WO2022099006A1 - Évaluation de cancers pour le traitement avec un anticorps bite cd33/cd3 - Google Patents

Évaluation de cancers pour le traitement avec un anticorps bite cd33/cd3 Download PDF

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WO2022099006A1
WO2022099006A1 PCT/US2021/058254 US2021058254W WO2022099006A1 WO 2022099006 A1 WO2022099006 A1 WO 2022099006A1 US 2021058254 W US2021058254 W US 2021058254W WO 2022099006 A1 WO2022099006 A1 WO 2022099006A1
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amv564
administered
cell
cells
binding
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Victoria Smith
Aurelien SARDE
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Amphivena Therapeutics Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • T cell engagers a particular class of bispecific antibody, mediate binding between a target cell and a T cell resulting in T cell directed lysis and T cell activation, differentiation and proliferation. While T cell engagers have demonstrated impressive potency and anti-tumor activity in some settings, a barrier to broader therapeutic success in many cases is undesirable activity against normal cells expressing the target of interest. This "on target, off tumor" toxicity can be significant, and has been reported widely for engineered T cell approaches.
  • MDSC Myeloid derived suppressor cells
  • MDSC elicit a range of suppressive functions that inhibit normal T cell responses as well as causing unresponsiveness to immune checkpoint blockade.
  • a dominant function of MDSC is the suppression of T cell activity in a variety of manners that are pathology and context dependent.
  • the presence of MDSC is thought to be associated with poor outcomes and lack of response to certain therapies, e.g., therapies that activate T cells and therapies involving the use of a checkpoint inhibitor.
  • compensatory myelopoiesis in response to T cell activation can result in further generation of MDSC and recruitment to tumors.
  • Described herein is a method for treating a patient with a bispecific T cell engager having two CD33 binding domains and two CD3 binding domains, the method comprising: (a) providing a patient sample comprising CD33 expressing tumor cells; (b) measuring the binding of a CD33 antibody to the CD33 expressing cells; (c) measuring the binding of the bispecific T cell engager to the CD33 expressing cells; (d) selecting the patient for treatment with the bispecific T cell engager if the binding of the T cell engager to the CD33 expressing cells is equivalent to the binding of the CD33 antibody to the CD33 expressing cells; and (e) treating the CD33 expressing cells with the bispecific T cell engager.
  • AML acute myeloid leukemia
  • Also described is a method for stratifying patients for entry into a clinical trial for assessment of bispecific T cell engager having two CD33 binding domains and two CD3 binding domains for treatment of AML comprising: (a) providing a patient sample comprising CD33 expressing tumor cells; (b) measuring the binding of a CD33 antibody to the CD33 expressing cells; (c) measuring the binding of the bispecific T cell engager to the CD33 expressing cells; (d) selecting the patient for inclusion in the clinical trial if the binding of the T cell engager to the CD33 expressing cells is equivalent to the binding of the CD33 antibody to the CD33 expressing cells otherwise excluding the patient from the clinical trial.
  • the bispecific T cell engager having two CD33 binding domains and two CD3 binding domains is AMV564; the bispecific T cell engager having two CD33 binding domains and two CD3 binding domains is AMV564 and AMV564 is administered by subcutaneous injection; the dose of AMV564 injected is 5 - 150 mcg; AMV564 is administered on a least 7 days over a 14 day period; the AMV6564 is administered on 10 days over a 14 day period; AMV6564 is administered on 5 consecutive days on two occasions over a 14 day period; AMV6564 is administered on 5 consecutive days, is not administered on the following two days and is administered on the following 5 consecutive days; the AMV564 is administered over a 21 day cycle in which AMV564 is administered on at least 7 days over a 14 day period and is not administered over the subsequent 7 day period; the 21 day cycle is repeated at least two times; AMV564 is administered on at least 10 days over a 14 day period with administration on 5 consecutive days followed by 2 days
  • AMV564 a bispecific, bivalent molecule which binds to CD3 and CD33.
  • AMV564 is homodimeric protein (i.e., a homodimer of a polypeptide having the amino acid sequence of SEQ. ID NO: 1) having four single-chain variable fragment (scFv) binding sites, two that bind CD33 and two that bind CD3.
  • scFv single-chain variable fragment
  • a bivalent design can, in theory, restore selectivity to a T cell engager, directing preferential binding to regions of high local target density, such as found at sites of active signaling or associated with high receptor density or expression.
