WO2018027135A1 - Treatment of lymphoma using antibody-coupled t cell receptor - Google Patents

Treatment of lymphoma using antibody-coupled t cell receptor Download PDF

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Publication number
WO2018027135A1
WO2018027135A1 PCT/US2017/045503 US2017045503W WO2018027135A1 WO 2018027135 A1 WO2018027135 A1 WO 2018027135A1 US 2017045503 W US2017045503 W US 2017045503W WO 2018027135 A1 WO2018027135 A1 WO 2018027135A1
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actr
cells
subject
antibody
dose
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PCT/US2017/045503
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French (fr)
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Heather Huet
Michael Vasconcelles
Seth Ettenberg
Charles Wilson
Gregory MOTZ
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Unum Therapeutics Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/804—Blood cells [leukemia, lymphoma]

Definitions

  • Cancer immunotherapy including cell-based therapy, antibody therapy and cytokine therapy, is used to provoke immune responses attacking tumor cells while sparing normal tissues. It is a promising option for treating various types of cancer because of its potential to evade genetic and cellular mechanisms of drug resistance, and to target tumor cells while sparing normal tissues.
  • T-lymphocytes can exert major anti-tumor effects as demonstrated by results of allogeneic hematopoietic stem cell transplantation (HSCT) for hematologic malignancies, where T-cell-mediated graft-versus-host disease (GvHD) is inversely associated with disease recurrence, and immunosuppression withdrawal or infusion of donor lymphocytes can contain relapse.
  • HSCT allogeneic hematopoietic stem cell transplantation
  • GvHD T-cell-mediated graft-versus-host disease
  • Cell-based therapy may involve cytotoxic T cells having reactivity skewed toward cancer cells.
  • Eshhar et al. Proc. Natl. Acad. Sci. U. S. A.; 1993;90(2):720-724; Geiger et al., J Immunol.1999;162(10):5931-5939; Brentjens et al., Nat. Med.2003;9(3):279-286; Cooper et al., Blood.2003;101(4):1637-1644; and Imai et al., Leukemia.2004;18:676-684.
  • One approach is to express a chimeric antigen receptor having an antigen-binding domain (e.g., a single-chain antibody) fused to one or more T cell activation signaling domains. Binding of a cancer antigen via the antigen-binding domain results in T cell activation and triggers cytotoxicity. Recent results of clinical trials with infusions of chimeric receptor- expressing autologous T lymphocytes provided compelling evidence of their clinical potential. Pule et al., Nat. Med.2008;14(11):1264-1270; Porter et al., N Engl J Med;
  • an antigen-binding domain e.g., a single-chain antibody
  • Antibody-based immunotherapies such as monoclonal antibodies, antibody- fusion proteins, and antibody drug conjugates (ADCs) are used to treat a wide variety of diseases, including many types of cancer.
  • Such therapies may depend on recognition of cell surface molecules that are differentially expressed on cells for which elimination is desired (e.g., target cells such as cancer cells) relative to normal cells (e.g., non- cancer cells).
  • Binding of an antibody-based immunotherapy to a cancer cell can lead to cancer cell death via various mechanisms, e.g., antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or direct cytotoxic activity of the payload from an antibody-drug conjugate (ADC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • ADC antibody-drug conjugate
  • aspects of the present disclosure provide methods of treating lymphoma comprising administering to a subject in need thereof (i) an effective amount of one or more lymphodepleting agents; (ii) an anti-CD20 antibody after (i); and (iii) immune cells expressing an antibody-coupled T cell receptor (ACTR) no more than about 10 days after (ii), wherein the ACTR comprises (a) an Fc binding domain of CD16, (b) a co-stimulatory signaling domain of 4-1BB, and (c) a cytoplasmic signaling domain of CD3 ⁇ .
  • ACTR antibody-coupled T cell receptor
  • the ACTR further comprises a transmembrane domain and/or a hinge domain.
  • the ACTR comprises, from N-terminus to C-terminus, (a) the Fc binding domain of CD16, (b) the transmembrane domain, (c) the co-stimulatory domain of 4-1BB, and (d) the cytoplasmic signaling domain of CD3 ⁇ .
  • the ACTR further comprises a hinge domain, which is located between (a) and (b).
  • the ACTR further comprises a signal peptide.
  • the CD16 is the CD16V isoform.
  • the ACTR comprises the amino acid sequence of SEQ ID NO: 1.
  • the subject is a human patient having a relapsed or refractory CD20+ lymphoma, for example diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary mediastinal B cell lymphoma (PMBCL), grade 3b follicular lymphoma (Gr3b-FL), and transformed histology follicular lymphoma (TH-FL).
  • DLBCL diffuse large B-cell lymphoma
  • MCL mantle cell lymphoma
  • PMBCL primary mediastinal B cell lymphoma
  • Gr3b-FL grade 3b follicular lymphoma
  • TH-FL transformed histology follicular lymphoma
  • the one or more lymphodepleting agent is fludarabine and cyclophosphamide. In some embodiments, the one or more lymphodepleting agent is administered to the subject at a frequency of one dose per day for three consecutive days. In some embodiments, the first dose of the lymphodepleting agent is about 6-15 days (e.g., 6-10 days) before administering the immune cells expressing an ACTR. In some examples, the one or more lymphodepleting agent is fludarabine, which is administered to the subject by intravenous injection at a daily dose of about 30 mg/m 2 , and cyclophosphamide, which is administered to the subject by intravenous injection at a daily dose of about 500 mg/m 2 .
  • the anti-CD20 antibody is administered no more than 7 days (e.g., within about 24-48 hours) prior to administering the immune cells expressing ACTR.
  • the anti-CD20 antibody is rituximab, and for example, is administered to the subject at a dose of about 375 mg/m 2 .
  • the rituximab is administered to the subject at multiple doses, wherein the first dose is no more than about 7 days prior to administration of the immune cells expressing ACTR.
  • the first dose of rituximab is no more than 7 days prior to the infusion of the immune cells expressing ACTR.
  • the first dose of the antibody may be about 24-48 hours prior to administering the immune cells expressing ACTR.
  • the rituximab is administered to the subject at a frequency of one dose every three weeks for up to 8 doses.
  • the immune cells are T cells, for example T cells collected from the subject.
  • the T cells expressing the ACTR are administered to the subject at a dose of about 0.5 x 10 6 to about 5 x 10 6 T-cells/kg.
  • the dose of T cells expressing ACTR is up to about 0.5 x 10 6 T-cells/kg, about 0.5 x 10 6 to about 1.5 x 10 6 T-cells/kg, or about 1.5 x 10 6 to about 5 x 10 6 T-cells/kg.
  • the subject is a human patient having DLBCL or PMBCL and the dose of the immune cells expressing the ACTR is about 5 x 10 6 cells/kg.
  • the subject is a human patient having MCL, Gr3b-FL, or TH-FL and the dose of the immune cells expressing the ACTR is about 5 x 10 6 cells/kg. In some embodiments, the subject is a human patient having DLBCL, MCL, PMBCL, Gr3b-FL, or TH-FL and the dose of the immune cells expressing the ACTR is below 5 x 10 6 cells/kg.
  • the immune cells expressing the ACTR are prepared by introducing a vector for expressing the ACTR into immune cells collected from the subject about 30-60 days prior to administration of the ACTR-expressing immune cells.
  • the subject is free from alemtuzumab within 6 months prior to collecting the immune cells; fludarabine, cladribine, or clofarabine within 3 months prior to collecting the immune cells; external beam radiation, administration of a monoclonal antibody, or lymphotoxic chemotherapy within two weeks prior to collecting the immune cells; or an experimental agent within three half-lives of the experimental agent prior to collecting the immune cells.
  • the subject has been subject to a prior chemo-immunotherapy (before the treatment of the one or more lymphodepleting agents).
  • the chemo-immunotherapy can comprise an anti-CD20 antibody (e.g., rituximab) and a chemotherapeutic agent such as anthracycline agent.
  • the present disclosure provides a kit for treating lymphoma, comprising (i) one or more lymphodepleting agents; (ii) an anti-CD20 antibody; and (iii) immune cells expressing an antibody-coupled T cell receptor, which comprises (a) an Fc binding domain of CD16, (b) a co-stimulatory signaling domain of 4-1BB, and (c) a cytoplasmic signaling domain of CD3 ⁇ .
  • compositions comprising immune cells that express any of the ACTR constructs for use in treating a lymphoma, wherein the pharmaceutical composition is administered to a subject in need of the treatment no more than about 7 days after the subject is treated with an anti- CD20 antibody (e.g., rituximab) by the first dose, and wherein prior to the anti-CD20 antibody treatment, the subject has been treated with one or more lymphodepleting agents.
  • the first dose of the lymphodepleting agent treatment is about 6-15 days (e.g., 6-10 days) prior to the treatment of the ACTR-expressing immune cells.
  • the subject being treated by the immune cells are subject to prior treatments of the anti-CD20 antibody and the one or more lymphodepleting agents as described herein following the regimen schedules also
  • Figure 1 is an exemplary dosing regimen comprising collecting immune cells from the subject (leukapheresis) and using the collected immune cells to produce the immune cells expressing the ACTR. After administration of lymphodepleting agents, the subjects receive a dose of rituximab followed by an infusion the immune cells expressing the ACTR one day later. Rituximab is subsequently administered every three weeks for up to 7 additional doses. DETAILED DESCRIPTION OF DISCLOSURE
  • Antibody-based immunotherapies are used to treat a wide variety of diseases, including many types of cancer. Such a therapy often depends on recognition of cell surface molecules that are differentially expressed on cells for which elimination is desired (e.g., target cells such as cancer cells) relative to normal cells (e.g., non-cancer cells) (Weiner et al. Cell (2012) 148(6): 1081-1084).
  • target cells such as cancer cells
  • normal cells e.g., non-cancer cells
  • ADCC antibody-dependent cell- mediated cytotoxicity of target cells (e.g. cancer cells), and for some it is generally considered that this is the mechanism of action in vivo, as well.
  • ADCC is a cell- mediated innate immune mechanism whereby an effector cell of the immune system, such as natural killer (NK) cells, T cells, monocyte cells, macrophages, or eosinophils, actively lyses target cells (e.g., cancer cells) recognized by specific antibodies.
  • NK natural killer
  • regimen schedules for treating a lymphoma by immune cells such as T cells expressing an antibody-coupled T cell receptor coupled with an anti-CD20 antibody.
  • Such a regimen schedule comprises a conditioning regimen
  • lymphodepleting chemotherapy which comprises one or more doses of one or more lymphodepleting agents
  • a treatment regimen anti-CD20/ACTR treatment
  • the subject may undergo a pre- treatment period prior to the conditioning regimen, in which the subject receives anti- cancer therapy such as chemotherapy and/or radiation.
  • Immune cells may be collected from the subject during this period for preparing the immune cells expressing an ACTR as described herein.
  • the methods described herein are based at least in part on the finding that the combination of immune cells expressing ACTRs and the anti-CD20 antibodies results in proliferation and activation of the immune cells in response to the antibodies binding of target cells expressing CD20 (e.g., cancer cells), and that the proliferation and activation are antibody-dependent and self-limiting.
  • CD20 e.g., cancer cells
  • the dependence on adequate exposure to the anti-CD20 antibody indicates that the activity of the immune cells expressing the ACTRs can be modulated by the antibody dose and dosing schedule, providing an advantage of the methods described herein over CAR T cells previously used.
  • “about” or“approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system.
  • “about” can mean within an acceptable standard deviation, per the practice in the art.
  • “about” can mean a range of up to ⁇ 20%, preferably up to ⁇ 10%, more preferably up to ⁇ 5%, and more preferably still up to ⁇ 1% of a given value.
  • the term can mean within an order of magnitude, preferably within 2-fold, of a value.
  • the terms“treat”,“treatment”, and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition.
  • the term “treat” also denotes to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease. For example, in burden, or prevent, delay or inhibit metastasis, etc.
  • the term“therapeutically effective” applied to dose or amount refers to that quantity of a compound, molecule, composition, pharmaceutical composition, cells (e.g., one or more lymphodepleting agents, an anti-CD20 antibody, immune cells such as T lymphocytes and/or NK cells, expressing an ACTR of the disclosure) that is sufficient to result in a desired activity upon administration to a subject in need thereof.
  • the terms“therapeutically effective” and“effective” may be used interchangeably throughout.
  • a subject may be administered an effective amount of one or more lymphodepleting agents, which refers to an amount of the one or more lymophodepleting agents that is sufficient to deplete or reduce the number of endogenous lymphocytes in the subject.
  • the term“therapeutically effective” refers to that quantity of a compound or pharmaceutical composition that is sufficient to delay the manifestation, arrest the progression, relieve or alleviate at least one symptom of a disorder treated by the methods of the present disclosure. Note that when a combination of active ingredients is administered the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually.
  • a subject to be treated by the treatment regimens described herein may be a human lymphoma patient who has been subject to prior treatment, for example, radiation, chemotherapy, immunotherapy, or a combination thereof.
  • the human patient has undergone a chemo-immunotherapy, which may comprise an anti-CD20 antibody such as rituximab and a chemotherapeutic agent, such as an anthracycline agent (e.g., daunorubicin, doxorubicin, epirubicin, or idarubicin).
  • a chemo-immunotherapy which may comprise an anti-CD20 antibody such as rituximab and a chemotherapeutic agent, such as an anthracycline agent (e.g., daunorubicin, doxorubicin, epirubicin, or idarubicin).
  • a subject Prior to the Anti-CD20/ACTR treatment, a subject such as a human lymphoma patient may receive a lymphodepleting therapy to reduce or deplete the endogenous lymphocyte of the subject.
  • the term“subject” refers to any mammal, such as a human, monkey, mouse, rabbit, or domestic mammal.
  • the subject is a human.
  • the subject is a human patient suffering from a lymphoma such as a relapsed or refractory CD20 + lymphoma.
  • a lymphoma refers to a group of blood cell tumors that develop from lymphatic cells Hodgkin lymphoma and non-Hodgkin lymphoma are the two major types of lymphomas.
  • a lymphoma may be considered“refractory” if lymphoma cells are present in the bone marrow of the subject after having undergone a treatment for the lymphoma.
  • a lymphoma is considered“relapsed” if a return of lymphoma cells is detected in the bone marrow and there is a decrease in the number of normal blood cells after remission of the
  • the relapsed or refractory CD20+ lymphoma is a non-Hodgkin’s lymphoma.
  • the CD20+ B-cell lymphoma is diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary mediastinal B cell lymphoma (PMBCL), grade 3b follicular lymphoma (Gr3b-FL), or transformed histology follicular lymphoma (TH-FL).
  • DLBCL diffuse large B-cell lymphoma
  • MCL mantle cell lymphoma
  • PMBCL primary mediastinal B cell lymphoma
  • Gr3b-FL grade 3b follicular lymphoma
  • TH-FL transformed histology follicular lymphoma
  • Lymphodepletion refers to the destruction of endogenous lymphocytes and/or T cells, which is commonly used prior to immunotransplantation and immunotherapy.
  • Lymphodepletion can be achieved by irradiation and/or chemotherapy.
  • lymphodepleting agent can be any molecule capable of reducing, depleting, or eliminating endogenous lymphocytes and/or T cells when administered to a subject.
  • the lymphodepleting agents are administered in an amount effective in reducing the number of lymphocytes by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 96%, 97%, 98%, or at least 99% as compared to the number of lymphocytes prior to administration of the agents.
  • the lymphodepleting agents are administered in an amount effective in reducing the number of lymphocytes by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 96%, 97%, 98%, or at least 99% as compared to the number of lymphocytes prior to administration of the agents.
  • the lymphodepleting agents are administered in an amount effective in reducing the number of lymphocytes by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%
  • lymphodepleting agents are administered in an amount effective in reducing the number of lymphocytes such that the number of lymphocytes in the subject is below the limits of detection.
  • the subject is administered at least one (e.g., 2, 3, 4, 5 or more) lymphodepleting agents.
  • the lymphodepleting agents are cytotoxic agents that specifically kill lymphocytes.
  • lymphodepleting agents include, without limitation, fludarabine, cyclophosphamide, bendamustin, 5-fluorouracil, gemcitabine, methotrexate, dacarbazine, melphalan, doxorubicin, vinblastine, cisplatin, oxaliplatin, paclitaxel, docetaxel, irinotecan, etopside phosphate, mitoxantrone, cladribine, denileukin diftitox, or DAB-IL2.
  • the lymphodepleting agent may be accompanied with low-dose irradiation. The lymphodepletion effect of the conditioning regimen can be monitored via routine practice.
  • the method described herein involves a conditioning cyclophosphamide.
  • a subject to be treated by the method described herein may receive multiple doses of the one or more lymphodepleting agents for a suitable period (e.g., 2-5 days) in the conditioning stage.
  • the first dose of the lymphodepleting agent may be about 6-15 days (e.g., 6-10days inclusive, for example, 6, 7, 8, 9, 10 days) before the infusion of the ACTR-expressing immune cells.
  • the subject receives fludarabine at about 20-50 mg/m 2 (e.g., 30 mg/m 2 ) per day for 2-4 days (e.g., 3 days) and cyclophosphamide at about 300-600 mg/m 2 (e.g., 500 mg/m 2 ) per day for 2-4 days (e.g., 3 days).
  • fludarabine at about 20-50 mg/m 2 (e.g., 30 mg/m 2 ) per day for 2-4 days (e.g., 3 days) and cyclophosphamide at about 300-600 mg/m 2 (e.g., 500 mg/m 2 ) per day for 2-4 days (e.g., 3 days).
  • an anti-CD20/ACTR treatment regimen which comprises administration of an anti-CD20 antibody such as rituximab and infusion of immune cells (e.g., T cells) expressing an ACTR.
  • an anti-CD20 antibody such as rituximab
  • immune cells e.g., T cells
  • An anti-CD20 treatment can be performed on the subject as described herein prior to the treatment of ACTR-expressing immune cells, for example about 10 days before the immune cell treatment.
  • the first dose of the anti-CD20 treatment is no more than 7 days (e.g., 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day) before the immune cell infusion as described herein.
  • CD-20 is a B lymphocyte antigen expressed on the surface of B cells of all stages. CD20 positive cells are found in cases of Hodgkins disease, myeloma, and thymoma. Human CD20 is encoded by the MS4A1 gene. Any anti-CD20 antibody known in the art may be used in the methods provided herein. An anti-CD20 antibody is an
  • immunoglobulin molecule capable of specific binding to a CD20 molecule, for example, a CD20 molecule expressed on the surface of B cells.
  • antibody encompasses not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab', F(ab') 2 , or Fv), single chain (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, linear
  • an antibody for use with the described methods, kits, and compositions may be an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof). Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes.
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • any of the antibodies described herein can be either monoclonal or polyclonal.
