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WO2018005775A1 - Cd47 blockade enhances therapeutic activity of antibodies to low density cancer epitopes - Google Patents

Cd47 blockade enhances therapeutic activity of antibodies to low density cancer epitopes Download PDF

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Publication number
WO2018005775A1
WO2018005775A1 PCT/US2017/039964 US2017039964W WO2018005775A1 WO 2018005775 A1 WO2018005775 A1 WO 2018005775A1 US 2017039964 W US2017039964 W US 2017039964W WO 2018005775 A1 WO2018005775 A1 WO 2018005775A1
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Prior art keywords
tcrm
antibody
cell
composition
density
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PCT/US2017/039964
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French (fr)
Inventor
David A. Scheinberg
Melissa MATHIAS
K. Christopher Garcia
Jonathan Thomas SOCKOLOSKY
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Memorial Sloan-Kettering Cancer Center
The Board Of Trustees Of The Leland Stanford Junior University
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Application filed by Memorial Sloan-Kettering Cancer Center, The Board Of Trustees Of The Leland Stanford Junior University filed Critical Memorial Sloan-Kettering Cancer Center
Publication of WO2018005775A1 publication Critical patent/WO2018005775A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/505Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507Comprising a combination of two or more separate antibodies

Definitions

  • the present disclosure relates generally to the use of monoclonal antibodies (mAb), including T Cell Receptor mimic (TCRm) antibodies (abs) to reduce solid and liquid tumors in a subject. More particularly, the present disclosure relates to the use of abs in combination with CD47 blockade when the density of peptide epitopes from undruggable intracellular proteins presented in the context of major histocompatibility complex (MHC) molecules is much lower than the density of typical antigenic targets of therapeutic antibodies.
  • mAb monoclonal antibodies
  • TCRm T Cell Receptor mimic
  • abs in combination with CD47 blockade when the density of peptide epitopes from undruggable intracellular proteins presented in the context of major histocompatibility complex (MHC) molecules is much lower than the density of typical antigenic targets of therapeutic antibodies.
  • MHC major histocompatibility complex
  • TCR mimic mAbs TCR mimic mAbs
  • mAbs In addition to the inability of mabs to bind intracellular antigens, of the many FDA approved mAbs for the treatment of cancer, most do not operate effectively alone by simply directing human effector cells to kill the cancer cells via antibody dependent cell mediated cytoxcity (ADCC) or antibody dependent cellular phagocytosis (ADCP). 7"9 mAbs require additional mechanisms to improve their effectiveness. These mechanisms include conjugation of the mAbs to drugs or radioisotopes, additional blockade of signaling pathways, upregulation of death pathways, or broad inactivation of T cell suppression. 10 ' 11 12 For some mAbs, multiple mechanisms are employed by the single IgG. 13
  • Low antigen density may further play a role in limiting therapeutic potential and as cancer cells are heterogeneous, cells within the population may express highly variable amounts of target antigen, allowing escape from mAb therapy.
  • the present disclosure is an extension of the discovery and development of T cell receptor mimic (TCRm) antibodies specific to peptides from previously undruggable intracellular protein targets to treat cancer.
  • TCRm T cell receptor mimic
  • the present disclosure is based on the observation that the efficacy of TCRm antibodies targeted to previously undruggable intracellular proteins can be enhanced by combining TCRm antibodies, that is, antibodies that recognize an epitope comprising a peptide of the intracellular protein in the context of an HLA molecule, with CD47 blockade.
  • the present disclosure relates to a composition
  • a composition comprising a T cell receptor mimic (TCRm) antibody and a CD47 antagonist.
  • TCRm T cell receptor mimic
  • the disclosure relates to a composition
  • a composition comprising a T cell receptor mimic (TCRm) antibody and a CD47 antagonist wherein the TCRm antibody comprises an antigen binding region specific for a human leukocyte antigen (HLA) A2 -restricted intracellular protein-derived peptide.
  • HLA human leukocyte antigen
  • WT1 Wilms Tumor gene
  • PRAME Preferentially Expressed Antigen in Melanoma
  • the composition further comprises a CD47 antagonist, which in one embodiment is a signal-regulatory protein a (SIRPa) or a variant thereof, such as the consensus variant 1 (CV1 ).
  • a CD47 antagonist is a CD47 antibody, homolog or CD47-binding fragment thereof, which are known in the art for example, B6H12 or an antibody that binds the same epitope as B6H12.
  • the disclosure relates to a method for killing tumor cells, reducing tumor burden and/or increasing survival, particularly in acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML), the method comprising coadministering to a subject in need thereof a therapeutically effective amount of a T cell receptor mimic antibody, homolog or fragment thereof and a CD47 antagonist either as a combination formulation or as individual formulations.
  • ALL acute lymphocytic leukemia
  • AML acute myeloid leukemia
  • the TCRm antibody comprises an antigen binding region specific for a human leukocyte antigen (HLA) A2 -restricted intracellular protein-derived peptide.
  • HLA human leukocyte antigen
  • the TCRm antibody is specific for a WT1 peptide
  • RMFPNAPYL SEQ ID NO: 1
  • ALYVDSLFFL SEQ ID NO: 2
  • PRAME Preferentially Expressed Antigen in Melanoma
  • the Fc of the TCRm antibody is afucosylated.
  • FIGS 1A-1 D show the results of experiments to assess the antigen-dependent cellular phagocytosis (ADCP) of leukemia cells in vitro.
  • PBMC's peripheral blood mononuclear cells
  • CSFE carboxyfluorescein succinimidyl ester
  • Figures 2A-2D show the effects of interferon- ⁇ on antigen expression and ADCP in vitro.
  • A) AML cell line was pretreated with 100 ng/mL IFNy for 72 hours. Treated (right peak) displayed a doubling of HLA expression compared to untreated controls (left peak).
  • B) Treated (right peak) showed a 10-fold increase in Pr20 binding compared to untreated controls (left peak).
  • Isolated human macrophages were incubated with pretreated AML cell line in the presence of 1 ) PBS, 2) CV1 alone, 3) Pr20M alone, 4) combination therapy with Pr20M and CV1 , 5) positive control B6H12 (previously described), 6) B6H12 with CV1 7) irrelevant control mAb, and 8) irrelevant control mAb with CV1 . All groups showed an increase in ADCP with IFNy pretreatment. Increase was most significant in combination therapy, B6H12, and B6H12 with Pr20M.
  • D) BV173 cell line was pretreated with 100 ng/uL of IFNy for 72 hours. Isolated human macrophages were incubated with pretreated BV173 cell line as above. All groups show an increase in ADCP with IFNy pretreatment. Increase is significant in combination therapy, positive control, and positive control with Pr20. These experiments were performed in duplicate with consistent results.
  • FIGS 3A-3D show the results of CV1 dose-response effects in vivo.
  • mice were engrafted via tail vein injection with 3 million cells/mouse of BV173 transfected with Luciferase gene. Mice were imaged via BLI on day 6. Mice were randomized to have equal group mean engraftment. Starting on day 6 after engraftment, mice were treated with either 200 g, 150 g, or 100 g of CV1 daily.
  • E) Mice were engrafted via tail vein injection with 3 million cells/mouse of AML14 transfected with Luciferase gene. Mice were imaged via BLI on day 6. Mice were randomized to have equal group mean engraftment.
  • mice were treated with 100 g of CV1 either daily or on Monday Wednesday Friday. Mice were imaged once a week for 3 weeks.
  • F Graph showing mean flux in photons/second of mice at days 6, 13, and 20.
  • G Mice were engrafted via tail vein injection with 3 million cells/mouse of BV173 transfected with Luciferase gene. Mice were imaged via BLI on day 6. Mice were randomized to have equal group mean engraftment. Starting on day 6 after engraftment, mice were treated with 100 g of CV1 either daily or on Monday Wednesday Friday. Mice were imaged once a week for 3 weeks.
  • H Graph showing mean flux in photons/second of mice at days 6, 13, and 20.
  • FIGS 4A-4D show CV1 schedule effects in vivo.
  • AML14 burden remains lower than engraftment 3 weeks after the end of therapy. At 67 days, 1 mouse in combination group had lymphomatous growth requiring sacrifice. The 4 remaining animals had lower tumor burden via BLI than on day of engraftment.
  • Figures 5A-5C show the results of therapy of human Ph+ALL in mice.
  • FIGS. 6A-6C show the results of therapy of human AML in mice
  • Figures 7A-7D show lnterferon- ⁇ release in vivo.
  • Luminex assay was done on mice serum for mouse cytokines IFNy, TGFp, M- CSF, and IL-1 B. At 12 hours we see an upward trend in concentration of IFNy (pg/mL) in groups treated with CV1 alone and combination therapy.
  • co-administration refers to the administration of a TCRm antibody and CD47 antagonist as one single formulation or as two separate formulations.
  • the co-administration can be simultaneous or sequential in either order, wherein there is a time period while both (or all) active agents simultaneously exert their biological activities.
  • One embodiment provides a pharmaceutical composition for combination therapy for preventing and/or treating of a cancer, comprising or consisting essentially of a TCRm antibody and CD47 antagonist as active ingredients.
  • the pharmaceutical composition for combination therapy may be a mixed formulation (e.g., a single composition comprising two or more active ingredients) of a TCRm antibody and CD47 antagonist.
  • the TCRm antibody and CD47 antagonist may be included in any amount that is pharmaceutically effective when used together.
  • the composition thus formulated can be used for simultaneous administration of the two active ingredients.
  • each of the TCRm antibody and CD47 antagonist can be formulated in a separate composition and the two active ingredients can be separately administered simultaneously or sequentially.
  • a first pharmaceutical composition including a pharmaceutically effective amount of a TCRm antibody as an active ingredient and a second pharmaceutical composition including a pharmaceutically effective amount of the CD47 antagonist as an active ingredient can be administered simultaneously (within 0-20 minutes) or sequentially (after 20 minutes).
  • any order of administration may be used.
  • the agents are administered sequentially and spaced apart by hours or days.
  • TCRm antibody is administered to the subject at time 0; subsequently, the CD47 antagonist is administered anywhere from 21 minutes to 21 days following the administration of TCRm antibody. In one embodiment, CD47 antagonist is administered anywhere from 30 minutes to 20 days following the
  • TCRm antibody in one embodiment CD47 antagonist is administered anywhere from 45 minutes to 15 days following the administration of TCRm antibody; in one embodiment CD47 antagonist is administered anywhere from 1 hour to 10 days following the administration of TCRm antibody; in one embodiment CD47 antagonist is administered anywhere from 5 hours to 5 days following the administration of TCRm antibody; in one embodiment CD47 antagonist is administered anywhere from 12 hours to 2 days following the administration of TCRm antibody.
  • the timing of the sequential administration will be based on the duration of the effect of each agent alone with the recipient subject. For example, if a particular TCRm is active for 7 days, and the CD47 antagonist is active for 2 days, then the CD47 antagonist can be administered within that time frame, so as to overlap in function with the activity of the TCRm. In one embodiment, therefore, the CD47 antagonist can be administered from 2 days prior to administration of TCRm and up to 7 days after.
  • kits useful for preventing and/or treating a leukemia comprising or consisting essentially of a first pharmaceutical composition including a TCRm antibody as an active ingredient, a second pharmaceutical
  • composition including a CD47 antagonist as an active ingredient.
  • the TCRm antibody and CD47 antagonist may be used in amounts that are pharmaceutically effective when combined, which amount may be determined by the skilled medical practitioner or medical researcher.
  • the pharmaceutically effective amount refers to an amount of active ingredient that can exert pharmaceutically significant effects (e.g., an amount sufficient to prevent or treat leukemia in a subject).
  • Monoclonal antibodies are potent cancer therapeutic agents, but exclusively recognize cell-surface targets whereas most cancer-associated proteins are found intracellularly.
  • potential cancer therapy targets such as over-expressed self-proteins, activated oncogenes, mutated tumor suppressors, and translocated gene products are not accessible to traditional mAb therapy.
  • An emerging approach to target these epitopes is the use of TCR mimic mAbs (TCRm) that recognize epitopes similar to those of T cell receptors (TCR).
  • T-cell receptor mimic (TCRm) antibodies or "T-cell receptor-like antibodies” therefore refers to antibodies that recognize an epitope similar to the epitope recognized by the receptor on the surface of a T-cell that causes activation of the T-cell when it binds an MHC (HLA)-restricted ligand on the surface of a tumor cell or
  • TCRm antigens are composed of a linear peptide sequence derived from degraded (processed) intracellular proteins and presented in the context of cell-surface MHC molecules. TCRm combine the specificity of TCR recognition with the potency, pharmacologic properties, and versatility of mAbs.
  • low-density and “ultra low-density” with respect to cancer antigens refer to the relative number of antigens on the surface of a cell with which macrophages might interact.
  • the density of peptide epitopes from undruggable intracellular proteins presented in the context of major histocompatibility complex (MHC) molecules is much lower than the density of typical cell surface antigenic targets of therapeutic antibodies (for example HER2/net/).
  • MHC major histocompatibility complex
  • the number of antigens on the surface of a cell with low density TCRm mAb targets may have as few as 1 % of the mAb binding sites as other targets.
  • Ultra-low-density applies to cells with about 900 to 2500 Pr20m mAb binding sites per cell.
  • T cell receptor mimic TCRm antibodies specific to peptides from previously undruggable intracellular protein targets to treat cancer.
  • the antibody is a TCRm antibody, for example a TCRm antibody (mAb ESKm) that is specific for Wilms tumor gene 1 product (VVT1 ) -derived peptide, RMFPNAPYL (SEQ ID NO: 1 ) in the context of HLA-A * 02:01.
  • VVT1 is an oncofetal antigen, a zinc finger transcription factor that is rare in normal tissues but seen overexpressed in a variety of multiple solid and liquid malignancies including mesothelioma and ovarian cancer. 15"17 .
