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WO2025097011A2 - Thérapies pour le traitement de malignités myéloïdes - Google Patents

Thérapies pour le traitement de malignités myéloïdes Download PDF

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Publication number
WO2025097011A2
WO2025097011A2 PCT/US2024/054203 US2024054203W WO2025097011A2 WO 2025097011 A2 WO2025097011 A2 WO 2025097011A2 US 2024054203 W US2024054203 W US 2024054203W WO 2025097011 A2 WO2025097011 A2 WO 2025097011A2
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Prior art keywords
antibody
antigen binding
antibodies
binding fragment
pharmaceutical composition
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WO2025097011A3 (fr
Inventor
Jeffrey Joel MOLLDREM
Helen Hong HE
Anna Sergeeva
Lisa Susanne ST. JOHN
Karen CLISE-DWYER
Qing Ma
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • 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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • a myeloid malignancy in a subject comprising: (a) administering to the subject a therapeutically effective amount of a first antibody, an antibody corresponding to the first antibody, a variant of the first antibody or the antibody Attorney Docket No.: 090723-1472570 MDA24-017PCT corresponding to the first antibody, or an antigen binding fragment of the first antibody or the antibody corresponding to the first antibody, wherein the first antibody is Hu8F4 or 8F4 antibody; and (b) administering to the subject a therapeutically effective amount of (1) a second antibody, an antibody corresponding to the second antibody, a variant of the second antibody or the antibody corresponding to the second antibody, or an antigen binding fragment of the second antibody or the antibody corresponding to the second antibody, wherein the second antibody is an antibody blocking CD47-SIRP ⁇ axis, such as an anti- CD47 antibody or an anti-CD172a antibody, or (2) a therapeutic capable of blocking
  • the patient may be a patient who has relapsed.
  • the steps (a) and (b) may be performed simultaneously or not performed simultaneously.
  • the methods according to the embodiments of the present disclosure may further comprise administering at an additional anti-cancer therapy to the patient.
  • the additional anti-cancer therapy may be a chemotherapy, molecular targeted therapy, immunotherapy, radiotherapy, radioimmunotherapy, phototherapy, gene therapy, surgery, hormonal therapy, epigenetic modulation, anti-angiogenic therapy or cytokine therapy.
  • (a) may be the antigen binding fragment of the first antibody.
  • (b)(1) may be the antigen binding fragment of the second antibody.
  • the antigen binding fragment of the first antibody and/or the antigen binding fragment of the second antibody may be a monovalent scFv (single chain fragment variable) antibody, divalent scFv, Fab fragment, F(ab’)2 fragment, F(ab’)3 fragment, Fv fragment, or single chain antibody.
  • the therapeutic (b)(2) may comprise one or more molecules shown in Table 3.
  • compositions comprising: (a) a nucleic acid encoding a first antibody, an antibody corresponding to the first antibody, a variant of the first antibody or the antibody corresponding to the first antibody, or an antigen binding fragment of the first antibody or the antibody corresponding to the first antibody, wherein the first antibody is Hu8F4 or 8F4 antibody; and, (b) a nucleic acid encoding (1) a second antibody, an antibody corresponding to the second antibody, a variant of the second antibody or the antibody corresponding to the second antibody, or an antigen binding fragment of the second antibody or the antibody corresponding to the second antibody, wherein the second antibody is an antibody blocking CD47-SIRP ⁇ axis, such as an anti-CD47 antibody or an anti-CD172a antibody, , or (2) a therapeutic capable of blocking CD47-SIRP ⁇ axis.
  • the second antibody may be a neutralizing anti-CD47 F(ab)’2 antibody, Magrolimab (also known as Hu5F9-G4), TJ011133, or TTI-622.
  • (a) may be antigen binding fragment of the first antibody.
  • (b)(1) may the antigen binding fragment of the second antibody.
  • the antigen binding fragment of the first Attorney Docket No.: 090723-1472570 MDA24-017PCT antibody and/or the antigen binding fragment of the second antibody may be a monovalent scFv (single chain fragment variable) antibody, divalent scFv, Fab fragment, F(ab’)2 fragment, F(ab’)3 fragment, Fv fragment, or single chain antibody.
  • the therapeutic (b)(2) may comprise one or more molecules shown in Table 3.
  • (a) and (b) may be incorporated in one or more vectors.
  • (a) and (b) may be (incorporated in one or more cells.
  • kits comprising: (a) a first antibody, an antibody corresponding to the first antibody, a variant of the first antibody or the antibody corresponding to the first antibody, or an antigen binding fragment of the first antibody or the antibody corresponding to the first antibody, wherein the first antibody is Hu8F4 or 8F4 antibody; and (b)(1) a second antibody, an antibody corresponding to the second antibody, a variant of the second antibody or the antibody corresponding to the second antibody, or an antigen binding fragment of the second antibody or the antibody corresponding to the second antibody, wherein the second antibody is an antibody blocking CD47-SIRP ⁇ axis, such as an anti-CD47 antibody or an anti-CD172a antibody, or (2) a therapeutic capable of blocking CD47-SIRP ⁇ axis.
  • kits may further comprise one or more of: (c) a pharmaceutical package; (d) a chemotherapeutic agent; (e) a cytotoxic agent; (f) a radiotherapeutic agent, or (g) an immunotherapeutic agent.
  • kits comprising: (a) a nucleic acid encoding a first antibody, an antibody corresponding to the first antibody, a variant of the first antibody or the antibody corresponding to the first antibody, or an antigen binding fragment of the first antibody or the antibody corresponding to the first antibody, wherein the first antibody is Hu8F4 or 8F4 antibody; and, (b) a nucleic acid encoding (1) a second antibody, an antibody corresponding to the second antibody, a variant of the second antibody or the antibody corresponding to the second antibody, or an antigen binding fragment of the second antibody or the antibody corresponding to the second antibody, wherein the second antibody is an antibody blocking CD47-SIRP ⁇ axis, such as an anti-CD47 antibody or an anti-CD172a
  • (b)(2) may comprise one or more molecules shown in Table 3.
  • the second antibody may be a neutralizing anti-CD47 F(ab)’2 antibody, Magrolimab (also known as Hu5F9-G4), TJ011133, or TTI-622.
  • the present disclosure includes the following figures. The figures are intended to illustrate certain embodiments and/or features of the compositions and methods, and to supplement any description(s) of the compositions and methods. The figures do not limit the scope of the compositions and methods, unless the written description expressly indicates that such is the case.
  • FIG.1 is a line plot illustrating that the loss of CD47 expression delays the growth of AML.
  • the plot shows that CD47 KO in THP1 AML cells slows the growth of THP1 AML cells in both NSG and NSGS mouse models.
  • NSGTM and NSGS mice were inoculated with THP1 wild type (WT) cells or THP1 KO cells.
  • FIG.2 is a line plot illustrating that the loss of CD47 expression delays the growth of AML.
  • FIG.9A, 9B, 9C, and 9D schematically illustrate the experiments showing that Hu8F4-induced phagocytosis by mouse bone marrow-derived monocytes (BMDM) requires CD47 blockade.
  • FIG.10 are bar graphs illustrating that anti-CD47 blockade acts synergistically with Hu8F4 to promote efficient phagocytosis of AML.
  • FIG.11 is a bar graph illustrating that H8F4-induced ADCP of target cells in presence of differentiated NSG mouse bone marrow-derived macrophages (BMDM) required inactivation of CD47.
  • FIG.12 are bar graphs illustrating that Blockage with either anti-CD47 or anti- SIRP ⁇ showe synergistic ADCP activity with h8F4.