  • a “monoclonal antibody” refers to a homogenous antibody population and a“polyclonal antibody” refers to a heterogeneous antibody population. These two terms do not limit the source of an antibody or the manner in which it is made.
  • the antibody used in the methods described herein is a humanized antibody.
  • Humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementary determining region
  • donor antibody non-human species
  • the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.
  • Antibodies may have Fc regions modified as described in WO
  • humanized antibodies have one or more CDRs (e.g., one, two, three, four, five, or six) which are altered with respect to the original antibody. These CDRs are also described as being "derived from” one or more CDRs from the original antibody. Humanized antibodies may also involve affinity maturation.
  • the antibody described herein is a chimeric antibody, which can include a heavy constant region and a light constant region from a human antibody.
  • Chimeric antibodies refer to antibodies having a variable region or part of variable region from a first species and a constant region from a second species.
  • the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant portions are homologous to the sequences in antibodies derived from another mammal such as human.
  • amino acid modifications can be made in the variable region and/or the constant region.
  • the anti-CD20 antibodies described herein have a suitable binding affinity to a CD20 molecule such as a human CD20 or an antigenic epitope thereof.
  • binding affinity refers to the apparent association constant or K A .
  • the K A is the reciprocal of the dissociation constant (K D ).
  • the antibody described herein may have a binding affinity (K D ) of at least 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 M, or lower.
  • An increased binding affinity corresponds to a decreased K D .
  • Higher affinity binding of an antibody to a first target relative to a second target can be indicated by a higher K A (or a smaller numerical value K D ) for binding the first target than the K A (or numerical value K D ) for binding the second target.
  • the antibody has specificity for the first target (e.g., a protein in a first conformation or mimic thereof) relative to the second target (e.g., the same protein in a second conformation or mimic thereof; or a second protein).
  • Differences in binding affinity can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 10 5 fold.
  • Binding affinity can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or binding affinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v/v) Surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration.
  • the concentration of bound binding protein [Bound]) is related to the concentration of free target protein ([Free]) and the concentration of binding sites for the binding protein on the target where (N) is the number of binding sites per target molecule by the following equation:
  • K A it is not always necessary to make an exact determination of K A , though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to K A , and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay (such as, e.g., an in vitro or in vivo assay).
  • a functional assay such as, e.g., an in vitro or in vivo assay.
  • Antibodies capable of binding CD20 as described herein can be made by any method known in the art. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.
  • the anti-CD20 antibody is a therapeutic antibody that is capable of treating, alleviating, or reducing the symptoms of any disease or disorder associated with CD20+ cells.
  • the anti-CD20 antibody binds to the cell surface antigen CD20, which is differentially expressed on cancer cells and indicate that the cell expressing the antigen should be subjected to ADCC.
  • the anti-CD20 antibody is a polyclonal antibody. In some embodiments, the anti-CD20 antibody is a monoclonal antibody.
  • anti-CD20 antibodies include, without limitation, rituximab, Y90-ibritumomab (Biogen/IDEC), I31- tositumomab (GSK), ofatumumab (Genmab AC/GSK), ocrelizumab
  • the anti- CD20 antibody is rituximab.
  • any of the anti-CD20 antibodies described herein can be mixed with a composition for use in the treatment regimen described herein.“Acceptable” means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated.
  • Pharmaceutically acceptable excipients including buffers, which are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.
  • a pharmaceutical composition described herein contains more than one anti-CD20 antibodies that recognize different epitopes/residues of the target antigen.
  • compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
  • polyvinylpyrrolidone amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN TM ,
  • Pharmaceutically acceptable excipients are further described herein.
  • compositions can also be administered via other conventional routes, e.g., administered parenterally.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, or infusion techniques.
  • Injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like).
  • various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like).
  • water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a
  • physiologically acceptable excipients is infused.
  • Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer’s solution or other suitable excipients.
  • Intramuscular preparations e.g., a sterile formulation of a suitable soluble salt form of the antibody, can be dissolved and administered in a pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or 5% glucose solution.
  • the anti-CD20 antibody is administered to the subject in one or more doses of about 100-500 mg/m 2 , 200- 400 mg/m 2 , or 300-400 mg/m 2 . In some embodiments, the anti-CD20 antibody is administered to the subject in one or more doses of about 375 mg/m 2 .
  • the method involves administering the anti-CD20 antibody (e.g., rituximab) to the subject in one dose. In some embodiments, the method involves administering the anti-CD20 antibody (e.g., rituximab) to the subject in multiple dose (e.g., at least 2, 3, 4, 5, 6, 7, or 8 doses). In some embodiments, the anti-CD20 antibody (e.g., rituximab) is administered to the subject in multiple doses, with the first dose of the anti-CD20 antibody administered to the subject about 1, 2, 3, 4, 5, 6, or 7 days prior to administration of the immune cells expressing ACTR. In some embodiments, the first dose of the anti-CD20 antibody is administered to the subject between about 24-48 hours prior to the administration of the immune cells expressing ACTR.
  • the anti-CD20 antibody e.g., rituximab
  • the anti-CD20 antibody is administered to the subject prior to administration of the immune cells expressing the ACTR and then subsequently about every three weeks. In some embodiments, the anti-CD20 antibody is administered to the subject about every three weeks, following administration of the immune cells, for up to 8 total doses.
  • the timing of the administration of the following the indicated day encompasses administration on day 18, day 19, day 20, day 21, day 22, day 23, or day 24).
  • the efficacy of the methods described herein may be assessed by any method known in the art and would be evident to a skilled medical professional.
  • the efficacy of the antibody-based immunotherapy may be assessed by survival of the subject or cancer burden in the subject or tissue or sample thereof.
  • the antibody based immunotherapy is assessed based on the safety or toxicity of the therapy (e.g., administration of the anti-CD20 antibody and the immune cells expressing the ACTRs) in the subject, for example by the overall health of the subject and/or the presence of adverse events or severe adverse events.
  • the therapy e.g., administration of the anti-CD20 antibody and the immune cells expressing the ACTRs
  • ACTR-expressing immune cells such as T cells via, e.g., infusion.
  • Antibody-coupled T-cell receptor is a non-naturally-occurring chimeric receptor comprising an Fc binding domain of CD16 with binding affinity and specificity for an Fc fragment, a co-stimulatory signaling domain of a co-stimulatory factor such as 4- 1BB, and a cytoplasmic signaling domain of a cell surface receptor such as CD3 ⁇ .
  • the ACTR may optionally further comprise a transmembrane domain and/or a hinge domain.
  • the ACTRs are configured such that, when expressed on a host cell, the Fc binding domain of CD16 is located extracellularly for binding to a target molecule (e.g., an antibody or a Fc-fusion protein) and the co-stimulatory signaling domain and the cytoplasmic signaling domain are located in the cytoplasm for triggering activation and/or effector signaling.
  • a ACTR construct as described herein comprises, from N-terminus to C-terminus, the Fc binding domain of CD16, the transmembrane domain, the co-stimulatory signaling domain of 4-1BB, and the cytoplasmic signaling domain of CD3 ⁇ .
  • any of the ACTRs described herein may further comprise a hinge domain, which may be located at the C-terminus of the Fc binding domain and the N-terminus of the transmembrane domain.
  • the ACTR constructs described herein may contain two or more co-stimulatory signaling domains, which may link to each other or be separated by the CD3 ⁇ cytoplasmic signaling domain.
  • the extracellular Fc-binding fragment of CD16, transmembrane domain, co-stimulatory signaling domain(s), and CD3 ⁇ cytoplasmic signaling domain in an ACTR construct may be linked to each other directly, or via a peptide linker.
  • the Fc-binding fragment in any of the ACTR constructs may be derived from any naturally occurring CD16 (e.g., CD16A or CD16B) receptor, including naturally- occurring polymorphism variants. Examples include CD16 F158 and CD16 V158.
  • the ACTR construct comprises the Fc-binding fragment of CD16 V158, which may have the amino acid sequence of SEQ ID NO: 6:
  • GMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYR CQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIP KATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQ Alternatively, it may be a functional variant of a wild-type counterpart, which carry one or more mutations (e.g., up to 10 amino acid residue substitutions) that alter the binding affinity to the Fc portion of an Ig molecule. In some instances, the mutation may alter the glycosylation pattern of the Fc receptor and thus the binding affinity to Fc.
  • the Fc-binding fragment of CD16 may comprise an amino acid sequence that is at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99%) identical to the amino acid sequence of the Fc-binding fragment of a naturally- occurring CD16 (e.g., the Fc-binding fragment of CD 158V as described herein).
  • the “percent identity” of two amino acid sequences can be determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993.
  • Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res.25(17):3389-3402, 1997.
  • the default parameters of the respective programs e.g., XBLAST and Any of the Fc-binding fragment of CD16 in an ACTR construct described herein may have a suitable binding affinity for the Fc portion of a therapeutic IgG antibody.
  • “binding affinity” refers to the apparent association constant or K A .
  • the K A is the reciprocal of the dissociation constant, K D .
  • the Fc-binding fragment of CD16 may have a binding affinity K D of at least 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 M or lower for the Fc portion of antibody.
  • transmembrane domain of the ACTRs described herein can be in any form known in the art.
  • a“transmembrane domain” refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane.
  • Transmembrane domains compatible for use in the ACTRs used herein may be obtained from a naturally occurring protein. Alternatively, it can be a synthetic, non-naturally occurring protein segment, e.g., a hydrophobic protein segment that is thermodynamically stable in a cell membrane.
  • Transmembrane domains are classified based on the three dimensional structure of the transmembrane domain.
  • transmembrane domains may form an alpha helix, a complex of more than one alpha helix, a beta-barrel, or any other stable structure capable of spanning the phospholipid bilayer of a cell.
  • transmembrane domains may also or alternatively be classified based on the
  • transmembrane domain topology including the number of passes that the
  • transmembrane domain makes across the membrane and the orientation of the protein.
  • single-pass membrane proteins cross the cell membrane once
  • multi- pass membrane proteins cross the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7 or more times).
  • Membrane proteins may be defined as Type I, Type II or Type III depending upon the topology of their termini and membrane-passing segment(s) relative to the inside and outside of the cell.
  • Type I membrane proteins have a single membrane- spanning region and are oriented such that the N-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the C-terminus of the protein is present on the cytoplasmic side.
  • Type II membrane proteins also have a single membrane-spanning region but are oriented such that the C-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the N-terminus of the protein is present on the cytoplasmic side.
  • Type III membrane proteins have multiple membrane-spanning segments and may be further sub-classified based on the number of transmembrane segments and the location of N- and C-termini.
  • the transmembrane domain of the ACTR described herein is derived from a Type I single-pass membrane protein.
  • Single-pass membrane proteins include, but are not limited to, CD8 ⁇ , CD8 ⁇ , 4-1BB/CD137, CD28, CD34, CD4, Fc ⁇ RI ⁇ , CD16, OX40/CD134, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40,
  • CD40L/CD154 CD40L/CD154, VEGFR2, FAS, and FGFR2B.
  • the FGFR2B CD40L/CD154, VEGFR2, FAS, and FGFR2B.
  • transmembrane domain is from a membrane protein selected from the following:
  • the transmembrane domain is of CD8 ⁇ . In some examples, the transmembrane domain is of 4-1BB/CD137. In other examples, the transmembrane domain is of CD28 or CD34. In yet other examples, the transmembrane domain is not derived from human CD8 ⁇ . In some embodiments, the transmembrane domain of the ACTR is a single-pass alpha helix.
  • Transmembrane domains from multi-pass membrane proteins may also be compatible for use in the ACTRs described herein.
  • Multi-pass membrane proteins may comprise a complex (at least 2, 3, 4, 5, 6, 7 or more) alpha helices or a beta sheet structure.
  • the N-terminus and the C-terminus of a multi-pass membrane protein are present on opposing sides of the lipid bilayer, e.g., the N-terminus of the protein is present on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is present on the extracellular side.
  • Either one or multiple helix passes from a multi-pass membrane protein can be used for constructing the ACTR described herein.
  • Transmembrane domains for use in the ACTRs described herein can also comprise at least a portion of a synthetic, non-naturally occurring protein segment.
  • the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet.
  • the protein segment is at least approximately 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art, for example in U.S. Patent No.7,052,906 B1 and PCT Publication No.
  • the amino acid sequence of the transmembrane domain does not comprise cysteine residues. In some embodiments, the amino acid sequence of the transmembrane domain comprises one cysteine residue. In some embodiments, the amino acid sequence of the transmembrane domain comprises two cysteine residues. In some embodiments, the amino acid sequence of the transmembrane domain comprises more than two cysteine residues (e.g., 3, 4, 5 or more).
  • the transmembrane domain may comprise a transmembrane region and a cytoplasmic region located at the C-terminal side of the transmembrane domain.
  • the cytoplasmic region of the transmembrane domain may comprise three or more amino acids and, in some embodiments, helps to orient the transmembrane domain in the lipid bilayer.
  • one or more cysteine residues are present in the transmembrane region of the transmembrane domain.
  • one or more cysteine residues are present in the cytoplasmic region of the transmembrane domain.
  • the cytoplasmic region of the transmembrane domain comprises positively charged amino acids.
  • the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine, and lysine.
  • the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the
  • transmembrane region comprises mostly hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine.
  • the transmembrane region is hydrophobic.
  • the transmembrane region comprises a poly-leucine-alanine sequence.
  • hydropathy, or hydrophobic or hydrophilic characteristics of a protein or protein segment can be assessed by any method known in the art, for example the Kyte and Doolittle hydropathy analysis.
  • the ACTR construct described herein comprises the co-stimulatory signaling domain of a co-stimulatory receptor such as 4-1BB (CD137).
  • co-stimulatory signaling domain refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response such as an effector function.
  • the co-stimulatory signaling domain of the ACTR described herein can be a cytoplasmic signaling domain from a co-stimulatory protein, which transduces a signal macrophages, neutrophils, or eosinophils.
  • the co-stimulatory domain (e.g., the co- stimulatory domain of 4-1BB) may be co-used with a co-stimulatory from another co- stimulatory receptor.
  • the 4-1BB co-stimulatory signaling domain for use in an ACTR construct comprises the amino acid sequence of:
  • variants of a naturally occurring 4-1BB receptor e.g., variants that include up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5, or 8) as compared to a wild-type counterpart.
  • Mutation of amino acid residues of the co-stimulatory signaling domain may result in an increase in signaling transduction and enhanced stimulation of immune responses relative to co-stimulatory signaling domains that do not comprise the mutation. Mutation of amino acid residues of the co-stimulatory signaling domain may result in a decrease in signaling transduction and reduced stimulation of immune responses relative to co-stimulatory signaling domains that do not comprise the mutation.
  • the co-stimulatory signaling domain is a variant of the 4-1BB costimulatory signaling domain.
  • the ACTRs may comprise more than one co-stimulatory signaling domain (e.g., 2, 3 or more). In some embodiments, the ACTR comprises two or more of the same co-stimulatory signaling domains, for example, two copies of the co-stimulatory signaling domain of 4-1BB. In some embodiments, the ACTR comprises two or more co-stimulatory signaling domains from different co-stimulatory proteins, such as any two or more co-stimulatory proteins described herein.
  • the type(s) of co-stimulatory signaling domains may be based on factors such as the type of host cells to be used with the ACTRs (e.g., immune cells such as T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function.
  • the ACTR comprises two co-stimulatory signaling domains.
  • the two co-stimulatory signaling domains are CD28 and 4-1BB.
  • the two co-stimulatory signaling domains are CD28 LL ⁇ GG variant and 4-1BB.
  • the ACTR described herein further comprises a cytoplasmic signaling domain of CD3 ⁇ .
  • the cytoplasmic signaling domain of CD3 ⁇ comprises the amino acid sequence of: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 8).
  • the ACTR described herein may further comprise a hinge domain that is located between the Fc binding domain and the transmembrane domain.
  • a hinge domain is an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the extracellular ligand-binding domain of an Fc receptor relative to the transmembrane domain of the ACTRs can be used.
  • the hinge domain may contain about 10-100 amino acids, e.g., 15-150 amino acids, 20-100 amino acids, or 30-60 amino acids. In some embodiments, the hinge domain may be of 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids in length.
  • the hinge domain is a hinge domain of a naturally occurring protein. Hinge domains of any protein known in the art to comprise a hinge domain are compatible for use in the ACTRs described herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to the ACTR. In some embodiments, the hinge domain is of CD8 ⁇ . In some embodiments, the hinge domain is a portion of the hinge domain of CD8 ⁇ , e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8 ⁇ . In one example, the ACTR described herein comprises a hinge/transmembrane domain from CD8 ⁇ , which may comprise the amino acid sequence of:
  • the hinge domain is the hinge domain that joins the constant domains CH1 and CH2 of an antibody.
  • the hinge domain is of an antibody and comprises the hinge domain of the antibody and one or more constant regions of the antibody.
  • the hinge domain comprises the hinge domain of an antibody and the CH3 constant region of the antibody.
  • the hinge domain comprises the hinge domain of an antibody and the CH2 and CH3 constant regions of the antibody.
  • the antibody is an IgG, IgA, IgM, IgE, or IgD antibody.
  • the antibody is an IgG antibody.
  • the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.
  • the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and the CH3 constant region of an IgG1 antibody.
  • Non-naturally occurring peptides may also be used as hinge domains for the ACTRs described herein.
  • the hinge domain between the C- terminus of the extracellular ligand-binding domain of an Fc receptor and the N- terminus of the transmembrane domain is a peptide linker, such as a (Gly x Ser) n linker, wherein x and n, independently can be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.
  • the hinge domain is (Gly 4 Ser) n (SEQ ID NO: 50), wherein n can be an integer between 3 and 60, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more.
  • the hinge domain is (Gly 4 Ser) 3 (SEQ ID NO: 51).
  • the hinge domain is (Gly 4 Ser) 6 (SEQ ID NO: 52).
  • the hinge domain is (Gly 4 Ser) 9 (SEQ ID NO: 53).
  • the hinge domain is (Gly 4 Ser) 12 (SEQ ID NO: 54).
