry JB, Carroll JA, Gross ML (2011) Fast photochemical oxidation of proteins for epitope mapping. Anal Chem 83:7657-61. Kabat EA, Wu TT, Bilofsky H, Reid-Miller M, Perry H (1983) Sequences of Proteins of Immunological Interest. Bethesda: National Institute of Health; 1983. 323 Kabat EA, Wu TT, Perry H, Gottesman K, Foeller C (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242. Kabat EA, Wu TT, Reid-Miller M, Perry H, Gottesman K (1987) Sequences of Proteins of Immunological Interest, Fourth Edition, U.S. Government Printing Office, NIH Publication No. 165-492. Kamta J, Chaar M, Ande A, Altomare DA, Ait-Oudhia S (2017) Advancing cancer therapy with present and emerging immuno-oncology approaches. Front Oncol 18(7):64. Kaufman RJ, Sharp PA (1982) Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary DNA gene. Mol Biol 159:601-21. Lefranc MP, Pommie C, Ruiz M, Giudicelli V, Foulquier E et al. (2003) IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol 27:55-77. Lesokhin AM, Callahan MK, Postow MA, Wolchok JD (2015) On being less tolerant: enhanced cancer immunosurveillance enabled by targeting checkpoints and agonists of T cell activation. Sci Transl Med 7(280):280sr1. Lipson EJ, Sharfman WH, Drake CG, Wollner I, Taube JM et al. (2013) Durable cancer regression off-treatment and effective reinduction therapy with an anti-PD-1 antibody. Clin Cancer Res 19:462-8. Liu SY, Wu YL (2017) Ongoing clinical trials of PD-1 and PD-L1 inhibitors for lung cancer in China. J Hematol Oncol 10(1):136. Mahoney KM, Rennert PD, Freeman GJ (2015) Combination cancer immunotherapy and new immunomodulatory targets. Nat Rev Drug Discov 14(8):561-84. Martin A, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: a combined algorithm. Proc Natl Acad Sci USA 86(23):9268-72. MacCallum RM., Martin ACR, Thornton JT (1996) Antibody-antigen interactions: contact analysis and binding site topography. J Mol Biol 262:732-45. Mellman I, Coukos G, Dranoff (2011) Cancer immunotherapy comes of age. Nature 480: 480-9. Munitic I, Kuka M, Allam A et al. (2013) CD70 deficiency impairs effector CD8 T cell generation and viral clearance but is dispensable for the recall response to lymphocytic choriomeningitis virus. J Immunol 190:1169-79. Olafsen T, Wu AM (2010) Antibody vectors for imaging. Semin Nucl Med 40(3):167-81. Ott PA, Hodi FS, Kaufman HL, Wigginton JM, Wolchok JD (2017) Combination immunotherapy: a road map. J Immunother Cancer 5:16. Pardoll DM (2012) The blockage of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 12: 252-64. Organon NV, PCT Publication WO 2008 / 156712, published December 24, 2008. Amplimmune, Inc., PCT Publication WO 2012 / 145493, published October 26, 2012. Bristol-Myers Squibb Co., PCT Publication WO 2013 / 173223, published November 21, 2013. AnaptysBio, Inc., PCT Publication WO 2014 / 179664, published November 6, 2014. Sorrento Therapeutics, Inc., PCT Publication WO 2014 / 194302, published December 4, 2014. PCT Publication WO 2014 / 206107, published December 31, 2014, by Shanghai Junshi Biosciences Inc. Beigene, Ltd., PCT Publication WO 2015 / 035606, published March 19, 2015. PCT Publication WO 2015 / 085847, published June 18, 2015, by Shanghai Hengrui Pharmaceutical Co., Ltd. Regeneron Pharmaceuticals, Inc., PCT Publication WO 2015 / 112800, published July 30, 2015. PCT Publication WO 2015 / 112900, published July 30, 2015 by Dana-Farber Cancer Institute, Inc. and Novartis AG PCT Publication WO 2016 / 106159 to Enumeral Biomedical Holdings, Inc., published June 30, 2016. PCT Publication WO 2006 / 121168, published November 16, 2006 by ONO Pharmaceutical Co., Ltd. and Medarex, Inc. Cytomx Therapeutics, Inc., PCT Publication WO 2016 / 149201, published September 22, 2016. PCT Publication WO 2016 / 197367, published December 15, 2016 by Wuxi Biologics (Shanghai) Co. Ltd. PCT Publication WO 2017 / 020291, published February 9, 2017, by Wuxi Biologics (Shanghai) Co. Ltd. PCT Publication WO 2017 / 020858, published February 9, 2017, by Wuxi Biologics (Shanghai) Co. Ltd. PCT Publication WO 2017 / 024465 by Innovent Biologics (Suzhou) Co., Ltd., published February 16, 2017 Wuxi Biologics (Cayman) Inc. PCT Publication WO 2017 / 024515, published February 16, 2017. PCT Publication WO 2017 / 025016, published February 16, 2017, by Innovent Biologics (Suzhou) Co., Ltd. Wuxi Biologics (Cayman) Inc. PCT Publication WO 2017 / 025051, published February 16, 2017. Eli Lilly and Co. PCT Publication WO 2017 / 034916, published March 2, 2017. Agenus Inc., PCT Publication WO 2017 / 040790, published March 9, 2017. Macrogenics, Inc., PCT Publication WO 2017 / 106061, published June 22, 