  • VVT1 antibodies are known in the art and are described for example, in US 9,040,669, US 9,074,000 and US 9,540,448 (the contents of each is hereby incorporated by reference into the present application).
  • the antibody is a TCRm antibody (mAb Pr20) that is specific for the "Preferentially Expressed Antigen in Melanoma" (PRAME) -derived peptide ALYVDSLFFL (SEQ ID NO: 2) in the context of HLA-A * 02:01 .
  • PRAME Preferentially Expressed Antigen in Melanoma
  • ALYVDSLFFL SEQ ID NO: 2
  • PRAME is a cancer testes antigen found in many cancers and leukemias, and expressed in normal ovaries, testes and endometrium with limited expression in other healthy tissues.
  • PRAME antibodies are described in WO2016191246 (the contents of which is hereby incorporated by reference into the present application.)
  • the Fc of the TCRm antibodies is engineered to be afucosylated so as to enhance effector cell recruitment and killing (see US 9,540, 448).
  • the TCRm antibodies alone which act via antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), show therapeutic activity in murine models, although the responses are incomplete and relapse is common in these models 17"19 .
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CD47 is a trans-membrane protein that transduces an anti-phagocytic signal via binding to its cognate ligand, SIRPa, in macrophages.
  • CD47 is expressed on normal host tissue and is upregulated on cancer tissue to inhibit macrophage-mediate phagocytosis.
  • 21 22 CD47 inhibitory mAbs are in clinical trials. However, to date the mAb have significant toxicities perhaps because of the ubiquitous expression of CD47 on normal tissue, especially red blood cells. Anemia is therefore a frequently seen adverse effect with currently available antibodies. 10 23
  • a truncated SIRPa protein variant, known as consensus variant 1 (CV1 ) 11 , that blocks the interaction between the SIRPa on the cancer cell and CD47 on the macrophage has been shown to increase macrophage mediated killing of cancer cells in vitro and in vivo without toxicity 10 .
  • CV1 has shown an additive effect in murine models of human cancers when combined with mAb against /?/g/?-density targets on both solid and liquid tumors. 0 24 25
  • TCRm antibodies were designed and produced at Memorial Sloan-Kettering Cancer Center (MSK) and Eureka Therapeutics (Emeryville CA) in their afucosylated IgG form. They included Pr20M, designed to react with PRAME peptide ALYVDSLFFL (SEQ ID NO: 2)/HLA-A * 02:01 ; ESKM prepared to react with VVT1 peptide RMFPNAPYL (SEQ ID NO: 1 )/HLA-A * 02:01 1 and irrelevant control IgG 17 not reactive with human antigens. 27 ' 44 CV1 was prepared as described. 41
  • Proteins were stored in phosphate buffered saline at 4° C.
  • B6H12 was purchased from Biolegend (San Diego, CA).
  • phagocytosis (ADCP) assays were as previously described12,40. Human blood was obtained from the Stanford Blood Center using IRB approved protocols and monocytes were isolated via CD14 positive selection (Miltenyi) and magnetic isolation. Recovered CD14+ monocytes were plated at a density of 10,000,000 monocytes per 150 mm TC treated dishes in IMDM + GlutaMAX media (Gibco) supplemented with 10% human serum and 1 % P/S and cultured for 7 days at 37 °C to yield human monocyte derived macrophages (MDM0). Phagocytosis assays were repeated in duplicate.
  • Mouse macrophages were derived from bone marrow (BMDMs).
  • Mouse bone marrow cells were flushed with a syringe from the tibia and femurs of NSG mice into IMDM + GlutaMAX supplemented with 10% FBS and 1 % P/S.
  • Cells were collected by centrifugation followed by RBC lysis with ACK buffer for 3 - 5 mins (Gibco), quenched with complete media, and filtered through a 70 ⁇ cell strainer.
  • Cells were pelleted by centrifugation, re-suspended in media containing 10 ng/mL M-CSF (Peprotech) and plated on 3 x 10 cm untreated pitri dishes per mouse in 10 mL media and cultured for 7 days without replenishing or changing media to derive BMDMs.
  • M-CSF M-CSF
  • tumor cells were harvested, labeled with carboxyfluorescein succinimidyl ester (CFSE), washed with serum free IMDM + GlutaMAX, and plated at a density of 100,000 cells/well in 25 ⁇ in a 96-well ultra low attachment round bottom plate (Costar, Cat. 7007) on ice. Tumor cells were opsonized by addition of 25 ⁇ _ of various antitumor antibodies, CD47 blocking reagents, and/or controls for 30 min on ice.
  • CFSE carboxyfluorescein succinimidyl ester
  • Macrophages were harvested by enzymatic dissociation and cell scraping, pelleted, washed in serum free IMDM, and added to opsonized tumor cells at a density of 50,000 cells/well in 50 ⁇ _ media for a final assay volume of 100 ⁇ _ and an effector to tumor cell ratio of 1 :2.
  • ADCP was measured after incubation for 2 hr at 37 °C with the following test groups: M0 + cancer cells alone, M0 + cancer cells opsonized with TCRm or control antibody alone (10 pg/mL mAb final), M0 + cancer cells opsonized with CV1 alone (100 nM final), and M0 + cancer cells opsonized with the combination (10 g/mL mAb + 100 nM CV1 ).
  • Phagocytosis was quantified gating based on SSC-A and FSC-A, singlets, live/dead (DAPI negative/low), and phagocytosis quantified as the percentage of F4/80-APC or CD206-Alexa647 positive macrophages that are also CFSE positive.
  • human macrophage-mediated ADCP To determine the role of IFNy exposure on human macrophage-mediated ADCP in vitro, leukemia cells and/or macrophages were cultured with human IFNy (Peprotech) at a concentration of 100 ng/mL for 72 hours prior to harvest for ADCP assays as described above.
  • Human macrophage phagocytosis assays were repeated at least twice with different human blood donors, anti-human CD200 mAb (Biolegend) served as an irrelevant isotype control for BV173 cell lines and anti-human CD33 mAbs as an irrelevant isotype control for AML14 cell lines.
  • NSG mouse BMDM phagocytosis assays were repeated in duplicate.
  • BV173 Ph+ ALL
  • AML14 AML14
  • PR20M could bind these lines maximally at an ultra-low-density, with about 900 and 2500 Pr20 mAb binding sites per cell, respectively.
  • BV173 has previously been reported to have about 700 binding sites per cell for the ESK1 TCRm 22
  • the lines were transduced with a Luciferase-green fluorescent protein (GFP) retrovirus and expanded in complete RPMI Medium for use in NSG animal models. Cell lines were obtained from ATCC® or MSKCC stocks.
  • GFP Luciferase-green fluorescent protein
  • Human xenograft models were prepared as described above using tail vein injection with 3 million AML14 cells. Mice were divided into 4 treatment groups with equal engraftment measured by mean fluorescent intensity via BLI. Groups were 1 ) control without treatment, 2) CV1 alone, 3) Pr20M alone, and 4) CV1 and Pr20M. Mice were bled before treatment and at 12 hours and 24 hours after treatment. Serum was analyzed on a Milliplex Luminex platform to detect the mouse cytokines IFNy, IL- ⁇ ⁇ , M-CSF, and TNFa.
  • AUC area under the curve
  • Tumor burden expressed as AUCs were compared between groups using the Kruskal-Wallis test. When the Kruskal-Wallis test indicated significant differences among the groups (p ⁇ 0.05), subsequent pairwise comparison were conducted. Survival studies were done using bilateral hind leg paralysis as surrogate for death or using predetermined morbidity characteristics. Overall survival was estimated by the Kaplan-Meier approach and compared among groups using the log-rank test, ns is defined as p > 0.05. * is defined as p ⁇ 0.05. ** is defined as p ⁇ 0.01 . *** is defined as p ⁇ _0.001 . All statistical tests were conducted using a permutation test procedure. 43 Statistical testing were performed using R 3.3.1 (R Core Team, Vienna Austria).
  • Leukemic burden was assessed by bioluminescence imaging, recorded as flux of protons per second and repeatedly measured at days 6, 13, 20, and 27, post
  • mice were treated twice a week beginning on day 6 through 27 for a total of six doses of TCRm and/or 21 daily doses of CV1.
  • CV1 and TCRm antibody show additive effects in vitro.
  • the ability of CV1 , an engineered SIRPa variant that potently antagonizes CD47, to enhance TCRm mAb- dependent macrophage phagocytosis of leukemia cells in vitro was evaluated utilizing the HLA-A * 02:01 positive human acute myeloid leukemia cell line AML14, and human acute lymphoblastic leukemia cell line BV173 as models, which express the target antigen of the TCRm mAb Pr20M (PRAME).
  • the HLA-A * 02:01 negative cell line HL60 was used as a control.
  • Blockade of leukemia cell CD47 with CV1 alone did not promote macrophage phagocytosis of AML14, BV173, or HL60 ( Figure 1 ).
  • Pr20M alone did not promote ADCP of HL60 or BV173, but significantly increased phagocytosis of AML14 ( Figure 1 A, B).
  • the combination of TCRm mAb and CV1 significantly increased macrophage phagocytosis of AML14 and BV173, but not the HLA-A * 02:01 negative cell line HL60 ( Figure 1A, B), indicating the effect is TCRm mAb antigen specific.
  • an anti- CD47 blocking antibody B6H12 a mouse monoclonal to CD47 (Abeam, Cambridge MA) induced a significant increase in macrophage phagocytosis of all three leukemia cell lines, and potentiated TCRm mAb-mediated phagocytosis of AML14 and BV173 ( Figure 1A, B).
  • IFNy leads to increased expression of HLA A02:01, increased leukemia cell binding of mAbs Pr20 and ESK, and enhanced macrophage-mediated ADCP in vitro.
  • IFNy is a potent immunocytokine with pleiotropic effects, including induction of MHC Class I and II expression and increased antigen processing and presentation.
  • Anti-CD47 mAb therapy triggers a phagocyte type I and II interferon (IFN) response in the tumor microenvironment that presumably increases tumor cell surface peptide- MHC (pMHC) density.
  • IFN interferon
  • pMHC tumor cell surface peptide- MHC
  • IFNy significantly increased expression of TCRm mAb epitopes of interest and increased macrophage-mediated ADCP of both AML14 and BV173 in vitro.
  • CV1 dose titrations in mice CV1 is effective at doses of 200ug daily in mice.
  • the therapeutic window of CV1 for use as an adjuvant to mAb therapy is unknown.
  • NSG mice bearing disseminated BV173 or AML14 leukemia cells were injected daily with either 200 g, 150 g, or 100 of CV1 beginning on day 6 post engraftment and anti-leukemia effects were measured by BLI and clinically.
  • CV1 was similarly effective at all three dose levels evaluated.
  • CV1 administered three times per week beginning on day 6 post engraftment was also effective at suppressing leukemia growth, but the effect varied among mice treated with this dosing schema, particularly against AML14 leukemias. As with daily dosing, leukemia escaped at the end of the 2 week treatment period (Figure 3 E-H).
  • CD47 blockade enhances the antitumor activity of antibodies that target overexpressed tumor antigens by mobilizing the innate and adaptive immune system. (12, 33) To determine if CD47 blockade also enhanced the antitumor activity of TCRm mAbs that target ultra low-density pMHC tumor antigens, mice engrafted with disseminated AML14 or BV173 leukemias were treated with TCRm mAb, CV1 , or the combination beginning on day 6 post engraftment.
  • TCRm mAb and CV1 monotherapy significantly reduced leukemia burden by 5-10 fold in the AML14 model and 5-100 fold in the BV173 model compared to control treated animals ( Figure 4, 5).
  • leukemias escaped in all single agent therapy groups in both models.
  • Combination therapy had a greater than additive effect compared to either agent alone, with a 3 log reduction in leukemia burden relative to control untreated mice and a 5-10 fold reduction relative to the single agent groups (Figure 4, 5).
  • the differences between the combination therapy and monotherapy were more pronounced in the AML14 model than in the BV173 Ph+ALL model, as CV1 alone was more effective in the latter.
  • mice bearing disseminated AML14 leukemias were treated on day 6 after engraftment with a vehicle control, CV1 alone, Pr20M alone, or the combination of CV1 and Pr20M.
  • the mouse cytokines IFNy, IL-1 ⁇ , M-CSF, and TNFa were quantified in serum before and after the various treatments.
  • IFNy alone is not cytotoxic to human leukemia cell lines.
  • increased tumor kill could also be secondary to direct cytotoxic effects of IFNy independent of the actions of CV1 or TCRm
  • We saw no increase in cell death with exposure to IFNy ( Figure 7C,D). Indeed, there was a small growth promoting effect. This further supports the hypothesis that IFNy can work indirectly as an adjuvant to this specific combination of CD47/SIRP alpha blockade and the TCRm via upregulation of the epitope and activation of phagocytosis.
  • CD47 blockade with high affinity SIRPa variants has shown beneficial therapeutic effects when administered in combination with Fc receptor engaging monoclonal antibodies that target overexpressed tumor antigens.
  • Some of this enhanced therapeutic activity may be related to this release of IFNy, which not only can activate phagocytosis further, but also causes a feed-forward mechanism unique to these TCRm antigenic systems in which the peptide epitope presentation on the target cells is up-regulated by IFNy.
  • NSG mice are B cell, T cell, and NK cell deficient, and although they have intact IFNy-dependent signaling, they have defective innate immunity and cytokine signaling pathways.
  • 49, 50 While it is difficult to draw parallels between human and mouse systems, in the human, a greater variety of more potent effectors and an immunocompetent host that responds to pro-inflammatory signaling could allow even greater efficacy of this drug combination in the human patient.
  • NSG mice have low circulating IgG levels that could compete with TCRm for Fc receptor interactions.
  • IFNy secretion caused by CV1 is likely contributing indirectly through a new mechanism to the therapeutic effects seen.
  • IFNy secreted by NK cells, T cells, dendritic cells and macrophages contributes directly to the innate and adaptive immune response and indirectly through its interplay with other cytokines.