  • AMV564 binds CD33, which is broadly expressed across the myeloid lineage, it can be dosed in a manner that that provides a desirable therapeutic index, with selective binding of MDSC.
  • AMV564 is very selective over a wide dose range, without being bound by any particular theory, this may be due to some combination of: bivalency, the affinity of the scFv and the geometry of the homodimer.
  • the structure of AMV564 may allow it to bind clusters of dimerized CD33.
  • AMV564 has dual activity: it induces T cell mediated killing of MDSC and drives T cell activation, promoting favorable polarization (e.g., Thl CD4 T cells and effector CD8 T cells).
  • AMV564 has an EC50 for MDSC that is less than about 3 pM. Dosed appropriately, AMV564 largely spares neutrophils, monocytes and many differentiated myeloid cells while directing killing of MDSC, thereby inhibiting MDSC-suppressive pathways.
  • Peripheral MDSC may play important roles in T cell suppression and T cell trafficking to tumor sites, a potentially rate-limiting factor for T cell activation-based therapies.
  • a 15 mcg dose of AMV564 can, in some circumstance, achieve depletion of MDSC populations.
  • peripheral depletion may enable sufficient control to benefit anti-tumor immunity over a dosing timeframe that exceeds the limited longevity of tissue-resident MDSC.
  • AMV564 in the tumor microenvironment could target MDSC at tumor sites while promoting expansion of local T cells.
  • AMV564 delivery or entry into draining lymph nodes in addition to the periphery could achieve de-repression of anti-tumor T cells and restore antigen presentation and immune homeostasis.
  • Subcutaneous delivery of AMV564, for example at doses of 5, 15, or 50 mcg provides a direct mechanism of initial distribution in the lymphatic system, including tumor draining lymph nodes. With subcutaneous administration AMV564 is effective at lower doses, likely due to the access to the lymph system.
  • AMV564 can both relieve immune suppression and activate T cell effector function in cancer patients. AMV564 can do so by relieving immunosuppression via targeted depletion of myeloid derived suppressor cells (MDSC) and by directly activating/repolarizing T cells and improved T effector function.
  • MDSC myeloid derived suppressor cells
  • subcutaneous administration of AMV564 facilitates immune activation by targeting the lymphatic system.
  • the target cell is a leukemic blast
  • a dosing regimen that establishes a steady-state exposure of 30 - 70 pM AMV564 is used to reduce leukemic blasts in AML, Myelodysplastic Syndrome (MDS) and other cancers with high CD33 expression.
  • MDS Myelodysplastic Syndrome
  • Such a regimen could be achieved by dosing at 50 - 125 mcg ("micrograms”) by continuous intravenous infusion (CIV), for example, 50 mcg, 75 mcg, 100 mcg.
  • Also described herein is a method of for controlling both MDSC by administering 5 - 125 mcg (for example, 50 mcg, 75 mcg, 100 mcg) of AMV564 by intravenous infusion.
  • 5 - 125 mcg for example, 50 mcg, 75 mcg, 100 mcg
  • a 5, 15, 30, 50 mcg AMV564 can be administered, e.g., by intravenous infusion.
  • AMV564 a polypeptide having the amino acid sequence of SEQ. ID NO: 1
  • AMV564 is administered by subcutaneous injection; the dose of AMV564 injected is 5 - 150 mcg (micrograms); the AMV564 is administered on a least 7 days (8, 9, 10, 11, 12, 13 or 14 days) over a 14 day period; the AMV564 is administered daily (e.g., at 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 mcg/day) subcutaneously; the AMV6564 is administered on 10 days over a 14 day period; the AMV6564 is administered on 5 consecutive days on two occasions over a 14 day period; the AMV564 (a polypeptide having the amino acid sequence of SEQ. ID NO: 1) to the patient.
  • AMV564 is administered by subcutaneous injection; the dose of
  • AMV564 is administered by subcutaneous injection; the dose of AMV564 injected is 5 - 150 mcg (micrograms); the AMV564 is administered on a least 7 days (8, 9, 10, 11, 12, 13 or 14 days) over a 14 day period; the AMV564 is administered daily (e.g., at 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 mcg/day) subcutaneously; the AMV6564 is administered on 10 days over a 14 day period; the AMV6564 is administered on 5 consecutive days on two occasions over a 14 day period; the AMV6564 is administered on 5 consecutive days, is
  • CD33 also known as Siglec-3, is transmembrane protein is expressed on cells of myeloid lineage.