  • n can be an integer between 3 and 60, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32
  • the hinge domain is (Gly 4 Ser) 15 (SEQ ID NO: 55). In some embodiments, the hinge domain is (Gly 4 Ser) 15 (SEQ ID NO: 55). In some embodiments, the hinge domain is (Gly 4 Ser) 15 (SEQ ID NO: 55). In some embodiments, the hinge domain is (Gly 4 Ser) 15 (SEQ ID NO: 55). In some embodiments, the hinge domain is (Gly 4 Ser) 15 (SEQ ID NO: 55). In some combination thereof
  • the hinge domain is (Gly 4 Ser) 30 (SEQ ID NO: 56). In some embodiments, the hinge domain is (Gly 4 Ser) 30 (SEQ ID NO: 56). In some embodiments, the hinge domain is (Gly 4 Ser) 30 (SEQ ID NO: 56). In some embodiments, the hinge domain is (Gly 4 Ser) 30 (SEQ ID NO: 56). In some embodiments, the hinge domain is (Gly 4 Ser) 30 (SEQ ID NO: 56). In some combination thereof
  • the hinge domain is (Gly 4 Ser) 45 (SEQ ID NO: 57). In some embodiments, the hinge domain is (Gly 4 Ser) 45 (SEQ ID NO: 57). In some embodiments, the hinge domain is (Gly 4 Ser) 45 (SEQ ID NO: 57). In some embodiments, the hinge domain is (Gly 4 Ser) 45 (SEQ ID NO: 57). In some embodiments, the hinge domain is (Gly 4 Ser) 45 (SEQ ID NO: 57). In some
  • the hinge domain is (Gly 4 Ser) 60 (SEQ ID NO: 58).
  • the hinge domain is an extended recombinant polypeptide (XTEN), which is an unstructured polypeptide consisting of hydrophilic residues of varying lengths (e.g., 10-200 amino acid residues, 20-150 amino acid residues, 30-100 amino acid residues, or 40-80 amino acid residues). Amino acid sequences of XTEN peptides will be evident to one of skill in the art and can be found, for example, in U.S. Patent No.8,673,860, which is herein incorporated by reference.
  • the hinge domain is an XTEN peptide and comprises 60 amino acids.
  • the hinge domain is an XTEN peptide and comprises 30 amino acids.
  • the hinge domain is an XTEN peptide and comprises 45 amino acids.
  • the hinge domain is an XTEN peptide and comprises 15 amino acids.
  • the ACTR may further comprise a signal peptide (also known as a signal sequence) at the N-terminus of the polypeptide.
  • signal sequences are peptide sequences that target a polypeptide to the desired site in a cell.
  • the signal sequence targets the ACTR to the secretory pathway of the cell and will allow for integration and anchoring of the ACTR into the lipid bilayer.
  • Signal sequences including signal sequences of naturally occurring proteins or synthetic, non- naturally occurring signal sequences, that are compatible for use in the ACTRs described herein will be evident to one of skill in the art.
  • the signal sequence from CD8 ⁇ for example, amino acid sequence of MALPVTALLLPLALLLHAARP (SEQ ID NO: 49).
  • the signal sequence is from CD28.
  • the signal sequence is from the murine kappa chain.
  • the signal sequence is from CD16.
  • the ACTRs described herein would confer a number of advantages. For example, via the extracellular domain that binds Fc, the ACTR constructs described herein can bind to the Fc portion of antibodies or other Fc-containing molecules, rather than directly binding a specific target antigen (e.g., a cancer antigen). Thus, immune cells expressing the ACTR constructs described herein would be able to induce cell death of any type of cells that are bound by an antibody or another Fc-containing molecule.
  • a specific target antigen e.g., a cancer antigen
  • Table 1 below provides exemplary ACTR constructs described herein. This exemplary constructs have, from N-terminus to C-terminus in order, the signal sequence, the Fc binder (e.g., an extracellular domain of an Fc receptor), the hinge domain, and the transmembrane, while the positions of the co-stimulatory domain and the cytoplasmic signaling domain can be switched.
  • Table 1 Exemplary ACTRs.
  • any of the above exemplary ACTRs may be removed or replaced with another suitable signal peptide without affecting the function of the ACTR.
  • Such variants of the exemplary ACTRs are also within the scope of the present disclosure.
  • any of the ACTRs described herein can be prepared by a routine method, such as recombinant technology.
  • Methods for preparing the ACTRs herein involve generation of a nucleic acid that encodes a polypeptide comprising each of the domains of the ACTR construct as described herein.
  • the nucleic acid sequence encodes any one of the exemplary ACTRs provided by SEQ ID NO: 1-5, and 9-47.
  • Sequences of each of the components of the ACTRs may be obtained via routine technology, e.g., PCR amplification from any one of a variety of sources known in the art.
  • sequences of one or more of the components of the ACTRs are obtained from a human cell.
  • the sequences of one or more components of the ACTRs can be synthesized.
  • Sequences of each of the components e.g., domains
  • the nucleic acid encoding the ACTR may be synthesized.
  • the nucleic acid is DNA.
  • the nucleic acid is RNA.
  • Host cells expressing the ACTR described herein provide a specific population of cells that can recognize target cells bound by antibodies (e.g., therapeutic antibodies) or Fc-fusion proteins. Engagement of the Fc binding domain of an ACTR construct expressed on such host cells (e.g., immune cells) with the Fc portion of an antibody transmits an activation signal to the co-stimulatory signaling domain(s) and the cytoplasmic signaling domain of the ACTR construct, which in turn activates cell proliferation and/or effector functions of the host cell, such as ADCC effects triggered by the host cells.
  • the combination of co-stimulatory signaling domain(s) and the cytoplasmic signaling domain may allow for robust activation of multiple signaling pathways within the cell.
  • the host cells are immune cells, such as T cells, NK cells, macrophages, neutrophils, eosinophils, or any combination thereof.
  • the immune cells are T cells.
  • the immune cells are NK cells.
  • the immune cells can be established cell lines, for example, NK-92 cells.
  • the cells are cells that can develop and/or differentiate into immune cells, for example progenitor cells.
  • expression vectors for stable or transient expression of the ACTR construct may be constructed via conventional methods as described herein and introduced into immune host cells.
  • nucleic acids encoding the ACTRs may be cloned into a suitable expression vector, such as a viral vector in operable linkage to a suitable promoter.
  • the nucleic acids and the vector may be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined with a ligase.
  • synthetic nucleic acid linkers can be ligated to the termini of the nucleic acid encoding the ACTRs.
  • the synthetic linkers may contain nucleic acid sequences that correspond to a particular restriction site in the vector.
  • vectors/plasmids/viral vectors would depend on the type of host cells for expression of the ACTRs, but should be suitable for integration and replication in eukaryotic cells.
  • promoters can be used for expression of the ACTRs described herein, including, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter.
  • CMV cytomegalovirus
  • viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR
  • SV40 simian virus 40
  • Additional promoters for expression of the ACTRs include any constitutively active promoter in an immune cell.
  • any regulatable promoter may be used, such that its expression can be modulated within an immune cell.
  • the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in host cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColE1 for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a“suicide switch” or“suicide gene” which when triggered causes cells carrying the vector to die (e.g., HSV thymidine kinase, an inducible caspase such as iCasp9), and reporter gene for assessing expression of the ACTR. See section VI below.
  • a selectable marker gene such as the neomycin gene for selection of stable or
  • the ACTR construct or the nucleic acid encoding said ACTR is a DNA molecule. In some embodiments, the ACTR construct or the nucleic acid encoding said ACTR is a transposon. In some embodiments, the ACTR construct or the nucleic acid encoding said ACTR is a plasmid. In some embodiments, the ACTR construct or the nucleic acid encoding said ACTR is a DNA plasmid may be electroporated into immune cells (see, e.g., Till, et al. Blood (2012) 119(17): 3940- 3950). In some embodiments, the nucleic acid encoding the ACTR is an RNA molecule, which may be electroporated into immune cells.
  • any of the vectors comprising a nucleic acid sequence that encodes an ACTR construct described herein is also within the scope of the present disclosure.
  • a vector may be delivered into host cells such as host immune cells by a suitable method. Methods of delivering vectors to immune cells are well known in the art and may include DNA electroporation, RNA electroporation, transfection reagents such as liposomes, or viral transduction.
  • the vectors for expression of the ACTRs are delivered to host cells by viral transduction. Exemplary viral methods for delivery include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos.
  • AAV adeno-associated virus
  • the vectors for expression of the ACTRs are retroviruses. In some embodiments, the vectors for expression of the ACTRs are lentiviruses. In some embodiments, the vectors for expression of the ACTRs are gamma-retroviruses. In some embodiments, the vectors for expression of the ACTRs are adeno-associated viruses (AAVs).
  • AAVs adeno-associated viruses
  • viral particles that are capable of infecting the immune cells and carry the vector may be produced by any method known in the art and can be found, for example in PCT Application No. WO 1991/002805A2, WO 1998/009271 A1, and U.S. Patent 6,194,191.
  • the viral particles are harvested from the cell culture supernatant and may be isolated and/or purified prior to contacting the viral particles with the immune cells.
  • the cells are cultured under conditions that allow for expression of the ACTR.
  • the nucleic acid encoding the ACTR is regulated by a regulatable promoter
  • the host cells are cultured in conditions wherein the regulatable promoter is activated.
  • the promoter is an inducible promoter and the immune cells are cultured in the presence of the inducing molecule or in conditions in which the inducing molecule is produced.
  • ACTR ACTR-encoding mRNA by quantitative reverse transcriptase PCR (qRT-PCR) or detection of the ACTR protein by methods including Western blotting, fluorescence microscopy, and flow cytometry.
  • qRT-PCR quantitative reverse transcriptase PCR
  • expression of the ACTR may take place in vivo after the immune cells are administered to a subject.
  • RNA molecules encoding the ACTR constructs can be prepared by in vitro transcription or by chemical synthesis.
  • the RNA molecules can then introduced into suitable host cells such as immune cells (e.g., T cells, NK cells, macrophages, neutrophils, eosinophils, or any combination thereof) by, e.g., electroporation.
  • immune cells e.g., T cells, NK cells, macrophages, neutrophils, eosinophils, or any combination thereof
  • electroporation e.g., electroporation.
  • RNA molecules can be synthesized and introduced into host immune cells following the methods described in Rabinovich et al., Human Gene Therapy, 17:1027-1035 and WO WO2013/040557.
  • Methods for preparing host cells expressing any of the ACTRs described herein may also comprise activating the host cells ex vivo.
  • Activating a host cell means stimulating a host cell into an activate state in which the cell may be able to perform effector functions (e.g., ADCC).
  • Methods of activating a host cell will depend on the type of host cell used for expression of the ACTRs.
  • T cells may be activated ex vivo in the presence of one or more molecule such as an anti-CD3 antibody, an anti-CD28 antibody, IL-2, or phytohemoagglutinin.
  • NK cells may be activated ex vivo in the presence of one or molecules such as a 4-1BB ligand, an anti-4-1BB antibody, IL-15, an anti-IL-15 receptor antibody, IL-2, IL12, IL- 21, and K562 cells.
  • the host cells expressing any of the ACTRs described herein are activated ex vivo prior to administration to a subject. Determining whether a host cell is activated will be evident to one of skill in the art and may include assessing expression of one or more cell surface markers associated with cell activation, expression or secretion of cytokines, and cell morphology.
  • the methods of preparing host cells expressing any of the ACTRs described herein may comprise expanding the host cells ex vivo. Expanding host cells may involve any method that results in an increase in the number of cells expressing ACTRs, for example, allowing the host cells to proliferate or stimulating the host cells to proliferate. Methods for stimulating expansion of host cells will depend on the type of host cell used for expression of the ACTRs and will be evident to one of skill in the art. In some embodiments, the host cells expressing any of the ACTRs described herein are expanded ex vivo prior to administration to a subject.
  • the host cells expressing the ACTRs are expanded and activated ex vivo prior to administration of the cells to the subject.
  • the ex vivo expansion and/or activation polarizes the host cells to a desired phenotype, for example, T cells or NK cells.
  • the immune cells can be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure.
  • an effective amount of the immune cells expressing any of the ACTR constructs described herein can be administered into a subject after the first dose of the anti-CD20 antibody (e.g., no less than about 10 days after the first dose of the anti-CD20 antibody).
  • the immune cells expressing the ACTRs are administered to the subject about 1 day after administration of the anti-CD20 antibody.
  • the immune cells expressing the ACTRs are administered to the subject about 1, 2, 3, 4, 5, 6, or 7 days after administration of the anti-CD20 antibody.
  • the immune cells may be autologous to the subject, i.e., the immune cells are obtained from the subject in need of the treatment, genetically engineered for expression of the ACTR constructs, and then administered to the same subject.
  • the host cells are allogeneic cells, i.e., the cells are obtained from a first subject, genetically engineered for expression of the ACTR construct, and administered to a second subject that is different from the first subject but of the same species.
  • allogeneic immune cells may be derived from a human donor and administered to a human recipient who is different from the donor.
  • the immune cells expressing the ACTRs are are
  • the immune cells expressing the ACTRs are administered to the subject via infusion in a dose of up to about 0.5x10 6 cells/kg.
  • the immune cells expressing the ACTRs are administered to the subject in a dose of less than about 5.0x10 6 cells/kg.
  • the immune cells expressing the ACTRs are administered to the subject in a dose between 0.5x10 6 cells/kg - 5.0x10 6 cells/kg, for example from about 0.5x10 6 cells/kg to about 1.5x10 6 cells/kg or from about 1.5x10 6 cells/kg to about 5.0x10 6 cells/kg.
  • the immune cells expressing the ACTRs are administered to the subject in a dose of about 0.1 x10 6 cells/kg, 0.2 x10 6 cells/kg, 0.3x10 6 cells/kg, 0.4 x10 6 cells/kg, 0.5 x10 6 cells/kg, 0.6 x10 6 cells/kg, 0.7 x10 6 cells/kg, 0.8 x10 6 cells/kg, 0.9 x10 6 cells/kg, 1.0 x10 6 cells/kg, 1.1 x10 6 cells/kg, 1.2 x10 6 cells/kg, 1.3 x10 6 cells/kg, 1.4 x10 6 cells/kg, 1.5 x10 6 cells/kg, 2.0 x10 6 cells/kg, 2.5 x10 6 cells/kg, 3.0 x10 6 cells/kg, 3.5 x10 6 cells/kg, 4.0 x10 6 cells/kg, 4.5 x10 6 cells/kg, or about 5.0 x10 6 cells/kg.
  • the dose of the immune cells expressing ACTR may be as high as 1 x 10 8 cells/kg, for example, 5.0 x10 6 cells/kg to 1 x 10 8 cells/kg, 1 x 10 7 cells/kg to 1 x 10 8 cells/kg, or 5.0 x 10 7 cells/kg to 1 x 10 8 cells/kg.
  • the amount of immune cells expressing the ACTRs administered to the subject may depend on factors such as the type of CD20+ B-cell lymphoma.
  • the subject has diffuse large B-cell lymphoma (DLBCL) or primary mediastinal B cell lymphoma (PMBCL) and the dose of the immune cells expressing the ACTR is about 5x10 6 cells/kg.
  • DLBCL diffuse large B-cell lymphoma
  • PMBCL primary mediastinal B cell lymphoma
  • the subject has mantle cell lymphoma (MCL), primary mediastinal B cell lymphoma (PMBCL), grade 3b follicular lymphoma (Gr3b-FL), and transformed histology follicular lymphoma (TH-FL) and the dose of the immune cells expressing the ACTR is about 5x10 6 cells/kg.
  • the subject has DLBCL, MCL, PMBCL, Gr3b-FL, TH-FL and the dose of the immune cells expressing the ACTR is less than about 5x10 6 cells/kg.
  • Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of
  • the effective amount alleviates, relieves,
  • the subject is a human. In some embodiments, the subject is a human patient having relapsed or refractory CD20+ lymphoma. III. Pre-Treatment Period
  • a subject may undergo a pre-treatment period, during which the subject may receive an anti-cancer therapy to control the disease, e.g., radiotherapy, chemotherapy, immunotherapy, and/or surgery.
  • an anti-cancer therapy to control the disease
  • examples include therapeutic vaccines (including but not limited to GVAX, DC-based vaccines, etc.), checkpoint inhibitors (including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.) or activators (including but not limited to agents that enhance 41BB, OX40, etc.).
  • Non-limiting examples of other therapeutic agents useful for combination with the immunotherapy of the disclosure include: (i) anti-angiogenic agents (e.g., TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitors of metalloproteases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptors, placental proliferin-related protein, as well as those listed by Carmeliet and Jain (2000)); (ii) a VEGF antagonist or a VEGF receptor antagonist such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinases and any combinations thereof; and (iii) chemotherapeut
  • cyclophosphamide cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethyhnelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not
  • immunosuppressives e.g., cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil
  • anti-angiogenic compounds e.g., TNP-470, genistein, and bevacizumab
  • growth factor inhibitors e.g., fibroblast growth factor (FGF) inhibitors
  • angiotensin receptor blocker e.g., trastuzumab
  • cell cycle inhibitors and differentiation inducers e.g., tretinoin
  • mTOR inhibitors topoisomerase inhibitors (e.g., doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxan
  • mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors. See also Example 1 below.
  • the subject may also undergo baseline assessments to determine whether the subject is suitable for the following conditioning and/or treatment regimens.
  • immune cells may be collected from the subject during the pre-treatment period, for example, about 30-60 days prior to the immune cell infusion. In some embodiments, the immune cells are collected from the subject by leukapheresis.
  • the population of immune cells can be obtained from any source, such as peripheral blood mononuclear cells (PBMCs), bone marrow, tissues such as spleen, lymph node, thymus, or tumor tissue.
  • PBMCs peripheral blood mononuclear cells
  • the population of immune cells is derived from PBMCs.
  • the population of immune cells is derived from a human patient having lymphoma.
  • the population of immune cells is collected from a human patient having lymphoma by leukapheresis.
  • the type of host cells desired may be expanded within the population of cells obtained by co-incubating the cells with stimulatory molecules, for example, IL-2, anti-CD3 and anti-CD28
  • antibodies may be used for expansion and/or stimulation of T cells.
  • the subject is free from alemtuzumab within 6 months prior to collecting the immune cells; fludarabine, cladribine, or clofarabine within 3 months of collecting the immune cells; external beam radiation, administration of a monoclonal antibody, or lymphotoxic chemotherapy within 2 weeks of collecting the immune cells; and experimental agents within 3 half-lives of the experimental agent collecting the immune cells.
  • the immune cells collected from the subject may be genetically engineered following the methods known in the art and/or described herein to introduce expression vectors for producing an ACTR construct on cell surface. Such ACTR-expressing immune cells can then be given to the subject at the treatment stage, following the anti- CD20 antibody treatment. IV. Kits for Therapeutic Use
  • kits for use of the immune cells expressing ACTRs and anti-CD20 antibodies in methods for treating lymphoma may include one or more containers comprising a first pharmaceutical composition that comprises one or more lymphodepleting agents, a second pharmaceutical composition that comprises an anti-CD20 antibody and a pharmaceutically acceptable carrier, and a third pharmaceutical composition that comprises any nucleic acid or host cells (e.g., immune cells such as those described herein), and a pharmaceutically acceptable carrier.
  • the kit can comprise instructions for use in any of the methods described herein.
  • the included instructions can comprise a description of administration of the first, second, and/or third pharmaceutical compositions to a subject to achieve the intended activity in a subject.