  • FIG.13 is a schematic illustration of experimental design. Adult female NSG mice were injected intravenously with 1 million THP-1 Luc/GFP leukemia cells via tail vein.
  • FIG.14 are line plots illustrating that that Anti-SIRP ⁇ (anti-CD172a) mediated anti-AML activity in vivo only in combination with Hu8F4. DETAILED DESCRIPTION I.
  • transitional phrase “consisting essentially of” is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the present disclosure or features of the claims. See, for example, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q.461, 463 (CCPA 1976) (emphasis in the original); see also MPEP ⁇ 2111.03.
  • nucleic acid As used throughout, the terms “nucleic acid,” “nucleic acid sequence,” “oligonucleotide,” “nucleotides,” or other grammatical equivalents as used herein mean at least two nucleotides, either deoxyribonucleotides or ribonucleotides, or analogs thereof, covalently linked together. Polynucleotides are polymers of any length, including, e.g., 20, 50, 100, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc.
  • modifications to the nucleotide structure may be imparted Attorney Docket No.: 090723-1472570 MDA24-017PCT before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • the term also includes both double- and single-stranded molecules. Unless otherwise specified or required, the term polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • Amino acid polymers may comprise entirely L-amino acids, entirely D-amino acids, or a mixture of L- and D-amino acids.
  • protein refers to either a polypeptide or a dimer (i.e., two) or multimer (i.e., three or more) of single chain polypeptides.
  • the single chain polypeptides of a protein may be joined by a covalent bond, e.g., a disulfide bond, or non-covalent interactions.
  • a “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith & Waterman, 1981, Add. APL. Math.2:482, by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol.48:443, by the Attorney Docket No.: 090723-1472570 MDA24-017PCT search for similarity method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. (U.S.A.) 85:2444, by computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.
  • Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, J. Mol. Biol.215: 403-10 and Altschul et al., 1977, Nucleic Acids Res.25: 3389-402, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site.
  • the algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al.
  • BLAST algorithm One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of Attorney Docket No.: 090723-1472570 MDA24-017PCT the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10 -5 , and most preferably less than about 10 -20 .
  • Calreticulin on the other hand, is anchored to the cancer cell surface through binding with membrane glycans, where it then interacts with the prolow- density lipoprotein receptor-related protein 1 (LRP1) receptor expressed on phagocytes.
  • LRP1 prolow- density lipoprotein receptor-related protein 1
  • tumour cells rely on the expression of ‘don’t eat me’ signals, including CD47, programmed cell death 1 ligand 1 (PD- L1), ⁇ 2-microglobulin (B2M) and other unidentified ligands that bind to leukocyte immunoglobulin-like receptor 2 (LILRB2) to inhibit their phagocytotic clearance by phagocytes; these molecules respectively bind to signal-regulatory protein ⁇ (SIRP ⁇ ), programmed cell death 1 (PD-1) and leukocyte immunoglobulin-like receptor 1 (LILRB1) on phagocytes to inhibit tumor cell phagocytosis.
  • SIRP ⁇ signal-regulatory protein ⁇
  • PD-1 programmed cell death 1
  • LILRB1 leukocyte immunoglobulin-like receptor 1
  • Therapeutic antibodies targeting some of these receptor–ligand interactions have been investigated as potential immunotherapy approaches for multiple cancer types.
  • Hu8F4 anti-PR1/HLA-A2 humanized IgG1
  • FcR Fc receptors
  • the “don’t eat me” signal is prevented by anti-CD47 F(ab)’2 that block CD47 binding to SIRP ⁇ on phagocytes.
  • Anti-CD47 F(ab)’2 lacks the Fc domain and cannot bind to FcR and therefore cannot mediate an “eat me” signal, which mimics the mechanism of action of Magrolimab (which is an IgG4 isotype and therefore also Attorney Docket No.: 090723-1472570 MDA24-017PCT does not bind to FcR).
  • Hu8F4 directs phagocytosis only against AML cells and not normal cells, because PR1/HLA-A2 is differentially expressed on AML, which provides a potent “eat me” signal to phagocytes. It is envisioned that combining Hu8F4 or its variants with anti-CD47 antibodies increases phagocytosis of AML by blocking the “don’t eat me” signal, which mediates maximal phagocytosis of AML and elimination of AML in vitro and in preclinical models.
  • anti-peptide/MHC-I TCR mimic antibodies such as, but not limited, to those that bind to VLQELNVTV (SEQ ID NO:45) epitope, when bound by an HLA- A2, receptor, can be used in combination with “don’t eat me” blockade.
  • VLQELNVTV SEQ ID NO:45
  • examples of such antibodies are described in International Patent Publications WO2014011489A2 abd WO2010065962A2
  • It is envisioned that such antibodies can block the “don’t eat me” interaction of leukocyte immunoglobulin like receptor B1( LILRB1) on the phagocyte by interacting with the peptide/MHC-I.
  • CD47 blockade was studied as a potential strategy to enhance Hu8F4 activity.
  • an HLA-A2 transfected leukemia cell line, U937 (U937-A2), and an endogenously expressing HLA-A2 cell line THP1 were used as targets (T).
  • NSG mouse bone marrow derived macrophages (BMDM) were used as effectors (E).
  • Hu8F4 alone (10 ⁇ g/ml) was insufficient to prevent growth of U937-A2 and THP1.
  • addition of neutralizing anti-CD47 F(ab)’2 antibody significantly slowed leukemia growth and eliminated both target cells by day 5.
  • CRISPR/Cas9 CD47 KO U937-A2 and THP1 cells lines were created, confirming the role of CD47 in the 8F4-mediated ADCP. Complete phagocytosis of both CD47KO cell lines was only observed in the presence, but not the absence of Hu8F4.
  • Myeloid malignancies can be categorized as five types: (1) acute myeloid leukemia (AML); (2) myelodysplastic syndromes (MDS); (3) myeloproliferative neoplasms (MPN); (4) myelodysplastic and myeloproliferative (MDS/MPN) neoplasms; and (5) myeloid neoplasms associated with eosinophilia and abnormalities of growth factor receptors derived from platelets or fibroblasts.
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndromes
  • MPN myeloproliferative neoplasms
  • MDS/MPN myelodysplastic and myeloproliferative
  • myeloid malignancies that express HLA-A2 allele include, for example, AML, MDS, and MPN.
  • Compositions and methods according to the present disclosure can treat, alleviate, or otherwise be useful for myeloid malignancies, in particular those characterized by expression of HLA-A2 allele.
  • Leukemia cells overexpress the “don’t eat me” membrane protein CD47.
  • CD47 on AML binds SIRP ⁇ on macrophages and neutrophils to inhibit phagocytosis. Blocking this interaction can allow antibodies to exert powerful phagocytic activity.
  • CD47 may play in the anti-tumor activity of TCR-mimic antibodies. IV.
  • compositions and methods for treating or alleviating myeloid malignancies for example in a subject having or suspected of having a myeloid malignancy, an animal model, an in vitro tissue culture model, and the like), such as, but not limited to, AML.
  • Antibodies or antigen binding portions thereof that specifically or selectively bind antigen targets related to myeloid malignancies are provided herein.
  • antibodies as described herein are humanized antibodies.
  • antibodies (or their fragments or variants, such as corresponding or synthetic antibodies, or other variations described in the present disclosure) specific to PR1/HLA-A2 tumor antigen complex are used.
  • antibody may be encoded by any nucleic acid sequence that encodes the appropriate amino acid sequence, such as those in SEQ ID NOs: 3, 60, 5, 8, 9, 10 (heavy CDRs 1, 2 and 3; light CDRs 1 and 2, 3, respectively), and SEQ ID NO:16, which includes the heavy CDRs and framework regions 1, 2 and 3, which flank upstream of heavy CDRs 1, 2 and 3, respectively, and SEQ ID NOs: 19 or 20, which includes the light CDRs and framework regions 1, 2 and 3, which flank upstream of light CDRs 1, 2 and 3, respectively.