  • CD33 has long been regarded as an appealing target for acute myeloid leukemia (AML), due to both high prevalence and high expression on leukemic blasts.
  • AML acute myeloid leukemia
  • the function of CD33 is not well understood, but activation of CD33 signaling on early lineage myeloid cells, such as immune- suppressive myeloid-derived suppressor cells (MDSC), has been shown to result in expansion of MDSC and production of suppressive cytokines and factors.
  • MDSC immune- suppressive myeloid-derived suppressor cells
  • CD33 expression is used as one component of cell surface marker sets to identify these immune-suppressive monocytic and granulocytic cells (for example, by use of flow cytometry for cellular immune phenotyping). It is unclear what role, if any, that CD33 plays on more differentiated myeloid lineage cells such as mature monocytes, neutrophils, macrophages and dendritic cells. Indeed, knockout of CD33 in human cells using CRISPR technology has demonstrated that CD33 is not required for lineage differentiation, but it nonetheless continues to be expressed at varying levels on most of these cells, rendering it as a challenging target for a non-selective T cell engager, with respect to both safety and efficacy.
  • T cell engagers function by acting as a bridge between an antigen on a target cell and different antigen on a T cell, forming a ternary complex between the drug and two different cell types, thereby mimicking the formation of a natural T cell-target cell synapse that is generated in the course of an adaptive immune response. It is thought that between 10 - 100 molecules of drug must be appropriately bound in order to activate T cell killing, and T cells must be available also. This active state thus brings additional considerations for receptor occupancy and dosing beyond a standard model of, for example, inhibiting a ligand or receptor via biologic drug binding, where dosing strategies aim for maximum target coverage until unacceptable toxicity is reached.
  • T cell engagers that target a broadly expressed antigen, such as CD33.
  • a broadly expressed antigen such as CD33.
  • this cellular population can be very large (e.g. there are up to 100 billion neutrophils generated daily in a human body) and efficacy could thus be compromised due to insufficiently suitable drug distribution and an inadequate supply of T cells to accomplish killing.
  • the targeting of other populations of cells such as leukemic blasts or the even rarer immune-suppressive MDSC could thus be hampered by a broad distribution of drug across CD33-positive normal myeloid cells and the need for sufficient associated T cells to achieve killing. For these reasons, determine the appropriate dose, dose schedule and routed of administration for a T cell engager is unusually challenging, far more so than for monovalent, monospecific agents. Binding properties of AMV564 and dose selection
  • AMV564 The bivalent design of AMV564 is reflected in its physical properties.
  • AMV564 is very potent and demonstrates cellular killing at low receptor occupancy, with elimination of CD33 target cells demonstrated ex vivo or in vitro with picomolar or sub-picomolar EC50 values.
  • AMV564 is a potent agonist, which can elicit biological activity at low receptor occupancy or low target binding levels. Binding studies using flow cytometry show that there is no binding of neutrophils, polymorphonuclear (PMN) leukocytes or monocytes at 1 or 10 pM AMV564 as compared to MDSC and leukemic blast cell line KG1, which are both potently bound at 1 and 10 pM (FIGS 1F-1G). At concentrations of 1 and 10 pM, AMV564 is thus highly selective for MDSC as compared to other abundant CD33 expressing cells The
  • an optimal therapeutic window for both safety and anti-tumor (leukemic blasts) and/or antisuppressor (MDSC) cell activity might be obtained when engagement of target cells by AMV564 is sufficient for activity, but binding of other myeloid cells is minimal.
  • AMV564 anti-tumor (leukemic blasts) and/or antisuppressor (MDSC) cell activity
  • MDSC critical cellular effectors of the suppressive tumor microenvironment
  • the critical cellular effectors of the suppressive tumor microenvironment are MDSC, which are associated with immune dysfunction, repression of anti-tumor immunity and poor response to immunotherapy.
  • MDSC suppress T cell and NK cell responses via a variety of cytokines, active species and pathways.
  • they repress effective antigen presentation by dendritic cells in tumor draining lymph nodes.
  • AMV564 depletes MDSC in the periphery and bone marrow of AML patients at low doses.
