Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
  • A61K2039/507—Comprising 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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