  • the antibody comprises a heavy chain variable region comprising CDRs comprising amino acid sequences SEQ ID NOs: 3, 60 and 5, respectively, and a light chain variable region comprising CDRs comprising amino acid sequences SEQ ID NOs: 8, 9 and 10, respectively.
  • the antibody comprises a heavy chain variable region comprising CDRs consisting of amino acid sequences SEQ ID NOs: 3, 60 and 5, respectively, and a light chain variable region comprising CDRs consisting of amino acid sequences SEQ ID NOs: 8, 9 and 10, respectively.
  • the antibody comprises a light chain comprising a light chain variable region at least 90% identity (e.g., Attorney Docket No.: 090723-1472570 MDA24-017PCT 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to amino acid sequence SEQ ID NO:20.
  • 90% identity e.g., Attorney Docket No.: 090723-1472570 MDA24-017PCT 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity
  • the antibody comprises a heavy chain variable region comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to amino acid sequence SEQ ID NO:16, and a light chain variable region comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to amino acid sequences SEQ ID NOs: 19 or 20.
  • the antibody comprises a heavy chain variable region consisting of amino acid sequence SEQ ID NO:16, and a light chain variable region consisting of amino acid sequences SEQ ID NOs: 19 or 20.
  • the antibody comprises a heavy chain comprising a heavy chain variable region comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to amino acid sequence SEQ ID NO:16, and a light chain comprising a light chain variable region at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to amino acid sequence SEQ ID NO:19.
  • 90% identity e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity
  • the antibody comprises a heavy chain constant region that is a human gamma-1 heavy chain constant region and a light chain constant region that is a human kappa light chain constant region.
  • the human gamma-1 heavy chain constant region comprises at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to residues 144-473 in amino acid sequence SEQ ID NO:38.
  • the human gamma-1 heavy chain constant region comprises at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to residues 144-473 in amino acid sequence SEQ ID NO:38
  • the human kappa light chain constant region comprises at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) residues 128-234 in amino acid sequence SEQ ID NO:42.
  • the human gamma-1 heavy chain constant region is residues 144-473 in amino acid sequence SEQ ID NO:38, and the human kappa light chain constant region is residues 128-234 in amino acid sequence SEQ ID NO:42.
  • the antibody comprises a heavy chain comprising a heavy chain variable region comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, Attorney Docket No.: 090723-1472570 MDA24-017PCT 95%, 96%, 97%, 98%, 99% or 100% identity) to amino acid sequence SEQ ID NO:16 and a heavy chain constant region which is a human gamma-1 heavy chain constant region, and a light chain comprising a light chain variable region at least 90% identity to amino acid sequence SEQ ID NO:19 and a light chain constant region which is a human kappa light chain constant region.
  • the antibody comprises a heavy chain comprising a heavy chain variable region comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to amino acid sequence SEQ ID NO:16 and the heavy chain constant region comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to residues 144-473 in the amino acid sequence SEQ ID NO:38, and a light chain comprising a light chain variable region comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to amino acid sequence SEQ ID NO:19 and the light chain constant region comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity
  • the antibody can have a light chain sequence comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to any one of SEQ ID NOs: 40 or 42 and a heavy chain sequence comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to of any one of SEQ ID NOs: 38 or 44.
  • a light chain sequence comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to any one of SEQ ID NOs: 38 or 44.
  • the antibody can have a light chain sequence comprising at least 90% identity to SEQ ID NO:40 and a heavy chain sequence comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID NO:38.
  • a light chain sequence comprising at least 90% identity to SEQ ID NO:40
  • a heavy chain sequence comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID NO:38.
  • the antibody can have a light chain sequence comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID NO:42 and a heavy chain sequence comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID NO:38.
  • a light chain sequence comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID NO:42 and a heavy chain sequence comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID NO:38.
  • the antibody can have a light chain sequence comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID NO:42 and a heavy chain sequence comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID NO:44.
  • a light chain sequence comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID NO:44.
  • the antibody is fused to a non-antibody peptide or polypeptide segment.
  • the antibody is linked to a diagnostic reagent or a therapeutic reagent.
  • the diagnostic reagent is a fluorophore, a chromophore, a dye, a radioisotope, a chemilluminescent molecule, a paramagnetic ion, or a spin-trapping reagent.
  • the therapeutic reagent is a cytokine, a chemotherapeutic, a radiotherapeutic, a hormone, an antibody Fc fragment, a TLR agonist, a CpG-containing molecule, or an immune co-stimulatory molecule.
  • the antibody is a bispecific antibody.
  • a bispecific antibody may have, in addition to binding affinity for PR1/HLA-A2 complex, binding affinity for B cells (CD19, CD20), NK cells, phagocytes (CD16), or monocytes (CD14).
  • the codons selected for encoding each amino acid may be modified to optimize expression of the nucleic acid in the host cell of interest.
  • the term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids. Codon preferences for various species of host cell are well known in the art. Codons preferred for use in humans, are well known to those of skill in the art (Wada et al., 1990). Codon preferences for other organisms also are well known to those of skill in the art (Wada et al., 1990, included herein in its entirety by reference). TABLE 2. Codon table.
  • the affinity of 4-1BB-specific antibodies or antigen binding fragments thereof may be optimized through CRISPR or other site-directed mutagenesis methods to increase or decrease affinity as desired based on one or more of the known characteristics of the binding interaction with 4-1BB, the structure of either or both of the antibodies or antigen binding fragments thereof, or the 4-1BB protein.
  • Antibodies according to the present disclosure may also be engineered into various modalities, such as engineered chimeric antigen receptors (CAR), also known as chimeric immunoreceptors, chimeric T cell receptors, or artificial T cell receptors, such as, for example CAR-T, CAR-NK, or CAR macrophage.
  • CARs for CAR T, CAR NK and CAR macrophage have similar structures: the extracellular domain including the antigen binding domain and a spacer that is involved in engagement of target cells; a transmembrane domain that docks CAR to immune cells and is also involved in other functions of CAR, such as stability and interaction with other membrane proteins; and an intracellular signaling domain that is involved in signaling transduction and activation of immune cells.
  • the following expressions - specifically binds to specific for, selectively binds and selective for a myeloid malignancy antigen or an epitope on a protein related to a myeloid malignancy - mean binding that is measurably different from a non- specific or non-selective interaction.
  • Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule.
  • Specific binding can also be determined by competition with a control molecule that is similar to the target, such as an excess of non-labeled target. In that case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by the excess non-labeled target.
  • An antibody can refer to an intact antibody (e.g., an intact immunoglobulin) and antibody fragment, for example, an antigen binding fragment, or a bispecific antibody.
  • Antigen binding fragments can comprise at least one antigen binding domain.
  • One example of an antigen binding domain is an antigen binding domain formed by a VH-VL dimer.
  • Antibodies and antigen binding fragments can be described by the antigen to which they specifically bind.
  • the V H and V L regions can be further subdivided into regions of hypervariability (hypervariable regions (HVRs), also called complementarity determining regions (CDRs)) interspersed with regions that are more conserved. The more conserved regions are called framework regions (FRs).
  • HVRs hypervariability
  • CDRs complementarity determining regions
  • Each V H and V L generally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1 - CDR1 - FR2 - CDR2 Attorney Docket No.: 090723-1472570 MDA24-017PCT - FR3 - CDR3 - FR4.