  • the rapid decline observed at low, lead-in doses of AMV546 indicates potent binding and depletion of both monocytic and granulocytic MDSC populations.
  • MDSC are rare in the periphery of a healthy adult and become significantly elevated in cancer patients. However, they are nonetheless relatively rare cells as compared to the mature myeloid lineage in general, and the efficacy of their depletion and control could be reduced at higher doses when overall receptor occupancy is unfavorable for selectivity for a bivalent T cell engager such as AMV564. Depletion of MDSC in addition to leukemic blasts is beneficial in AML with respect to maintaining T cell activation and proliferation, and may promote more durable benefits such as sustained responses and restoration or induction of T cell memory.
  • Doses of 50 - 125 mcg of AMV564 by CIV route could maintain control of both MDSC and leukemic blasts. This dosing regimen could also be effective for MDS. In MDS patients with a lower blast burden, where MDSC likely play a more critical role in disease progression, an even lower dose may be effective (e.g. 5, 15, 30, 50 mcg by CIV route).
  • AMV564 In solid tumor patients, administration of AMV564 at doses of 5-50 mcg, generating approximate steady state exposures ranging from 0.1 - 5 pM, can be effective in depleting MDSC and promoting a favorable CD4 and CD8 T cell activation profile and cytokine milieu to promote restoration of antitumor immunity. Higher doses of 50-75 mcg or 75 - 150 mcg would also yield exposures that remain within the selective range for MDSC depletion.
  • Circulating MDSC are a pharmacodynamic biomarker of AMV564 and T cell responses.
  • MDSC are known to be induced by T cell activation, they are induced as a consequence of the T cell activation stimulated by AMV564.
  • the MDSC reflect engagement of AMV564 with target cells (MDSC) and depletion of such cells, and in relationship to dose for a bivalent, bispecific T cell engager such as AMV564, they reflect effective dosing within the optimal therapeutic index of the drug, to enable effective depletion of these comparatively rare cells as compared to the rest of the CD33 positive myeloid lineage.
  • AMV564 depletes MDSC
  • treatment with AMV564 for example, under the dosing regimens described herein, can be useful for depleting MDSC in a variety of contexts.
  • AMV564 can be used to deplete MDSC in patients being treated with a therapy that activates T cells or involves administration of activated T cells.
  • CRS Cytokine Release Syndrome
  • CRS While not fully understood, appears to be related to T cell activation and subsequent activation of macrophages and other myeloid cells to generate and secrete IL-6, IL-1B and other cytokines.
  • CRS is commonly associated with T cell engaging therapies such as T cell engaging bispecific antibodies and CAR-T therapy.
  • CRS is most apparent at initiation of dosing.
  • administration of AMV564 by subcutaneous route in solid tumor patients at doses, for example, of 15 - 50 mcg results in robust T cell activation as assessed by various metrics including up to 10 - 40X increases from baseline in detectable peripheral Interferon gamma ( I FNy ) in the first cycle of dosing.
  • IL-6 is comparatively modest (the two cytokines are around 1:1 or favor higher I FNy) (see FIGS 7A-7E) and IL-1B is not detected at significant levels.
  • This favorable profile is consistent with the absence of CRS observed in patients in this clinical study.
  • This favorable profile of demonstrably strong T cell activation with lack of CRS could reflect a combination of features including the depletion of MDSC (which can produce inflammatory cytokines), bivalent T cell engagement by AMV564 (which may more closely resemble a more native T cell receptor engagement) and the lymphatic delivery and distribution kinetics associated with subcutaneous injection of AMV564.
  • AMV564 has a favorable therapeutic index that is amenable to chronic dosing, these properties should also assist in the mitigation of CRS after lead in dosing to target dose is completed.
  • Combination strategies include but are not limited to, in solid tumors, checkpoint blockade (PD-1 or PDL-1 blocking agents), T cell activators and expanders such as cytokines IL-2, IL- 10 and IL-15, dual targeting agents such as those targeting checkpoint (e.g. PD-1 or PDL-1) and immune repression (e.g. TGF ), CAR-T therapies, NK activating therapies, or standard of care chemotherapy.
  • therapies could also be used in combination, along with other established agents in AML such as hypomethylating agents (e.g. azacytidine, decitabine), differentiation agents (e.g. targeting I DH 1/2), targeted agents (e.g. against FLT3), agents targeting anti-apoptotic proteins such as BCL2 (e.g. venetoclax), BCL-XL, or MCL1, or lenalidomide.