2017. Armo Biosciences, Inc., PCT Publication WO 2017 / 123557, published July 20, 2017. Innovent Biologics (Suzhou) Co., Ltd., PCT Publication WO 2017 / 132827, published August 10, 2017. Innovent Biologics (Suzhou) Co., Ltd., PCT Publication WO 2017 / 133540, published August 10, 2017. Pianko MJ, Liu Y, Bagchi S, Lesokhin AM (2017) Immune checkpoint blockade for hematologic malignancies: a review. Stem Cell Investig 4:32. Ramakrishna V, Sundarapandiyan K, Zhao B et al. (2015) Characterization of the human T cell response to in vitro CD27 costimulation with varlilumab. J Immunother Cancer 3:37. Roberts DJ, Franklin NA, Kingeter LM et al. (2010) Control of established melanoma by CD27 stimulation is associated with enhanced effector functionおよびpersistence,およびreduced PD-1 expression, of tumor infiltrating CD8+ T cells. J Immunother 33:769-79. Sakanishi T, Yagita H (2010) Anti-tumor effects of depletingおよびnon-depleting anti-CD27 mAbs in immune-competent mice. Biochem Biophys Res Commun 393:829-35. Salzer E, Daschkey S, Choo S et al. (2013) Combined immunodeficiency with life-threatening EBV-associated lymphoproliferative disorder in patients lacking functional CD27. Haematologica 98:473-8. Sanchez PJ, McWilliams JA, Haluszczak C et al. (2007) Combined TLR / CD40 stimulation mediates potent cellular immunity by regulating dendritic cell expression of CD70 in vivo. J Immunol 178:1564-72. Sanborn RE, Pishvaian MJ, Kluger HM, Callahan MK et al. (2017) Clinical results with combination of anti-CD27 agonist antibody, varlilumab, with anti-PD1 antibody nivolumab in advanced cancer patients. J Clin Oncol 35(15) Suppl.:3007. Taraban VY, Rowley TF, Al-Shamkhani A (2004) Cutting edge: a critical role for CD70 in CD8 T cell priming by CD40-licensed APCs. J Immunol 173:6542-6. U.S. Patent 6,808,710 to Wood et al., issued October 26, 2004 U.S. Patent 7,488,802 to Collins et al., published February 10, 2009 U.S. Patent 7,943,743, published May 17, 2011, to Korman et al. U.S. Patent 7,767,429, published August 3, 2010, to Bookbinder et al. U.S. Patent 8,008,449 to Korman et al., published August 30, 2011 U.S. Patent 8,168,757, published May 1, 2012, to Finnefrock et al. U.S. Patent 8,217,149, published July 10, 2012, to Irving et al. U.S. Patent 8,354,509 to Carven et al., published January 15, 2013 U.S. Patent 8,779,108, published July 15, 2014, to Queva et al. U.S. Patent 9,175,082 to Zhou et al., published November 3, 2015 U.S. Patent 9,205,148 to Langermann et al., published December 8, 2015 U.S. Patent 9,624,298 to Nastri et al., published April 18, 2017 U.S. Patent 9,938,345 to Papadopoulos et al., published April 10, 2018 U.S. Patent 9,987,500 to Papadopoulos et al. published June 5, 2018 U.S. Publication 2015 / 0079109 by Li et al., published March 19, 2015 US Publication 2016 / 0272708 by Chen et al., published September 22, 2016 van Gisbergen KP, Klarenbeek PL, Kragten NA, et al. (2011) The costimulatory molecule CD27 maintains clonally diverse CD8 (+) T cell responses of low antigen affinity to protect against viral variants. Immunity 35:97-108. van Montfrans JM, Hoepelman AI, Otto S et al. (2012) CD27 deficiency is associated with combined immunodeficiency and persistent symptomatic EBV viremia. Allergy Clin Immunol 129:787-93. Wajant H (2016) Therapeutic targeting of CD70 and CD27. Expert Opin Ther Targets 15:1-15. Wang C, Thudium KB, Han M, Wang XT et al. (2014) In vitro characterization of the anti-PD-1 antibody nivolumab, BMS-936558, and in vivo toxicology in non-human primates. Cancer Imm Res 2(9):846-56. Weber J (2010) Immune checkpoint proteins: a new therapeutic paradigm for cancer-preclinical background: CTLA-4 and PD-1 blockade. Semin Oncol 37(5): 430-9. Wei H, Mo J, Tao L, Russell RG, Tymiak AA et al. (2014) Hydrogen / deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications. Drug Discov Today 19:95-102. Wolchok JD, Weber JS, Maio M, Neyns B, Harmankaya K et al. (2013) Four-year survival rates for patients with metastatic melanoma who received ipilimumab in phase II clinical trials. Ann Oncol 24(8):2174-80. Wu TT, Kabat EA (1970) An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J Exp Med 132:211-50. Yan Y, Chen G, Wei H, Huang R, Mo J et al. (2014) Fast photochemical oxidation of proteins (FPOP) maps the epitope of EGFR binding to adnectin. J Am Soc Mass Spec 25:2084-92. Yao S, Zhu Y, Chen L (2013) Advances in targeting cell surface signalling molecules for immune modulation. Nature Rev Drug Discov 12:130-46. Zhang F, Wei H, Wang X, Bai Y, Wang P et al. (2017) Structural basis of a novel PD-L1 nanobody for immune checkpoint blockade. Cell Discov 3:17004.