  • NSG mice have no T or NK cells.
  • IFNy-dependent signaling enhanced TCRm mAb dependent, macrophage-mediated phagocytosis.
  • WT1 Wilms tumor gene

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Abstract

The present disclosure describes the use of monoclonal T Cell Receptor mimic (TCRm) antibodies (abs) in combination with CD47 blockade to kill tumor cells, reduce tumor burden and/or increase survival, particularly in acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML) or when the density of peptide epitopes from previously undruggable intracellular proteins presented in the context of major histocompatibility complex (MHC) molecules is lower than the density of typical antigenic targets of therapeutic antibodies to reduce solid and liquid tumors in a subject.

Description

CD47 BLOCKADE ENHANCES THERAPEUTIC ACTIVITY OF
ANTIBODIES TO LOW DENSITY CANCER EPITOPES
Cross-Reference to Related Applications
[0001] This application claims priority to U.S. provisional application number 62/356,073 filed June 29, 2016, the contents of which are incorporated by reference in their entirety into the present disclosure.
Statement of Rights Under Federally-Sponsored Research
[0002] This invention was made with government support under grants CA055349, CA023766 and CA177684 awarded by the National Institutes of Health. The government has certain rights in the invention.
Sequence Listing
[0003] The instant application contains a Sequence Listing, created on June 28, 2016; the file, in ASCII format, is designated 3314078AWO_seqlisting_ST25.txt and is 896 bytes in size. The file is hereby incorporated by reference in its entirety into the instant application.
Technical Field
[0004] The present disclosure relates generally to the use of monoclonal antibodies (mAb), including T Cell Receptor mimic (TCRm) antibodies (abs) to reduce solid and liquid tumors in a subject. More particularly, the present disclosure relates to the use of abs in combination with CD47 blockade when the density of peptide epitopes from undruggable intracellular proteins presented in the context of major histocompatibility complex (MHC) molecules is much lower than the density of typical antigenic targets of therapeutic antibodies.
Background of the Disclosure
[0005] Promising immunotherapeutic agents against leukemias include chimeric antigen receptor T cells, monoclonal antibodies, adoptive T cells, and checkpoint blockade.1' 2 Monoclonal antibody (mAb) therapy has shown substantial clinical success in several tumor types, especially in B cell lymphomas, chronic leukemias and pediatric
leukemias. (3_6) Monoclonal antibodies, however, exclusively recognize cell-surface targets whereas most cancer-associated proteins are found intracellular^. Potential cancer therapy targets such as over-expressed self-proteins, activated oncogenes, mutated tumor suppressors, and translocated gene products are not accessible to traditional mAb therapy. An emerging approach to target low-density and intracellular protein epitopes is the use of TCR mimic mAbs (TCRm) that recognize epitopes similar to those of T cell receptors (TCR).
[0006] In addition to the inability of mabs to bind intracellular antigens, of the many FDA approved mAbs for the treatment of cancer, most do not operate effectively alone by simply directing human effector cells to kill the cancer cells via antibody dependent cell mediated cytoxcity (ADCC) or antibody dependent cellular phagocytosis (ADCP).7"9 mAbs require additional mechanisms to improve their effectiveness. These mechanisms include conjugation of the mAbs to drugs or radioisotopes, additional blockade of signaling pathways, upregulation of death pathways, or broad inactivation of T cell suppression.10' 11 12 For some mAbs, multiple mechanisms are employed by the single IgG.13
[0007] Causes for mAb failure are multiple. Patients with cancer, especially those with hematopoietic cancers, are immunocompromised, due to reduced numbers of, or malfunctioning immune effector cells. These deficiencies are a result of disease marrow infiltration, as well as side effects of immunosuppressive and lymphodepleting
chemotherapy, antibodies, and radiation.14 15 Human tumors have innate
immunosuppressive characteristics.16' 17
[0008] Low antigen density may further play a role in limiting therapeutic potential and as cancer cells are heterogeneous, cells within the population may express highly variable amounts of target antigen, allowing escape from mAb therapy. Some of the most clinically effective mAbs, such as trastuzumab and rituximab, target hundreds of thousands of cell surface antigens and mediate cytotoxicity via mechanisms, including ADCC, ADCP and complement-dependent cytotoxicity (CDC).18. Complement-mediated killing also is abrogated below 100,000 sites per cell.19 Summary of the Disclosure
[0009] The present disclosure is an extension of the discovery and development of T cell receptor mimic (TCRm) antibodies specific to peptides from previously undruggable intracellular protein targets to treat cancer. The present disclosure is based on the observation that the efficacy of TCRm antibodies targeted to previously undruggable intracellular proteins can be enhanced by combining TCRm antibodies, that is, antibodies that recognize an epitope comprising a peptide of the intracellular protein in the context of an HLA molecule, with CD47 blockade.
[0010] In one aspect, the present disclosure relates to a composition comprising a T cell receptor mimic (TCRm) antibody and a CD47 antagonist. These agents together, administered either simultaneously or sequentially, were superior in combination therapy as compared to either drug alone in suppressing leukemia growth in the liver, spleen and bone marrow and promoting survival benefits.
[0011] In one aspect, the disclosure relates to a composition comprising a T cell receptor mimic (TCRm) antibody and a CD47 antagonist wherein the TCRm antibody comprises an antigen binding region specific for a human leukocyte antigen (HLA) A2 -restricted intracellular protein-derived peptide. Examples include but are not limited to antibodies specific for Wilms Tumor gene (WT1 ) and antibodies specific for Preferentially Expressed Antigen in Melanoma (PRAME). The combination provides superior tumor killing than either agent alone, suppressed growth of several tumor models, and resulted in significantly increased survival in two tumor types.
[0012] The composition further comprises a CD47 antagonist, which in one embodiment is a signal-regulatory protein a (SIRPa) or a variant thereof, such as the consensus variant 1 (CV1 ).10 In one embodiment, the CD47 antagonist is a CD47 antibody, homolog or CD47-binding fragment thereof, which are known in the art for example, B6H12 or an antibody that binds the same epitope as B6H12.
[0013] In another aspect, the disclosure relates to a method for killing tumor cells, reducing tumor burden and/or increasing survival, particularly in acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML), the method comprising coadministering to a subject in need thereof a therapeutically effective amount of a T cell receptor mimic antibody, homolog or fragment thereof and a CD47 antagonist either as a combination formulation or as individual formulations.
[0014] In one embodiment, the TCRm antibody comprises an antigen binding region specific for a human leukocyte antigen (HLA) A2 -restricted intracellular protein-derived peptide. In one embodiment, the TCRm antibody is specific for a WT1 peptide,
RMFPNAPYL (SEQ ID NO: 1 ) or ALYVDSLFFL (SEQ ID NO: 2), a peptide derived from Preferentially Expressed Antigen in Melanoma (PRAME). In one embodiment, the Fc of the TCRm antibody is afucosylated. These antibodies are known in the art.
Brief Description of the Drawings
[0015] Figures 1A-1 D show the results of experiments to assess the antigen-dependent cellular phagocytosis (ADCP) of leukemia cells in vitro. A) Human macrophage phagocytosis of AML 14, BV173, and HL60 treated with various combinations of TCRm and CV1 were quantified by flow cytometry. Human macrophages were isolated from human peripheral blood mononuclear cells (PBMC's) using CD14 positive selection beads. Macrophages were cultured and then incubated with carboxyfluorescein succinimidyl ester (CSFE) labelled tumor cells. Macrophages were labelled with Alexa- 647. Flow cytometry detected double positive cells showing macrophages engulfing tumor cells. B) Flow cytometry schema depicting percentage of macrophages that have engulfed CFSE positive tumor cells. C) NSG-derived mouse macrophage ADCP of CSFE labeled AML14 cells quantified by flow cytometry. D) Phagocytosis of BV173 increases with increasing concentrations of PR20M. BV173 was incubated with CV1 and human macrophages and incubated with various doses of PR20M. Phagocytosis EC50 was determined showing the potency and specificity of the approach. EC50 ~ 0.6pg/ml.
[0016] Figures 2A-2D show the effects of interferon-γ on antigen expression and ADCP in vitro. A) AML cell line was pretreated with 100 ng/mL IFNy for 72 hours. Treated (right peak) displayed a doubling of HLA expression compared to untreated controls (left peak). B) Treated (right peak) showed a 10-fold increase in Pr20 binding compared to untreated controls (left peak). C) AML cell line was pretreated with 100 ng/uL of IFNy for 72 hours. Isolated human macrophages were incubated with pretreated AML cell line in the presence of 1 ) PBS, 2) CV1 alone, 3) Pr20M alone, 4) combination therapy with Pr20M and CV1 , 5) positive control B6H12 (previously described), 6) B6H12 with CV1 7) irrelevant control mAb, and 8) irrelevant control mAb with CV1 . All groups showed an increase in ADCP with IFNy pretreatment. Increase was most significant in combination therapy, B6H12, and B6H12 with Pr20M. D) BV173 cell line was pretreated with 100 ng/uL of IFNy for 72 hours. Isolated human macrophages were incubated with pretreated BV173 cell line as above. All groups show an increase in ADCP with IFNy pretreatment. Increase is significant in combination therapy, positive control, and positive control with Pr20. These experiments were performed in duplicate with consistent results.
[0017] Figures 3A-3D show the results of CV1 dose-response effects in vivo. A) Mice were engrafted via tail vein injection with 3 million cells/mouse of AML14 transfected with Luciferase gene. Mice were imaged via BLI on day 6. Mice were randomized to have equal group mean engraftment. Starting on day 6 after engraftment, mice were treated with either 200 g, 150 g, or 100 g of CV1 daily. Mice were imaged once a week for 3 weeks. B) Graph showing individually normalized mean flux in photons/second of mice at days 6, 12, and 18. C) Mice were engrafted via tail vein injection with 3 million cells/mouse of BV173 transfected with Luciferase gene. Mice were imaged via BLI on day 6. Mice were randomized to have equal group mean engraftment. Starting on day 6 after engraftment, mice were treated with either 200 g, 150 g, or 100 g of CV1 daily. D) Graph showing individually normalized mean flux in photons/second of mice at days 6, 12, and 18. E) Mice were engrafted via tail vein injection with 3 million cells/mouse of AML14 transfected with Luciferase gene. Mice were imaged via BLI on day 6. Mice were randomized to have equal group mean engraftment. Starting on day 6 after engraftment, mice were treated with 100 g of CV1 either daily or on Monday Wednesday Friday. Mice were imaged once a week for 3 weeks. F) Graph showing mean flux in photons/second of mice at days 6, 13, and 20. G) Mice were engrafted via tail vein injection with 3 million cells/mouse of BV173 transfected with Luciferase gene. Mice were imaged via BLI on day 6. Mice were randomized to have equal group mean engraftment. Starting on day 6 after engraftment, mice were treated with 100 g of CV1 either daily or on Monday Wednesday Friday. Mice were imaged once a week for 3 weeks. H) Graph showing mean flux in photons/second of mice at days 6, 13, and 20.
[0018] Figures 4A-4D show CV1 schedule effects in vivo. A) Mice were engrafted via tail vein injection with 3million cells/mouse of AML14 transfected with Luciferase gene. Mice were imaged via BLI on day 6. Mice were randomized into 5 groups of 5 mice each to have equal group mean engraftment. The 5 groups were: 1 ) Control, 2) PR20 alone 3) CV1 alone 4) CV1 plus Isotype TCRm MAGE antibody and 5) CV1 +PR20. Treatment started on day 6. PR20 was administered retro-orbitally biweekly at 50pg. CV1 was administered intraperitoneal^ daily at 100pg. Mice were imaged once a week for 3 weeks. B) Graph showing tumor burden as measured by mean flux in photons/second of mice at days 6, 13, and 20, and 27. C) Kaplan-Meier curve showing survival. Control and single treated groups had 100% death within 50 days. Experiment was truncated at 100 days at which time 4 of 5 mice in combination were alive. Log-rank test among all 5 groups indicated significant differences in overall survival among the groups (p<0.001 ). D) AML14 burden remains lower than engraftment 3 weeks after the end of therapy. At 67 days, 1 mouse in combination group had lymphomatous growth requiring sacrifice. The 4 remaining animals had lower tumor burden via BLI than on day of engraftment.
[0019] Figures 5A-5C show the results of therapy of human Ph+ALL in mice. A) Mice were engrafted via tail vein injection with 3 million cells/mouse of BV173 transfected with Luciferase gene. Mice were imaged via BLI on day 6. Mice were randomized into 5 groups of 5 mice each to have equal group mean engraftment. The 5 groups were: 1 ) Control, 2) PR20M alone 3) CV1 alone 4) CV1 plus Isotype TCRm MAGE antibody 5) CV1 +PR20M. Treatment started on day 6. PR20M was administered retro-orbitally biweekly at 50 g. CV1 was administered intraperitoneally daily at 100 g. Mice were imaged once a week for 3 weeks. B) Graph showing mean flux in photons/second of mice at days 6, 13, and 20, and 27. C) Kaplan-Meier curve showing overall survival. Control and single treated groups had 100% death within 100 days. Experiment was truncated at 141 days at which time 2 of 5 mice were alive. Log-rank test among all 5 groups indicated significant differences in overall survival among the groups (p<0.001 ).
[0020] Figures 6A-6C show the results of therapy of human AML in mice A) Mice were engrafted via tail vein injection with 3 million cells/mouse of BV173 transfected with Luciferase gene. Mice were imaged via BLI on day 6. Mice were randomized into 5 groups of 5 mice each to have equal group mean engraftment. The 5 groups were: 1 ) Control, 2) ESK alone 3) CV1 alone 4) CV1 plus Isotype TCRm MAGE antibody 5) CV1 +ESKM. Treatment started on day 6. ESK was administered retro-orbitally biweekly at 50 g. CV1 was administered intraperitoneally daily at 100 g. Mice were imaged once a week for 3 weeks. B) Graph showing mean flux in photons/second of mice at days 6, 13, and 20, and 27. C) Kaplan-Meier curve showing overall survival. Log-rank test among all 5 groups indicated significant differences in overall survival among the groups (p<0.001 ).