  • hypomethylating agents e.g. azacytidine, decitabine
  • differentiation agents e.g. targeting I DH 1/2
  • targeted agents e.g. against FLT3
  • agents targeting anti-apoptotic proteins such as BCL2 (e.g. venetoclax), BCL-XL, or MCL1, or lenalidomide.
  • BCL2 e.g. venetoclax
  • BCL-XL e.g. venetoclax
  • MCL1 lenalidomide
  • AMV564 can be used alone or in combination to treat melanoma (e.g., patients with unresectable or metastatic melanoma, melanoma with involvement of lymph node(s) following complete resection); non-Small Cell Lung Cancer (NSCLC) (e.g., metastatic non-squamous NSCLC, III NSCLC, metastatic NSCLC expressing PD-L1); head and Neck Squamous Cell Cancer (HNSCC); Classical Hodgkin Lymphoma (cHL); Primary Mediastinal Large B-Cell Lymphoma (PMBCL); urothelial Carcinoma (e.g., locally advanced or metastatic urothelial carcinoma expressing PD-L1); Microsatellite Instability-High Cancer (e.g., unresectable or metastatic, microsatellite instability-high (MSI-H) or mismatch repair deficient; solid tumors that have progressed following prior treatment; Gastric Cancer (e
  • FIGS 1A-1G present data showing that AMV264 depletes MDSC and activates T cells ex vivo and that AMD564 binds MSC and KG-1 cells at 1 and 10 pm, but does not bind monocytes at these concentrations.
  • FIGS 2A-2F present data showing that that AMV264 depletes MDSC and activates T cells in patients.
  • the lighter bar indicates the days of the lead in dose and the darker bar indicates the days of the target dose.
  • FIGS 3A-3C present data showing the impact of AMV264 on peripheral blood MDSC, bone marrow MDSC, peripheral blood T cells.
  • the lighter bar indicates the days of the lead in dose and the darker bar indicates the days of the target dose.
  • FIGS 4A-4D present data showing that AMV564 is a selective an potent conditional agonist.
  • the single open circles and triangles show the result of a CD3/CD28 stimulation in the absence of AMV564.
  • FIGS 5A-5E present data showing the MDSC control is associated with Treg control in solid tumor patients.
  • the filled squared are G-MDSC and the filled circles are M-MDSC.
  • the bars of the axis indicate AMV564 dosing days.
  • FIGS 6A-6G present data showing that CD8:Treg ratio improves on AMV564 therapy in solid tumor patients
  • the dotted line indicates the baseline ratio; bars along the x-axis indicate dosing days and the broad bar indicates the ratio for healthy controls.
  • FIGS 7A-7E present data showing that AMV564 promotes favorable CD4 and CD8 T cell polarization in an ovarian cancer patient.
  • the dotted line indicates the baseline ratio; bars along the x-axis indicate dosing days and the broad bar indicates the ratio for healthy controls.
  • FIGS 8A-8F show I FNy Cycle 1, 1 FNy Cycle 2, IL-6 Cycle 1 and IL-6 Cycle 2 levels in six solid tumor patients treated with AMV564.
  • FIGS 9A-9D show the results of M-MDSC and G-MDSC measurements in four solid tumor patients treated with AMV564 in combination pembrolizumab.
  • the filled squared are G-MDSC and the filled circles are M-MDSC.
  • the bars of the axis indicate AMV564 dosing days.
  • FIGS 10A-10D show the impact of AMV564 in combination pembrolizumab on T-Bet and granzyme B positive CD8 cells and CD8/Treg ratio in two solid tumor patients.
  • the dotted line indicates the baseline ratio; bars along the x-axis indicate AMV564 dosing days and the broad bar indicates the ratio for healthy controls.
  • FIGS 11A-11B show the impact of AMV564 in combination pembrolizumab treat on CD8 cell proliferation and activation in two solid tumor patients.
  • FIGS 12A-12C show differences in the binding of AMV564 and a CD33 antibody to KG-1 and KG-la cells.
  • AMV564 is a homodimer of SEQ ID NO: 1.
  • AMV564 is described in US 9212225 (Diabody 16; SEQ ID NO: 1.
  • a pharmaceutical composition of AMV564 comprises a polypeptide having the amino acid sequence of
  • SEQ ID NO:1 SEQ ID NO:1 and a pharmaceutically acceptable carrier or excipient.