Claims

1. 1. An agonistic monoclonal antibody or antigen-binding portion thereof that specifically binds to human CD27, the sequence of which is set forth in SEQ ID NO:1, as measured by surface plasmon resonance (SPR) or flow cytometry, and does not block binding of CD27 to its CD70 ligand, said antibody or antigen-binding portion thereof comprising: (a) a heavy chain variable region (V) comprising a CDR1 comprising consecutively linked amino acids having the sequence set forth in SEQ ID NO:2, a CDR2 comprising consecutively linked amino acids having the sequence set forth in SEQ ID NO:3, and a CDR3 comprising consecutively linked amino acids having the sequence set forth in SEQ ID NO:4; Ｈ ), and (b) a light chain variable region (V) comprising a CDR1 having consecutively linked amino acids having the sequence set forth in SEQ ID NO:5, a CDR2 having consecutively linked amino acids having the sequence set forth in SEQ ID NO:6, and a CDR3 having consecutively linked amino acids having the sequence set forth in SEQ ID NO:7; Ｌ ) An agonist monoclonal antibody or an antigen-binding portion thereof, comprising:

2. The agonistic monoclonal antibody or antigen-binding portion thereof of claim 1, which specifically binds to an epitope comprising two discontinuous binding regions of human CD27, TPAPKSCPERHYWAQGKLCCQ (SEQ ID NO: 15) and DCDQHR (SEQ ID NO: 18), as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and / or fast photochemical oxidation of proteins (FPOP) epitope mapping.