  • the kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment.
  • the instructions comprise a description of administering the first, second, and third pharmaceutical compositions to a subject who is in need of the treatment.
  • the instructions relating to the use of the ACTRs and the first, second, and third pharmaceutical compositions described herein generally include information as to dosage, dosing schedule, and route of administration for the intended treatment.
  • the containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.
  • Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert.
  • the label or package insert indicates that the pharmaceutical compositions are used for treating, delaying the onset, and/or alleviating a disease or disorder in a subject.
  • kits provided herein are in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like.
  • packages for use in combination with a specific device such as an inhaler, nasal administration device, or an infusion device.
  • a kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the container may also have a sterile access port.
  • At least one active agent in the pharmaceutical composition is a ACTR as described herein.
  • Kits optionally may provide additional components such as buffers and interpretive information.
  • the kit comprises a container and a label or package insert(s) on or associated with the container.
  • the disclosure provides articles of manufacture comprising contents of the kits described above.
  • Antibodies P. Finch, 1997; Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed.1985); Nucleic Acid Hybridization (B.D. Hames & S.J.
  • a phase 1, multi-center, single-arm, open label study is described to evaluate the safety and efficacy of infusion of a single dose of ⁇ -retrovirus transduced T cells expressing ACTR in combination with the anti-CD20 antibody rituximab, in subjects with relapsed or refractory CD20+ B-cell lymphoma, such as diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary mediastinal B-cell lymphoma
  • DLBCL diffuse large B-cell lymphoma
  • MCL mantle cell lymphoma
  • primary mediastinal B-cell lymphoma such as diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary mediastinal B-cell lymphoma
  • PMBCL grade 3b follicular lymphoma
  • Gr3b-FL grade 3b follicular lymphoma
  • TH-FL transformed histology follicular lymphoma
  • the study is separated into 2 sequential phases, a dose escalation phase and an expansion cohort phase with the intent of evaluating the safety of the ACTR-expressing T cells in combination with rituximab in subjects with relapsed or refractory CD20+ B-cell lymphomas.
  • Each study phase is comprised of a pre-treatment period, a treatment period, and a follow-up period. Study procedures are summarized in Figure 1. Subject Inclusion Criteria
  • CD20+ B-cell lymphoma defined as an anti-CD20 mAb in combination with an anthracycline-containing chemotherapy regimen (i.e. chemo-immunotherapy) and at least one of the following:
  • auto-HSCT autologous hematopoietic stem cell transplant
  • TH-FL relapsed or refractory disease following at least 2 prior regimens or following an auto-HSCT. At least 1 prior regimen with an anti-CD20 mAb in combination with chemotherapy is required following documented transformation
  • HIV Human Immunodeficiency Virus
  • HBsAg hepatitis B surface antigen
  • CNS disease such as history of a seizure disorder, dementia or cerebrovascular ischemia/hemorrhage with residual paresis or aphasia, cerebellar disease, Parkinson’s disease, psychosis or organic brain syndrome
  • corticosteroid therapy > 5 mg/day of prednisone or equivalent doses of other corticosteroids (topical, intranasal, and inhaled corticosteroids in standard doses and physiologic replacement for subjects with adrenal insufficiency are allowed)
  • Dose escalation phase The dose escalation phase of the study enrolls 9-18 subjects with CD20+ B cell lymphoma to evaluate the safety of escalating doses of ACTR-expressing T cells in combination with rituximab.
  • the dose escalation phase follows a traditional 3+3 model to determine the maximum tolerated dose (MTD) and the Recommended Phase 2 Dose (RP2D) for the ACTR- expressing T cells.
  • the dose-limiting toxicity evaluation period will be 28 days for each dose cohort, assessed from Day 0 (the day of administration of ACTR-expressing T cells).
  • ACTR-expressing T cell are generated and administered at the maximum dose within each dose range.
  • a target maximum dose of 5 x 10 6 ACTR T-cells/kg is planned.
  • the highest dose cohort studied may be concluded as the Maximal Feasible Dose, and the RP2D in this circumstance will be assessed accordingly.
  • additional dose cohorts may be investigated, with each target maximal dose increased by 1 ⁇ 2 log from the previous target maximal dose, as defined in the planned dose cohorts below.
  • the first cohort three subjects receive an initial target maximum dose of 0.5 x 10 6 ACTR T-cells/kg in combination with rituximab. If fewer ACTR T-cells than the maximum target dose for a subject are obtained during the production process, the cells available are infused and the dose given is recorded. If the initial dose cohort is not found to be safe, as determined by dose limiting toxicities (DLT) assessment, then a lower maximal target dose than 0.5 x 10 5 ACTR T-cells/kg in combination with rituximab may be evaluated.
  • DLT dose limiting toxicities
  • the dose level will be escalated to the next target maximum dose of 1.5 x 10 6 ACTR T-cells/kg in combination with rituximab for the next 3 subjects. If fewer ACTR T-cells than the maximum target dose in the second planned dose cohort are obtained for a subject during the production process, the cells available are infused and the dose given is recorded.
  • the dose level is escalated to the next target maximum dose of 5 x 10 6 ACTR T-cells/kg in combination with rituximab. If fewer ACTR T-cells than the maximum target dose for a subject are obtained during the production process, the cells available are infused and the dose given is recorded. Cohort expansion phase
  • the cohort expansion phase enrolls up to an additional 36 subjects with CD20+ B cell lymphoma in 3 cohorts of 10 to 12 subjects each:
  • FIG. 1 shows a diagram of the study including a pre-treatment period involving screening eligible subjects, enrolling the subjects, performing leukapheresis to obtain immune cells from the subjects and modifying the immune cells to express ACTR.
  • the pre- treatment period is followed by the treatment period in which subjected undergo a conditioning regimen (lymphodepleting regimen) involving administration of fludararbine and cyclophosphoamide for three days.
  • a conditioning regimen lymphodepleting regimen
  • the subjects are then administered rituximab (cycle 1) on day -1 (the day prior to administration of the ACTR-expressing T cells), followed by the ACTR-expressing T cells on day 0.
  • Rituximab may be subsequently administered every three weeks for up to 7 additional doses (cycles).
  • the study further includes a follow-up period in which the subjects are assessed and may enter a long term follow up study involving administration of ACTR-expressing T cells.
  • eligible subjects Upon completion of screening procedures and study enrollment, eligible subjects enter the pre-treatment period, which continues until the treatment period commences.
  • the minimum target nucleated cell count is 1.0 x 10 9 to be collected in a single leukapheresis session, which may be repeated to meet this minimum cell count requirement.
  • Culturing of collected PBMCs will proceed in a CliniMACS Prodigy cell expansion device (Miltenyi Biotech) with IL-2 and agonist antibodies directed to CD3 and CD28, followed by transduction of the expanded cells with a ⁇ -retrovirus containing an expression construct of ACTR (CD16V-4-1BB-CD3 ⁇ ), and apportionment for infusion into subjects.
  • CliniMACS Prodigy cell expansion device Miltenyi Biotech
  • the subject may be administered anti-cancer chemotherapy, such as a low dose single agent chemotherapy (e.g., vincristine or cyclophosphamide) after leukapheresis prior to the initiation of the conditioning regimen (lymphodepleting chemotherapy).
  • a low dose single agent chemotherapy e.g., vincristine or cyclophosphamide
  • lymphodepleting chemotherapy No therapy involving administration of monoclonal antibodies (including rituximab), nitrosoureas, or mitomycin C is allowed.
  • Local irradiation to a single lesion or subset of lesions may be administered, so long as un-radiated positron emission tomography (PET)-positive lymphoma lesions remain.
  • PET positron emission tomography
  • Subjects receive a 3-day lymphodepleting chemotherapy regimen with fludarabine and cyclophosphamide, starting 6 to 10 days prior to the planned day of infusion ACTR- expressing T cells.
  • the lymphodepleting chemotherapy regimen will be as follows: Table 7: Lymphodepleting Chemotherapy Regimen
  • the lymphodepleting chemotherapy regimen can be initiated as early as Day -10 before the planned infusion of ACTR-expressing T cells, for ease of scheduling at the site.
  • Serum creatinine is measured on the planned first day of administration of lymphodepleting chemotherapy; chemotherapy should be withheld if serum creatinine is ⁇ 1.5 times the age- adjusted upper limits of normal (ULN). Re-assessment of inclusion/exclusion criteria is required if lymphodepleting chemotherapy is delayed by more than 14 days.
  • Once lymphodepleting chemotherapy is initiated if dose(s) on the second or third day are omitted, the schedule of administration for all outstanding doses of chemotherapy may only be delayed for one day beyond the 3-day schedule.
  • rituximab is administered followed by dosing with ACTR-expressing T cells, as follows: Table 8: Rituximab and ACTR-expressing T cells
  • ACTR-expressing T cells are not expected to proliferate or persist in the absence of rituximab. Therefore, to maximize the potential therapeutic window for activity, rituximab will be given 24-48 hours prior to infusion of ACTR-expressing T cells so that rituximab serum levels are near maximum during infusion of the ACTR-expressing T cells.
  • this window may be extended to no more than 7 days. After 7 days, the rituximab serum levels begin to approach trough levels and may not support sufficient expansion of ACTR T-cells.
  • Subjects will be admitted as inpatients on the day of or the day before ACTR T-cell product infusion. Subjects will stay as inpatients for a minimum of 3 days after the ACTR T- cell product infusion. Subjects may be discharged to home on Day +3, if they are clinically stable. After subjects are discharged from the hospital and up through Day 20 after infusion of ACTR-expressing T cells, subjects will take and record their temperature daily. Subjects will return to the clinical site weekly for vital sign testing, adverse event assessments, concomitant medications, physical exams and performance status, and laboratory testing throughout the assessment period (to Day 28). Investigators will be asked to assess whether any adverse event is related to lymphodepleting chemotherapy, rituximab, or the ACTR- expressing T cells. Additional cycles of rituximab (cycles 2-8)
  • rituximab cycle 1
  • ACTR-expressing T cells subjects will receive up to 7 additional cycles of rituximab for a total of 8 cycles of rituximab.
  • the interval including an administration of rituximab, assessment of subject response, and approval for the next administration of rituximab is referred to as one treatment cycle.
  • the total number of rituximab cycles for a particular study subject, up to a maximum of 8, will be determined by tolerability of the treatment regimen, the absence of progression of the subject’s underlying disease, and the ongoing willingness of the subject to participate in the study.
  • rituximab will be administered every 21 days ⁇ 2 days, unless delayed or discontinued due to toxicities.
  • PET/CT response assessments will occur at the end of every 2 cycles of rituximab (i.e., within 3 days prior to rituximab doses 3, 5, and 7), and at the end of the treatment period. PET scans are no longer required after a subject achieves CR, unless PD is suspected on a follow-up CT scan. If a bone marrow aspirate and/or biopsy or other radiographic assessment for extranodal disease was obtained at the baseline disease assessment (at the discretion of the investigator), this will be repeated within 2 weeks of a documented radiographic response to confirm the response.
  • Subjects with documented stable disease, partial response, or complete response following treatment with ACTR-expressing T cells and rituximab will be followed according to the schedule of follow-up assessments. Subjects with documented disease progression will be encouraged to undergo an end-of-study visit at the time of documented disease
  • the post-treatment follow-up period is defined as the period immediately after the end-of-study visit through study subject discontinuation. All surviving subjects will be monitored for a total of 15 years after infusion of the ACTR-expressing T cells to assess them for survival, general health, and potential long-term toxicity of ACTR-expressing T cells (with particular attention to development of leukemia or other secondary malignancies and autoimmune/rheumatologic, neurologic, or hematologic disorders).

Abstract

Disclosed herein are immune cells expressing antibody-coupled T cell receptors (ACTRs) and methods of using such in combination with anti-CD20 antibodies in treating a lymphoma.

Description

TREATMENT OF LYMPHOMA USING
ANTIBODY-COUPLED T CELL RECEPTOR CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of the filing date of U.S. Provisional
Application No.62/371,160, filed August 4, 2016, the entire contents of which are incorporated by reference herein. BACKGROUND OF DISCLOSURE
Cancer immunotherapy, including cell-based therapy, antibody therapy and cytokine therapy, is used to provoke immune responses attacking tumor cells while sparing normal tissues. It is a promising option for treating various types of cancer because of its potential to evade genetic and cellular mechanisms of drug resistance, and to target tumor cells while sparing normal tissues. T-lymphocytes can exert major anti-tumor effects as demonstrated by results of allogeneic hematopoietic stem cell transplantation (HSCT) for hematologic malignancies, where T-cell-mediated graft-versus-host disease (GvHD) is inversely associated with disease recurrence, and immunosuppression withdrawal or infusion of donor lymphocytes can contain relapse. Weiden et al., NEnglJ Med.1979;300(19):1068-1073; Porter et al., NEnglJ Med.1994;330(2):100-106; Kolb et al., Blood.1995;86(5):2041-2050; Slavin et al., Blood.1996;87(6):2195-2204; and Appelbaum, Nature.2001;411(6835):385-389.
Cell-based therapy may involve cytotoxic T cells having reactivity skewed toward cancer cells. Eshhar et al., Proc. Natl. Acad. Sci. U. S. A.; 1993;90(2):720-724; Geiger et al., J Immunol.1999;162(10):5931-5939; Brentjens et al., Nat. Med.2003;9(3):279-286; Cooper et al., Blood.2003;101(4):1637-1644; and Imai et al., Leukemia.2004;18:676-684. One approach is to express a chimeric antigen receptor having an antigen-binding domain (e.g., a single-chain antibody) fused to one or more T cell activation signaling domains. Binding of a cancer antigen via the antigen-binding domain results in T cell activation and triggers cytotoxicity. Recent results of clinical trials with infusions of chimeric receptor- expressing autologous T lymphocytes provided compelling evidence of their clinical potential. Pule et al., Nat. Med.2008;14(11):1264-1270; Porter et al., N Engl J Med;
2011; 25;365(8):725-733; Brentjens et al., Blood.2011;118(18):4817-4828; Till et al., Blood.2012;119(17):3940-3950; Kochenderfer et al., Blood.2012;119(12):2709-2720; and Brentjens et al., Sci Transl Med.2013;5(177):177ra138.
Antibody-based immunotherapies, such as monoclonal antibodies, antibody- fusion proteins, and antibody drug conjugates (ADCs) are used to treat a wide variety of diseases, including many types of cancer. Such therapies may depend on recognition of cell surface molecules that are differentially expressed on cells for which elimination is desired (e.g., target cells such as cancer cells) relative to normal cells (e.g., non- cancer cells). Binding of an antibody-based immunotherapy to a cancer cell can lead to cancer cell death via various mechanisms, e.g., antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or direct cytotoxic activity of the payload from an antibody-drug conjugate (ADC). SUMMARY OF DISCLOSURE
Aspects of the present disclosure provide methods of treating lymphoma comprising administering to a subject in need thereof (i) an effective amount of one or more lymphodepleting agents; (ii) an anti-CD20 antibody after (i); and (iii) immune cells expressing an antibody-coupled T cell receptor (ACTR) no more than about 10 days after (ii), wherein the ACTR comprises (a) an Fc binding domain of CD16, (b) a co-stimulatory signaling domain of 4-1BB, and (c) a cytoplasmic signaling domain of CD3ζ.
In some embodiments, the ACTR further comprises a transmembrane domain and/or a hinge domain. In some embodiments, the ACTR comprises, from N-terminus to C-terminus, (a) the Fc binding domain of CD16, (b) the transmembrane domain, (c) the co-stimulatory domain of 4-1BB, and (d) the cytoplasmic signaling domain of CD3ζ. In some embodiments, the ACTR further comprises a hinge domain, which is located between (a) and (b). In some embodiments, the ACTR further comprises a signal peptide.
In some embodiments, the CD16 is the CD16V isoform. In some examples, the ACTR comprises the amino acid sequence of SEQ ID NO: 1.
In some embodiments, the subject is a human patient having a relapsed or refractory CD20+ lymphoma, for example diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary mediastinal B cell lymphoma (PMBCL), grade 3b follicular lymphoma (Gr3b-FL), and transformed histology follicular lymphoma (TH-FL).
In some embodiments, the one or more lymphodepleting agent is fludarabine and cyclophosphamide. In some embodiments, the one or more lymphodepleting agent is administered to the subject at a frequency of one dose per day for three consecutive days. In some embodiments, the first dose of the lymphodepleting agent is about 6-15 days (e.g., 6-10 days) before administering the immune cells expressing an ACTR. In some examples, the one or more lymphodepleting agent is fludarabine, which is administered to the subject by intravenous injection at a daily dose of about 30 mg/m2, and cyclophosphamide, which is administered to the subject by intravenous injection at a daily dose of about 500 mg/m2.
In some embodiments, the anti-CD20 antibody is administered no more than 7 days (e.g., within about 24-48 hours) prior to administering the immune cells expressing ACTR. In some embodiments, the anti-CD20 antibody is rituximab, and for example, is administered to the subject at a dose of about 375 mg/m2. In some embodiments, the rituximab is administered to the subject at multiple doses, wherein the first dose is no more than about 7 days prior to administration of the immune cells expressing ACTR. In some embodiments, the first dose of rituximab is no more than 7 days prior to the infusion of the immune cells expressing ACTR. For example, the first dose of the antibody may be about 24-48 hours prior to administering the immune cells expressing ACTR. In some embodiments, the rituximab is administered to the subject at a frequency of one dose every three weeks for up to 8 doses.
In any of the embodiments described herein, the immune cells are T cells, for example T cells collected from the subject. In some embodiments, the T cells expressing the ACTR are administered to the subject at a dose of about 0.5 x 106 to about 5 x 106 T-cells/kg. In some embodiments, the dose of T cells expressing ACTR is up to about 0.5 x 106 T-cells/kg, about 0.5 x 106 to about 1.5 x 106 T-cells/kg, or about 1.5 x 106 to about 5 x 106 T-cells/kg. In some examples, the subject is a human patient having DLBCL or PMBCL and the dose of the immune cells expressing the ACTR is about 5 x 106 cells/kg. In some examples, the subject is a human patient having MCL, Gr3b-FL, or TH-FL and the dose of the immune cells expressing the ACTR is about 5 x 106 cells/kg. In some embodiments, the subject is a human patient having DLBCL, MCL, PMBCL, Gr3b-FL, or TH-FL and the dose of the immune cells expressing the ACTR is below 5 x 106 cells/kg.
In some embodiments, the immune cells expressing the ACTR are prepared by introducing a vector for expressing the ACTR into immune cells collected from the subject about 30-60 days prior to administration of the ACTR-expressing immune cells.