  • the CDRs are involved in antigen binding and confer antigen specificity and binding affinity to the antibody.
  • CDR sequences on the heavy chain (VH) may be designated as CDRH1, 2, 3, while CDR sequences on the light chain (Vv) may be designated as CDRL1, 2, 3.
  • mAbs monoclonal antibodies
  • recombinant/chimeric antibodies i.e., synthetic antibodies derived from synthetic nucleic acid constructs, such as viral vectors, that may also contain the sequences of the monoclonal antibodies disclosed herein).
  • the affinity of myeloid malignancy antigen-specific antibodies or antigen binding fragments thereof may be optimized through mutations to increase or decrease affinity as desired based on one or more of the known characteristics of the binding interaction with the cognant myeloid malignancy antigen, the structure of either or both of the antibodies or fragments thereof, or the myeloid malignancy antigen.
  • the mutations permit facile elution of purified antibodies or fragments thereof under desirable elution conditions during isolation and purification.
  • the present disclosure also encompasses antibodies or fragments thereof that bind to the same epitope of myeloid malignancy antigens as the antibodies disclosed herein. Such antibodies can be identified using routine techniques known in the art, including, for example, competitive binding assays.
  • epitope as used herein, means a component of an antigen capable of specific binding to an antibody or antigen binding fragment thereof. Such components optionally comprise one or more contiguous amino acid residues and/or one or more non- contiguous amino acid residues.
  • Epitopes frequently consist of surface-accessible amino acid residues and/or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non- conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • An epitope can comprise amino acid residues that Attorney Docket No.: 090723-1472570 MDA24-017PCT are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding.
  • the epitope to which an antigen binding protein binds can be determined using known techniques for epitope determination such as, for example, testing for antigen binding protein binding to antigen variants with different point mutations.
  • the present disclosure also provides chimeric antibodies.
  • the term chimeric antibody refers to an antibody in which a component of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • a human antibody is one that possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e.g., obtained from human sources, genetically modified non-human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.
  • the antibody or antigen binding fragment thereof provided herein can include a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL).
  • VH heavy chain variable domain sequence
  • VL light chain variable domain sequence
  • an antibody molecule comprises or consists of a heavy chain and a light chain (referred to as a half antibody).
  • an antibody molecule in another example, includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequence, thereby forming two antigen binding sites, such as Fab, Fab′, F(ab′) 2 , Fc, Fd, Fd′, Fv, single chain antibodies (scFv, for example), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by the modification of whole antibodies or synthesized de novo using recombinant DNA technologies. These functional antibody fragments retain the ability to selectively bind with their respective antigen.
  • Antibodies and antibody fragments can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4) of antibodies.
  • the preparation of antibody molecules can be monoclonal or polyclonal.
  • An antibody molecule can also be a human, humanized, CDR- grafted, or an in vitro generated antibody.
  • the antibody can have a heavy chain constant region chosen from, e.g., IgG1, IgG2, IgG3, or IgG4.
  • the antibody can also have a light chain chosen from either kappa or lambda light chains.
  • the term monoclonal antibody refers to an antibody from a population of substantially homogeneous antibodies.
  • a population of substantially homogeneous antibodies comprises antibodies that are the same or substantially similar and Attorney Docket No.: 090723-1472570 MDA24-017PCT that bind the same epitope(s), except for variants that can normally arise during production of the monoclonal antibody. Such variants are generally present in only minor amounts.
  • a monoclonal antibody is typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies.
  • the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of yeast clones, phage clones, bacterial clones, mammalian cell clones, hybridoma clones, or other recombinant DNA clones.
  • the selected antibody can be further altered, for example, to improve affinity for the target, for example, by affinity maturation, to humanize the antibody, to improve its production in cell culture, and/or to reduce its immunogenicity in a subject.
  • Antigen binding fragments of an antibody molecule are well known in the art, and include, for example, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv) (see e.g., Bird et al.
  • antibodies and antibody compositions as used in the embodiments of the present disclosure are distinguishable from naturally occurring antibodies and compositions in one or more respects.
  • Such distinguishable antibodies and compositions may be referred to as “synthetic,” or may be identified by the proviso that the antibody or composition “is not naturally occurring” or affirmatively as “non-naturally occurring.”
  • corresponding antibody and “corresponding to” describes the relationship between (1) an antibody characterized by six specific CDR sequences of the antibodies according to the present disclosure and (2) a synthetic antibody comprising the same six CDR sequences.
  • Synthetic antibodies of this disclosure may differ in structure from naturally occurring antibodies with the same CDRs. That is, synthetic antibodies identified by specified CDRs may be structurally different from antibodies comprising the specified CDRs. Possible differences for synthetic antibodies include variable region sequences that differ corresponding naturally occurring antibodies, different light chain sequences (i.e.
  • the synthetic antibody is an engineered polypeptide, also referred to as a recombinant polypeptide, that is made using conventional protein and antibody engineering molecular biology, chemical, and biochemical methods.
  • the antibody comprises a heavy chain variable region sequence and a light chain variable region sequence that are derived from an immunoglobulin producing human B cell, and further comprises a kappa or lambda light chain constant region.
  • the light chain constant region is from the same type of light chain (i.e., kappa or lambda) as the light chain variable region that was derived from the immunoglobulin producing human B cell; as a non-limiting example, if an IgE- producing human B cell comprises a kappa light chain, then the antibody that is produced can comprise the light chain variable region from the IgE-producing B cell and further comprises a kappa light chain constant region.
  • the antibody comprises a heavy chain variable region sequence and a light chain variable region sequence that are derived from an immunoglobulin-producing human B cell, and further comprises a heavy chain constant region having an IgG isotype (e.g., IgG4), an IgA isotype (e.g., IgA1), an IgM isotype, an IgD isotype, or that is derived from an IgG, IgA, IgM, or IgD isotype (e.g., is a modified IgG4 constant region).
  • IgG isotype
  • IgA1 an IgA isotype
  • IgM isotype
  • IgD IgD isotype
  • the different heavy chain isotypes (IgA, IgD, IgE, IgG, and IgM) have different effector functions that are mediated by the heavy chain constant region, and that for certain uses it may be desirable to have an antibody that has the effector function of a particular isotype (e.g., IgG).
  • the antibody comprises a native (i.e., wild-type) human IgG, IgA, IgM, or IgD constant region.
  • the antibody comprises a native human IgG1 constant region, a native human IgG2 constant region, a native human IgG3 constant region, a native human IgG4 constant region, a native human IgA1 constant region, a native human IgA2 constant region, a native human IgM constant region, or a native human IgD constant region.
  • the antibody comprises a heavy chain constant region that comprises one or more modifications. It will be appreciated by a person of ordinary skill in the art that modifications such as amino acid substitutions can be made at one or more residues within the heavy chain constant region that modulate effector function.
  • the modification reduces effector function, e.g., results in a reduced ability to induce certain biological functions upon binding to an Fc receptor expressed on an Attorney Docket No.: 090723-1472570 MDA24-017PCT effector cell that mediates the effector function.
  • the modification e.g., amino acid substitution
  • the antibody comprises a native (i.e., wild-type) human IgM constant region, human IgD constant region, human IgG constant region that is derived from IgG1, IgG2, IgG3, or IgG4, or human IgA constant region that is derived from IgA1 or IgA2 and comprises one or more modifications that modulate effector function.
  • the antibody comprises a human IgM constant region, human IgD constant region, human IgG constant region that is derived from IgG1, IgG2, IgG3, or IgG4, or human IgA constant region that is derived from IgA1 or IgA2.