  • DQMTQSPSS LSASVGDRVT ITCRSSTGAV TTSNYANWVQ QKPGKAPKAL IGGTNKRAPG VPSRFSGSLI GDKATLTI SS LQPEDFATYY CALWYSNLWV FGQGTKVEIK GGSGGSQVQL VQSGAEVKKP GASVKVSCKA SGYTFTSYDI NWVRQAPGQG LEWMGWMNPN SGNTGFAQKF QGRVTMTRDT STSTVYMELS SLRSEDTAVY YCARDRANTD YSLGMDVWGQ GTLVTVSSGG SGQSVLTQPP SASGTPGQRV TI SCSGSRSN IGSNTVNWYQ QLPGTAPKLL IYGNNQRPSG VPDRFSGSKS GTSASLAI SG LQSEDEADYY CATWDDSLIG WVFGGGTKLT VLGGSGGSEV QLVESGGGLV QPGGSLRLSC AASGFTFSTY AMNWVR
  • Example 1 AMV564 Depletes MDSC and Activates T Cells ex vivo
  • FIG 1A sows that treatment of PBMC with CD33 ligand S100A9 results in expansion of MDSC and increase in CD33 expression.
  • FIGS 1B-1E it can be seen that exposure to AMV564: negates reactive oxygen species (ROS) produced in response to S100A9 stimulation of PBMC to expand MDSC (FIG IB), causes selective depletion of MDSC (FIG 1C), and increases in CD8 T cell (FIG ID) and CD4 T cell (FIG IE) numbers and activation state (as assessed by I FNy positive fraction).
  • ROS reactive oxygen species
  • PBMC peripheral blood mononuclear cells
  • AMV564 binds to MDSC (and leukemic blast line KG1) at 1 and 10 pM. However, at these concentrations, there is essentially no evidence of binding to monocytes, neutrophils and polymorphonuclear leukocytes (PMN). These concentrations are within the range of exposure observed for dosing of AMV564 by subcutaneous route at doses of 5 - 15 - 50 mcg (about 0.1 - 5 pM). As shown in FIG 1G.
  • Example 2 AMV564 Depletes MDSC and Activates T Cells in Patients
  • FIGS 2A-2F show that AMV564 treatment leads to depletion of peripheral blood MDSC and bone marrow MDSC.
  • FIGS 2A-2F show that AMV564 treatment resulted in depletion of AML blasts with no decrease in neutrophils.
  • rapid depletion of both monocytic and granulocytic MDSCs is apparent with little or no impact on circulating neutrophil or monocytes populations.
  • Evidence of early T cell activation is apparent with rapid re-distribution/margination of T cells (this apparent transient lymphopenia is a consequence of T cell activation and migration to lymph nodes and tissues).
  • FIGS. 2A-2F the period of lead-in AMV564 dosing (15 mcg/kg; 3 days) is indicated by the lighter colored bar and the target AMV564 dosing (100 mcg/kg) is indicated by the darker colored bar).
  • FIGS. 2A and 2B it can be seen the peripheral blood MDSC are depleted.
  • FIG 2C it can be seen the bone marrow MDSC are depleted.
  • FIGS 2D-2E respectively, show the impact of AMV564 treatment on peripheral blood T cells, peripheral blood neutrophils, and peripheral blood blasts.
  • Example 3 AMV564 Depletes MDSC in solid tumor patients
  • FIGS 3A-C show an initial increase in peripheral blood MDSC in response to T cell activation in lead in dosing (Days 1-3 in some patients).
  • FIG 3C shows the rapid redistribution/margination of peripheral blood T cells, consistent with T cell activation, as T cells transmigrate to lymph nodes and tissues.
  • peripheral MDSC are controlled. Bone marrow MDSC are also substantially decreased when assessed at day 15 relative to baseline. However, both bone marrow and peripheral blood MDSC can rebound once AMV564 treatment is stopped.
  • Example 4 AMV564 is a Selective and Potent Conditional Agonist
  • FIGS 4A-4D AMV564 induces potent dose-dependent cell death of KG1 (FIG 4A), at a maximum level similar to a CD3-CD28 stimulation.
  • FIG 4B This is accompanied by an increase in daughter cells, reflective of T cell proliferation (FIG 4B) at levels equivalent to or exceeding the CD3-CD28 reference stimulation.