In some embodiments, the subject is free from alemtuzumab within 6 months prior to collecting the immune cells; fludarabine, cladribine, or clofarabine within 3 months prior to collecting the immune cells; external beam radiation, administration of a monoclonal antibody, or lymphotoxic chemotherapy within two weeks prior to collecting the immune cells; or an experimental agent within three half-lives of the experimental agent prior to collecting the immune cells.
In any of the methods described herein, the subject has been subject to a prior chemo-immunotherapy (before the treatment of the one or more lymphodepleting agents). The chemo-immunotherapy can comprise an anti-CD20 antibody (e.g., rituximab) and a chemotherapeutic agent such as anthracycline agent.
In another aspect, the present disclosure provides a kit for treating lymphoma, comprising (i) one or more lymphodepleting agents; (ii) an anti-CD20 antibody; and (iii) immune cells expressing an antibody-coupled T cell receptor, which comprises (a) an Fc binding domain of CD16, (b) a co-stimulatory signaling domain of 4-1BB, and (c) a cytoplasmic signaling domain of CD3ζ.
Also within the scope of the present disclosure are pharmaceutical compositions comprising immune cells that express any of the ACTR constructs for use in treating a lymphoma, wherein the pharmaceutical composition is administered to a subject in need of the treatment no more than about 7 days after the subject is treated with an anti- CD20 antibody (e.g., rituximab) by the first dose, and wherein prior to the anti-CD20 antibody treatment, the subject has been treated with one or more lymphodepleting agents. In some embodiments, the first dose of the lymphodepleting agent treatment is about 6-15 days (e.g., 6-10 days) prior to the treatment of the ACTR-expressing immune cells. Also provided herein are uses of the immune cells for manufacturing a medicament for use in treating a lymphoma, wherein the subject being treated by the immune cells are subject to prior treatments of the anti-CD20 antibody and the one or more lymphodepleting agents as described herein following the regimen schedules also The details of one of more embodiments of the disclosure are set forth in the description below. Other features or advantages of the present disclosure will be apparent from the detailed description of several embodiments and also from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS
The following drawing forms part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Figure 1 is an exemplary dosing regimen comprising collecting immune cells from the subject (leukapheresis) and using the collected immune cells to produce the immune cells expressing the ACTR. After administration of lymphodepleting agents, the subjects receive a dose of rituximab followed by an infusion the immune cells expressing the ACTR one day later. Rituximab is subsequently administered every three weeks for up to 7 additional doses. DETAILED DESCRIPTION OF DISCLOSURE
Antibody-based immunotherapies are used to treat a wide variety of diseases, including many types of cancer. Such a therapy often depends on recognition of cell surface molecules that are differentially expressed on cells for which elimination is desired (e.g., target cells such as cancer cells) relative to normal cells (e.g., non-cancer cells) (Weiner et al. Cell (2012) 148(6): 1081-1084). Several antibody-based
immunotherapies have been shown in vitro to facilitate antibody-dependent cell- mediated cytotoxicity of target cells (e.g. cancer cells), and for some it is generally considered that this is the mechanism of action in vivo, as well. ADCC is a cell- mediated innate immune mechanism whereby an effector cell of the immune system, such as natural killer (NK) cells, T cells, monocyte cells, macrophages, or eosinophils, actively lyses target cells (e.g., cancer cells) recognized by specific antibodies.
Described herein are regimen schedules for treating a lymphoma by immune cells such as T cells expressing an antibody-coupled T cell receptor coupled with an anti-CD20 antibody. Such a regimen schedule comprises a conditioning regimen
(lymphodepleting chemotherapy), which comprises one or more doses of one or more lymphodepleting agents, and a treatment regimen (anti-CD20/ACTR treatment), which comprises treatment of an anti-CD20 antibody such as rituximab followed by treatment of the immune cells that express an ACTR. Optionally, the subject may undergo a pre- treatment period prior to the conditioning regimen, in which the subject receives anti- cancer therapy such as chemotherapy and/or radiation. Immune cells may be collected from the subject during this period for preparing the immune cells expressing an ACTR as described herein.
The methods described herein are based at least in part on the finding that the combination of immune cells expressing ACTRs and the anti-CD20 antibodies results in proliferation and activation of the immune cells in response to the antibodies binding of target cells expressing CD20 (e.g., cancer cells), and that the proliferation and activation are antibody-dependent and self-limiting. The dependence on adequate exposure to the anti-CD20 antibody indicates that the activity of the immune cells expressing the ACTRs can be modulated by the antibody dose and dosing schedule, providing an advantage of the methods described herein over CAR T cells previously used.
The term“about” or“approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example,“about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively,“about” can mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and more preferably still up to ±1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term“about” is implicit and in this context means within an acceptable error range for the particular value.
In the context of the present disclosure insofar as it relates to any of the disease conditions recited herein, the terms“treat”,“treatment”, and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition. Within the meaning of the present disclosure, the term “treat” also denotes to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease. For example, in burden, or prevent, delay or inhibit metastasis, etc.
As used herein the term“therapeutically effective” applied to dose or amount refers to that quantity of a compound, molecule, composition, pharmaceutical composition, cells (e.g., one or more lymphodepleting agents, an anti-CD20 antibody, immune cells such as T lymphocytes and/or NK cells, expressing an ACTR of the disclosure) that is sufficient to result in a desired activity upon administration to a subject in need thereof. The terms“therapeutically effective” and“effective” may be used interchangeably throughout. For example, a subject may be administered an effective amount of one or more lymphodepleting agents, which refers to an amount of the one or more lymophodepleting agents that is sufficient to deplete or reduce the number of endogenous lymphocytes in the subject. Within the context of the present disclosure, the term“therapeutically effective” refers to that quantity of a compound or pharmaceutical composition that is sufficient to delay the manifestation, arrest the progression, relieve or alleviate at least one symptom of a disorder treated by the methods of the present disclosure. Note that when a combination of active ingredients is administered the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually.
A subject to be treated by the treatment regimens described herein may be a human lymphoma patient who has been subject to prior treatment, for example, radiation, chemotherapy, immunotherapy, or a combination thereof. In some examples, the human patient has undergone a chemo-immunotherapy, which may comprise an anti-CD20 antibody such as rituximab and a chemotherapeutic agent, such as an anthracycline agent (e.g., daunorubicin, doxorubicin, epirubicin, or idarubicin). I. Conditioning Regimen (Lymphodepleting Therapy)
Prior to the Anti-CD20/ACTR treatment, a subject such as a human lymphoma patient may receive a lymphodepleting therapy to reduce or deplete the endogenous lymphocyte of the subject.
As used herein, the term“subject” refers to any mammal, such as a human, monkey, mouse, rabbit, or domestic mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a human patient suffering from a lymphoma such as a relapsed or refractory CD20+ lymphoma. A lymphoma refers to a group of blood cell tumors that develop from lymphatic cells Hodgkin lymphoma and non-Hodgkin lymphoma are the two major types of lymphomas. A lymphoma may be considered“refractory” if lymphoma cells are present in the bone marrow of the subject after having undergone a treatment for the lymphoma. Alternatively, a lymphoma is considered“relapsed” if a return of lymphoma cells is detected in the bone marrow and there is a decrease in the number of normal blood cells after remission of the
lymphoma. In some embodiments, the relapsed or refractory CD20+ lymphoma is a non-Hodgkin’s lymphoma. In some embodiments, the CD20+ B-cell lymphoma is diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary mediastinal B cell lymphoma (PMBCL), grade 3b follicular lymphoma (Gr3b-FL), or transformed histology follicular lymphoma (TH-FL).
Lymphodepletion refers to the destruction of endogenous lymphocytes and/or T cells, which is commonly used prior to immunotransplantation and immunotherapy.
Lymphodepletion can be achieved by irradiation and/or chemotherapy. A
“lymphodepleting agent” can be any molecule capable of reducing, depleting, or eliminating endogenous lymphocytes and/or T cells when administered to a subject. In some embodiments, the lymphodepleting agents are administered in an amount effective in reducing the number of lymphocytes by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 96%, 97%, 98%, or at least 99% as compared to the number of lymphocytes prior to administration of the agents. In some embodiments, the
lymphodepleting agents are administered in an amount effective in reducing the number of lymphocytes such that the number of lymphocytes in the subject is below the limits of detection. In some embodiments, the subject is administered at least one (e.g., 2, 3, 4, 5 or more) lymphodepleting agents.
In some embodiments, the lymphodepleting agents are cytotoxic agents that specifically kill lymphocytes. Examples of lymphodepleting agents include, without limitation, fludarabine, cyclophosphamide, bendamustin, 5-fluorouracil, gemcitabine, methotrexate, dacarbazine, melphalan, doxorubicin, vinblastine, cisplatin, oxaliplatin, paclitaxel, docetaxel, irinotecan, etopside phosphate, mitoxantrone, cladribine, denileukin diftitox, or DAB-IL2. In some instances, the lymphodepleting agent may be accompanied with low-dose irradiation. The lymphodepletion effect of the conditioning regimen can be monitored via routine practice.
In some embodiments, the method described herein involves a conditioning cyclophosphamide. A subject to be treated by the method described herein may receive multiple doses of the one or more lymphodepleting agents for a suitable period (e.g., 2-5 days) in the conditioning stage. The first dose of the lymphodepleting agent may be about 6-15 days (e.g., 6-10days inclusive, for example, 6, 7, 8, 9, 10 days) before the infusion of the ACTR-expressing immune cells.
In one example, the subject receives fludarabine at about 20-50 mg/m2 (e.g., 30 mg/m2) per day for 2-4 days (e.g., 3 days) and cyclophosphamide at about 300-600 mg/m2 (e.g., 500 mg/m2) per day for 2-4 days (e.g., 3 days). II. Anti-CD20/ACTR Treatment Regimen
Following the conditioning regimen (lymphodepleting therapy), the subject is subject to an anti-CD20/ACTR treatment regimen, which comprises administration of an anti-CD20 antibody such as rituximab and infusion of immune cells (e.g., T cells) expressing an ACTR. (a) Anti-CD20 Treatment
An anti-CD20 treatment can be performed on the subject as described herein prior to the treatment of ACTR-expressing immune cells, for example about 10 days before the immune cell treatment. In some examples, the first dose of the anti-CD20 treatment is no more than 7 days (e.g., 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day) before the immune cell infusion as described herein.
(i) Anti-CD20 Antibodies
CD-20 is a B lymphocyte antigen expressed on the surface of B cells of all stages. CD20 positive cells are found in cases of Hodgkins disease, myeloma, and thymoma. Human CD20 is encoded by the MS4A1 gene. Any anti-CD20 antibody known in the art may be used in the methods provided herein. An anti-CD20 antibody is an
immunoglobulin molecule capable of specific binding to a CD20 molecule, for example, a CD20 molecule expressed on the surface of B cells.
As used herein, the term“antibody” encompasses not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, or Fv), single chain (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, linear
tib di i l h i tib di lti ifi tib di ( bi ifi tib di ) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. An antibody for use with the described methods, kits, and compositions may be an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof). Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
Any of the antibodies described herein can be either monoclonal or polyclonal. A “monoclonal antibody” refers to a homogenous antibody population and a“polyclonal antibody” refers to a heterogeneous antibody population. These two terms do not limit the source of an antibody or the manner in which it is made.
In one example, the antibody used in the methods described herein is a humanized antibody. Humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human
immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human
immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies may have Fc regions modified as described in WO
99/58572. Other forms of humanized antibodies have one or more CDRs (e.g., one, two, three, four, five, or six) which are altered with respect to the original antibody. These CDRs are also described as being "derived from" one or more CDRs from the original antibody. Humanized antibodies may also involve affinity maturation.
In another example, the antibody described herein is a chimeric antibody, which can include a heavy constant region and a light constant region from a human antibody. Chimeric antibodies refer to antibodies having a variable region or part of variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant portions are homologous to the sequences in antibodies derived from another mammal such as human. In some embodiments, amino acid modifications can be made in the variable region and/or the constant region.
In some embodiments, the anti-CD20 antibodies described herein have a suitable binding affinity to a CD20 molecule such as a human CD20 or an antigenic epitope thereof. As used herein, "binding affinity" refers to the apparent association constant or KA. The KA is the reciprocal of the dissociation constant (KD). The antibody described herein may have a binding affinity (KD) of at least 10-5, 10-6, 10-7, 10-8, 10-9, 10-10 M, or lower. An increased binding affinity corresponds to a decreased KD. Higher affinity binding of an antibody to a first target relative to a second target can be indicated by a higher KA (or a smaller numerical value KD) for binding the first target than the KA (or numerical value KD) for binding the second target. In such cases, the antibody has specificity for the first target (e.g., a protein in a first conformation or mimic thereof) relative to the second target (e.g., the same protein in a second conformation or mimic thereof; or a second protein). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 105 fold.
Binding affinity can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or binding affinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v/v) Surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. The concentration of bound binding protein ([Bound]) is related to the concentration of free target protein ([Free]) and the concentration of binding sites for the binding protein on the target where (N) is the number of binding sites per target molecule by the following equation:
[Bound] = [N][Free]/(Kd+[Free])
It is not always necessary to make an exact determination of KA, though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to KA, and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay (such as, e.g., an in vitro or in vivo assay).
Antibodies capable of binding CD20 as described herein can be made by any method known in the art. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.
In some embodiments, the anti-CD20 antibody is a therapeutic antibody that is capable of treating, alleviating, or reducing the symptoms of any disease or disorder associated with CD20+ cells. In such therapy, the anti-CD20 antibody binds to the cell surface antigen CD20, which is differentially expressed on cancer cells and indicate that the cell expressing the antigen should be subjected to ADCC.
In some embodiments, the anti-CD20 antibody is a polyclonal antibody. In some embodiments, the anti-CD20 antibody is a monoclonal antibody. Examples of anti-CD20 antibodies include, without limitation, rituximab, Y90-ibritumomab (Biogen/IDEC), I31- tositumomab (GSK), ofatumumab (Genmab AC/GSK), ocrelizumab
(Genentech/Roche/Biogen), TRU-015 (Trubion Pharma/Wyeth), veltuzumab/IMMU-106 (Immunomedics), AME-133v (Applied Molecular Evolution/Eli Lilly), PRO131921 humanized (Genentech), and GA101 (Glycart/Roche). In some embodiments, the anti- CD20 antibody is rituximab.
(ii) Therapeutic Applications
Any of the anti-CD20 antibodies described herein can be mixed with a composition for use in the treatment regimen described herein.“Acceptable” means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated. Pharmaceutically acceptable excipients (carriers) including buffers, which are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover. In one example, a pharmaceutical composition described herein contains more than one anti-CD20 antibodies that recognize different epitopes/residues of the target antigen.
The pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEENTM,
PLURONICSTM or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.
Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the anti-CD20 antibody-containing pharmaceutical composition to the subject in need of the treatment. This composition can also be administered via other conventional routes, e.g., administered parenterally. The term“parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, or infusion techniques.
Injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injection, water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a
physiologically acceptable excipients is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer’s solution or other suitable excipients. Intramuscular preparations, e.g., a sterile formulation of a suitable soluble salt form of the antibody, can be dissolved and administered in a pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or 5% glucose solution.
The particular dosage regimen, i.e., dose, timing and repetition, used in the method described herein will depend on the particular subject and that subject's medical history. In some embodiments, the anti-CD20 antibody is administered to the subject in one or more doses of about 100-500 mg/m2, 200- 400 mg/m2, or 300-400 mg/m2. In some embodiments, the anti-CD20 antibody is administered to the subject in one or more doses of about 375 mg/m2.
In some embodiments, the method involves administering the anti-CD20 antibody (e.g., rituximab) to the subject in one dose. In some embodiments, the method involves administering the anti-CD20 antibody (e.g., rituximab) to the subject in multiple dose (e.g., at least 2, 3, 4, 5, 6, 7, or 8 doses). In some embodiments, the anti-CD20 antibody (e.g., rituximab) is administered to the subject in multiple doses, with the first dose of the anti-CD20 antibody administered to the subject about 1, 2, 3, 4, 5, 6, or 7 days prior to administration of the immune cells expressing ACTR. In some embodiments, the first dose of the anti-CD20 antibody is administered to the subject between about 24-48 hours prior to the administration of the immune cells expressing ACTR.
In some embodiments, the anti-CD20 antibody is administered to the subject prior to administration of the immune cells expressing the ACTR and then subsequently about every three weeks. In some embodiments, the anti-CD20 antibody is administered to the subject about every three weeks, following administration of the immune cells, for up to 8 total doses.
In any of the embodiments described herein, the timing of the administration of the following the indicated day (e.g., administration every three weeks encompasses administration on day 18, day 19, day 20, day 21, day 22, day 23, or day 24).
The efficacy of the methods described herein may be assessed by any method known in the art and would be evident to a skilled medical professional. For example, the efficacy of the antibody-based immunotherapy may be assessed by survival of the subject or cancer burden in the subject or tissue or sample thereof. In some embodiments, the antibody based immunotherapy is assessed based on the safety or toxicity of the therapy (e.g., administration of the anti-CD20 antibody and the immune cells expressing the ACTRs) in the subject, for example by the overall health of the subject and/or the presence of adverse events or severe adverse events. (b) ACTR Treatment
Following the anti-CD20 treatment (e.g., about 6-10 days after the first dose of the anti-CD20 antibody), the subject receives ACTR-expressing immune cells such as T cells via, e.g., infusion. (i) ACTR Construct
Antibody-coupled T-cell receptor (ACTR) is a non-naturally-occurring chimeric receptor comprising an Fc binding domain of CD16 with binding affinity and specificity for an Fc fragment, a co-stimulatory signaling domain of a co-stimulatory factor such as 4- 1BB, and a cytoplasmic signaling domain of a cell surface receptor such as CD3ζ. The ACTR may optionally further comprise a transmembrane domain and/or a hinge domain. The ACTRs are configured such that, when expressed on a host cell, the Fc binding domain of CD16 is located extracellularly for binding to a target molecule (e.g., an antibody or a Fc-fusion protein) and the co-stimulatory signaling domain and the cytoplasmic signaling domain are located in the cytoplasm for triggering activation and/or effector signaling. In some embodiments, a ACTR construct as described herein comprises, from N-terminus to C-terminus, the Fc binding domain of CD16, the transmembrane domain, the co-stimulatory signaling domain of 4-1BB, and the cytoplasmic signaling domain of CD3ζ.
Any of the ACTRs described herein may further comprise a hinge domain, which may be located at the C-terminus of the Fc binding domain and the N-terminus of the transmembrane domain. Alternatively or in addition, the ACTR constructs described herein may contain two or more co-stimulatory signaling domains, which may link to each other or be separated by the CD3ζ cytoplasmic signaling domain. The extracellular Fc-binding fragment of CD16, transmembrane domain, co-stimulatory signaling domain(s), and CD3ζ cytoplasmic signaling domain in an ACTR construct may be linked to each other directly, or via a peptide linker.