  • the antibody comprises a native (i.e., wild-type) human IgM constant region, human IgD constant region, human IgG constant region that is derived from IgG1, IgG2, IgG3, or IgG4, or human IgA constant region that is derived from IgA1 or IgA2 and comprises one, two, three, four, five, six, seven, eight, nine, ten or more modifications (e.g., amino acid substitutions).
  • the constant region includes variations (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions) that reduce effector function.
  • Synthetic antibodies of this disclosure may comprise variations in heavy chain constant regions to change the properties of the synthetic antibody relative to the corresponding naturally occurring antibody.
  • Exemplary changes include mutations to modulate antibody effector function (e.g., complement-based effector function or Fc ⁇ R-based effector function), alter half-like, modulate co-engagement of antigen and Fc ⁇ Rs, introduce or remove glycosylation motifs (glyco-engineering).
  • the heavy chain variable region and/or the light chain variable region of the antibody has an identical sequence to the heavy chain variable region and/or the light chain variable region encoded by the immunoglobulin producing single B cell Attorney Docket No.: 090723-1472570 MDA24-017PCT from the human subject having a myeloid malignancy.
  • the heavy chain variable region and/or the light chain variable region of the antibody comprises one or more modifications, e.g., amino acid substitutions, deletions, or insertions.
  • the heavy chain variable region sequence and/or light chain variable region sequence of an antibody described herein can be engineered to comprise one or more variations in the heavy chain variable region sequence and/or light chain variable region sequence.
  • the engineered variation(s) improves the binding affinity of the antibody for a myeloid malignancy. In some embodiments, the engineered variation(s) improves the cross-reactivity of the antibody for a second myeloid malignancy.
  • the engineered variation is a variation in one or more CDRs, e.g., an amino acid substitution in a heavy chain CDR and/or a light chain CDR as described herein. In some embodiments, the engineered variation is a variation in one or more framework regions, e.g., an amino acid substitution in a heavy chain framework region and/or a light chain framework region.
  • the engineered variation is a reversion of a region of the heavy chain and/or light chain sequence to the inferred na ⁇ ve sequence.
  • Methods for determining an inferred na ⁇ ve immunoglobulin sequence are described in the art. See, e.g., Magnani et al., PLoS Negl Trop Dis, 2017, 11:e0005655, doi:10.1371/ journal.pntd.0005655 [0086]
  • affinity maturation is used to engineer further mutations that enhance the binding affinity of the antibody for a myeloid malignancy or enhance the cross- reactivity of the antibody for a second myeloid malignancy or other non-myeloid related pathology.
  • Antibody molecules can also be single domain antibodies.
  • Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies.
  • Single domain antibodies may be any of the art, or any future single domain antibodies.
  • Single domain antibodies may be derived from any species including, but not limited to mouse, rat, guinea, pig, human, camel, llama, fish, shark, goat, rabbit, and bovine. Single domain antibodies are described, for example, in International Application Publication No. WO 94/04678. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to Attorney Docket No.: 090723-1472570 MDA24-017PCT distinguish it from the conventional VH of four chain immunoglobulins.
  • VHH molecule can be derived from antibodies raised in Camelidae species (e.g., camel, llama, dromedary, alpaca and guanaco) or other species besides Camelidae.
  • an antigen binding fragment can also be or can also comprise, e.g., a non-antibody, scaffold protein. These proteins are generally obtained through combinatorial chemistry-based adaptation of preexisting antigen-binding proteins.
  • the binding site of human transferrin for human transferrin receptor can be diversified using the system described herein to create a diverse library of transferrin variants, some of which have acquired affinity for different antigens. See, e.g., Ali et al.
  • Synthetic antibodies of this disclosure may differ from naturally occurring compositions in at least one or more of the following respects: (i) composition comprises antibodies that are purified, i.e., separated from tissue or cellular material with which they are associated in the human body, and optionally in an manufactured excipient or medium; and/or (ii) antibody compositions according to the present disclosure contain a single species of antibody (are monoclonal) such that all antibodies in the composition have the same structure and specificity. V. THERAPEUTICS THAT BLOCK THE CD47-SIRP ⁇ AXIS [0090] Embodiments of the present disclosure are envisioned to use various therapeutics that block the CD47-SIRP ⁇ axis or, when appropriate, nucleic acid molecules encoding such therapeutics.
  • a therapeutic that blocks the CD47-SIRP ⁇ axis may be or comprise any molecule that blocks the CD47-SIRP ⁇ axis.
  • Non-limiting examples of such molecules include, but are not limited, antibodies described elsewhere in the present disclosure (such as a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure, Magrolimab, TJ011133, or TTI-622), or antigen binding fragments of such antibodies, or variants of any thereof, such as corresponding or synthetic antibodies (or other variations described in the present disclosure).
  • Non-limiting examples of such molecules are small molecules, large molecules, peptides, proteins, etc.
  • the present disclosure provides a method of treating a subject with a myeloid malignancy, such as AML, comprising administering to the subject a therapeutically effective amount of a Hu8F4 or 8F4 antibody, or an antigen binding fragment thereof, or variant of any thereof, such as corresponding or synthetic antibodies (or other variations described in the present disclosure) and a therapeutically effective amount a molecule that blocks the CD47-SIRP ⁇ axis.
  • molecules that block the CD47-SIRP ⁇ axis are anti-CD47 antibodies or anti-CD172a antibodies, such as, but not limited to a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure, Magrolimab, TJ011133, or TTI-622, or antigen binding fragments of such anti-CD47 antibodies, or variant of any thereof, such as corresponding or synthetic antibodies (or other variations described in the present disclosure).
  • Some examples of molecules that block the CD47-SIRP ⁇ axis are shown in in Table 3. In some embodiments, the subject has or is determined to have a myeloid malignancy, such as AML.
  • compositions described herein are useful in, inter alia, methods for treating a myeloid malignancy, such as AML, in a subject.
  • the term subject means a mammalian subject. Exemplary subjects include, but are not limited to humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats and sheep.
  • the subject is a human.
  • the subject has or is suspected to have a myeloid malignancy, such as AML.
  • the subject is diagnosed with a myeloid malignancy.
  • the subject is a human that is suspected of having a myeloid malignancy, for example, AML.
  • administer or administration refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., a Hu8F4 or 8F4 antibody, or a corresponding antibody, or an antigen binding fragment thereof, and an antibody capable of blocking CD47-SIRP ⁇ axis, such as an anti-CD47 or an anti-CD172a antibody, including, but not limited to a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure, Magrolimab, TJ011133, TTI-622, or a corresponding antibody, or an antigen binding fragment thereof, or a construct or constructs encoding same) into a patient, such as by mucosal, intradermal, intravenous, intramuscular, subcutaneous Attorney Docket No.: 090723-1472570 MDA24-017PCT delivery and/or any other method of physical delivery described herein or known in the art.
  • a substance as it exists outside the body e.g., a Hu8F4
  • compositions can be administered to a subject, e.g., a human subject, using a variety of methods that depend, in part, on the route of administration.
  • the route can be, e.g., intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection, intramuscular injection (IM), intradermal injection (ID), subcutaneous, transdermal, intracavity, oral, intracranial injection, or intrathecal injection (IT).
  • the injection can be in a bolus or a continuous infusion.
  • Techniques for preparing injectate or infusate delivery systems containing antibodies are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the antibodies, such as the paratope binding capacity (see, for example, Remington's Pharmaceutical Sciences, 18th edition, 1990, Mack Publishing). Those of skill in the art can readily determine the various parameters and conditions for producing antibody injectates or infusates without resort to undue experimentation. [0095] Administration can be achieved by, e.g., topical administration, local infusion, injection, or by means of an implant.