  • FIG 4C there is no evidence of AMV564 promoting significant cell death for autologous monocytes or neutrophils (FIG 4C), and similarly, no evidence of any induction of T cell proliferation (FIG 4D) with these cell populations, unlike general T cell stimulation using CD3-CD28.
  • cytokine profile consistent with activation of T cells including CD4 Thl helper cells, antigen presenting cells, and improved T cell trafficking to tissues such as tumor tissues (increase in I FNy, IL-15, IL-18, soluble granzyme B and CXCL10). While strong T cell activation was observed, there were no episodes of cytokine release syndrome.
  • Example 6 MDSC Control is Associated with Treg Control in Solid Tumor Patients
  • FIGS. 5A-5D show that M-MDSC and G-MDSC are controlled in patients with solid tumors (FIG 5A: ovarian - 15 mcg AMV564; FIG 5B: cutaneous 50 mcg AMV564; FIG 5C: small bowel- 15 mcg AMV564; FIG 5D: gastroesohageal junction - 15 mcg AMV564) that were treated with AMV564 (once a day by subcutaneous injection on days 1-5 and days 8-12 of a 21-day cycle).
  • FIGS 6A-6G show that there an increase in the CD8/Treg ratio (for most subjects) while solid tumor patients (FIG 6A: Small Bowel - Stable Disease; FIG 6B: Ovarian - Complete Response; FIG 6C: GE Junction - Progressive Disease; FIG 6D: Endometrial - Stable Disease; FIG 6E: Colorectal - Progressive Disease; FIG 6F: Cutaneous - Stable Disease; FIG 6G: Appendiceal- Stable Disease) were on AMV564 therapy (once a day by subcutaneous injection on days 1-5 and days 8-12 of a 21-day cycle (FIGS 6A- 6C 15 mcg; FIG 6D-6G 50 mcg). The dotted line indicates the baseline ratio; bars along the x-axis indicate dosing days and the broad bar indicates the ratio for healthy controls whose peripheral blood samples were processed in the same flow based assay.
  • Example 8 AMV564 Promotes Favorable CD4 and CD8 T Cell Polarization in an Ovarian Cancer Patient
  • This patient exhibited an ongoing increase in CD8/Treg, increase in % CD8, maintained or increased effector CD8 (TBX21 and/or granzyme B positive) and memory CD8 cells, a dynamic increases in TBX21-positive CD4 T helper cells and dynamic modulation of PD1 positive CD8 fraction but without substantial overall increase as shown in FIGS 7A-7E.
  • Example 9 Patients Treated with AMV564 Exhibit Signs of T Cell Activation without CRS
  • FIGS 8A-8F (Patients 1, 2, 3, 11, 9 and 14, respectively) show the results of I FNy and IL-6 measurements in six solid tumor patients treated with AMV564 (once a day by subcutaneous injection on days 1-5 and days 8-12 of a 21-day cycle). As can be seen there is clear evidence of systemic I FNy production without excessive IL-6 production (ratio of I FNy : IL-6 was about 1:1 or better for most patients).
  • FIGS 9A-9D show the results of M-MDSC and G-MDSC measurements in four solid tumor patients treated with AMV564 once a day by subcutaneous injection on days 1-5 and days 8-12 of a 21-day cycle (5 mcg/day (FIG 9A and FIG 9B) or 15 mcg/day (FIG 9C and FIG 9D)) in combination pembrolizumab administered intravenously at 200 mg every 3 week (Q3W).
  • the AMV564 administration days are indicated by a bar along the x-axis and the pembrolizumab treatment days are indicated with an asterisk. As can be seen, very good MDSC control was observed.
  • FIGS. 10A-10D shows data from two patients (FIG 10A and FIG 10C: Patient 15; FIG 10B and FIG 10D: Patient 16) treated with AMV 564 (15 mcg once a day by subcutaneous injection on days 1-5 and days 8-12 of a 21-day cycle in combination pembrolizumab (administered intravenously at 200 mg Q3W).
  • AMV 564 15 mcg once a day by subcutaneous injection on days 1-5 and days 8-12 of a 21-day cycle in combination pembrolizumab (administered intravenously at 200 mg Q3W).
  • This data shows evidence of a substantial increase in CD8 effector cell fraction in cycles 1 - 2 and a substantial increase in CD8/Treg ratio.