The Fc-binding fragment in any of the ACTR constructs may be derived from any naturally occurring CD16 (e.g., CD16A or CD16B) receptor, including naturally- occurring polymorphism variants. Examples include CD16 F158 and CD16 V158. In one example, the ACTR construct comprises the Fc-binding fragment of CD16 V158, which may have the amino acid sequence of SEQ ID NO: 6:
GMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYR CQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIP KATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQ Alternatively, it may be a functional variant of a wild-type counterpart, which carry one or more mutations (e.g., up to 10 amino acid residue substitutions) that alter the binding affinity to the Fc portion of an Ig molecule. In some instances, the mutation may alter the glycosylation pattern of the Fc receptor and thus the binding affinity to Fc.
In some embodiments, the Fc-binding fragment of CD16 may comprise an amino acid sequence that is at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99%) identical to the amino acid sequence of the Fc-binding fragment of a naturally- occurring CD16 (e.g., the Fc-binding fragment of CD 158V as described herein). The “percent identity” of two amino acid sequences can be determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol.215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences
homologous to the protein molecules of the disclosure. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res.25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and Any of the Fc-binding fragment of CD16 in an ACTR construct described herein may have a suitable binding affinity for the Fc portion of a therapeutic IgG antibody. As used herein,“binding affinity” refers to the apparent association constant or KA. The KA is the reciprocal of the dissociation constant, KD. The Fc-binding fragment of CD16 may have a binding affinity KD of at least 10-5, 10-6, 10-7, 10-8, 10-9, 10-10M or lower for the Fc portion of antibody.
The transmembrane domain of the ACTRs described herein can be in any form known in the art. As used herein, a“transmembrane domain” refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. Transmembrane domains compatible for use in the ACTRs used herein may be obtained from a naturally occurring protein. Alternatively, it can be a synthetic, non-naturally occurring protein segment, e.g., a hydrophobic protein segment that is thermodynamically stable in a cell membrane.
Transmembrane domains are classified based on the three dimensional structure of the transmembrane domain. For example, transmembrane domains may form an alpha helix, a complex of more than one alpha helix, a beta-barrel, or any other stable structure capable of spanning the phospholipid bilayer of a cell. Furthermore, transmembrane domains may also or alternatively be classified based on the
transmembrane domain topology, including the number of passes that the
transmembrane domain makes across the membrane and the orientation of the protein. For example, single-pass membrane proteins cross the cell membrane once, and multi- pass membrane proteins cross the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7 or more times).
Membrane proteins may be defined as Type I, Type II or Type III depending upon the topology of their termini and membrane-passing segment(s) relative to the inside and outside of the cell. Type I membrane proteins have a single membrane- spanning region and are oriented such that the N-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the C-terminus of the protein is present on the cytoplasmic side. Type II membrane proteins also have a single membrane-spanning region but are oriented such that the C-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the N-terminus of the protein is present on the cytoplasmic side. Type III membrane proteins have multiple membrane-spanning segments and may be further sub-classified based on the number of transmembrane segments and the location of N- and C-termini.
In some embodiments, the transmembrane domain of the ACTR described herein is derived from a Type I single-pass membrane protein. Single-pass membrane proteins include, but are not limited to, CD8α, CD8β, 4-1BB/CD137, CD28, CD34, CD4, FcεRIγ, CD16, OX40/CD134, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRζ, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40,
CD40L/CD154, VEGFR2, FAS, and FGFR2B. In some embodiments, the
transmembrane domain is from a membrane protein selected from the following:
CD8α, CD8β, 4-1BB/CD137, CD28, CD34, CD4, FcεRIγ, CD16, OX40/CD134, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, and FGFR2B. In some examples, the transmembrane domain is of CD8α. In some examples, the transmembrane domain is of 4-1BB/CD137. In other examples, the transmembrane domain is of CD28 or CD34. In yet other examples, the transmembrane domain is not derived from human CD8α. In some embodiments, the transmembrane domain of the ACTR is a single-pass alpha helix.
Transmembrane domains from multi-pass membrane proteins may also be compatible for use in the ACTRs described herein. Multi-pass membrane proteins may comprise a complex (at least 2, 3, 4, 5, 6, 7 or more) alpha helices or a beta sheet structure. Preferably, the N-terminus and the C-terminus of a multi-pass membrane protein are present on opposing sides of the lipid bilayer, e.g., the N-terminus of the protein is present on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is present on the extracellular side. Either one or multiple helix passes from a multi-pass membrane protein can be used for constructing the ACTR described herein.
Transmembrane domains for use in the ACTRs described herein can also comprise at least a portion of a synthetic, non-naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment is at least approximately 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art, for example in U.S. Patent No.7,052,906 B1 and PCT Publication No. WO 2000/032776 A2, the relevant disclosures of which are incorporated by In some embodiments, the amino acid sequence of the transmembrane domain does not comprise cysteine residues. In some embodiments, the amino acid sequence of the transmembrane domain comprises one cysteine residue. In some embodiments, the amino acid sequence of the transmembrane domain comprises two cysteine residues. In some embodiments, the amino acid sequence of the transmembrane domain comprises more than two cysteine residues (e.g., 3, 4, 5 or more).
The transmembrane domain may comprise a transmembrane region and a cytoplasmic region located at the C-terminal side of the transmembrane domain. The cytoplasmic region of the transmembrane domain may comprise three or more amino acids and, in some embodiments, helps to orient the transmembrane domain in the lipid bilayer. In some embodiments, one or more cysteine residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain comprises positively charged amino acids. In some embodiments, the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine, and lysine.
In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the
transmembrane region comprises mostly hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a poly-leucine-alanine sequence.
The hydropathy, or hydrophobic or hydrophilic characteristics of a protein or protein segment, can be assessed by any method known in the art, for example the Kyte and Doolittle hydropathy analysis.
The ACTR construct described herein comprises the co-stimulatory signaling domain of a co-stimulatory receptor such as 4-1BB (CD137). The term“co-stimulatory signaling domain,” as used herein, refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response such as an effector function. The co-stimulatory signaling domain of the ACTR described herein can be a cytoplasmic signaling domain from a co-stimulatory protein, which transduces a signal macrophages, neutrophils, or eosinophils. The co-stimulatory domain (e.g., the co- stimulatory domain of 4-1BB) may be co-used with a co-stimulatory from another co- stimulatory receptor.
In one example, the 4-1BB co-stimulatory signaling domain for use in an ACTR construct comprises the amino acid sequence of:
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 7)
Also within the scope of the present disclosure are variants of a naturally occurring 4-1BB receptor, e.g., variants that include up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5, or 8) as compared to a wild-type counterpart.
Mutation of amino acid residues of the co-stimulatory signaling domain may result in an increase in signaling transduction and enhanced stimulation of immune responses relative to co-stimulatory signaling domains that do not comprise the mutation. Mutation of amino acid residues of the co-stimulatory signaling domain may result in a decrease in signaling transduction and reduced stimulation of immune responses relative to co-stimulatory signaling domains that do not comprise the mutation. In some embodiments, the co-stimulatory signaling domain is a variant of the 4-1BB costimulatory signaling domain.
In some embodiments, the ACTRs may comprise more than one co-stimulatory signaling domain (e.g., 2, 3 or more). In some embodiments, the ACTR comprises two or more of the same co-stimulatory signaling domains, for example, two copies of the co-stimulatory signaling domain of 4-1BB. In some embodiments, the ACTR comprises two or more co-stimulatory signaling domains from different co-stimulatory proteins, such as any two or more co-stimulatory proteins described herein. Selection of the type(s) of co-stimulatory signaling domains may be based on factors such as the type of host cells to be used with the ACTRs (e.g., immune cells such as T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function. In some embodiments, the ACTR comprises two co-stimulatory signaling domains. In some embodiments, the two co-stimulatory signaling domains are CD28 and 4-1BB. In some embodiments, the two co-stimulatory signaling domains are CD28LL→GG variant and 4-1BB.
The ACTR described herein further comprises a cytoplasmic signaling domain of CD3ζ. In one example, the cytoplasmic signaling domain of CD3ζ comprises the amino acid sequence of: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 8).
In some embodiments, the ACTR described herein may further comprise a hinge domain that is located between the Fc binding domain and the transmembrane domain. A hinge domain is an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the extracellular ligand-binding domain of an Fc receptor relative to the transmembrane domain of the ACTRs can be used.
The hinge domain may contain about 10-100 amino acids, e.g., 15-150 amino acids, 20-100 amino acids, or 30-60 amino acids. In some embodiments, the hinge domain may be of 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids in length.
In some embodiments, the hinge domain is a hinge domain of a naturally occurring protein. Hinge domains of any protein known in the art to comprise a hinge domain are compatible for use in the ACTRs described herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to the ACTR. In some embodiments, the hinge domain is of CD8α. In some embodiments, the hinge domain is a portion of the hinge domain of CD8α, e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8α. In one example, the ACTR described herein comprises a hinge/transmembrane domain from CD8α, which may comprise the amino acid sequence of:
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC
(SEQ ID NO: 48)
Hinge domains of antibodies, such as an IgG, IgA, IgM, IgE, or IgD antibodies, are also compatible for use in the ACTRs described herein. In some embodiments, the hinge domain is the hinge domain that joins the constant domains CH1 and CH2 of an antibody. In some embodiments, the hinge domain is of an antibody and comprises the hinge domain of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH3 constant region of the antibody In some embodiments the hinge domain comprises the hinge domain of an antibody and the CH2 and CH3 constant regions of the antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some
embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and the CH3 constant region of an IgG1 antibody.
Non-naturally occurring peptides may also be used as hinge domains for the ACTRs described herein. In some embodiments, the hinge domain between the C- terminus of the extracellular ligand-binding domain of an Fc receptor and the N- terminus of the transmembrane domain is a peptide linker, such as a (GlyxSer)n linker, wherein x and n, independently can be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more. In some embodiments, the hinge domain is (Gly4Ser)n (SEQ ID NO: 50), wherein n can be an integer between 3 and 60, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more. In some embodiments, the hinge domain is (Gly4Ser)3 (SEQ ID NO: 51). In some embodiments, the hinge domain is (Gly4Ser)6 (SEQ ID NO: 52). In some embodiments, the hinge domain is (Gly4Ser)9 (SEQ ID NO: 53). In some embodiments, the hinge domain is (Gly4Ser)12 (SEQ ID NO: 54). In some
embodiments, the hinge domain is (Gly4Ser)15 (SEQ ID NO: 55). In some
embodiments, the hinge domain is (Gly4Ser)30 (SEQ ID NO: 56). In some
embodiments, the hinge domain is (Gly4Ser)45 (SEQ ID NO: 57). In some
embodiments, the hinge domain is (Gly4Ser)60 (SEQ ID NO: 58).
In other embodiments, the hinge domain is an extended recombinant polypeptide (XTEN), which is an unstructured polypeptide consisting of hydrophilic residues of varying lengths (e.g., 10-200 amino acid residues, 20-150 amino acid residues, 30-100 amino acid residues, or 40-80 amino acid residues). Amino acid sequences of XTEN peptides will be evident to one of skill in the art and can be found, for example, in U.S. Patent No.8,673,860, which is herein incorporated by reference. In some embodiments, the hinge domain is an XTEN peptide and comprises 60 amino acids. In some embodiments, the hinge domain is an XTEN peptide and comprises 30 amino acids. In some embodiments, the hinge domain is an XTEN peptide and comprises 45 amino acids. In some embodiments, the hinge domain is an XTEN peptide and comprises 15 amino acids.
Moreover, the ACTR may further comprise a signal peptide (also known as a signal sequence) at the N-terminus of the polypeptide. In general, signal sequences are peptide sequences that target a polypeptide to the desired site in a cell. In some embodiments, the signal sequence targets the ACTR to the secretory pathway of the cell and will allow for integration and anchoring of the ACTR into the lipid bilayer. Signal sequences including signal sequences of naturally occurring proteins or synthetic, non- naturally occurring signal sequences, that are compatible for use in the ACTRs described herein will be evident to one of skill in the art. In some embodiments, the signal sequence from CD8α, for example, amino acid sequence of MALPVTALLLPLALLLHAARP (SEQ ID NO: 49). In some embodiments, the signal sequence is from CD28. In other embodiments, the signal sequence is from the murine kappa chain. In yet other embodiments, the signal sequence is from CD16.
The ACTRs described herein would confer a number of advantages. For example, via the extracellular domain that binds Fc, the ACTR constructs described herein can bind to the Fc portion of antibodies or other Fc-containing molecules, rather than directly binding a specific target antigen (e.g., a cancer antigen). Thus, immune cells expressing the ACTR constructs described herein would be able to induce cell death of any type of cells that are bound by an antibody or another Fc-containing molecule.
Table 1 below provides exemplary ACTR constructs described herein. This exemplary constructs have, from N-terminus to C-terminus in order, the signal sequence, the Fc binder (e.g., an extracellular domain of an Fc receptor), the hinge domain, and the transmembrane, while the positions of the co-stimulatory domain and the cytoplasmic signaling domain can be switched. Table 1: Exemplary ACTRs.
Figure imgf000026_0001
24 4901170
Figure imgf000027_0001
25 4901170
Figure imgf000028_0001
Amino acid sequences of the example ACTRs are provided below (signal peptide italicized). SEQ ID NO:1
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPED NSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPR WVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGL VGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQPLSLRPEAC RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRP VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR SEQ ID NO: 2:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIISFFLALTSTALLFLLFFLTLRFSVVKRGKR GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 3:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKKR GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 4:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDLIALVTSGALLAVLGITGYFLMNRKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 5:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDLLAALLALLAALLALLAALLARSKKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 9:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLL HSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEE DGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR SEQ ID NO: 10:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLV ITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 11:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQGGSPAGSPTSTEEGTSESA TPESGPGTSTEPSEGSAPGSPAGSPTIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 13:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRGG HSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEE DGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR SEQ ID NO: 14:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDMALIVLGGVAGLLLFIGLGIFFCVRKRGRKKL LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNE LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 15:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDMALIVLGGVAGLLLFIGLGIFFCVRRSKRSRG GHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 16:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDLCYILDAILFLYGIVLTLLYCRLKKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 17:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDLLLILLGVLAGVLATLAALLARSKKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 18:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDITLGLLVAGVLVLLVSLGVAIHLCKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 19:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDVSFCLVMVLLFAVDTGLYFSVKTNKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 20:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDVAAILGLGLVLGLLGPLAILLALYKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 21:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDLCYLLDGILFIYGVILTALFLRVKKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 22:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDVMSVATIVIVDICITGGLLLLVYYWSKNRKRG RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 23:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDGFLFAEIVSIFVLAVGVYFIAGQDKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 24:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDGIIVTDVIATLLLALGVFCFAGHETKRGRKKL LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNE LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 25:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDVIGFRILLLKVAGFNLLMTLRLWKRGRKKLLY IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELN LGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG KGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 26:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIIVAVVIATAVAAIVAAVVALIYCRKKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYN ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 27:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDVLFYLAVGIMFLVNTVLWVTIRKEKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 28:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIIILVGTAVIAMFFWLLLVIILRTKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 29:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDLGWLCLLLLPIPLIVWVKRKKRGRKKLLYIFK QPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 30:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIAIYCIGVFLIACMVVTVILCRMKKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 31:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 32:
MALPVTALLLPLALLLHAARPQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSS STQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVS SRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGTYHC SGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLY FSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQ LPTPVWFHIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR SEQ ID NO: 33:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLV ITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 34:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQEPKSCDKTHTCPGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLL SLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSA DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 35:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQEPKSCDKTHTCPIYIWAPL AGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 36:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEAFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQ EEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPR SEQ ID NO: 37:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPFACDIYIW APLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 38:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQGGGGSGGGGSGGGGSIYIW APLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 39:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQ EEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPR SEQ ID NO: 40:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 41:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSIYIWAPLAGTCGVLLLS LVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 42:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQGGSPAGSPTSTEEGTSESA TPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAIYIWAPLAGTCGVLLLS LVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 43:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQGGSPAGSPTSTEEGTSESA TPESGPGTSTEIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQ EEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPR SEQ ID NO: 44:
MALPVTALLLPLALLLHAARPGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQGGSPAGSPTSTEEGTIYIW APLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 45:
MLRLLLALNLFPSIQVTGGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDN STQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAP RWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFC RGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQPLSL RPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIF KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLG RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 46:
METDTLLLWVLLLWVPGSTGDGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSP EDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLL QAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGS YFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 47:
MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNST QWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRW VFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRG LVGSKNVSSETVNITITQGLAVSTISSFFPPGYQTTTPAPRPPTPAPTIASQPLSLRP EACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQ PFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR
It would be appreciated by a skilled person that the signal peptide of any of the above exemplary ACTRs may be removed or replaced with another suitable signal peptide without affecting the function of the ACTR. Such variants of the exemplary ACTRs are also within the scope of the present disclosure.
Any of the ACTRs described herein can be prepared by a routine method, such as recombinant technology. Methods for preparing the ACTRs herein involve generation of a nucleic acid that encodes a polypeptide comprising each of the domains of the ACTR construct as described herein. In some embodiments, the nucleic acid sequence encodes any one of the exemplary ACTRs provided by SEQ ID NO: 1-5, and 9-47.
Sequences of each of the components of the ACTRs may be obtained via routine technology, e.g., PCR amplification from any one of a variety of sources known in the art. In some embodiments, sequences of one or more of the components of the ACTRs are obtained from a human cell. Alternatively, the sequences of one or more components of the ACTRs can be synthesized. Sequences of each of the components (e.g., domains) can be joined directly or indirectly (e.g., using a nucleic acid sequence encoding a peptide linker) to form a nucleic acid sequence encoding the ACTR, using methods such as PCR amplification or ligation. Alternatively, the nucleic acid encoding the ACTR may be synthesized. In some embodiments, the nucleic acid is DNA. In other embodiments, the nucleic acid is RNA. (ii) Immune Cells Expressing ACTRs
Host cells expressing the ACTR described herein provide a specific population of cells that can recognize target cells bound by antibodies (e.g., therapeutic antibodies) or Fc-fusion proteins. Engagement of the Fc binding domain of an ACTR construct expressed on such host cells (e.g., immune cells) with the Fc portion of an antibody transmits an activation signal to the co-stimulatory signaling domain(s) and the cytoplasmic signaling domain of the ACTR construct, which in turn activates cell proliferation and/or effector functions of the host cell, such as ADCC effects triggered by the host cells. The combination of co-stimulatory signaling domain(s) and the cytoplasmic signaling domain may allow for robust activation of multiple signaling pathways within the cell. In some embodiments, the host cells are immune cells, such as T cells, NK cells, macrophages, neutrophils, eosinophils, or any combination thereof. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells. In other embodiments, the immune cells can be established cell lines, for example, NK-92 cells. In some embodiments, the cells are cells that can develop and/or differentiate into immune cells, for example progenitor cells.