Figures 7A-7D show lnterferon-γ release in vivo. A) Mice were engrafted via tail vein injection with 3 million cells/mouse of AML14 transfected with Luciferase gene. Mice were divided into 4 groups with equal group mean engraftment. The 4 groups were: 1 ) control, 2) Pr20M alone, 3) CV1 , and 4) CV1 + Pr20M (n=5). Treatment started on day 6. Pr20M was administered retro-orbital ly at 50 g. CV1 was administered intraperitoneally at 100 g. Mice were bled before treatment and at 12 hours after treatment. Serum was separated. Luminex assay was done on mice serum for mouse cytokines IFNy, TGFp, M- CSF, and IL-1 B. At 12 hours we see an upward trend in concentration of IFNy (pg/mL) in groups treated with CV1 alone and combination therapy. B) Distribution of CV1 versus non-CV1 treated mice. As an exploratory analysis based on the distribution of IFNy among the groups, we combined the four groups into non-CV1 (control and Pr20M only) and CV1 (CV1 -only and CV1 + Pr20M) groups. Results from the stratified Kruskal-Wallis test (stratified by Pr20M status) indicate significant differences between the non-CV1 and CV1 groups (medians, non-CV1 = 4.64, CV1 = 6.64, p=0.006). C) IFNy was incubated with BV173cell line at concentrations of 1 ng/ml_, 10ng/ml_ and 100ng/ml_. Cell viability was assessed at 24, 48, and 72 hours with Cell Viability Glo Assay. IFNy alone did not cause cell toxicity nor block cell proliferation. These experiments were completed in five replicates. D) IFNy was incubated with AML14 at varying concentrations as described above. Cell viability was assessed at time points as described above. IFNy alone did not cause cell toxicity nor block cell proliferation. These experiments were completed in five replicates.
Detailed Description of the Disclosure
[0021] All patents, publications, published applications and other references cited herein are hereby incorporated in their entirety into the present application.
[0022] In practicing the present invention, many conventional techniques in molecular biology, microbiology, cell biology, biochemistry, and immunology are used, which are within the skill of the art. These techniques are described in greater detail in, for example, Molecular Cloning: a Laboratory Manual 3rd edition, J.F. Sambrook and D.W. Russell, ed. Cold Spring Harbor Laboratory Press 2001 ; Recombinant Antibodies for Immunotherapy, Melvyn Little, ed. Cambridge University Press 2009; "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987);
"Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F. M. Ausubel et al., eds., 1987, and periodic updates); "PCR: The Polymerase Chain Reaction", (Mullis et al., ed., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001 ). The contents of these references and other references containing standard protocols, widely known to and relied upon by those of skill in the art, including manufacturers' instructions are hereby incorporated by reference as part of the present disclosure.
[0023] In the description that follows, certain conventions will be followed as regards the usage of terminology. Generally, terms used herein are intended to be interpreted consistently with the meaning of those terms as they are known to those of skill in the art.
[0024] The terms "co-administration" or co-administering" refer to the administration of a TCRm antibody and CD47 antagonist as one single formulation or as two separate formulations. The co-administration can be simultaneous or sequential in either order, wherein there is a time period while both (or all) active agents simultaneously exert their biological activities.
[0025] One embodiment provides a pharmaceutical composition for combination therapy for preventing and/or treating of a cancer, comprising or consisting essentially of a TCRm antibody and CD47 antagonist as active ingredients.
[0026] The pharmaceutical composition for combination therapy may be a mixed formulation (e.g., a single composition comprising two or more active ingredients) of a TCRm antibody and CD47 antagonist. The TCRm antibody and CD47 antagonist may be included in any amount that is pharmaceutically effective when used together. The composition thus formulated can be used for simultaneous administration of the two active ingredients.
[0027] Alternatively, each of the TCRm antibody and CD47 antagonist can be formulated in a separate composition and the two active ingredients can be separately administered simultaneously or sequentially. For instance, a first pharmaceutical composition including a pharmaceutically effective amount of a TCRm antibody as an active ingredient and a second pharmaceutical composition including a pharmaceutically effective amount of the CD47 antagonist as an active ingredient can be administered simultaneously (within 0-20 minutes) or sequentially (after 20 minutes). In the case of the sequential administration, any order of administration may be used.
[0028] In one embodiment, the agents are administered sequentially and spaced apart by hours or days. For example, TCRm antibody is administered to the subject at time 0; subsequently, the CD47 antagonist is administered anywhere from 21 minutes to 21 days following the administration of TCRm antibody. In one embodiment, CD47 antagonist is administered anywhere from 30 minutes to 20 days following the
administration of TCRm antibody; in one embodiment CD47 antagonist is administered anywhere from 45 minutes to 15 days following the administration of TCRm antibody; in one embodiment CD47 antagonist is administered anywhere from 1 hour to 10 days following the administration of TCRm antibody; in one embodiment CD47 antagonist is administered anywhere from 5 hours to 5 days following the administration of TCRm antibody; in one embodiment CD47 antagonist is administered anywhere from 12 hours to 2 days following the administration of TCRm antibody.
[0029] The timing of the sequential administration will be based on the duration of the effect of each agent alone with the recipient subject. For example, if a particular TCRm is active for 7 days, and the CD47 antagonist is active for 2 days, then the CD47 antagonist can be administered within that time frame, so as to overlap in function with the activity of the TCRm. In one embodiment, therefore, the CD47 antagonist can be administered from 2 days prior to administration of TCRm and up to 7 days after.
[0030] Another embodiment provides a kit useful for preventing and/or treating a leukemia, comprising or consisting essentially of a first pharmaceutical composition including a TCRm antibody as an active ingredient, a second pharmaceutical
composition including a CD47 antagonist as an active ingredient. The TCRm antibody and CD47 antagonist may be used in amounts that are pharmaceutically effective when combined, which amount may be determined by the skilled medical practitioner or medical researcher. [0031] The term "the pharmaceutically effective amount" as used in this specification refers to an amount of active ingredient that can exert pharmaceutically significant effects (e.g., an amount sufficient to prevent or treat leukemia in a subject).
[0032] Monoclonal antibodies (mAbs) are potent cancer therapeutic agents, but exclusively recognize cell-surface targets whereas most cancer-associated proteins are found intracellularly. As a result, potential cancer therapy targets such as over-expressed self-proteins, activated oncogenes, mutated tumor suppressors, and translocated gene products are not accessible to traditional mAb therapy. An emerging approach to target these epitopes is the use of TCR mimic mAbs (TCRm) that recognize epitopes similar to those of T cell receptors (TCR).
[0033] The term "T-cell receptor mimic (TCRm) antibodies" or "T-cell receptor-like antibodies" therefore refers to antibodies that recognize an epitope similar to the epitope recognized by the receptor on the surface of a T-cell that causes activation of the T-cell when it binds an MHC (HLA)-restricted ligand on the surface of a tumor cell or
pathogenic organism. TCRm antigens are composed of a linear peptide sequence derived from degraded (processed) intracellular proteins and presented in the context of cell-surface MHC molecules. TCRm combine the specificity of TCR recognition with the potency, pharmacologic properties, and versatility of mAbs.
[0034] The terms "low-density" and "ultra low-density" with respect to cancer antigens refer to the relative number of antigens on the surface of a cell with which macrophages might interact. The density of peptide epitopes from undruggable intracellular proteins presented in the context of major histocompatibility complex (MHC) molecules is much lower than the density of typical cell surface antigenic targets of therapeutic antibodies (for example HER2/net/). The number of antigens on the surface of a cell with low density TCRm mAb targets may have as few as 1 % of the mAb binding sites as other targets. Ultra-low-density applies to cells with about 900 to 2500 Pr20m mAb binding sites per cell. As another example, BV173 cells have previously been reported to have about 700 binding sites per cell for the ESK1 TCRm. [0035] The present disclosure is an extension of the discovery and development of T cell receptor mimic (TCRm) antibodies specific to peptides from previously undruggable intracellular protein targets to treat cancer.
TCRm Antibodies
[0036] In one embodiment, the antibody is a TCRm antibody, for example a TCRm antibody (mAb ESKm) that is specific for Wilms tumor gene 1 product (VVT1 ) -derived peptide, RMFPNAPYL (SEQ ID NO: 1 ) in the context of HLA-A*02:01. VVT1 is an oncofetal antigen, a zinc finger transcription factor that is rare in normal tissues but seen overexpressed in a variety of multiple solid and liquid malignancies including mesothelioma and ovarian cancer.15"17. VVT1 antibodies are known in the art and are described for example, in US 9,040,669, US 9,074,000 and US 9,540,448 (the contents of each is hereby incorporated by reference into the present application).
[0037] In one embodiment, the antibody is a TCRm antibody (mAb Pr20) that is specific for the "Preferentially Expressed Antigen in Melanoma" (PRAME) -derived peptide ALYVDSLFFL (SEQ ID NO: 2) in the context of HLA-A*02:01 . 11< 12 13 PRAME is a cancer testes antigen found in many cancers and leukemias, and expressed in normal ovaries, testes and endometrium with limited expression in other healthy tissues.14 PRAME antibodies are described in WO2016191246 (the contents of which is hereby incorporated by reference into the present application.)
[0038] In some embodiments, the Fc of the TCRm antibodies is engineered to be afucosylated so as to enhance effector cell recruitment and killing (see US 9,540, 448).18_ 20 The TCRm antibodies alone, which act via antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), show therapeutic activity in murine models, although the responses are incomplete and relapse is common in these models 17"19.
[0039] In addition to drugging intracellular proteins, these mAb can address highly specific, yet low- and ultra-low-density targets. The combination of ESKm with tyrosine kinase inhibitors to cytoreduce leukemic burden has shown promising results with Ph+ leukemias.19 CD47 inhibitors
[0040] CD47 is a trans-membrane protein that transduces an anti-phagocytic signal via binding to its cognate ligand, SIRPa, in macrophages. CD47 is expressed on normal host tissue and is upregulated on cancer tissue to inhibit macrophage-mediate phagocytosis. 21 22 CD47 inhibitory mAbs are in clinical trials. However, to date the mAb have significant toxicities perhaps because of the ubiquitous expression of CD47 on normal tissue, especially red blood cells. Anemia is therefore a frequently seen adverse effect with currently available antibodies.10 23
[0041] A truncated SIRPa protein variant, known as consensus variant 1 (CV1 )11 , that blocks the interaction between the SIRPa on the cancer cell and CD47 on the macrophage has been shown to increase macrophage mediated killing of cancer cells in vitro and in vivo without toxicity 10. In addition, CV1 has shown an additive effect in murine models of human cancers when combined with mAb against /?/g/?-density targets on both solid and liquid tumors. 0 24 25
[0042] There are however, no data to suggest that blockade of the CD47 signaling axis would have similar effects on low density TCRm mAb targets, which have as few as 1 % of the mAb binding sites as other targets, with which macrophages might interact. Furthermore, there may be a threshold below which ADCP would not be active. Therefore, whether TCRm antibodies providing a pro-phagocytic signal combined with CV1 inhibition of the CD47 anti-phagocytic signal could enhance immunotherapy for leukemias was evaluated. These two agents together resulted in much greater than additive therapeutic effects in several human tumor xenograft model systems. The combination of these therapies also allowed CV1 to be used in half of the previously reported doses. Combination therapy led to superior tumor killing than either agent alone, suppressed growth of several tumor models, and resulted in significantly increased survival in two tumor types. In addition, in models of human myeloid leukemia, combination therapy for three weeks led to tumor burden lower than at engraftment weeks after the stop of therapy. This was not previously seen with the TCRm alone. EXAMPLES
Example 1: Methods
TCRm antibodies and SIRPa inhibitors
[0043] TCRm antibodies were designed and produced at Memorial Sloan-Kettering Cancer Center (MSK) and Eureka Therapeutics (Emeryville CA) in their afucosylated IgG form. They included Pr20M, designed to react with PRAME peptide ALYVDSLFFL (SEQ ID NO: 2)/HLA-A*02:01 ; ESKM prepared to react with VVT1 peptide RMFPNAPYL (SEQ ID NO: 1 )/HLA-A*02:01 1 and irrelevant control IgG17 not reactive with human antigens.27' 44 CV1 was prepared as described. 41
[0044] Proteins were stored in phosphate buffered saline at 4° C. [0045] B6H12 was purchased from Biolegend (San Diego, CA).
ADCP assays
[0046] Human and mouse macrophage-mediated antibody-dependent cellular
phagocytosis (ADCP) assays were as previously described12,40. Human blood was obtained from the Stanford Blood Center using IRB approved protocols and monocytes were isolated via CD14 positive selection (Miltenyi) and magnetic isolation. Recovered CD14+ monocytes were plated at a density of 10,000,000 monocytes per 150 mm TC treated dishes in IMDM + GlutaMAX media (Gibco) supplemented with 10% human serum and 1 % P/S and cultured for 7 days at 37 °C to yield human monocyte derived macrophages (MDM0). Phagocytosis assays were repeated in duplicate.
[0047] Mouse macrophages were derived from bone marrow (BMDMs). Mouse bone marrow cells were flushed with a syringe from the tibia and femurs of NSG mice into IMDM + GlutaMAX supplemented with 10% FBS and 1 % P/S. Cells were collected by centrifugation followed by RBC lysis with ACK buffer for 3 - 5 mins (Gibco), quenched with complete media, and filtered through a 70 μΜ cell strainer. Cells were pelleted by centrifugation, re-suspended in media containing 10 ng/mL M-CSF (Peprotech) and plated on 3 x 10 cm untreated pitri dishes per mouse in 10 mL media and cultured for 7 days without replenishing or changing media to derive BMDMs.