  • the implant can be of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • the implant can be configured for sustained or periodic release of the composition to the subject. See, e.g., U.S. Patent Application Publication No.20080241223; U.S. Patent Nos.5,501,856; 5,164,188; 4,863,457; and 3,710,795.
  • composition can be delivered to the subject by way of an implantable device based on, e.g., diffusive, erodible, or convective systems, e.g., osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmosis systems, vapor pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion- based systems, or electromechanical systems.
  • an implantable device based on, e.g., diffusive, erodible, or convective systems, e.g., osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmosis systems, vapor pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion- based systems, or electromechanical systems.
  • a therapeutically effective amount of a Hu8F4 or 8F4 antibody, or a corresponding antibody, or an antigen binding fragment thereof, and a therapeutically effective amount of an antibody capable of blocking CD47-SIRP ⁇ axis such as an anti-CD47 or an anti-CD172a antibody, including, but not limited to a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure or Magrolimab, TJ011133, TTI-622, or a corresponding antibody, or an antigen binding fragment thereof, are delivered to a subject by way of local administration.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and the like.
  • Treating or treatment of any disease or disorder refers to ameliorating a disease or disorder that exists in a subject or a symptom thereof.
  • the term ameliorating refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a myeloid malignancy, lessening in the severity or progression, promoting remission or durations of remission, or curing thereof.
  • treating or treatment includes ameliorating at least one physical parameter or symptom.
  • Treating or treatment includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. Treating or treatment includes delaying or preventing metastasis.
  • treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition.
  • a method for treating a myeloid malignancy in a subject by administering an antibody as described in this disclosure is considered to be a treatment if there is a 10% reduction in one or more symptoms of the cancer in a subject as compared to a control.
  • the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.
  • the principal symptoms of myeloid malignancy can include (without intending to be limiting) bone pain, nausea, constipation, loss of appetite, mental fogginess or confusion, fatigue, frequent infections, weight loss, weakness or numbness in the legs, excessive thirst, easily fractured or broken bones, anemia, leukopenia, thrombocytopenia, excessive urination, hypercalcemia, spinal cord compression, kidney dysfunction, hyperviscosity, and the like.
  • the term “therapeutically effective amount” or effective amount refers to an amount of a Hu8F4 or 8F4 antibody, or a corresponding antibody, or an antigen binding fragment thereof, and an antibody capable of blocking CD47-SIRP ⁇ axis, such as an anti-CD47 or an anti-CD172a antibody, including, but not limited to a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure, Magrolimab, TJ011133, TTI-622, or a corresponding antibody, or an antigen binding fragment thereof, that, when administered to a subject, is effective to treat a disease or disorder such that the symptoms of myeloid malignancy disease are ameliorated, or the likelihood of myeloid malignancy developing or progressing is decreased.
  • a therapeutically effective amount is not, however, a dosage so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like.
  • a suitable dose of an antibody or fragment thereof described herein, which dose is capable of treating a myeloid malignancy in a subject can depend on a variety of factors including the particular construct used and whether it is used concomitantly with other therapeutic agents. For example, a different dose of a whole antibody may be required to treat a subject with a myeloid malignancy as compared to the dose of a fragment of an antibody (e.g., Fab’ antibody fragment) required to treat the same subject.
  • Fab fragment of an antibody
  • a subject that has had a previous myeloid malignancy may require administration of a different dosage than a subject who has not previously had a myeloid malignancy.
  • a therapeutically effective amount may vary with the subject’s age, condition, and sex, as well as the extent of the disease in the subject and can be determined by one of skill in the art.
  • Other factors can include, e.g., other medical disorders concurrently or previously affecting the subject, the general health of the subject, the genetic disposition of the subject, diet, time of administration, rate of excretion, drug combination, and any other additional therapeutics that are administered to the subject.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.
  • the dosage of the therapeutically effective amount may be adjusted by the individual physician or veterinarian in the event of any complication.
  • a therapeutically effective amount may vary from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 20 mg/kg, most preferably from about 0.2 mg/kg to about 2 mg/kg, in one or more dose administrations daily, for one or several days.
  • a pharmaceutical composition according to the present disclosure can include therapeutically effective amounts of antibodies according to the present disclosure. Such effective amounts can be readily determined by one of ordinary skill in the art as described above. Considerations include the effect of the administered antibodies according to the present disclosure with one or more additional active agents, if more than one agent is used in or with the pharmaceutical composition. In certain aspects, the doses can be about 1, about 0.5, about 0.1, about 0.05, or about 0.01 mg/kg, or any intervening dose between about 0.01 mg/kg and 1 mg/kg. [0101] Suitable human doses of any of the antibodies according to the present disclosure can further be evaluated in, e.g., Phase I dose escalation studies.
  • Toxicity and therapeutic efficacy of antibodies according to the present disclosure can be determined by known pharmaceutical procedures in cell cultures or experimental animals (e.g., animal models of any of the cancers described herein). These procedures can be used, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD 50 /ED 50 .
  • An antibody thereof that exhibits a high therapeutic index is preferred. While constructs that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such constructs to the site of affected tissue and to minimize potential damage to normal cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of the antibodies according to the present disclosure lies generally within a range of circulating concentrations of the the antibodies according to the present disclosure that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the EC 50 (i.e., the concentration of the construct – e.g., antibody – which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • EC 50 i.e., the concentration of the construct – e.g., antibody – which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be Attorney Docket No.: 090723-1472570 MDA24-017PCT measured, for example, by high performance liquid chromatography.
  • cell culture or animal models can be used to determine a dose required to achieve a therapeutically effective concentration within the local site.
  • the antibodies according to the present disclosure can be administered to a subject simultaneously. That is, a therapeutically effective amount of a Hu8F4 or 8F4 antibody, or a corresponding antibody, or an antigen binding fragment thereof, is administered simultaneously with and an antibody capable of blocking CD47-SIRP ⁇ axis, such as an anti-CD47 or an anti-CD172a antibody, including, but not limited to a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure, Magrolimab, TJ011133, TTI-622, or a corresponding antibody, or an antigen binding fragment thereof.
  • the antibodies according to the present disclosure can be administered separately in any order.
  • a therapeutically effective amount of a Hu8F4 or 8F4 antibody, or a corresponding antibody, or an antigen binding fragment thereof is administered separately, before or after, a therapeutically effective amount of an antibody capable of blocking CD47-SIRP ⁇ axis, such as an anti-CD47 or an anti-CD172a antibody, including, but not limited to a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure, Magrolimab, TJ011133, TTI-622, or a corresponding antibody, or an antigen binding fragment thereof.
  • an antibody capable of blocking CD47-SIRP ⁇ axis such as an anti-CD47 or an anti-CD172a antibody, including, but not limited to a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure, Magrolimab, TJ011133, TTI-622, or a corresponding antibody, or an antigen binding fragment thereof.
  • an antibody capable of blocking CD47-SIRP ⁇ axis
  • each or both of the antibodies according to the present disclosure can be administered to a subject at the same time, prior to, or after, a second therapy.
  • each or both of the antibodies according to the present disclosure and the one or more additional active agents are administered at the same time.
  • the antibodies according to the present disclosure are administered first in time and the one or more additional active agents are administered second in time.
  • the one or more additional active agents are administered first in time and the antibodies according to the present disclosure is administered second in time.
  • the each or both of the antibodies according to the present disclosure and the one or more additional agents are administered simultaneously in the same or different routes.
  • the antibodies according to the present disclosure can replace or augment a previously or currently administered therapy.
  • administration of the one or more additional active agents can cease or diminish, e.g., be administered at lower Attorney Docket No.: 090723-1472570 MDA24-017PCT levels or dosages.