3. (a) a K for human CD27 of 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 1 nM to 100 nM, 10 nM to 70 nM, 10 nM to 50 nM, or 40 nM to 45 nM Ｄ and / or (b) induces NF-κB and MAPK signaling with an EC50 of 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.005 nM to 0.5 nM, 0.01 nM to 0.4 nM, or 0.02 nM to 0.25 nM in naive human T cells stimulated with anti-CD3 and anti-CD28 antibodies; 3. An agonistic monoclonal antibody or an antigen-binding portion thereof according to claim 1 or 2.

4. V comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:8 Ｈ and V comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:

9. Ｌ The agonist monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 3, comprising:

5. An agonistic monoclonal antibody or antigen-binding portion thereof that specifically binds to human CD27 as measured by surface plasmon resonance (SPR) or flow cytometry, does not block binding of CD27 to its CD70 ligand, and cross-competes with the agonistic monoclonal antibody or antigen-binding portion thereof described in claim 4 for binding to human CD27.

6. (a) does not specifically bind to rat CD27 and / or mouse CD27 as measured by SPR or flow cytometry; (b) does not specifically bind to one or more TNF receptor superfamily members selected from the group consisting of CD30, HVEM, DR5, 4-1BB, CD40, OX40, GITR, and any combination thereof; (c) does not specifically bind to one or more human tissues at a concentration of up to 10 μg / mL, wherein the human tissue is selected from the group consisting of thyroid, lung, skin, uterus, prostate, liver, kidney, pancreas, adrenal gland, pituitary gland, placenta, testis, cerebrum, cerebellum, heart, peripheral nerve, and any combination thereof; (d) it can induce NF-κB and MAPK signaling in naive and preactivated human T cells stimulated with anti-CD3 and anti-CD28 antibodies; (e) capable of inducing proliferation and / or IFN-γ secretion in a CHO-svCD3-CD32A assay; (f) CD4 ＋ CD45RO ＋ It can induce the proliferation of memory T cells; (g) is capable of increasing IL-2 release from Staphylococcal enterotoxin B (SEB)-stimulated human peripheral blood mononuclear cells (PBMC); (h) capable of increasing IL-2 release by at least 2-fold when combined with an anti-PD-1 antibody; (i) Monocyte-derived dendritic cells (MDDCs) and CD4+ cells co-cultured in the presence of soluble OKT3 ＋ Reverse Treg-mediated suppression of responder T cells; and (j) the ability to enhance human T cell proliferation and induction of IFN-γ secretion by soluble CD70, or an antigen-binding portion thereof, of any one of claims 1 to 5.

7. A monoclonal antibody or an antigen-binding portion thereof: (a) binds to human T cells with an EC50 of 0.1 nM or less, 0.09 nM or less, 0.08 nM or less, 0.07 nM or less, 0.06 nM or less, 0.05 nM or less, 0.04 nM or less, 0.01 nM to 0.1 nM, 0.025 nM to 0.075 nM, or 0.03 nM to 0.06 nM; (b) binds to cynomolgus T cells with an EC50 of 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, 0.1 nM or less, 0.01 nM to 0.5 nM, 0.025 nM to 0.4 nM, 0.04 to 0.3 nM, or 0.06 to 0.2 nM; and / or (c) comprising a heavy chain constant region that is of a human IgG1, IgG2, IgG3, or IgG4 isotype; An agonistic monoclonal antibody or an antigen-binding portion thereof according to any one of claims 1 to 6.