To construct the immune cells that express any of the ACTR constructs described herein, expression vectors for stable or transient expression of the ACTR construct may be constructed via conventional methods as described herein and introduced into immune host cells. For example, nucleic acids encoding the ACTRs may be cloned into a suitable expression vector, such as a viral vector in operable linkage to a suitable promoter. The nucleic acids and the vector may be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the termini of the nucleic acid encoding the ACTRs. The synthetic linkers may contain nucleic acid sequences that correspond to a particular restriction site in the vector. The selection of expression
vectors/plasmids/viral vectors would depend on the type of host cells for expression of the ACTRs, but should be suitable for integration and replication in eukaryotic cells.
A variety of promoters can be used for expression of the ACTRs described herein, including, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter.
Additional promoters for expression of the ACTRs include any constitutively active promoter in an immune cell. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within an immune cell.
Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in host cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColE1 for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a“suicide switch” or“suicide gene” which when triggered causes cells carrying the vector to die (e.g., HSV thymidine kinase, an inducible caspase such as iCasp9), and reporter gene for assessing expression of the ACTR. See section VI below. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Examples of the preparation of vectors for expression of ACTRs can be found, for example, in
US2014/0106449, herein incorporated in its entirety by reference.
In some embodiments, the ACTR construct or the nucleic acid encoding said ACTR is a DNA molecule. In some embodiments, the ACTR construct or the nucleic acid encoding said ACTR is a transposon. In some embodiments, the ACTR construct or the nucleic acid encoding said ACTR is a plasmid. In some embodiments, the ACTR construct or the nucleic acid encoding said ACTR is a DNA plasmid may be electroporated into immune cells (see, e.g., Till, et al. Blood (2012) 119(17): 3940- 3950). In some embodiments, the nucleic acid encoding the ACTR is an RNA molecule, which may be electroporated into immune cells.
Any of the vectors comprising a nucleic acid sequence that encodes an ACTR construct described herein is also within the scope of the present disclosure. Such a vector may be delivered into host cells such as host immune cells by a suitable method. Methods of delivering vectors to immune cells are well known in the art and may include DNA electroporation, RNA electroporation, transfection reagents such as liposomes, or viral transduction. In some embodiments, the vectors for expression of the ACTRs are delivered to host cells by viral transduction. Exemplary viral methods for delivery include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat. Nos.5,219,740 and 4,777,127; GB Patent No.2,200,651; and EP Patent No.0345242), alphavirus-based vectors, and adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). In some embodiments, the vectors for expression of the ACTRs are retroviruses. In some embodiments, the vectors for expression of the ACTRs are lentiviruses. In some embodiments, the vectors for expression of the ACTRs are gamma-retroviruses. In some embodiments, the vectors for expression of the ACTRs are adeno-associated viruses (AAVs).
In examples in which the vectors encoding ACTRs are introduced to the host cells using a viral vector, viral particles that are capable of infecting the immune cells and carry the vector may be produced by any method known in the art and can be found, for example in PCT Application No. WO 1991/002805A2, WO 1998/009271 A1, and U.S. Patent 6,194,191. The viral particles are harvested from the cell culture supernatant and may be isolated and/or purified prior to contacting the viral particles with the immune cells.
Following introduction into the host cells a vector encoding any of the ACTRs provided herein, the cells are cultured under conditions that allow for expression of the ACTR. In examples in which the nucleic acid encoding the ACTR is regulated by a regulatable promoter, the host cells are cultured in conditions wherein the regulatable promoter is activated. In some embodiments, the promoter is an inducible promoter and the immune cells are cultured in the presence of the inducing molecule or in conditions in which the inducing molecule is produced. Determining whether the ACTR is expressed will be evident to one of skill in the art and may be assessed by any known method, for example, detection of the ACTR-encoding mRNA by quantitative reverse transcriptase PCR (qRT-PCR) or detection of the ACTR protein by methods including Western blotting, fluorescence microscopy, and flow cytometry.
Alternatively, expression of the ACTR may take place in vivo after the immune cells are administered to a subject.
Alternatively, expression of an ACTR construct in any of the immune cells disclosed herein can be achieved by introducing RNA molecules encoding the ACTR constructs. Such RNA molecules can be prepared by in vitro transcription or by chemical synthesis. The RNA molecules can then introduced into suitable host cells such as immune cells (e.g., T cells, NK cells, macrophages, neutrophils, eosinophils, or any combination thereof) by, e.g., electroporation. For example, RNA molecules can be synthesized and introduced into host immune cells following the methods described in Rabinovich et al., Human Gene Therapy, 17:1027-1035 and WO WO2013/040557.
Methods for preparing host cells expressing any of the ACTRs described herein may also comprise activating the host cells ex vivo. Activating a host cell means stimulating a host cell into an activate state in which the cell may be able to perform effector functions (e.g., ADCC). Methods of activating a host cell will depend on the type of host cell used for expression of the ACTRs. For example, T cells may be activated ex vivo in the presence of one or more molecule such as an anti-CD3 antibody, an anti-CD28 antibody, IL-2, or phytohemoagglutinin. In other examples, NK cells may be activated ex vivo in the presence of one or molecules such as a 4-1BB ligand, an anti-4-1BB antibody, IL-15, an anti-IL-15 receptor antibody, IL-2, IL12, IL- 21, and K562 cells. In some embodiments, the host cells expressing any of the ACTRs described herein are activated ex vivo prior to administration to a subject. Determining whether a host cell is activated will be evident to one of skill in the art and may include assessing expression of one or more cell surface markers associated with cell activation, expression or secretion of cytokines, and cell morphology.
The methods of preparing host cells expressing any of the ACTRs described herein may comprise expanding the host cells ex vivo. Expanding host cells may involve any method that results in an increase in the number of cells expressing ACTRs, for example, allowing the host cells to proliferate or stimulating the host cells to proliferate. Methods for stimulating expansion of host cells will depend on the type of host cell used for expression of the ACTRs and will be evident to one of skill in the art. In some embodiments, the host cells expressing any of the ACTRs described herein are expanded ex vivo prior to administration to a subject.
In some embodiments, the host cells expressing the ACTRs are expanded and activated ex vivo prior to administration of the cells to the subject. In some
embodiments, the ex vivo expansion and/or activation polarizes the host cells to a desired phenotype, for example, T cells or NK cells. (iii) Therapeutic Applications
The immune cells can be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure.
To perform the methods described herein, an effective amount of the immune cells expressing any of the ACTR constructs described herein can be administered into a subject after the first dose of the anti-CD20 antibody (e.g., no less than about 10 days after the first dose of the anti-CD20 antibody). In some embodiments, the immune cells expressing the ACTRs are administered to the subject about 1 day after administration of the anti-CD20 antibody. In some embodiments, the immune cells expressing the ACTRs are administered to the subject about 1, 2, 3, 4, 5, 6, or 7 days after administration of the anti-CD20 antibody.
The immune cells may be autologous to the subject, i.e., the immune cells are obtained from the subject in need of the treatment, genetically engineered for expression of the ACTR constructs, and then administered to the same subject.
Administration of autologous cells to a subject may result in reduced rejection of the host cells as compared to administration of non-autologous cells. Alternatively, the host cells are allogeneic cells, i.e., the cells are obtained from a first subject, genetically engineered for expression of the ACTR construct, and administered to a second subject that is different from the first subject but of the same species. For example, allogeneic immune cells may be derived from a human donor and administered to a human recipient who is different from the donor.
In some embodiments, the immune cells expressing the ACTRs are
administered to the subject via infusion in a dose of up to about 0.5x106 cells/kg. In some embodiments, the immune cells expressing the ACTRs are administered to the subject in a dose of less than about 5.0x106 cells/kg. In some embodiments, the immune cells expressing the ACTRs are administered to the subject in a dose between 0.5x106 cells/kg - 5.0x106 cells/kg, for example from about 0.5x106 cells/kg to about 1.5x106 cells/kg or from about 1.5x106 cells/kg to about 5.0x106 cells/kg. In some embodiments, the immune cells expressing the ACTRs are administered to the subject in a dose of about 0.1 x106 cells/kg, 0.2 x106 cells/kg, 0.3x106 cells/kg, 0.4 x106 cells/kg, 0.5 x106 cells/kg, 0.6 x106 cells/kg, 0.7 x106 cells/kg, 0.8 x106 cells/kg, 0.9 x106 cells/kg, 1.0 x106 cells/kg, 1.1 x106 cells/kg, 1.2 x106 cells/kg, 1.3 x106 cells/kg, 1.4 x106 cells/kg, 1.5 x106 cells/kg, 2.0 x106 cells/kg, 2.5 x106 cells/kg, 3.0 x106 cells/kg, 3.5 x106 cells/kg, 4.0 x106 cells/kg, 4.5 x106 cells/kg, or about 5.0 x106 cells/kg. In some examples, the dose of the immune cells expressing ACTR may be as high as 1 x 108 cells/kg, for example, 5.0 x106 cells/kg to 1 x 108 cells/kg, 1 x 107 cells/kg to 1 x 108 cells/kg, or 5.0 x 107 cells/kg to 1 x 108 cells/kg.
As will be evident to one of skill in the art, the amount of immune cells expressing the ACTRs administered to the subject may depend on factors such as the type of CD20+ B-cell lymphoma. In some embodiments, the subject has diffuse large B-cell lymphoma (DLBCL) or primary mediastinal B cell lymphoma (PMBCL) and the dose of the immune cells expressing the ACTR is about 5x106 cells/kg. In some embodiments, the subject has mantle cell lymphoma (MCL), primary mediastinal B cell lymphoma (PMBCL), grade 3b follicular lymphoma (Gr3b-FL), and transformed histology follicular lymphoma (TH-FL) and the dose of the immune cells expressing the ACTR is about 5x106 cells/kg. In some embodiments, the subject has DLBCL, MCL, PMBCL, Gr3b-FL, TH-FL and the dose of the immune cells expressing the ACTR is less than about 5x106 cells/kg.
Determination of whether an amount of the cells or compositions described herein achieved the therapeutic effect would be evident to one of skill in the art.
Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of
administration and like factors within the knowledge and expertise of the health practitioner. In some embodiments, the effective amount alleviates, relieves,
ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject. In some embodiments, the subject is a human. In some embodiments, the subject is a human patient having relapsed or refractory CD20+ lymphoma. III. Pre-Treatment Period
Prior to the conditioning regimen as described herein, a subject may undergo a pre-treatment period, during which the subject may receive an anti-cancer therapy to control the disease, e.g., radiotherapy, chemotherapy, immunotherapy, and/or surgery. Examples include therapeutic vaccines (including but not limited to GVAX, DC-based vaccines, etc.), checkpoint inhibitors (including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.) or activators (including but not limited to agents that enhance 41BB, OX40, etc.).
Non-limiting examples of other therapeutic agents useful for combination with the immunotherapy of the disclosure include: (i) anti-angiogenic agents (e.g., TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitors of metalloproteases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptors, placental proliferin-related protein, as well as those listed by Carmeliet and Jain (2000)); (ii) a VEGF antagonist or a VEGF receptor antagonist such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinases and any combinations thereof; and (iii) chemotherapeutic compounds such as, e.g., pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine), purine analogs, folate antagonists and related inhibitors (e.g., mercaptopurine, thioguanine, pentostatin and 2- chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (e.g., vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (e.g., paclitaxel and docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (e.g., etoposide and teniposide), DNA damaging agents (e.g., actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin,
cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethyhnelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (e.g.,
mechlorethamine, cyclophosphamide and analogs, melphalan, or chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (e.g., carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (e.g., cisplatin and carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (e.g., estrogen, tamoxifen, goserelin, bicalutamide, and nilutamide) and aromatase inhibitors (e.g., letrozole and anastrozole); anticoagulants (e.g., heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (e.g., breveldin);
immunosuppressives (e.g., cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (e.g., TNP-470, genistein, and bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (e.g., trastuzumab); cell cycle inhibitors and differentiation inducers (e.g., tretinoin); mTOR inhibitors, topoisomerase inhibitors (e.g., doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, and irinotecan), corticosteroids (e.g., cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors;
mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors. See also Example 1 below.
During the pre-treatment period, the subject may also undergo baseline assessments to determine whether the subject is suitable for the following conditioning and/or treatment regimens.
In some embodiments, immune cells may be collected from the subject during the pre-treatment period, for example, about 30-60 days prior to the immune cell infusion. In some embodiments, the immune cells are collected from the subject by leukapheresis.
The population of immune cells can be obtained from any source, such as peripheral blood mononuclear cells (PBMCs), bone marrow, tissues such as spleen, lymph node, thymus, or tumor tissue. A source suitable for obtaining the type of host cells desired would be evident to one of skill in the art. In some embodiments, the population of immune cells is derived from PBMCs. In some embodiments, the population of immune cells is derived from a human patient having lymphoma. In some embodiments, the population of immune cells is collected from a human patient having lymphoma by leukapheresis. The type of host cells desired (e.g., immune cells such as T cells, NK cells, macrophages, neutrophils, eosinophils, or any combination thereof) may be expanded within the population of cells obtained by co-incubating the cells with stimulatory molecules, for example, IL-2, anti-CD3 and anti-CD28
antibodies may be used for expansion and/or stimulation of T cells. In some embodiments, the subject is free from alemtuzumab within 6 months prior to collecting the immune cells; fludarabine, cladribine, or clofarabine within 3 months of collecting the immune cells; external beam radiation, administration of a monoclonal antibody, or lymphotoxic chemotherapy within 2 weeks of collecting the immune cells; and experimental agents within 3 half-lives of the experimental agent collecting the immune cells.
The immune cells collected from the subject may be genetically engineered following the methods known in the art and/or described herein to introduce expression vectors for producing an ACTR construct on cell surface. Such ACTR-expressing immune cells can then be given to the subject at the treatment stage, following the anti- CD20 antibody treatment. IV. Kits for Therapeutic Use
The present disclosure also provides kits for use of the immune cells expressing ACTRs and anti-CD20 antibodies in methods for treating lymphoma. Such kits may include one or more containers comprising a first pharmaceutical composition that comprises one or more lymphodepleting agents, a second pharmaceutical composition that comprises an anti-CD20 antibody and a pharmaceutically acceptable carrier, and a third pharmaceutical composition that comprises any nucleic acid or host cells (e.g., immune cells such as those described herein), and a pharmaceutically acceptable carrier.
In some embodiments, the kit can comprise instructions for use in any of the methods described herein. The included instructions can comprise a description of administration of the first, second, and/or third pharmaceutical compositions to a subject to achieve the intended activity in a subject. The kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment. In some embodiments, the instructions comprise a description of administering the first, second, and third pharmaceutical compositions to a subject who is in need of the treatment.
The instructions relating to the use of the ACTRs and the first, second, and third pharmaceutical compositions described herein generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical compositions are used for treating, delaying the onset, and/or alleviating a disease or disorder in a subject.
The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device, or an infusion device. A kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port. At least one active agent in the pharmaceutical composition is a ACTR as described herein.
Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above. General techniques
The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed.1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed.1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds.1994); Current Protocols in
Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997);
Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed.1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985»; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984»; Animal Cell Culture (R.I. Freshney, ed. (1986»; Immobilized Cells and Enzymes (lRL Press, (1986»; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).
Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein. EXAMPLES
A phase 1, multi-center, single-arm, open label study is described to evaluate the safety and efficacy of infusion of a single dose of γ-retrovirus transduced T cells expressing ACTR in combination with the anti-CD20 antibody rituximab, in subjects with relapsed or refractory CD20+ B-cell lymphoma, such as diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary mediastinal B-cell lymphoma
(PMBCL), grade 3b follicular lymphoma (Gr3b-FL) or transformed histology follicular lymphoma (TH-FL).
The study is separated into 2 sequential phases, a dose escalation phase and an expansion cohort phase with the intent of evaluating the safety of the ACTR-expressing T cells in combination with rituximab in subjects with relapsed or refractory CD20+ B-cell lymphomas. Each study phase is comprised of a pre-treatment period, a treatment period, and a follow-up period. Study procedures are summarized in Figure 1. Subject Inclusion Criteria
1) Age: 18 to 75 years at the time of informed consent
2) Signed written informed consent obtained prior to study procedures
3) Histologically-confirmed relapsed or refractory CD20+ B-cell lymphoma of one of the following types, with documented disease progression or recurrence following the immediate prior therapy:
-DLBCL, regardless of cell of origin or underlying molecular genetics -MCL
-PMBCL
-Gr3b-FL
-TH-FL (Subjects with TH-FL include only those who had a prior diagnosis of FL before transforming to DLBCL)
4) Biopsy-confirmed CD20+ expression of the underlying malignancy by
immunohistochemical staining or flow cytometry between the most recent dose of an anti- CD20 monoclonal antibody (mAb) and study enrollment
5) At least 1 measurable lesion on imaging. Lesions that have been previously irradiated will be considered measurable only if progression has been documented following completion of radiation therapy
6) Must have received adequate prior therapy for the underlying CD20+ B-cell lymphoma, defined as an anti-CD20 mAb in combination with an anthracycline-containing chemotherapy regimen (i.e. chemo-immunotherapy) and at least one of the following:
-biopsy-proven refractory disease after frontline chemo-immunotherapy -relapse within 1 year from frontline chemo-immunotherapy and ineligible for autologous hematopoietic stem cell transplant (auto-HSCT)
-For subjects with DLBCL, PMBCL, and Gr3b-FL: relapsed or refractory disease following at least 2 prior regimens or following an auto-HSCT
-For subjects with TH-FL: relapsed or refractory disease following at least 2 prior regimens or following an auto-HSCT. At least 1 prior regimen with an anti-CD20 mAb in combination with chemotherapy is required following documented transformation
-For subjects with MCL (confirmed with cyclin D1 expression or evidence of t(11;14) by cytogenetics, fluorescent in situ hybridization (FISH) or polymerase chain reaction (PCR)): relapsed or refractory disease after at least 1 prior regimen with chemo- immunotherapy (prior auto-HSCT is allowable)
7) Karnofsky performance scale≥ 60% (See Appendix A)
8) Life expectancy of at least 6 months
9) Absolute neutrophil count (ANC) > 1000/µL
10) Platelet count > 50,000/µL
11) For women of childbearing potential (defined as physiologically capable of becoming pregnant), agreement to use of highly effective contraception for at least 1 year following infusion of ACTR-expressing T cells. For men with partners of childbearing potential, agreement to use effective barrier contraception for at least 1 year following infusion of ACTR-expressing T cells. Subject Exclusion Criteria
1) Known active central nervous system (CNS) involvement by malignancy.