  • administration of the previous therapy can be maintained.
  • a previous therapy is maintained until the level of the antibodies according to the present disclosure reach a level sufficient to provide a therapeutic effect.
  • these methods comprise using the CAR to redirect the specificity of an immune effector cell (e.g., a T cell) to target a cancer cell (e.g., a myeloid malignancy antigen-expressing cancer cell).
  • an immune effector cell e.g., a T cell
  • a cancer cell e.g., a myeloid malignancy antigen-expressing cancer cell.
  • “stimulating” an immune cell refers to eliciting an effector cell-mediated response (such as a T cell-mediated immune response), which is different from activating an immune cell.
  • CAR-expressing effector cells described herein can be infused to a subject in need of treatment (e.g., a cancer patient). In some embodiments, the infused cell is able to kill (or lead to the killing of) cancer cells in the subject. Formulations and methods for making CAR-expressing effector cells and using them in therapeutic methods are known in the art (see, e.g., Feins et al., 2019, Am. J. Hematol.94(S1):S3-S9).
  • Monitoring a subject for an improvement of myeloid malignancy, as defined herein, means evaluating the subject for a change in a disease parameter, e.g., a reduction in one or more symptoms of myeloid malignancy exhibited by the subject.
  • the evaluation is performed at least one (1) hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after an administration.
  • the subject can be evaluated in one or more of the following periods: prior to beginning of treatment; during the treatment; or after one or more elements of the treatment have been administered. Evaluation can include evaluating the need for further treatment, e.g., evaluating whether a dosage, frequency of administration, or duration of treatment should be altered. It can also include evaluating the need to add or drop a selected therapeutic modality, e.g., adding or dropping any of the treatments for a myeloid malignancy described herein. [0108] In some instances, each or both of the antibodies according to the present disclosure can be administered via virus-like particles.
  • Virus-like particles comprise viral Attorney Docket No.: 090723-1472570 MDA24-017PCT protein(s) derived from the structural proteins of a virus. Methods for making and using virus like particles are described in, for example, Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004). [0109] In some instances, each or both of the antibodies according to the present disclosure can be administered by subviral dense bodies (DBs). DBs transport proteins into target cells by membrane fusion. Methods for making and using DBs are described in, for example, Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003).
  • DBs subviral dense bodies
  • each or both of the antibodies according to the present disclosure can be administered by tegument aggregates. Methods for making and using tegument aggregates are described in International Publication No. WO 2006/110728.
  • a method of treating a subject with cancer the method comprising administering to the patient cells that have been genetically engineered, using methods such as those described herein, to express and secrete the antibodies according to the present disclosure.
  • a method of treating a subject with cancer the method comprising administering to the patient immune cells that express a CAR comprising each or both of the antibodies according to the present disclosure.
  • compositions and methods which can be used to deliver the nucleic acid molecules and/or polypeptides to cells, either in vitro or in vivo via, for example, expression vectors. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein.
  • plasmid or viral vectors are agents that transport the disclosed nucleic acids into the cell without undesired degradation and include a promoter yielding expression of the nucleic acid molecule and/or adapter polypeptide in the cells into which it is delivered.
  • Viral vectors are, for example, Adenovirus, Adeno-associated virus, herpes virus, Vaccinia virus, Polio virus, Sindbis, and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors.
  • Retroviral vectors in general are described by Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997), which Attorney Docket No.: 090723-1472570 MDA24-017PCT is incorporated by reference herein for the vectors and methods of making them.
  • the construction of replication-defective adenoviruses has been described (Berkner et al., J. Virology 61:1213-20 (1987); Massie et al., Mol. Cell. Biol.6:2872-83 (1986); Haj-Ahmad et al., J. Virology 57:267-74 (1986); Davidson et al., J.
  • viruses as vectors are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infections viral particles.
  • Recombinant adenoviruses have been shown to achieve high efficiency after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and a number of other tissue sites.
  • Other useful systems include, for example, replicating and host- restricted non-replicating vaccinia virus vectors.
  • Non-viral based delivery methods can include expression vectors comprising nucleic acid molecules and nucleic acid sequences encoding the adapter polypeptides, wherein the nucleic acids are operably linked to an expression control sequence.
  • Suitable vector backbones include, for example, those routinely used in the art such as plasmids, artificial chromosomes, BACs, YACs, or PACs.
  • Vectors typically contain one or more regulatory regions. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5’ and 3’ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns.
  • Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters (e.g., ⁇ -actin promoter or EF1 ⁇ promoter), or from hybrid or chimeric promoters (e.g., CMV promoter fused to the ⁇ -actin promoter).
  • viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters (e.g., ⁇ -actin promoter or EF1 ⁇ promoter), or from hybrid or chimeric promoters (e.g., CMV promoter fuse
  • Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5’ or 3’ to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. Attorney Docket No.: 090723-1472570 MDA24-017PCT They are usually between 10 and 300 bp in length, and they function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers can also contain response elements that mediate the regulation of transcription. While many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression.
  • the promoter and/or the enhancer can be inducible (e.g., chemically or physically regulated).
  • a chemically regulated promoter and/or enhancer can, for example, be regulated by the presence of alcohol, tetracycline, a steroid, or a metal.
  • a physically regulated promoter and/or enhancer can, for example, be regulated by environmental factors, such as temperature and light.
  • the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize the expression of the region of the transcription unit to be transcribed.
  • the promoter and/or enhancer region can be active in a cell type specific manner.
  • the promoter and/or enhancer region can be active in all eukaryotic cells, independent of cell type.
  • Preferred promoters of this type are the CMV promoter, the SV40 promoter, the beta-actin promoter, the EF1 ⁇ promoter, and the retroviral long terminal repeat (LTR).
  • the vectors also can include, for example, origins of replication and/or markers.
  • a marker gene can confer a selectable phenotype, e.g., antibiotic resistance, on a cell.
  • the marker product is used to determine if the vector has been delivered to the cell and once delivered is being expressed.
  • selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure.
  • Other markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase.
  • an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide.
  • Tag sequences such as GFP, glutathione S- transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAGTM tag (Kodak; New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide.
  • GFP glutathione S- transferase
  • polyhistidine polyhistidine
  • c-myc hemagglutinin
  • FLAGTM tag FLAGTM tag
  • the effective amount of a pharmaceutical composition comprising each or both of the antibodies according to the present disclosure, for example, upon the therapeutic context and objectives.
  • the appropriate dosage levels for treatment vary depending, in part, upon the molecule delivered, the indication for which a each or both of the antibodies according to the present disclosure are being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient. The clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
  • the clinician also selects the frequency of dosing, taking into account the pharmacokinetic parameters of the each or both of the antibodies according to the present disclosure in the formulation used.
  • pharmacokinetic parameters are well known in the art, i.e., the rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol.58:611-617; Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci.84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin.
  • a clinician administers the composition until a dosage is reached that achieves the desired effect.
  • the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via, for example, an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them.
  • appropriate dosages can be ascertained through use of appropriate dose-response data.
  • the dosage (of the active component) ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 20 mg/kg, of the patient’s body weight.
  • dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, 10 mg/kg body weight or within the range of 0.1-20 mg/kg.
  • each or both of the antibodies according to the present disclosure can be administered at a dose of 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, or 5 mg/kg once every other day at least four times.
  • An exemplary treatment regime may include administration once per day, once per week, twice a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every three to 6 months.
  • the treatment comprises administering each or both of the antibodies according to the present disclosure according to one of the aforementioned dosing regimens for a first period and another of the aforementioned dosing regimens for a second period.