8. (a) is a human antibody or fragment thereof; (b) is a humanized antibody or fragment thereof; (c) is a chimeric antibody or fragment thereof; (d) a human CD27 agonist; (e) does not specifically bind to rat CD27 and / or mouse CD27 as measured by SPR or flow cytometry; (f) does not specifically bind to one or more TNF receptor superfamily members selected from the group consisting of CD30, HVEM, DR5, 4-1BB, CD40, OX40, GITR, and any combination thereof; (g) does not specifically bind to one or more human tissues at a concentration of up to 10 μg / mL, wherein the human tissue is selected from the group consisting of thyroid, lung, skin, uterus, prostate, liver, kidney, pancreas, adrenal gland, pituitary gland, placenta, testis, cerebrum, cerebellum, heart, peripheral nerve, and any combination thereof; (h) induces NF-κB and MAPK signaling in naive and pre-activated human T cells stimulated with anti-CD3 and anti-CD28 antibodies with an EC50 of 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.005 nM to 0.5 nM, 0.01 nM to 0.4 nM, or 0.02 to 0.25 nM; (i) induces proliferation and / or IFN-γ secretion in a CHO-svCD3-CD32A assay with an EC50 of 0.05 nM or less, 0.04 nM or less, 0.03 nM or less, 0.0005 nM to 0.05 nM, 0.0005 nM to 0.04 nM, 0.0005 nM to 0.03 nM, 0.001 nM to 0.05 nM, 0.001 nM to 0.04 nM, or 0.001 nM to 0.03 nM; (j) CD4 ＋ CD45RO ＋ induces memory T cell proliferation with an EC50 of 0.01 nM or less, 0.009 nM or less, 0.008 nM or less, 0.007 nM or less, 0.006 nM or less, 0.005 nM or less, 0.001 nM to 0.01 nM, 0.002 nM to 0.008 nM, 0.003 nM to 0.007 nM, or 0.004 nM to 0.006 nM; (k) increasing IL-2 release from SEB-stimulated human PBMCs by more than 50% in the presence of a crosslinker or at least 2-fold when combined with an anti-PD-1 antibody; (l) Co-culture CD4 in the presence of MDDC and soluble OKT3 ＋ reverse Treg-mediated suppression of responder T cells by at least 70%; or (m) enhancing the induction of human T cell proliferation and IFN-γ secretion by soluble CD70 with an EC50 of 0.01 nM or less, 0.009 nM or less, 0.008 nM or less, 0.007 nM or less, 0.006 nM or less, 0.005 nM or less, 0.001 nM to 0.01 nM, 0.002 nM to 0.008 nM, or 0.003 nM to 0.005 nM, and enhancing the induction of IFN-γ secretion with an EC50 of 0.01 nM or less, 0.009 nM or less, 0.008 nM or less, 0.007 nM or less, 0.001 nM to 0.01 nM, 0.002 nM to 0.008 nM, or 0.005 nM to 0.007 nM; An agonistic monoclonal antibody or an antigen-binding portion thereof according to any one of claims 1 to 7.

9. (a) a full-length antibody of the human IgG1, IgG2, IgG3 or IgG4 isotype; or (b) is an antibody fragment or a single chain antibody; 9. The antigen-binding portion of any one of claims 1 to 8, wherein the antibody fragment is selected from the group consisting of Fab, F(ab')2, Fd and Fv fragments, and the single chain antibody is selected from the group consisting of single chain variable fragment (scFv), bivalent scFv (di-scFv) and bivalent scFv (bi-scFv), diabodies, minibodies, and any combination thereof.

10. An agonistic monoclonal antibody described in any one of claims 1 to 8, comprising a heavy chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO: 12 and / or a light chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:

13.

11. An agonistic monoclonal antibody which specifically binds to human CD27 and does not block binding of CD27 to its CD70 ligand, as measured by surface plasmon resonance (SPR) or flow cytometry, said monoclonal antibody comprising a heavy chain comprising consecutively linked amino acids having the sequence set forth in SEQ ID NO:12 and a light chain comprising consecutively linked amino acids having the sequence set forth in SEQ ID NO:13, the monoclonal antibody designated BMS-986215.

12. 12. An immunoconjugate comprising an agonist monoclonal antibody, or an antigen-binding portion thereof, according to any one of claims 1 to 11 conjugated to a therapeutic agent.

13. The immunoconjugate of claim 12, wherein the therapeutic agent is a cytotoxin or a radioisotope.

14. A bispecific molecule comprising an agonistic monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 11, linked to a binding domain having a binding specificity different from that of the monoclonal antibody or antigen-binding portion thereof.

15. (a) an agonistic monoclonal antibody or an antigen-binding portion thereof according to any one of claims 1 to 11; (b) an immunoconjugate according to claim 12 or 13; or (c) the bispecific molecule of claim 14 . and a pharma- ceutically acceptable carrier.