Subjects with prior CNS involvement with their lymphoma must have completed effective treatment of their CNS disease at least 3 months prior to enrollment with no evidence of disease clinically and at least stable findings on relevant CNS imaging
2) Prior treatment as follows:
-alemtuzumab within 6 months of enrollment
-fludarabine, cladribine, or clofarabine within 3 months of enrollment -external beam radiation within 2 weeks of enrollment
-mAb (including rituximab) within 2 weeks of enrollment
-other lymphotoxic chemotherapy (including steroids except as below) within 2 weeks of enrollment
-experimental agents within 3 half-lives prior to enrollment, unless progression is documented on therapy
3) Serum creatinine≥ 1.5 x age-adjusted upper limits of normal (ULN)
4) Pulse oximetry < 92% on room air
5) Direct bilirubin≥ 3.0 mg/dL (50 mmol/L)
6) Alanine transaminase (ALT)≥ 3 times the ULN, unless determined to be directly due to lymphoma
7) Aspartate transaminase (AST)≥ 3 times the ULN, unless determined to be directly due to lymphoma 8) Class III or IV heart failure as defined by the New York Heart Association (NYHA), history of cardiac angioplasty or stenting, documented myocardial infarction or unstable angina within 6 months prior to enrollment, cardiac ejection fraction of < 45%, or other clinically significant cardiac disease
9) Clinical history of, prior diagnosis of, or overt evidence of autoimmune disease, regardless of severity
10) Clinically significant, active infection, in the judgment of the investigator 11) Pregnancy (negative serum pregnancy test to be obtained within 6 days prior to enrollment for subjects of childbearing potential)
12) Breastfeeding
13) Primary immunodeficiency
14) Seropositive for Human Immunodeficiency Virus (HIV) 1 or HIV 2, or positive hepatitis B surface antigen (HBsAg) or hepatitis C antibody
15) Will need or has needed active treatment of a second malignancy within the prior 3 years before enrollment, other than FL, non-melanoma skin cancers, localized prostate cancer treated with curative intent, or cervical carcinoma in situ
16) Is unable to receive any of the agents used in this study due a history of severe immediate hypersensitivity reaction (e.g. hypersensitivity to dimethyl sulfoxide (DMSO)) 17) History of prior allogeneic HSCT
18) History of Richter’s transformation from chronic lymphocytic leukemia (CLL) 19) Prior infusion of a genetically modified therapy
20) History or presence of clinically relevant CNS disease such as history of a seizure disorder, dementia or cerebrovascular ischemia/hemorrhage with residual paresis or aphasia, cerebellar disease, Parkinson’s disease, psychosis or organic brain syndrome
21) Current use of corticosteroid therapy > 5 mg/day of prednisone or equivalent doses of other corticosteroids (topical, intranasal, and inhaled corticosteroids in standard doses and physiologic replacement for subjects with adrenal insufficiency are allowed)
22) Concomitant participation in any other interventional studies for the treatment of the subject’s B-cell lymphoma Study Populations and Study Phases
Dose escalation phase The dose escalation phase of the study enrolls 9-18 subjects with CD20+ B cell lymphoma to evaluate the safety of escalating doses of ACTR-expressing T cells in combination with rituximab.
The dose escalation phase follows a traditional 3+3 model to determine the maximum tolerated dose (MTD) and the Recommended Phase 2 Dose (RP2D) for the ACTR- expressing T cells. The dose-limiting toxicity evaluation period will be 28 days for each dose cohort, assessed from Day 0 (the day of administration of ACTR-expressing T cells). There are three dose cohorts defined by a dose range (i.e. up to 0.5 x 106, 0.51– 1.5 x 106, and 1.51 – 5 x 106 ACTR T-cells/kg). During the dose escalation phase ACTR-expressing T cell are generated and administered at the maximum dose within each dose range. A target maximum dose of 5 x 106 ACTR T-cells/kg is planned. The highest dose cohort studied may be concluded as the Maximal Feasible Dose, and the RP2D in this circumstance will be assessed accordingly. However, should manufacturing constraints not be limiting to further dose escalation, additional dose cohorts may be investigated, with each target maximal dose increased by ½ log from the previous target maximal dose, as defined in the planned dose cohorts below.
In the first cohort, three subjects receive an initial target maximum dose of 0.5 x 106 ACTR T-cells/kg in combination with rituximab. If fewer ACTR T-cells than the maximum target dose for a subject are obtained during the production process, the cells available are infused and the dose given is recorded. If the initial dose cohort is not found to be safe, as determined by dose limiting toxicities (DLT) assessment, then a lower maximal target dose than 0.5 x 105 ACTR T-cells/kg in combination with rituximab may be evaluated.
If the first target maximum dose is tolerated by all subjects in the first cohort, the dose level will be escalated to the next target maximum dose of 1.5 x 106 ACTR T-cells/kg in combination with rituximab for the next 3 subjects. If fewer ACTR T-cells than the maximum target dose in the second planned dose cohort are obtained for a subject during the production process, the cells available are infused and the dose given is recorded.
If the second dose is tolerated, the dose level is escalated to the next target maximum dose of 5 x 106 ACTR T-cells/kg in combination with rituximab. If fewer ACTR T-cells than the maximum target dose for a subject are obtained during the production process, the cells available are infused and the dose given is recorded. Cohort expansion phase
The cohort expansion phase enrolls up to an additional 36 subjects with CD20+ B cell lymphoma in 3 cohorts of 10 to 12 subjects each:
- subjects with DLBCL or PMBCL treated with ACTR-expressing T-cells at the RP2D (i.e. maximal target dose and corresponding dose range
- subjects with MCL, Gr3b-FL, or TH-FL treated with ACTR-expressing T-cells at the RP2D (i.e., maximal target dose and corresponding dose range) - subjects with DLBCL, MCL, PMBCL, Gr3b-FL or TH-FL treated with ACTR- expressing T-cells below the RP2D dose range Figure 1 shows a diagram of the study including a pre-treatment period involving screening eligible subjects, enrolling the subjects, performing leukapheresis to obtain immune cells from the subjects and modifying the immune cells to express ACTR. The pre- treatment period is followed by the treatment period in which subjected undergo a conditioning regimen (lymphodepleting regimen) involving administration of fludararbine and cyclophosphoamide for three days. The subjects are then administered rituximab (cycle 1) on day -1 (the day prior to administration of the ACTR-expressing T cells), followed by the ACTR-expressing T cells on day 0. Rituximab may be subsequently administered every three weeks for up to 7 additional doses (cycles). The study further includes a follow-up period in which the subjects are assessed and may enter a long term follow up study involving administration of ACTR-expressing T cells. Pre-treatment period
Upon completion of screening procedures and study enrollment, eligible subjects enter the pre-treatment period, which continues until the treatment period commences.
During this period, additional baseline assessments are completed, the study subject receives anti-cancer chemotherapy or radiation for disease control (at the discretion of the
investigator), and leukapheresis are performed to collect PBMCs for production of the autologous ACTR-expressing T cells. The minimum target nucleated cell count is 1.0 x 109 to be collected in a single leukapheresis session, which may be repeated to meet this minimum cell count requirement.
Culturing of collected PBMCs will proceed in a CliniMACS Prodigy cell expansion device (Miltenyi Biotech) with IL-2 and agonist antibodies directed to CD3 and CD28, followed by transduction of the expanded cells with a γ-retrovirus containing an expression construct of ACTR (CD16V-4-1BB-CD3ζ), and apportionment for infusion into subjects.
During the pre-treatment period the subject may be administered anti-cancer chemotherapy, such as a low dose single agent chemotherapy (e.g., vincristine or cyclophosphamide) after leukapheresis prior to the initiation of the conditioning regimen (lymphodepleting chemotherapy). No therapy involving administration of monoclonal antibodies (including rituximab), nitrosoureas, or mitomycin C is allowed. Local irradiation to a single lesion or subset of lesions may be administered, so long as un-radiated positron emission tomography (PET)-positive lymphoma lesions remain. Conditioning Regimen / Lymphodepleting chemotherapy
Subjects receive a 3-day lymphodepleting chemotherapy regimen with fludarabine and cyclophosphamide, starting 6 to 10 days prior to the planned day of infusion ACTR- expressing T cells.
The lymphodepleting chemotherapy regimen will be as follows: Table 7: Lymphodepleting Chemotherapy Regimen
Figure imgf000055_0001
The lymphodepleting chemotherapy regimen can be initiated as early as Day -10 before the planned infusion of ACTR-expressing T cells, for ease of scheduling at the site. Serum creatinine is measured on the planned first day of administration of lymphodepleting chemotherapy; chemotherapy should be withheld if serum creatinine is≥ 1.5 times the age- adjusted upper limits of normal (ULN). Re-assessment of inclusion/exclusion criteria is required if lymphodepleting chemotherapy is delayed by more than 14 days. Once lymphodepleting chemotherapy is initiated, if dose(s) on the second or third day are omitted, the schedule of administration for all outstanding doses of chemotherapy may only be delayed for one day beyond the 3-day schedule. Rituximab administration and ACTR-expressing T cell infusion
After lymphodepleting chemotherapy, rituximab is administered followed by dosing with ACTR-expressing T cells, as follows: Table 8: Rituximab and ACTR-expressing T cells Administration
Figure imgf000056_0001
ACTR-expressing T cells are not expected to proliferate or persist in the absence of rituximab. Therefore, to maximize the potential therapeutic window for activity, rituximab will be given 24-48 hours prior to infusion of ACTR-expressing T cells so that rituximab serum levels are near maximum during infusion of the ACTR-expressing T cells. However, if medically necessary and deemed appropriate by the investigator (for example to allow subjects to recover from adverse events related to their lymphodepleting chemotherapy or rituximab, this window may be extended to no more than 7 days. After 7 days, the rituximab serum levels begin to approach trough levels and may not support sufficient expansion of ACTR T-cells.
Subjects will be admitted as inpatients on the day of or the day before ACTR T-cell product infusion. Subjects will stay as inpatients for a minimum of 3 days after the ACTR T- cell product infusion. Subjects may be discharged to home on Day +3, if they are clinically stable. After subjects are discharged from the hospital and up through Day 20 after infusion of ACTR-expressing T cells, subjects will take and record their temperature daily. Subjects will return to the clinical site weekly for vital sign testing, adverse event assessments, concomitant medications, physical exams and performance status, and laboratory testing throughout the assessment period (to Day 28). Investigators will be asked to assess whether any adverse event is related to lymphodepleting chemotherapy, rituximab, or the ACTR- expressing T cells. Additional cycles of rituximab (cycles 2-8)
Following dosing with rituximab (cycle 1) and ACTR-expressing T cells, subjects will receive up to 7 additional cycles of rituximab for a total of 8 cycles of rituximab. The interval including an administration of rituximab, assessment of subject response, and approval for the next administration of rituximab is referred to as one treatment cycle. The total number of rituximab cycles for a particular study subject, up to a maximum of 8, will be determined by tolerability of the treatment regimen, the absence of progression of the subject’s underlying disease, and the ongoing willingness of the subject to participate in the study.
Subjects will return to the clinical site 10 days ± 2 days after rituximab administration (7 days ± 1 day in cycle 2) for vital sign evaluation, targeted physical exams with a review of systems, and laboratory testing. In the absence of disease progression, rituximab will be administered every 21 days ± 2 days, unless delayed or discontinued due to toxicities.
PET/CT response assessments will occur at the end of every 2 cycles of rituximab (i.e., within 3 days prior to rituximab doses 3, 5, and 7), and at the end of the treatment period. PET scans are no longer required after a subject achieves CR, unless PD is suspected on a follow-up CT scan. If a bone marrow aspirate and/or biopsy or other radiographic assessment for extranodal disease was obtained at the baseline disease assessment (at the discretion of the investigator), this will be repeated within 2 weeks of a documented radiographic response to confirm the response.
Assessments in support of exploratory objectives (e.g., peripheral blood samples for ACTR persistence, inflammatory markers and cytokines, rituximab levels, product characteristics, and tumor or bone marrow biopsy samples) will be obtained.
Subjects with documented stable disease, partial response, or complete response following treatment with ACTR-expressing T cells and rituximab will be followed according to the schedule of follow-up assessments. Subjects with documented disease progression will be encouraged to undergo an end-of-study visit at the time of documented disease
progression. Post-treatment Follow-up period
The post-treatment follow-up period is defined as the period immediately after the end-of-study visit through study subject discontinuation. All surviving subjects will be monitored for a total of 15 years after infusion of the ACTR-expressing T cells to assess them for survival, general health, and potential long-term toxicity of ACTR-expressing T cells (with particular attention to development of leukemia or other secondary malignancies and autoimmune/rheumatologic, neurologic, or hematologic disorders).
Subjects who develop progressive disease (PD) at any time while enrolled in this study will undergo exit visit procedures and receive further follow-up. Subjects with SD, PR or CR will be followed under this study protocol until 2 years after the last study subject treated received the infusion of ACTR-expressing T cells. OTHER EMBODIMENTS
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one of skill in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

Claims

WHAT IS CLAIMED IS: 1. A method for treating lymphoma, comprising:
(i) administering to a subject in need thereof an effective amount of one or more lymphodepleting agents;
(ii) administering to the subject an anti-CD20 antibody after (i); and
(iii) administering to the subject immune cells expressing an antibody-coupled T cell receptor (ACTR) no more than about 10 days after (ii), wherein the ACTR comprises:
(a) an Fc binding domain of CD16,
(b) a co-stimulatory signaling domain of 4-1BB, and
(c) a cytoplasmic signaling domain of CD3ζ.
2. The method of claim 1, wherein the ACTR further comprises a
transmembrane domain and/or a hinge domain.
3. The method of claim 1 or 2, wherein the ACTR comprises, from N- terminus to C-terminus,
(a) the Fc binding domain of CD16,
(b) the transmembrane domain,
(c) the co-stimulatory domain of 4-1BB, and
(d) the cytoplasmic signaling domain of CD3ζ.
4. The method of claim 3, wherein the ACTR further comprises a hinge domain, which is located between (a) and (b).
5. The method of any one of claims 1-4, wherein the ACTR further comprises a signal peptide.
6. The method of any one of claims 1-5, wherein the CD16 is the CD16V isoform.
7. The method of any one of claims 1-6, wherein the ACTR comprises the amino acid sequence of SEQ ID NO: 1.
8. The method of any one of claims 1-7, wherein the subject is a human patient having a relapsed or refractory CD20+ lymphoma.
9. The method of claim 8, wherein the relapsed or refractory CD20+ B cell lymphoma is selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary mediastinal B cell lymphoma
(PMBCL), grade 3b follicular lymphoma (Gr3b-FL), and transformed histology follicular lymphoma (TH-FL).
10. The method of any one of claims 1-9, wherein the one or more
lymphodepleting agent is fludarabine and cyclophosphamide.
11. The method of any one of claims 1-10, wherein the one or more lymphodepleting agent is administered to the subject at a frequency of one dose per day for three consecutive days.
12. The method of claim 11, wherein the first dose of the lymphodepleting agent is about 6-15 days before (iii).
13. The method of claim 11 or claim 12, wherein the one or more
lymphodepleting agent is fludarabine, which is administered to the subject by intravenous injection at a daily dose of about 30 mg/m2, and cyclophosphamide, which is administered to the subject by intravenous injection at a daily dose of about 500 mg/m2.
14. The method of any one of claims 1-13, wherein in (ii), the anti-CD20 antibody is administered no later than 7 days prior to (iii)
15. The method of any one of claims 1-14, wherein in (ii), the anti-CD20 antibody is administered within about 24-48 hours prior to (iii).
16. The method of any one of claims 1-15, wherein the anti-CD20 antibody is rituximab.
17. The method of claim 16, wherein the rituximab is administered to the subject at a dose of about 375 mg/m2.
18. The method of claim 16 or claim 17, wherein the rituximab is administered to the subject at multiple doses, wherein the first dose is no more than about 7 days prior to (iii).
19. The method of claim 18, wherein the first dose of rituximab is about 24-48 hours prior to (iii).
20. The method of any one of claims 16-19, wherein the rituximab is administered to the subject at a frequency of one dose every three weeks for up to 8 doses.
21. The method of any one of claims 1-20, wherein the immune cells are T cells.
22. The method of claim 21, wherein the T cells expressing the ACTR are administered to the subject at a dose of about 0.5 x 106 to about 5 x 106 T-cells/kg.
23. The method of claim 22, wherein the dose of T cells expressing ACTR is up to about 0.5 x 106 T-cells/kg, about 0.5 x 106 to 1.5 x 106 about T-cells/kg, or about 1.5 x 106 to about 5 x 106 T-cells/kg.
24. The method of any one of claims 1-23, wherein the subject is a human patient having DLBCL or PMBCL and the dose of the immune cells expressing the ACTR is about 5 x 106 cells/kg.
25. The method of any one of claims 1-23, wherein the subject is a human patient having MCL, Gr3b-FL, or TH-FL and the dose of the immune cells expressing the ACTR is about 5 x 106 cells/kg.
26. The method of any one of claims 1-23, wherein the subject is a human patient having DLBCL, MCL, PMBCL, Gr3b-FL, or TH-FL and the dose of the immune cells expressing the ACTR is below 5 x 106 cells/kg.
27. The method of any one of claims 1-26, wherein the immune cells expressing the ACTR are prepared by introducing a vector for expressing the ACTR into immune cells collected from the subject about 30-60 days prior to step (iii).
28. The method of any one of claims 1-27, wherein the subject is free from alemtuzumab within 6 months prior to collecting the immune cells;
fludarabine, cladribine, or clofarabine within 3 months prior to collecting the immune cells;
external beam radiation, administration of a monoclonal antibody, or lymphotoxic chemotherapy within two weeks prior to collecting the immune cells; or
an experimental agent within three half-lives of the experimental agent prior to collecting the immune cells.
29. The method of any one of claims 1-28, wherein the subject has been subject to a chemo-immunotherapy prior to step (i).
30. The method of claim 29, wherein the chemo-immunotherapy comprises an anti-CD20 antibody and a chemotherapeutic agent.
31. A kit for treating lymphoma, comprising:
(i) one or more lymphodepleting agents;
(ii) an anti-CD20 antibody; and
(iii) immune cells expressing an antibody-coupled T cell receptor, which comprises:
(a) an Fc binding domain of CD16,
(b) a co-stimulatory signaling domain of 4-1BB, and
(c) a cytoplasmic signaling domain of CD3ζ.
32. The kit of claim 31, wherein the one or more lymphodepleting agents are fludarabine and cyclophosphamide.
33. The kit of claim 31 or claim 32, wherein the anti-CD20 antibody is rituximab.
34. The kit of any one of claims 31-33, wherein the ACTR comprises the amino acid sequence of SEQ ID NO:1.
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WO2019226894A1 (en) 2018-05-23 2019-11-28 Againchance Corporation Limited Bispecific t cell engager and uses thereof
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