[0048] To quantify macrophage-mediated ADCP, tumor cells were harvested, labeled with carboxyfluorescein succinimidyl ester (CFSE), washed with serum free IMDM + GlutaMAX, and plated at a density of 100,000 cells/well in 25 μί in a 96-well ultra low attachment round bottom plate (Costar, Cat. 7007) on ice. Tumor cells were opsonized by addition of 25 μΙ_ of various antitumor antibodies, CD47 blocking reagents, and/or controls for 30 min on ice. Macrophages were harvested by enzymatic dissociation and cell scraping, pelleted, washed in serum free IMDM, and added to opsonized tumor cells at a density of 50,000 cells/well in 50 μΙ_ media for a final assay volume of 100 μΙ_ and an effector to tumor cell ratio of 1 :2. ADCP was measured after incubation for 2 hr at 37 °C with the following test groups: M0 + cancer cells alone, M0 + cancer cells opsonized with TCRm or control antibody alone (10 pg/mL mAb final), M0 + cancer cells opsonized with CV1 alone (100 nM final), and M0 + cancer cells opsonized with the combination (10 g/mL mAb + 100 nM CV1 ). Cells were then pelleted and washed with autoMACS running buffer (Miltenyi), and stained with a 1 : 100 dilution of anti-mouse F4/80-APC (Biolegend) for mouse BMDMs or a 1 : 100 dilution of anti-human CD206-Alexa647 (Biolegend) for human MDM0 in autoMACS buffer for 1 hr at 4 °C. Cells were pelleted, washed, and re-suspended in a 1 : 10000 dilution of DAPI and analyzed by FACS using the CytoFLEX equipped with a high throughput sampler. Phagocytosis was quantified gating based on SSC-A and FSC-A, singlets, live/dead (DAPI negative/low), and phagocytosis quantified as the percentage of F4/80-APC or CD206-Alexa647 positive macrophages that are also CFSE positive.
[0049] To determine the role of IFNy exposure on human macrophage-mediated ADCP in vitro, leukemia cells and/or macrophages were cultured with human IFNy (Peprotech) at a concentration of 100 ng/mL for 72 hours prior to harvest for ADCP assays as described above. Human macrophage phagocytosis assays were repeated at least twice with different human blood donors, anti-human CD200 mAb (Biolegend) served as an irrelevant isotype control for BV173 cell lines and anti-human CD33 mAbs as an irrelevant isotype control for AML14 cell lines. NSG mouse BMDM phagocytosis assays were repeated in duplicate.
[0050] Ordinary one-way ANOVA with Turky's test for multiple comparisons for just looking at difference between proteins. 2 way ANOVA with Sidak's test for multiple comparisons when comparing IFNy untreated vs. treated for the numerous protein combinations. Flow Cytometry
[0051] Single color flow cytometry assays detected HLA-A*02:01 and binding of ESKM and Pr20M to epitopes of interest. These assays were repeated in the presence or absence of IFNy at a concentration of 100 ng/mL. IFNy was purchased from eBioscience (San Diego, CA)
Human Cell Lines
[0052] The human leukemia lines BV173 (Ph+ ALL) and AML14 (AML) cell lines were confirmed for CD47 expression by flow cytometry. Scatchard plot analysis confirmed PR20M could bind these lines maximally at an ultra-low-density, with about 900 and 2500 Pr20 mAb binding sites per cell, respectively. BV173 has previously been reported to have about 700 binding sites per cell for the ESK1 TCRm22 The lines were transduced with a Luciferase-green fluorescent protein (GFP) retrovirus and expanded in complete RPMI Medium for use in NSG animal models. Cell lines were obtained from ATCC® or MSKCC stocks.
CELLTITER-GLO® Assays
[0053] Human leukemia lines, BV173 and AML14, were incubated at 37°C with varying concentration of mouse IFNy. At 24, 48 and 72 hours, cell viability was ascertained through the quantification of ATP using CELLTITER-GLO® Assay.
Luminex Assays
[0054] Human xenograft models were prepared as described above using tail vein injection with 3 million AML14 cells. Mice were divided into 4 treatment groups with equal engraftment measured by mean fluorescent intensity via BLI. Groups were 1 ) control without treatment, 2) CV1 alone, 3) Pr20M alone, and 4) CV1 and Pr20M. Mice were bled before treatment and at 12 hours and 24 hours after treatment. Serum was analyzed on a Milliplex Luminex platform to detect the mouse cytokines IFNy, IL-Ι β, M-CSF, and TNFa.
Statistical Analysis
[0055] Due to sample size and anticipated non-linearity of the time effect, we analyzed the data as area under the curve (AUC) for leukemic burden per mouse. Tumor burden expressed as AUCs were compared between groups using the Kruskal-Wallis test. When the Kruskal-Wallis test indicated significant differences among the groups (p<0.05), subsequent pairwise comparison were conducted. Survival studies were done using bilateral hind leg paralysis as surrogate for death or using predetermined morbidity characteristics. Overall survival was estimated by the Kaplan-Meier approach and compared among groups using the log-rank test, ns is defined as p > 0.05. * is defined as p< 0.05. ** is defined as p<0.01 . *** is defined as p <_0.001 . All statistical tests were conducted using a permutation test procedure.43 Statistical testing were performed using R 3.3.1 (R Core Team, Vienna Austria).
Example 2
Trials of Pr20M and ESKM with CV1 in mice
[0056] Mouse research was conducted under protocols approved by the MSKCC institutional animal care and use committee. Therapeutic trials were conducted in human xenograft models in 6-week-old male non-obese/diabetic severe combined immunodeficient (NOD/SCID) mice with ΙΙ_2γ receptor negative (NSG) mice from Jackson Laboratory (Bar Harbor, ME). Mice were engrafted via tail vein injection with 3 million BV173 and AML14 cells transfected with the firefly luciferase gene.
[0057] Disseminated engraftment of leukemia was confirmed via bioluminescent imaging (BLI) in all mice before beginning treatment. Mice were randomly assorted into the following five treatment groups with equal mean BLI flux (photons/second): 1 ) control without treatment, 2) TCRm antibody alone, 3) CV1 alone, 4) CV1 and an afucosylated isotype control antibody, and 5) CV1 and TCRm antibody. TCRm antibodies were administered retro-orbital ly biweekly at doses of 50 g. For combination therapy studies CV1 was administered daily via intraperitoneal injection at doses of 100 g.
[0058] Leukemic burden was assessed by bioluminescence imaging, recorded as flux of protons per second and repeatedly measured at days 6, 13, 20, and 27, post
engraftment. Mice were treated twice a week beginning on day 6 through 27 for a total of six doses of TCRm and/or 21 daily doses of CV1.
[0059] Additional mouse or human PBMC's were not administered.
[0060] CV1 and TCRm antibody show additive effects in vitro. The ability of CV1 , an engineered SIRPa variant that potently antagonizes CD47, to enhance TCRm mAb- dependent macrophage phagocytosis of leukemia cells in vitro was evaluated utilizing the HLA-A*02:01 positive human acute myeloid leukemia cell line AML14, and human acute lymphoblastic leukemia cell line BV173 as models, which express the target antigen of the TCRm mAb Pr20M (PRAME). The HLA-A*02:01 negative cell line HL60 was used as a control. Blockade of leukemia cell CD47 with CV1 alone did not promote macrophage phagocytosis of AML14, BV173, or HL60 (Figure 1 ). Pr20M alone did not promote ADCP of HL60 or BV173, but significantly increased phagocytosis of AML14 (Figure 1 A, B). However, the combination of TCRm mAb and CV1 significantly increased macrophage phagocytosis of AML14 and BV173, but not the HLA-A*02:01 negative cell line HL60 (Figure 1A, B), indicating the effect is TCRm mAb antigen specific. As expected, an anti- CD47 blocking antibody B6H12, a mouse monoclonal to CD47 (Abeam, Cambridge MA) induced a significant increase in macrophage phagocytosis of all three leukemia cell lines, and potentiated TCRm mAb-mediated phagocytosis of AML14 and BV173 (Figure 1A, B).
[0061] These experiments, when repeated with NSG mouse macrophages as effectors, showed improved phagocytosis with CV1 as well, with more than additive effects when CV1 and the TCRm PR20 were combined (Figure 1 C). BV173 was incubated with CV1 and human macrophages and incubated with various doses of Pr20M. Phagocytosis ECso was determined showing the potency and specificity of the approach (Figure 1 D). Collectively, these results indicate that CD47 blockade is effective at improving the in vitro macrophage effector function of antibodies that target ultra-low density tumor antigens, such as TCRm mAbs.
[0062] IFNy leads to increased expression of HLA A02:01, increased leukemia cell binding of mAbs Pr20 and ESK, and enhanced macrophage-mediated ADCP in vitro.
IFNy is a potent immunocytokine with pleiotropic effects, including induction of MHC Class I and II expression and increased antigen processing and presentation. (43). Anti-CD47 mAb therapy triggers a phagocyte type I and II interferon (IFN) response in the tumor microenvironment that presumably increases tumor cell surface peptide- MHC (pMHC) density. (33) Given that the target of TCRm mAbs is presented by pMHC, promoting IFN signaling may boost TCRm mAb effector functions by increasing target antigen density on the tumor cell surface. To determine if IFN promotes expression of the pMHC target of Pr20M and ESKM, we treated AML14 and BV173 with IFNy for 72 hrs in vitro and measured cell surface levels of HLA, CD47, and the pMHC epitopes of Pr20M and ESKM by FACS. IFNy treatment increased HLA expression resulting in increased binding of Pr20 (Figure 2 A,B) and ESK1 (not shown) to AML14. IFNy significantly increased expression of TCRm mAb epitopes of interest and increased macrophage-mediated ADCP of both AML14 and BV173 in vitro. (Figure 2 C,D) Treatment of AML14 with IFNy prior to ADCP reactions significantly increased ADCP of AML14 opsonized with Pr20M alone, Pr20M and CV1 , and Pr20M and B6H12 (Figure 2C). While there was modest increase in phagocytosis of AML14 with IFNy alone, and IFNy with CV1 , there was a significantly higher increase of phagocytosis with combination therapy with TCRm, and CV1 and TCRm. In the BV173 model, there were small increases in phagocytosis with vehicle, CV1 alone or TCRm alone, in the presence of IFNy (Figure 2D)
[0063] CV1 dose titrations in mice. CV1 is effective at doses of 200ug daily in mice. However, the therapeutic window of CV1 for use as an adjuvant to mAb therapy is unknown. We observed a reduction in leukemia burden in mice treated with 200 g daily injections of CV1 alone. Therefore, we sought to further characterize dosing regimens for CV1 monotherapy against AML14 and BV172 leukemias. NSG mice bearing disseminated BV173 or AML14 leukemia cells were injected daily with either 200 g, 150 g, or 100
Figure imgf000020_0001
of CV1 beginning on day 6 post engraftment and anti-leukemia effects were measured by BLI and clinically. CV1 was similarly effective at all three dose levels evaluated. There was no significant difference in leukemia burden as measured by AUC based on BLI among the three dose groups (Figure 3 A-D). CV1 alone suppressed leukemia growth for the first week of treatment; however, leukemias escaped after 2 weeks of treatment at all doses.
[0064] CV1 administered three times per week beginning on day 6 post engraftment was also effective at suppressing leukemia growth, but the effect varied among mice treated with this dosing schema, particularly against AML14 leukemias. As with daily dosing, leukemia escaped at the end of the 2 week treatment period (Figure 3 E-H). A
comparison of leukemia burden as measured by AUC based on BLI showed a
statistically significant difference in leukemia burden between dosing schedules.
Therefore, a 100 g per day dose of CV1 was used for all subsequent combination therapy studies.
[0065] CV1 and TCRm antibody show greater than additive therapeutic effects in vivo. CD47 blockade enhances the antitumor activity of antibodies that target overexpressed tumor antigens by mobilizing the innate and adaptive immune system. (12, 33) To determine if CD47 blockade also enhanced the antitumor activity of TCRm mAbs that target ultra low-density pMHC tumor antigens, mice engrafted with disseminated AML14 or BV173 leukemias were treated with TCRm mAb, CV1 , or the combination beginning on day 6 post engraftment. TCRm mAb and CV1 monotherapy significantly reduced leukemia burden by 5-10 fold in the AML14 model and 5-100 fold in the BV173 model compared to control treated animals (Figure 4, 5). However, after two weeks of treatment, leukemias escaped in all single agent therapy groups in both models. Combination therapy had a greater than additive effect compared to either agent alone, with a 3 log reduction in leukemia burden relative to control untreated mice and a 5-10 fold reduction relative to the single agent groups (Figure 4, 5). The differences between the combination therapy and monotherapy were more pronounced in the AML14 model than in the BV173 Ph+ALL model, as CV1 alone was more effective in the latter. (Figure 4 A, B and Figure 5 A, B) In the AML14 model, all 5 mice treated with combination therapy had leukemic burden lower than at engraftment 3 weeks after therapy had stopped. (Figure 4D) Leukemias did not relapse while on combination therapy.