16. The present invention further includes an additional antibody or antigen-binding portion thereof, wherein the additional antibody or antigen-binding portion thereof is an anti-PD-1 antibody, an anti-programmed death-ligand-1 (PD-L1) antibody, an anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibody, an anti-lymphocyte activation gene-3 (LAG-3) antibody, an anti-B- and T-lymphocyte attenuator (BTLA) antibody, an anti-T-cell immunoglobulin and mucin domain-3 (TIM-3) antibody, an anti-killer immunoglobulin-like receptor (KIR) antibody, an anti-killer cell lectin-like receptor G1 (KLRG-1) antibody, an anti-adenosine A2a receptor (A2aR) antibody, an anti-natural killer cell receptor 2B4 (CD244) antibody, an anti-CD160 antibody, an anti-IL-1 antibody, an anti-IL-2 antibody, an anti-IL-3 antibody, an anti-IL-4 antibody, an anti-IL-5 antibody, an anti-IL-6 antibody, an anti-IL-7 antibody, an anti-IL-8 antibody, an anti-IL-9 antibody, an anti-IL-10 antibody, an anti-IL-11 antibody, an anti-IL-12 antibody, an anti-IL-13 antibody, an anti-IL-14 antibody, an anti-IL-15 antibody, an anti-IL-16 antibody, an anti-IL-17 antibody, an anti-IL-18 antibody, an anti-IL-19 antibody, an anti-IL-20 antibody, an anti-IL-21 antibody, an anti-IL-22 antibody, an anti-IL-23 antibody, an anti-IL-24 antibody, an anti-IL-25 antibody, an anti-IL-31 antibody, an anti-IL-19 antibody, an anti-IL-25 antibody, an anti-IL-32 antibody, an anti-IL-19 antibody, an anti-IL-25 antibody, an anti-IL-31 antibody, an anti-IL-19 16. The composition of claim 15, wherein the antibody is selected from the group consisting of a T cell immunoreceptor with g and ITIM domains (TIGIT) antibody, a V-domain Ig suppressor of T cell activation (VISTA) antibody, nivolumab, pembrolizumab, cemiplimab, atezolizumab, durvalumab, avelumab, an antibody designated BMS-936559, an anti-inducible T cell costimulatory molecule (ICOS) antibody, an anti-CD137 (4-1BB) antibody, an anti-CD134 (OX40) antibody, an anti-CD27 antibody, an anti-glucocorticoid-induced TNFR-related protein (GITR) antibody, an anti-herpes virus entry mediator (HVEM) antibody, and any combination thereof.

17. An isolated nucleic acid encoding the agonist monoclonal antibody or antigen-binding portion thereof of any one of claims 1 to 11.

18. 20. An expression vector comprising the isolated nucleic acid of claim 17.

19. A host cell comprising the expression vector of claim 18.

20. A transgenic mouse comprising human immunoglobulin heavy and light chain transgenes, the transgenic mouse expressing an agonist monoclonal antibody according to any one of claims 1 to 11.

21. A hybridoma obtained from the mouse of claim 20, which produces an agonistic monoclonal antibody of any one of claims 1 to 11, which antibody specifically binds to human CD27.

22. 20. A method for producing an anti-CD27 monoclonal antibody or antigen-binding portion thereof, comprising expressing the agonist monoclonal antibody or antigen-binding portion thereof in a host cell according to claim 19 and isolating the monoclonal antibody or antigen-binding portion thereof from the host cell.

23. 17. A pharmaceutical composition for treating a subject having cancer, comprising a therapeutically effective amount of an agonistic monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 11, an immunoconjugate according to claim 12 or 13, a bispecific molecule according to claim 14, or a composition according to claim 15 or 16.

24. 17. A pharmaceutical composition for inhibiting tumor cell proliferation in a subject, comprising a therapeutically effective amount of an agonistic monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 11, an immunoconjugate according to claim 12 or 13, a bispecific molecule according to claim 14, or a composition according to claim 15 or 16.

25. 25. The pharmaceutical composition of claim 23 or 24, wherein the subject is a human.