  • the data also show expansion of T-Bet and granzyme B positive CD8 cells.
  • FIGS 11A-11B shows data from two patients (FIG 11A: Patient 15; FIG 11B: Patient 16) treated with AMV 564 (15 mcg once a day by subcutaneous injection on days 1-5 and days 8-12 of a 21-day cycle in combination pembrolizumab (administered intravenously at 200 mg Q3W).
  • AMV 564 15 mcg once a day by subcutaneous injection on days 1-5 and days 8-12 of a 21-day cycle in combination pembrolizumab (administered intravenously at 200 mg Q3W).
  • This data shows evidence of significant increase in CD8 proliferation (assessed by CD8 Ki67) and activation (assessed by CD 8 CD38).
  • CD8 Ki67 proliferation
  • CD 8 CD38 activation
  • Example 11 AMV564 Exhibits Selectivity Against Different Leukemic Blast Cells
  • the structure of AMV564 may allow it to bind clusters of dimerized CD33.
  • the bivalent structure of AMV564 promotes more potent binding to clustered CD33 compared to a monovalent CD33 binding molecule, such as a CD33 antibody.
  • Clustered CD33 can be found in lipid raft regions or active signaling configurations. While CD33 is broadly expressed by leukemic blasts, the degree and homogeneity of expression can vary. The binding of AMV564 to leukemic blasts and primary human myeloid cells and impact on T cell mediated cytotoxicity was thus further explored, and novel assays for evaluation of patient samples were developed.
  • AML cell lines, primary human cells, and patient samples were analyzed using flow cytometry with appropriate marker panels including AMV564, which was directly labeled (phycoerythrin) or detected with labeled anti-AMV564 antibodies.
  • T cell cytotoxicity assays were conducted using primary human T cells and leukemic blasts or other target cells (3:1 ratio) for 48 or 72 hours.
  • Results from flow cytometry assays of cell binding by AMV564 were similar whether using phycoerythrin-labeled AMV564 or a labeled anti-AMV564 antibody.
  • AMV564 showed consistent binding characteristics for many leukemic blast lines (largely of M2 FAB subtype); however, substantial differences in binding were detected between the KG1 myeloid cell line (M2 FAB subtype) and the KGla cell line (MO FAB subtype) that represents a morphologically and functionally less mature state than the parental KG1 cell line.
  • M2 FAB subtype myeloid cell line
  • MO FAB subtype KGla cell line
  • a CD33 antibody had a similar binding profile for each cell line (Figure 12A).
  • the difference in AMV564 binding between these two cell lines is reflected in the cytotoxic potency of AMV564, with a 17.6-fold difference in EC50 (0.42 pM for KG1 vs 7.39 pM for KGla, Figure 12C).
  • AMV564 can engage CD8 and CD4 T cells to drive target-dependent killing and the difference in potency against KG1 vs KGla was observed using both CD8 and CD4 T cells.
  • purified naive CD8 T cells killed KG1 effectively with AMV564
  • T cell proliferation was proportional to cytotoxicity in these assays, with much larger increases in proliferating cells apparent with KG1 target cells.
  • the AMV564 binding assay is compatible with primary human samples.
  • the assay described above can be used to select patients in whom blasts are expressing CD33 in a predominantly clustered configuration, and thus identify patients most likely to experience deeper and more durable responses with AMV564 monotherapy.
  • AML identify patients likely to have a better response to AMV564 therapy by determining the subclass of the AML according to the French-American-British (FAB) classification system identifies immunotypes based on morphology (Table 1).
  • Subtypes in which CD33 is expressed in a predominantly clustered configuration e.g., M2, M3 or M4 may be more responsive to therapy with M254 than subtypes in which the CD33 is not expressed in a predominantly clustered configuration, e.g., Ml.
  • Table 1

Abstract

L'invention concerne des procédés d'évaluation de la possibilité de traitement de certains cancers avec un anticorps BiTE ayant deux domaines de liaison de CD33 et deux domaines de liaison de CD3. Les procédés comprennent l'évaluation du regroupement de CD33 sur des cellules tumorales, par exemple, un blaste leucémique.
PCT/US2021/058254 2020-11-05 2021-11-05 Évaluation de cancers pour le traitement avec un anticorps bite cd33/cd3 WO2022099006A1 (fr)

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