[0066] In the AML14 model, treatment was stopped on day 29, when the first control animal died; animals were followed off therapy and survival analysis was performed. In the BV173 model, combination treated leukemia growth escaped by day 27; however, leukemias did not relapse at the initial sites (bone marrow and spleen) but in lymphomatous nodules that have been described. (27) Kaplan Meier analysis indicates significantly improved survival for single agent therapy as compared to controls and further improvement in overall survival of mice treated with the combination therapy. (Figure 4C and 5C)
[0067] To determine if these effects were generalizable, we repeated these experiments with a second TCRm antibody (ESKM) directed to a different specificity, VVT1 , using the Ph+ ALL Line, BV173. Similar to results obtained with the TCRm mAb PR20M, ESKM and CV1 monotherapy was effective for one week with leukemia escape by two weeks. (Figure 6) Combination ESKM and CV1 therapy significantly reduced leukemic growth compared to monotherapy, leading to a greater than additive effect and prolonged survival. (Figure 6C) [0068] CV1 therapy increased serum concentrations of IFNy. A possible mechanism involving cytokine release for the greater than additive in vivo responses to the combination therapy was explored. IFNy exposure increases cell surface expression of HLA, target epitopes of interest (Figure 2 A,B), and ADCP in vitro (Figure 2 C,D); therefore, we asked if anti-CD47 therapy triggers a systemic cytokine response that could enhance the potency of TCRm mAbs through cytokine feed-forward signaling mechanisms. Mice (n=5 per group) bearing disseminated AML14 leukemias were treated on day 6 after engraftment with a vehicle control, CV1 alone, Pr20M alone, or the combination of CV1 and Pr20M. The mouse cytokines IFNy, IL-1 β, M-CSF, and TNFa were quantified in serum before and after the various treatments. Serum concentrations of IFNy were increased in CV1 treated mice 12 hours post treatment (Figure 7). It appeared that CV1 treated animals had elevated serum cytokines, independent of mAb administration. Therefore, an exploratory statistical analysis was conducted based on the distribution of IFNy among the groups by combining the two non-CV1 treated groups (control and Pr20M) compared to the CV1 treated groups (CV1 only and CV1 + Pr20M). The Kruskai-Wallis test, stratified by Pr20M status, indicated significant differences in serum IFNy concentrations between the non- CV1 and CV1 treated animals (median non CV1 4.64pg/ml_, median CV1 6.64pg/ml_ p=0.006) (Figure 7B). We observed no differences in serum concentrations of other cytokines evaluated, or at alternative time points, in response to CV1 , Pr20M, or the combination.
[0069] IFNy alone is not cytotoxic to human leukemia cell lines. To ascertain if increased tumor kill could also be secondary to direct cytotoxic effects of IFNy independent of the actions of CV1 or TCRm, we incubated mouse IFNy at varying concentrations with BV173 and AML14. We saw no increase in cell death with exposure to IFNy (Figure 7C,D). Indeed, there was a small growth promoting effect. This further supports the hypothesis that IFNy can work indirectly as an adjuvant to this specific combination of CD47/SIRP alpha blockade and the TCRm via upregulation of the epitope and activation of phagocytosis.
[0070] CD47 blockade with high affinity SIRPa variants has shown beneficial therapeutic effects when administered in combination with Fc receptor engaging monoclonal antibodies that target overexpressed tumor antigens. (40) We sought to determine if anti- CD47 adjuvant therapy could also improve the efficacy of TCR mimic antibodies that target previously undruggable low-density tumor antigens. While we found additive effects with the two drugs in vitro, surprisingly, we found that using half of the previously reported doses of CV1 , in combination with two TCRm mAb, showed greater than additive activity in several mouse models of human leukemias. Both decreased tumor burden and significantly prolonged survival were observed. In addition, we discovered that CV1 infusion caused elaboration of IFNy by the mice. Some of this enhanced therapeutic activity may be related to this release of IFNy, which not only can activate phagocytosis further, but also causes a feed-forward mechanism unique to these TCRm antigenic systems in which the peptide epitope presentation on the target cells is up-regulated by IFNy.
[0071] The dramatic therapeutic effects of combination CV1 and TCRm mAb therapy in vivo were surprising. Several factors may explain the potency in vivo. First, there are substantial differences between in vitro and in vivo microenvironments including cytokine and cellular networks that may alter macrophage effector function in vivo. Second, macrophages may not be the only immune effector in the mouse that contribute to anti- CD47 therapy. (44) Neutrophils express both SIRPa and Fc receptors and have been implicated in responses to anti-CD47 antibody treatment. SIRPa also contributes to neutrophil transmigration and blocking SIRPa signaling may alter neutrophil trafficking and therapeutic responses. (44, 45) Interestingly, relapses that occur in BV173 engrafted mice were outside sites with high neutrophil density. Third, the leukemias evaluated preferentially engraft in liver, spleen and bone marrow. These organs have high intrinsic numbers of phagocytic cells increasing the likelihood of making productive, cytotoxic contacts with leukemic cells. High local concentration of effectors may enhance leukemia clearance. (46) Fourth, cross-species differences in Fc receptor biology, as well as alternative xenogeneic ligand-receptor interactions between human tumor cells and mouse immune effectors may alter antibody and immune cell function. (47, 48) It is important to note that NSG mice are B cell, T cell, and NK cell deficient, and although they have intact IFNy-dependent signaling, they have defective innate immunity and cytokine signaling pathways. (49, 50) While it is difficult to draw parallels between human and mouse systems, in the human, a greater variety of more potent effectors and an immunocompetent host that responds to pro-inflammatory signaling could allow even greater efficacy of this drug combination in the human patient. In addition, NSG mice have low circulating IgG levels that could compete with TCRm for Fc receptor interactions. (50) [0072] Finally, IFNy secretion caused by CV1 is likely contributing indirectly through a new mechanism to the therapeutic effects seen. IFNy secreted by NK cells, T cells, dendritic cells and macrophages contributes directly to the innate and adaptive immune response and indirectly through its interplay with other cytokines. (51 ) However, NSG mice have no T or NK cells. (52) We found no other cytokines in the serum of CV1 treated mice that were up-regulated other than IFNy. Not surprisingly, we found that IFNy-dependent signaling enhanced TCRm mAb dependent, macrophage-mediated phagocytosis. This enhancement of phagocytosis was likely mediated through multiple mechanisms including IFNy-dependent macrophage activation, which is expected to enhance ADCP of any antitumor antibody with Fc effector function, as well as TCRm mAb specific mechanisms involving tumor cell intrinsic increases in HLA expression and target antigen presentation. Thus, strategies that promote an IFNy response could uniquely potentiate the activity of TCRm mAbs, beyond what might be seen with traditional mAbs. CD47 blockade has been shown to trigger a type I and II IFN response. (33) This is consistent with the increase in serum concentrations of IFNy we observed in mice treated with CV1. This increase in IFNy likely contributes to the remarkable in vivo synergy between TCRm mAb and CV1. Notably, IFNy alone did not cause cell cytotoxicity. Therefore, it is the combination of CV1 and TCRm in this specific in vivo milieu that leads to remarkable tumor kill. Although we only quantified systemic cytokine responses, it is likely that the local concentration of IFNy at sites where the targets and effectors are in contact is much higher, such as in the marrow, spleen and liver. It is also possible that alternative cytokines were also increased locally as a result of treatment, but not high enough to be detected in serum. Proinflammatory cytokine responses to CV1 monotherapy have not been reported. This novel feature of its activity may contribute to its usefulness as a cancer therapeutic. It will be important to determine if this effect is specific to leukemia, or applicable to alternative hematologic and solid tumors.
[0073] Synergy between CV1 and antitumor antibodies may be especially pronounced with TCRm mAbs compared to traditional mAbs since the targets of TCRm mAbs are presented by HLA and are thus regulated by cytokine signaling. The greater than additive effect of these agents in vivo is particularly unexpected given the low epitope density of PRAME and WT1 -derived peptide epitopes and the reduced dose of CV1 that was used. This further corroborates the potency of this drug combination. Although we demonstrate the therapeutic utility of antagonizing CD47 to potentiate the antitumor activity of TCRm mAbs, this approach may be applicable to any antibody that targets a low cell surface density tumor antigen. This strategy could turn poorly efficacious antibodies into powerful antitumor therapeutics and significantly expand the possible cancer antigen targets of monoclonal antibodies.
References
1. Kohrt HE, Tumeh PC, Benson D, Bhardwaj N, Brody J, Formenti S, et al.
Immunodynamics: a cancer immunotherapy trials network review of immune monitoring in immuno-oncology clinical trials. J Immunother Cancer. 2016;4:15.
2. Davila ML, Sadelain M. Biology and clinical application of CAR T cells for B cell malignancies. Int J Hematol. 2016;104(1):6-17.
3. Maury S, Chevret S, Thomas X, Heim D, Leguay T, Huguet F, et al. Rituximab in B- Lineage Adult Acute Lymphoblastic Leukemia. N Engl J Med. 2016;375(ll):1044-53.
4. Ribrag V, Koscielny S, Bosq J, Leguay T, Casasnovas 0, Fornecker LM, et al. Rituximab and dose-dense chemotherapy for adults with Burkitt's lymphoma: a randomised, controlled, open-label, phase 3 trial. Lancet. 2016;387(10036):2402-11.
5. Vedi A, Ziegler DS. Antibody therapy for pediatric leukemia. Front Oncol. 2014;4:82.
6. Blattman JN, Greenberg PD. Cancer immunotherapy: a treatment for the masses. Science. 2004;305(5681):200-5.
7. Safdari Y, Ahmadzadeh V, Farajnia S. CD20-targeting in B-cell malignancies: novel prospects for antibodies and combination therapies. Invest New Drugs. 2016;34(4):497- 512.
8. Robak T, Blonski JZ, Robak P. Antibody therapy alone and in combination with targeted drugs in chronic lymphocytic leukemia. Semin Oncol. 2016;43(2):280-90.
9. Batlevi CL, Matsuki E, Brentjens RJ, Younes A. Novel immunotherapies in lymphoid malignancies. Nat Rev Clin Oncol. 2016;13(l):25-40.
10. Kramer K, Humm JL, Souweidane MM, Zanzonico PB, Dunkel IJ, Gerald WL, et al. Phase I study of targeted radioimmunotherapy for leptomeningeal cancers using intra- Ommaya 131-I-3F8. J Clin Oncol. 2007;25(34):5465-70.
11. Gerber HP, Sapra P, Loganzo F, May C. Combining antibody-drug conjugates and immune-mediated cancer therapy: What to expect? Biochem Pharmacol. 2016;102: 1-6.
12. Sockolosky JT, Dougan M, Ingram JR, Ho CC, Kauke MJ, Almo SC, et al. Durable antitumor responses to CD47 blockade require adaptive immune stimulation. Proc Natl Acad Sci U S A. 2016;113(19):E2646-54.
13. Wang W, Wang EQ, Balthasar JP. Monoclonal antibody pharmacokinetics and pharmacodynamics. Clin Pharmacol Ther. 2008;84(5): 548-58.
14. Mackall CL, Fleisher TA, Brown MR, Andrich MP, Chen CC, Feuerstein IM, et al.
Distinctions between CD8+ and CD4+ T-cell regenerative pathways result in prolonged T- cell subset imbalance after intensive chemotherapy. Blood. 1997;89(10):3700-7.
15. Kotsakis A, Sarra E, Peraki M, Koukourakis M, Apostolaki S, Souglakos J, et al.
Docetaxel-induced lymphopenia in patients with solid tumors: a prospective phenotypic analysis. Cancer. 2000;89(6): 1380-6.
16. Gross S, Walden P. Immunosuppressive mechanisms in human tumors: why we still cannot cure cancer. Immunol Lett. 2008;116(1) :7-14.
17. Curiel TJ. Tregs and rethinking cancer immunotherapy. J Clin Invest.
2007;117(5):1167-74.
18. Shi Y, Fan X, Deng H, Brezski RJ, Rycyzyn M, Jordan RE, et al. Trastuzumab triggers phagocytic killing of high HER2 cancer cells in vitro and in vivo by interaction with Fcgamma receptors on macrophages. J Immunol. 2015;194(9):4379-86. 19. Caron PC, Co MS, Bull MK, Avdalovic NM, Queen C, Scheinberg DA. Biological and immunological features of humanized M195 (anti-CD33) monoclonal antibodies. Cancer Res. 1992;52(24):6761-7.
20. Chang AY, Gejman RS, Brea EJ, Oh CY, Mathias MD, Pankov D, et al. Opportunities and challenges for TCR mimic antibodies in cancer therapy. Expert Opin Biol Ther. 2016:1-9.
21. Chang AY, Dao T, Gejman RS, Jarvis CA, Scott A, Dubrovsky L, et al. A therapeutic T cell receptor mimic antibody to a proteasome-regulated PRAME peptide/HLA-1 complex. J Clinical Investigation, in press.
22. Menssen HD, Schmidt A, Bartelt S, Arjomand A, Thomsen H, Leben R, et al. Analysis of Wilms tumor gene (WT1) expression in acute leukemia patients with special reference to the differential diagnosis between eosinophilic leukemia and idiopathic hypereosinophilic syndromes. Leuk Lymphoma. 2000;36(3-4):285-94.
23. Menssen HD, Bertelmann E, Bartelt S, Schmidt RA, Pecher G, Schramm K, et al. Wilms' tumor gene (WT1) expression in lung cancer, colon cancer and glioblastoma cell lines compared to freshly isolated tumor specimens. J Cancer Res Clin Oncol. 2000;126(4):226-32.
24. Wadelin F, Fulton J, McEwan PA, Spriggs KA, Emsley J, Heery DM. Leucine-rich repeat protein PRAME: expression, potential functions and clinical implications for leukaemia. Mol Cancer. 2010;9:226.
25. Dao T, Yan S, Veomett N, Pankov D, Zhou L, Korontsvit T, et al. Targeting the intracellular WT1 oncogene product with a therapeutic human antibody. Sci Transl Med. 2013;5(176):176ra33.
26. Veomett N, Dao T, Liu H, Xiang J, Pankov D, Dubrovsky L, et al. Therapeutic efficacy of an Fc-enhanced TCR-like antibody to the intracellular WT1 oncoprotein. Clin Cancer Res. 2014;20(15):4036-46.
27. Dubrovsky L, Pankov D, Brea EJ, Dao T, Scott A, Yan S, et al. A TCR-mimic antibody to WT1 bypasses tyrosine kinase inhibitor resistance in human BCR-ABL+ leukemias. Blood. 2014;123(21) :3296-304.