26. 25. The pharmaceutical composition of claim 23 or 24, further comprising a therapeutically effective amount of an additional therapeutic agent for treating a subject with cancer or for inhibiting the growth of tumor cells in a subject, wherein the additional therapeutic agent is (a), (b), (c) or (d) of: (a) a compound that reduces inhibition or increases stimulation of the immune system; (b) a small molecule compound, a macrocyclic peptide, a fusion protein, or an antibody; (c) an antagonist antibody or antigen-binding portion thereof that specifically binds to a receptor for programmed death-1 (PD-1), programmed death-ligand-1 (PD-L1), cytotoxic T-lymphocyte antigen-4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), B- and T-lymphocyte attenuator (BTLA), T-cell immunoglobulin and mucin domain-3 (TIM-3), killer immunoglobulin-like receptor (KIR), killer cell lectin-like receptor G1 (KLRG-1), adenosine A2a receptor (A2aR), natural killer cell receptor 2B4 (CD244), CD160, T-cell immunoreceptor with Ig and ITIM domains (TIGIT), or V domain Ig suppressor of T-cell activation (VISTA), wherein the antibody or antigen-binding portion thereof that specifically binds to PD-1 or PD-L1 is: (i) disrupting the interaction between PD-1 and PD-L1 and inhibiting PD-1 / PD-L1 signaling; (ii) is a chimeric, humanized or human monoclonal antibody or an antigen-binding portion thereof; (iii) cross-competes with nivolumab for binding to human PD-1; (iv) specifically binds to PD-1 and is nivolumab or pembrolizumab; or (v) specifically binds to PD-L1 and is atezolizumab, durvalumab or avelumab; or (d) An agonistic antibody or an antigen-binding portion thereof that specifically binds to inducible T cell costimulatory molecule (ICOS), CD137 (4-1BB), CD134 (OX40), CD27, glucocorticoid-inducible TNFR-related protein (GITR), or herpes virus entry mediator (HVEM).

27. (a) the cancer is a solid tumor or the tumor cell is a cell of a solid tumor, and the solid tumor is selected from the group consisting of colon cancer, fibrosarcoma, squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous NSCLC, non-squamous NSCLC, head and neck cancer, breast cancer, esophageal cancer, gastric cancer, digestive cancer, small intestine cancer, liver cancer, hepatocellular carcinoma (HCC), pancreatic cancer (PAC), kidney cancer, renal cell carcinoma (RCC), bladder cancer, urethral cancer, ureteral cancer, colorectal cancer (CRC), colon cancer, anal cancer, endometrial cancer, prostate cancer, neuroblastoma, glioma, glioblastoma, germ cell tumor, pediatric sarcoma, sinus natural killer, black lung cancer, bladder cancer, bladder cancer, bladder cancer, ureter cancer, black lung cancer, or is a cancer selected from the group consisting of cancers of the skin, bone, cervical, uterine, endometrial, fallopian tube, ovarian, cervical, vaginal, vulvar, testicular, endocrine system, thyroid, parathyroid, adrenal, soft tissue sarcoma, penile, renal pelvis, central nervous system (CNS) neoplasms, primary CNS lymphomas, tumor angiogenesis, spinal axis tumors, brain, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid, squamous cell carcinomas, childhood solid tumors, environmentally induced cancers, virus-associated cancers, cancers of viral origin, advanced cancers, unresectable cancers, metastatic cancers, refractory cancers, recurrent cancers, and any combination thereof; or (b) the cancer is a hematopoietic tumor or the tumor cells are cells of a hematopoietic tumor, and the hematopoietic tumor is selected from T-cell lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), T-cell lymphoma, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), multiple myeloma, smoldering myeloma, monoclonal gammopathy of undetermined significance (MGUS), progressive, metastatic, refractory and / or recurrent hematopoietic tumors, and any combination of said hematopoietic tumors; The pharmaceutical composition according to any one of claims 23 to 26.

28. 1. A kit for treating a subject having cancer or for inhibiting the growth of tumor cells in a subject, comprising: (a) a dosage of one or more of the agonistic monoclonal antibody or antigen-binding portion thereof of any of claims 1 to 11, the immunoconjugate of claim 12 or 13, the bispecific molecule of claim 14, or the composition of claim 15 or 16 in the range of about 0.1 to about 20 mg / kg body weight; and (b) instructions for using one or more dosage amounts of the pharmaceutical composition according to any one of claims 23 to 27. Including the kit.

29. 29. The kit of claim 28, further comprising one or more doses of an additional therapeutic agent and instructions for using said one or more doses of an additional therapeutic agent in the pharmaceutical composition of claim 26 or 27.

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