28. Shinkawa T, Nakamura K, Yamane N, Shoji-Hosaka E, Kanda Y, Sakurada M, et al. The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgGl complex-type oligosaccharides shows the critical role of enhancing antibody- dependent cellular cytotoxicity. J Biol Chem. 2003;278(5):3466-73.
29. Brown EJ, Frazier WA. Integrin-associated protein (CD47) and its ligands. Trends Cell Biol. 2001;ll(3):130-5.
30. Babic I, Schallhorn A, Lindberg FP, Jirik FR. SHPS-1 induces aggregation of Ba/F3 pro- B cells via an interaction with CD47. J Immunol. 2000;164(7):3652-8.
31. Oldenborg PA, Zheleznyak A, Fang YF, Lagenaur CF, Gresham HD, Lindberg FP. Role of CD47 as a marker of self on red blood cells. Science. 2000;288(5473):2051-4.
32. Kong F, Gao F, Li H, Liu H, Zhang Y, Zheng R, et al. CD47: a potential immunotherapy target for eliminating cancer cells. Clin Transl Oncol. 2016.
33. Liu X, Pu Y, Cron K, Deng L, Kline J, Frazier WA, et al. CD47 blockade triggers T cell- mediated destruction of immunogenic tumors. Nat Med. 2015;21(10):1209-15.
34. Majeti R, Chao MP, Alizadeh AA, Pang WW, Jaiswal S, Gibbs KD, Jr., et al. CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells. Cell. 2009;138(2):286-99.
35. Willingham SB, Volkmer JP, Gentles AJ, Sahoo D, Dalerba P, Mitra SS, et al. The CD47- signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. Proc Natl Acad Sci U S A. 2012;109(17):6662-7. 36. Theocharides AP, Jin L, Cheng PY, Prasolava TK, Malko AV, Ho JM, et al. Disruption of SIRPalpha signaling in macrophages eliminates human acute myeloid leukemia stem cells in xenografts. J Exp Med. 2012;209(10): 1883-99.
37. Liu J, Wang L, Zhao F, Tseng S, Narayanan C, Shura L, et al. Pre-Clinical Development of a Humanized Anti-CD47 Antibody with Anti-Cancer Therapeutic Potential. PLoS One. 2015;10(9):e0137345.
38. Petrova PS, Viller NN, Wong M, Pang X, Lin GH, Dodge K, et al. TTI-621 (SIRPalphaFc): A CD47-Blocking Innate Immune Checkpoint Inhibitor with Broad Anti-Tumor Activity and Minimal Erythrocyte Binding. Clin Cancer Res. 2016.
39. van den Berg TK, van Bruggen R. Loss of CD47 Makes Dendritic Cells See Red.
Immunity. 2015;43(4):622-4.
40. Weiskopf K, Ring AM, Ho CC, Volkmer JP, Levin AM, Volkmer AK, et al. Engineered SIRPalpha variants as immunotherapeutic adjuvants to anticancer antibodies. Science.
2013;341(6141):88-91.
41. Weiskopf K, Ring AM, Schnorr PJ, Volkmer JP, Volkmer AK, Weissman IL, et al.
Improving macrophage responses to therapeutic antibodies by molecular engineering of SIRPalpha variants. Oncoimmunology. 2013;2(9):e25773.
42. Ho CC, Guo N, Sockolosky JT, Ring AM, Weiskopf K, Ozkan E, et al. "Velcro"
engineering of high affinity CD47 ectodomain as signal regulatory protein alpha (SIRPalpha) antagonists that enhance antibody-dependent cellular phagocytosis. J Biol Chem.
2015;290(20) :12650-63.
43. Parker BS, Rautela J, Hertzog PJ. Antitumour actions of interferons: implications for cancer therapy. Nat Rev Cancer. 2016;16(3):131-44.
44. Zhao XW, van Beek EM, Schornagel K, Van der Maaden H, Van Houdt M, Otten MA, et al. CD47-signal regulatory protein-alpha (SIRPalpha) interactions form a barrier for antibody-mediated tumor cell destruction. Proc Natl Acad Sci U S A. 2011;108(45): 18342-7.
45. Liu Y, Buhring HJ, Zen K, Burst SL, Schnell FJ, Williams IR, et al. Signal regulatory protein (SIRPalpha), a cellular ligand for CD47, regulates neutrophil transmigration. J Biol Chem. 2002;277(12): 10028-36.
46. Dubrovsky L, Dao T, Gejman RS, Brea EJ, Chang AY, Oh CY, et al. T cell receptor mimic antibodies for cancer therapy. Oncoimmunology. 2016;5(l):el049803.
47. Bruhns P. Properties of mouse and human IgG receptors and their contribution to disease models. Blood. 2012;119(24):5640-9.
48. Overdijk MB, Verploegen S, Ortiz Buijsse A, Vink T, Leusen JH, Bleeker WK, et al. Crosstalk between human IgG isotypes and murine effector cells. J Immunol.
2012;189(7):3430-8.
49. Ishikawa F, Yasukawa M, Lyons B, Yoshida S, Miyamoto T, Yoshimoto G, et al.
Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice. Blood. 2005;106(5):1565-73.
50. Shultz LD, Lyons BL, Burzenski LM, Gott B, Chen X, Chaleff S, et al. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol. 2005;174(10):6477-89.
51. Darwich L, Coma G, Pena R, Bellido R, Blanco EJ, Este JA, et al. Secretion of interferon- gamma by human macrophages demonstrated at the single-cell level after costimulation with interleukin (IL)-12 plus IL-18. Immunology. 2009;126(3):386-93.
52. Tanaka S, Saito Y, Kunisawa J, Kurashima Y, Wake T, Suzuki N, et al. Development of mature and functional human myeloid subsets in hematopoietic stem cell-engrafted
NOD/SCID/IL2rgammaKO mice. J Immunol. 2012;188(12):6145-55.

Claims

Claims We claim:
1. A composition comprising a monoclonal T-cell receptor mimic (TCRm) antibody to a low-density cancer antigen and a CD47 antagonist.
2. The composition of claim 1 , wherein the low-density cancer antigen is present on the cell surface at a density of from 10 to 10,000 binding sites per cell.
3. The composition of claim 1 or 2, wherein the low-density cancer antigen is present on the cell surface at a density of from 50 to 5,000 binding sites per cell.
4. The composition of claim 1 , 2 or 3, wherein the low-density cancer antigen is present on the cell surface at a density of from 100 to 2,500 binding sites per cell.
5. The composition of one of claims 1 -4, wherein the low-density cancer antigen is present on the cell surface at a density of from 500 to 1 ,000 binding sites per cell.
6. The composition of one of claims 1 -5, wherein said TCRm antibody comprises an antigen binding region specific for a human leukocyte antigen (HLA) A2 -restricted intracellular protein-derived peptide.
7. The composition of claim 6, wherein said human leukocyte antigen (HLA) A2- restricted intracellular protein-derived peptide has the amino acid sequence of SEQ ID NO: 1.
8. The composition of claim 6, wherein said human leukocyte antigen (HLA) A2- restricted intracellular protein-derived peptide has the amino acid sequence of SEQ ID NO: 2.
9. The composition of one of claims 1 -8, wherein said TCRm antibody is selected from WT 1 TCRm, ESKM antibody or homolog or fragment thereof and PRAME TCRm, Pr20M antibody or homolog or fragment thereof.
10. The composition of one of claims 1 -9, wherein the Fc of the TCRm antibody is afucosylated.
1 1 . The composition of one of claims 1 -10, wherein the CD47 antagonist is a signaling regulatory protein a (SIRPa) or a fragment or variant thereof.
12. The composition of claim 1 1 , wherein the SIRPa variant is consensus variant 1 (CV1 ).
13. The composition of one of claims 1 -1 1 , wherein the CD47 antagonist is an anti- CD47 antibody.
14. A method for killing tumor cells comprising contacting said cells with an effective amount of a T cell receptor mimic (TCRm) antibody and a CD47 antagonist.
15. A method for killing tumor cells comprising administering to a subject in whom the tumor cells reside a therapeutically effective amount of a T cell receptor mimic (TCRm) antibody and a CD47 antagonist.
16. A method of treating leukemia, the method comprising administering to a subject in need of such treatment, either simultaneously or sequentially, an effective amount of a T cell receptor mimic (TCRm) antibody and a CD47 antagonist.
17. The method of one of claims 15 or 16 wherein a T cell receptor mimic (TCRm) antibody and a CD47 antagonist are administered sequentially in either order.
18. The method of one of claims 14 to 17, wherein said TCRm antibody comprises an antigen binding region specific for a human leukocyte antigen (HLA) A2 -restricted intracellular protein-derived peptide.
19. The method of claim 18, wherein said human leukocyte antigen (HLA) A2- restricted intracellular protein-derived peptide has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
20. The method of one of claims 14-19, wherein the T cell receptor mimic (TCRm) antibody and CD47 antagonist are co-administered either as a combination formulation or as individual formulations administered simultaneously and/or sequentially on different schedules.
21 . The method of any one of claims 14 and 15, wherein said tumor cells are leukemia cells.
22. The method of claim 21 , wherein said leukemia cells are acute lymphocytic leukemia (ALL) or acute myeloid leukemia (AML).
23. Use of a composition comprising a monoclonal T-cell receptor mimic (TCRm) antibody to a low-density cancer antigen and a CD47 antagonist for the treatment of ALL or AML.
24. The use of claim 21 , wherein said TCRm antibody comprises an antigen binding region specific for a human leukocyte antigen (HLA) A2-restricted intracellular protein- derived peptide.
25. The use of claim 21 , wherein said TCRm antibody is selected from VVT1 TCRm, ESKM antibody, homolog or fragment thereof and PRAME TCRm, Pr20M antibody, homolog or fragment thereof.
26. The use of claim 24, wherein said human leukocyte antigen (HLA) A2-restricted intracellular protein-derived peptide has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
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WO2020102240A1 (en) * 2018-11-13 2020-05-22 Memorial Sloan Kettering Cancer Center Compositions and methods for adoptive cell therapy for cancer
US11591390B2 (en) 2018-09-27 2023-02-28 Celgene Corporation SIRP-α binding proteins and methods of use thereof
US12084499B2 (en) 2018-09-27 2024-09-10 Celgene Corporation SIRP-α binding proteins and methods of use thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2111869A1 (en) * 2008-04-23 2009-10-28 Stichting Sanquin Bloedvoorziening Compositions and methods to enhance the immune system
US9040669B2 (en) 2011-02-11 2015-05-26 Memorial Sloan Kettering Cancer Center HLA-restricted, peptide-specific antigen binding proteins
US9074000B2 (en) 2011-04-01 2015-07-07 Memorial Sloan Kettering Cancer Center T cell receptor-like antibodies specific for a WT1 peptide presented by HLA-A2
WO2016191246A2 (en) 2015-05-22 2016-12-01 Memorial Sloan-Kettering Cancer Center T cell receptor-like antibodies specific for a prame peptide

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2111869A1 (en) * 2008-04-23 2009-10-28 Stichting Sanquin Bloedvoorziening Compositions and methods to enhance the immune system
US9040669B2 (en) 2011-02-11 2015-05-26 Memorial Sloan Kettering Cancer Center HLA-restricted, peptide-specific antigen binding proteins
US9074000B2 (en) 2011-04-01 2015-07-07 Memorial Sloan Kettering Cancer Center T cell receptor-like antibodies specific for a WT1 peptide presented by HLA-A2
US9540448B2 (en) 2011-04-01 2017-01-10 Memorial Sloan Kettering Cancer Center T cell receptor-like antibodies specific for a WTI peptide presented by HLA-A2
WO2016191246A2 (en) 2015-05-22 2016-12-01 Memorial Sloan-Kettering Cancer Center T cell receptor-like antibodies specific for a prame peptide

Non-Patent Citations (63)

* Cited by examiner, † Cited by third party
Title
"Animal Cell Culture", 1987
"Current Protocols in Molecular Biology", 1987
"Methods in Enzymology", ACADEMIC PRESS, INC
"Molecular Cloning: a Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY PRESS
"Oligonucleotide Synthesis", 1984
"PCR: The Polymerase Chain Reaction", 1994
"Recombinant Antibodies for Immunotherapy", 2009, CAMBRIDGE UNIVERSITY PRESS
BABIC 1; SCHALLHORN A; LINDBERG FP; JIRIK FR.: "SHPS-1 induces aggregation of Ba/F3 pro-B cells via an interaction with CD47.", J IMMUNOL., vol. 164, no. 7, 2000, pages 3652 - 8
BARBAS ET AL.: "Phage Display: A Laboratory Manual", 2001
BATLEVI CL; MATSUKI E; BRENTJENS RJ; YOUNES A.: "Novel immunotherapies in lymphoid malignancies.", NAT REV CLIN ONCOL., vol. 13, no. 1, 2016, pages 25 - 40
BLATTMAN JN; GREENBERG PD.: "Cancer immunotherapy: a treatment for the masses.", SCIENCE, vol. 305, no. 5681, 2004, pages 200 - 5
BROWN EJ; FRAZIER WA.: "Integrin-associated protein (CD47) and its ligands.", TRENDS CELL BIOL, vol. 11, no. 3, 2001, pages 130 - 5
BRUHNS P.: "Properties of mouse and human IgG receptors and their contribution to disease models", BLOOD, vol. 119, no. 24, 2012, pages 5640 - 9
CARON PC; CO MS; BULL MK; AVDALOVIC NM; QUEEN C; SCHEINBERG DA.: "Biological and immunological features of humanized M195 (anti-CD33) monoclonal antibodies.", CANCER RES., vol. 52, no. 24, 1992, pages 6761 - 7
CHANG AY; DAO T; GEJMAN RS; JARVIS CA; SCOTT A; DUBROVSKY L ET AL.: "A therapeutic T cell receptor mimic antibody to a proteasome-regulated PRAME peptide/HLA-1 complex", J CLINICAL INVESTIGATION
CHANG AY; GEJMAN RS; BREA EJ; OH CY; MATHIAS MD; PANKOV D ET AL.: "Opportunities and challenges for TCR mimic antibodies in cancer therapy.", EXPERT OPIN BIOL THER., 2016, pages 1 - 9
CURIEL TJ.: "Tregs and rethinking cancer immunotherapy", J CLIN INVEST., vol. 117, no. 5, 2007, pages 1167 - 74
DAO T; YAN S; VEOMETT N; PANKOV D; ZHOU L; KORONTSVIT T ET AL.: "Targeting the intracellular WT1 oncogene product with a therapeutic human antibody.", SCI TRANSL MED., vol. 5, no. 176, 2013, pages 176ra33
DARWICH L; COMA G; PENA R; BELLIDO R; BLANCO EJ; ESTE JA ET AL.: "Secretion of interferon-gamma by human macrophages demonstrated at the single-cell level after costimulation with interleukin (IL)-12 plus IL-18", IMMUNOLOGY, vol. 126, no. 3, 2009, pages 386 - 93
DAVILA ML; SADELAIN M.: "Biology and clinical application of CAR T cells for B cell malignancies.", INTJ HEMATOL., vol. 104, no. 1, 2016, pages 6 - 17
DUBROVSKY L; DAO T; GEJMAN RS; BREA EJ; CHANG AY; OH CY ET AL.: "T cell receptor mimic antibodies for cancer therapy.", ONCOIMMUNOLOGY, vol. 5, no. l, 2016, pages el049803
DUBROVSKY L; PANKOV D; BREA EJ; DAO T; SCOTT A; YAN S ET AL.: "A TCR-mimic antibody to WT1 bypasses tyrosine kinase inhibitor resistance in human BCR-ABL+ leukemias", BLOOD, vol. 123, no. 21, 2014, pages 3296 - 304
GERBER HP; SAPRA P; LOGANZO F; MAY C.: "Combining antibody-drug conjugates and immune-mediated cancer therapy: What to expect?", BIOCHEM PHARMACOL, vol. 102, 2016, pages 1 - 6
GROSS S; WALDEN P.: "Immunosuppressive mechanisms in human tumors: why we still cannot cure cancer.", IMMUNOL LETT., vol. 116, no. 1, 2008, pages 7 - 14
HO CC; GUO N; SOCKOLOSKY JT; RING AM; WEISKOPF K; OZKAN E ET AL.: "Velcro'' engineering of high affinity CD47 ectodomain as signal regulatory protein alpha (SIRPalpha) antagonists that enhance antibody-dependent cellular phagocytosis", J BIOL CHEM., vol. 290, no. 20, 2015, pages 12650 - 63
ISHIKAWA F; YASUKAWA M; LYONS B; YOSHIDA S; MIYAMOTO T; YOSHIMOTO G ET AL.: "Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice.", BLOOD, vol. 106, no. 5, 2005, pages 1565 - 73
KOHRT HE; TUMEH PC; BENSON D; BHARDWAJ N; BRODY J; FORMENTI S ET AL.: "Immunodynamics: a cancer immunotherapy trials network review of immune monitoring in immuno-oncology clinical trials", J IMMUNOTHER CANCER., vol. 4, 2016, pages 15
KONG F; GAO F; LI H; LIU H; ZHANG Y; ZHENG R ET AL.: "CD47: a potential immunotherapy target for eliminating cancer cells.", CLIN TRANSL ONCOL., 2016
KOTSAKIS A; SARRA E; PERAKI M; KOUKOURAKIS M; APOSTOLAKI S; SOUGLAKOS J ET AL.: "Docetaxel-induced lymphopenia in patients with solid tumors: a prospective phenotypic analysis.", CANCER., vol. 89, no. 6, 2000, pages 1380 - 6
KRAMER K; HUMM JL; SOUWEIDANE MM; ZANZONICO PB; DUNKEL IJ; GERALD WL ET AL.: "Phase I study of targeted radioimmunotherapy for leptomeningeal cancers using intra-Ommaya 131-1-3F8", J CLIN ONCOL, vol. 25, no. 34, 2007, pages 5465 - 70
LIU J; WANG L; ZHAO F; TSENG S; NARAYANAN C; SHURA L ET AL.: "Pre-Clinical Development of a Humanized Anti-CD47 Antibody with Anti-Cancer Therapeutic Potential.", PLOS ONE, vol. 10, no. 9, 2015, pages e0137345
LIU X; PU Y; CRON K; DENG L; KLINE J; FRAZIER WA ET AL.: "CD47 blockade triggers T cell-mediated destruction of immunogenic tumors", NAT MED, vol. 21, no. 10, 2015, pages 1209 - 15
LIU Y; BUHRING HJ; ZEN K; BURST SL; SCHNELL FJ; WILLIAMS 1R ET AL.: "Signal regulatory protein (SIRPalpha), a cellular ligand for CD47, regulates neutrophil transmigration.", J BIOL CHEM., vol. 277, no. 12, 2002, pages 10028 - 36
MACKALL CL; FLEISHER TA; BROWN MR; ANDRICH MP; CHEN CC; FEUERSTEIN 1M ET AL.: "Distinctions between CD8+ and CD4+ T-cell regenerative pathways result in prolonged T-cell subset imbalance after intensive chemotherapy", BLOOD, vol. 89, no. 10, 1997, pages 3700 - 7
MAJETI R; CHAO MP; ALIZADEH AA; PANG WW; JAISWAL S; GIBBS KD, JR. ET AL.: "CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells.", CELL, vol. 138, no. 2, 2009, pages 286 - 99
MATHIAS MELISSA D ET AL: "CD47 Blockade Enhances Therapeutic Activity of TCR Mimic Antibodies to Ultra-Low Density Cancer Epitopes through Cytokine Feed Forward Mechanisms", BLOOD, AMERICAN SOCIETY OF HEMATOLOGY, US, vol. 128, no. 22, 2 December 2016 (2016-12-02), XP009195473, ISSN: 0006-4971 *
MAUIY S; CHEVRET S; THOMAS X; HEIM D; LEGUAY T; HUGUET F ET AL.: "Rituximab in B-Lineage Adult Acute Lymphoblastic Leukemia", N ENGL J MED, vol. 375, no. 11, 2016, pages 1044 - 53
MENSSEN HD; BERTELMANN E; BARTELT S; SCHMIDT RA; PECHER G; SCHRAMM K ET AL.: "Wilms' tumor gene (WT1) expression in lung cancer, colon cancer and glioblastoma cell lines compared to freshly isolated tumor specimens", J CANCER RES CLIN ONCOL., vol. 126, no. 4, 2000, pages 226 - 32
MENSSEN HD; SCHMIDT A; BARTELT S; ARJOMAND A; THOMSEN H; LEBEN R ET AL.: "Analysis of Wilms tumor gene (WT1) expression in acute leukemia patients with special reference to the differential diagnosis between eosinophilic leukemia and idiopathic hypereosinophilic syndromes.", LEUK LYMPHOMA., vol. 36, no. 3-4, 2000, pages 285 - 94
OLDENBORG PA; ZHELEZNYAK A; FANG YF; LAGENAUR CF; GRESHAM HD; LINDBERG FP.: "Role of CD47 as a marker of self on red blood cells", SCIENCE, vol. 288, no. 5473, 2000, pages 2051 - 4
OVERDIJK MB; VERPLOEGEN S; ORTIZ BUIJSSE A; VINK T; LEUSEN JH; BLEEKER WK ET AL.: "Crosstalk between human IgG isotypes and murine effector cells", J IMMUNOL., vol. 189, no. 7, 2012, pages 3430 - 8
PARKER BS; RAUTELA J; HERTZOG PJ.: "Antitumour actions of interferons: implications for cancer therapy.", NAT REV CANCER, vol. 16, no. 3, 2016, pages 131 - 44
PERBAL BERNARD V: "A Practical Guide to Molecular Cloning", 1988
PETROVA PS; VILLER NN; WONG M; PANG X; LIN GH; DODGE K ET AL.: "TTI-621 (SIRPalphaFc): A CD47-Blocking Innate Immune Checkpoint Inhibitor with Broad Anti-Tumor Activity and Minimal Erythrocyte Binding.", CLIN CANCER RES., 2016
RIBRAG V; KOSCIELNY S; BOSQ J; LEGUAY T; CASASNOVAS 0; FORNECKER LM ET AL.: "Rituximab and dose-dense chemotherapy for adults with Burkitt's lymphoma: a randomised, controlled, open-label, phase 3 trial.", LANCET, vol. 387, no. 10036, 2016, pages 2402 - 11
ROBAK T; BLONSKI JZ; ROBAK P.: "Antibody therapy alone and in combination with targeted drugs in chronic lymphocytic leukemia", SEMIN ONCOL, vol. 43, no. 2, 2016, pages 280 - 90
SAFDARI Y; AHMADZADEH V; FARAJNIA S.: "CD20-targeting in B-cell malignancies: novel prospects for antibodies and combination therapies", INVEST NEW DRUGS, vol. 34, no. 4, 2016, pages 497 - 512
SHI Y; FAN X; DENG H; BREZSKI RJ; RYCYZYN M; JORDAN RE ET AL.: "Trastuzumab triggers phagocytic killing of high HER2 cancer cells in vitro and in vivo by interaction with Fcgamma receptors on macrophages", J IMMUNOL., vol. 194, no. 9, 2015, pages 4379 - 86
SHINKAWA T; NAKAMURA K; YAMANE N; SHOJI-HOSAKA E; KANDA Y; SAKURADA M ET AL.: "The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgGl complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity.", J BIOL CHEM., vol. 278, no. 5, 2003, pages 3466 - 73
SHULTZ LD; LYONS BL; BURZENSKI LM; GOTT B; CHEN X; CHALEFF S ET AL.: "Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells", J IMMUNOL., vol. 174, no. 10, 2005, pages 6477 - 89
SOCKOLOSKY JT; DOUGAN M; INGRAM JR; HO CC; KAUKE MJ; ALMO SC ET AL.: "Durable antitumor responses to CD47 blockade require adaptive immune stimulation", PROC NATL ACAD SCI USA., vol. 113, no. 19, 2016, pages E2646 - 54
TANAKA S; SAITO Y; KUNISAWA J; KURASHIMA Y; WAKE T; SUZUKI N ET AL.: "Development of mature and functional human myeloid subsets in hematopoietic stem cell-engrafted NOD/SCID/IL2rgammaKO mice", J IMMUNOL., vol. 188, no. 12, 2012, pages 6145 - 55
THEOCHARIDES AP; JIN L; CHENG PY; PRASOLAVA TK; MALKO AV; HO JM ET AL.: "Disruption of SIRPalpha signaling in macrophages eliminates human acute myeloid leukemia stem cells in xenografts", J EXP MED., vol. 209, no. 10, 2012, pages 1883 - 99
VAN DEN BERG TK; VAN BRUGGEN R.: "Loss of CD47 Makes Dendritic Cells See Red", IMMUNITY, vol. 43, no. 4, 2015, pages 622 - 4
VEDI A; ZIEGLER DS.: "Antibody therapy for pediatric leukemia.", FRONT ONCOL., vol. 4, 2014, pages 82
VEOMETT N; DAO T; LIU H; XIANG J; PANKOV D; DUBROVSKY L ET AL.: "Therapeutic efficacy of an Fc-enhanced TCR-like antibody to the intracellular WT1 oncoprotein.", CLIN CANCER RES., vol. 20, no. 15, 2014, pages 4036 - 46
WADELIN F; FULTON J; MCEWAN PA; SPRIGGS KA; EMSLEY J; HEEIY DM.: "Leucine-rich repeat protein PRAME: expression, potential functions and clinical implications for leukaemia.", MOL CANCER., vol. 9, 2010, pages 226
WANG W; WANG EQ; BALTHASAR JP.: "Monoclonal antibody pharmacokinetics and pharmacodynamics.", CLIN PHARMACOL THER., vol. 84, no. 5, 2008, pages 548 - 58
WEISKOPF K; RING AM; HO CC; VOLKMER JP; LEVIN AM; VOLKMER AK ET AL.: "Engineered SIRPalpha variants as immunotherapeutic adjuvants to anticancer antibodies", SCIENCE, vol. 341, no. 6141, 2013, pages 88 - 91
WEISKOPF K; RING AM; SCHNORR PJ; VOLKMER JP; VOLKMER AK; WEISSMAN 1L ET AL.: "Improving macrophage responses to therapeutic antibodies by molecular engineering of SIRPalpha variants.", ONCOIMMUNOLOGY., vol. 2, no. 9, 2013, pages e25773
WEISKOPF KIPP ET AL: "Engineered SIRP alpha Variants as Immunotherapeutic Adjuvants to Anticancer Antibodies", SCIENCE (WASHINGTON D C), vol. 341, no. 6141, July 2013 (2013-07-01), pages 88 - 91, XP002774274 *
WILLINGHAM SB; VOLKMER JP; GENTLES AJ; SAHOO D; DALERBA P; MITRA SS ET AL.: "The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors.", PROC NATL ACAD SCI USA., vol. 109, no. 17, 2012, pages 6662 - 7
ZHAO XW; VAN BEEK EM; SCHORNAGEL K; VAN DER MAADEN H; VAN HOUDT M; OTTEN MA ET AL.: "CD47-signal regulatory protein-alpha (SIRPalpha) interactions form a barrier for antibody-mediated tumor cell destruction", PROC NATL ACAD SCI USA., vol. 108, no. 45, 2011, pages 18342 - 7

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11591390B2 (en) 2018-09-27 2023-02-28 Celgene Corporation SIRP-α binding proteins and methods of use thereof
US12084499B2 (en) 2018-09-27 2024-09-10 Celgene Corporation SIRP-α binding proteins and methods of use thereof
WO2020102240A1 (en) * 2018-11-13 2020-05-22 Memorial Sloan Kettering Cancer Center Compositions and methods for adoptive cell therapy for cancer
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