  • the treatment discontinues for a period of time before the same or a different dosing regimen resumes.
  • a patient may be on a dosing regimen of each or both of the antibodies according to the present disclosure for two weeks, off for a week, on for another two weeks, and so on.
  • Dosage regimens each or both of the antibodies according to the present disclosure include 0.1 mg/kg body weight, 0.3 mg/kg body weight, 2 mg/kg body weight, 3 mg/kg body weight, or 10 mg/kg via intravenous administration, with the each or both of the antibodies according to the present disclosure being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks.
  • the route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebral, intraventricular, intramuscular, subcutaneously, intra-ocular, intraarterial, intraportal, or intralesional routes, by sustained release systems or by implantation devices.
  • the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.
  • individual elements of a combination therapy may be administered by different routes.
  • the composition can be administered locally, e.g., during surgery or topically.
  • Optionally local administration is via implantation of a membrane, sponge, or another appropriate material onto which the desired molecule has been absorbed or encapsulated.
  • the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.
  • it can be desirable to use a pharmaceutical composition comprising each or both of the antibodies according to the present disclosure in an ex vivo manner. In such instances, cells that have been removed from a subject may be exposed to a pharmaceutical composition comprising each or both of the antibodies according to the present disclosure, after which the cells are subsequently implanted back into the subject.
  • the provided methods may include administering to the subject a each or both of the antibodies according to the present disclosure that is conjugated to a Attorney Docket No.: 090723-1472570 MDA24-017PCT therapeutic agent.
  • the therapeutic agent may be at least one of a cytotoxic agent, a chemotherapeutic agent, or an immunosuppressive agent. Such therapeutic agents are described below.
  • the provided methods may include administering the antibodies according to the present disclosure and a second form of cancer therapy to the subject.
  • the second form of cancer therapy may include a cytotoxic agent, a chemotherapeutic agent, an immunosuppressive agent (including immune checkpoint inhibitors), or radiation therapy.
  • the second form of cancer therapy is an antibody (e.g., a monoclonal antibody).
  • Monoclonal antibodies which may be administered as a second form of cancer therapy include, but are not limited to, rituximab (e.g., for treatment of B-cell lymphomas), trastuzumab (e.g., for treatment of breast cancer), and cetuximab (e.g., for treatment of lung cancer).
  • rituximab e.g., for treatment of B-cell lymphomas
  • trastuzumab e.g., for treatment of breast cancer
  • cetuximab e.g., for treatment of lung cancer.
  • each or both of the antibodies according to the present disclosure can be labeled, conjugated, or fused with a therapeutic agent or diagnostic agent (such as an imaging agent).
  • the linkage can be covalent or noncovalent (e.g., ionic).
  • antibody-drug conjugates Such antibodies and antibody fragments are referred to antibody-drug conjugates (ADC) or immunoconjugates.
  • ADC antibody-drug conjugates
  • the antibody conjugates are useful for the local delivery of therapeutic agents, particularly cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment of cancer allows targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, where systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated.
  • Therapeutic agents include but are not limited to toxins, including but not limited to plant and bacterial toxins, small molecules, peptides, polypeptides and proteins.
  • a target cell or target cells are myeloid malignancy antigen- positive cells.
  • each or both of the antibodies according to the present disclosure are conjugated to a moiety that specifically binds to an immune cell.
  • a bispecific antibody comprising one or both of the antibodies according to the present disclosure or antigen binding fragment thereof as described herein and an antibody or antigen binding fragment thereof that specifically binds to an immune cell.
  • the bispecific antibody comprises an antibody according to the present disclosure and an antibody moiety that specifically binds to T cells.
  • T cells Such a molecule Attorney Docket No.: 090723-1472570 MDA24-017PCT is referred to as a bispecific T cell engager and may induce T cell-mediated cytotoxicity of myeloid malignancy antigen-expressing cancer cells (see, e.g., Zhou et al., 2021, Biomarker Research 9:38).
  • the bispecific antibody comprises an antibody according to the present disclosure and an antibody moiety that specifically binds to natural killer cells (NK cells).
  • a NK cell engager Such a molecule is referred to as a NK cell engager and may induce NK cell-mediated cytotoxicity of myeloid malignancy antigen-expressing cancer cells (see, e.g., Demaria et al., 2021, European Journal of Immunology 51(8):1934-1942).
  • each or both of the antibodies according to the present disclosure and CARs (or cells comprising CARs as described herein) according to the present disclosure can be administered as a co-therapy with other therapeutic agents.
  • therapeutic agents include chemotherapeutic agents, a radiotherapeutic agent, and immunotherapeutic agent, as well as combinations thereof.
  • the antibody or peptide complex or CAR (or cell comprising a CAR) delivered to the subject can be multifunctional, in that it exerts one therapeutic effect by binding to the myeloid malignancy antigen protein and a second therapeutic effect by delivering a supplemental therapeutic agent.
  • the therapeutic agent can act extracellularly, for example by initiating or affecting an immune response, or it can act intracellularly, either directly by translocating through the cell membrane or indirectly by, for example, affecting transmembrane cell signaling.
  • the therapeutic agent is optionally cleavable from an antibody according to the present disclosure. Cleavage can be autolytic, accomplished by proteolysis, or affected by contacting the cell with a cleavage agent.
  • the therapeutic agent is a cytotoxic agent.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples of toxins or toxin moieties include diphtheria, ricin, streptavidin, and modifications thereof.
  • Additional examples include paclitaxel, cisplatin, carboplatin, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, colchicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6- thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.
  • antimetabolites e.g., methotrexate, 6-mercaptopurine, 6- thioguanine, cytarabine, 5-fluorouracil, decarbazine
  • alkylating agents e.g., mechlorethamine, thio
  • daunorubicin Attorney Docket No.: 090723-1472570 MDA24-017PCT (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
  • antibiotics e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)
  • anti-mitotic agents e.g., vincristine and vinblastine.
  • Cytotoxic peptides such as auristatin (antineoplastic) peptides auristatin E (AE) and monomethylauristatin (MMAE), which are synthetic analogs of dolastatin, may also be conjugated to each or both of the antibodies according to the present disclosure.
  • a chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
  • chemotherapeutic agents include erlotinib (such as TARCEVA ® , Genentech/OSI Pharm.), bortezomib (such as VELCADE ® , Millenium Pharm.), fulvestrant (such as FASLODEX ® , AstraZeneca), sutent (such as SU11248, Pfizer), letrozole (such as FEMARA ® , Novartis), imatinib mesylate (such as GLEEVEC ® , Novartis), PTK787/ZK222584 (Novartis), oxaliplatin (such as ELOXATIN ® , Sanofi), 5-fluorouracil (5- FU), leucovorin
  • Chemotherapeutic agents also refers to (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX ® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (such as FARESTON ® ); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (such as MEGASE ® ), exemestane (such as AROMASIN ® ), formestanie, fadrozole, vorozole (such as RI
  • the treatment methods provided herein may further comprise administering an immunosuppressive agent such as an immune checkpoint inhibitor as part of the method.
  • an immunosuppressive agent such as an immune checkpoint inhibitor as part of the method.
  • PD-1 inhibitors such as nivolumab and pembrolizumab
  • PD-L1 inhibitors such as atezolizumab, durvalumab, and avelumab
  • CTLA-4 inhibitors such as ipilimumab.
  • the second form of cancer therapy comprises a PD-L1 inhibitor, a PD-1 inhibitor, or a CTLA4 inhibitor.
  • combinations of such inhibitors can be administered.
  • the PD-L1 inhibitor, the PD-1 inhibitor, and/or the CTLA4 inhibitor may be an inhibitory antibody that binds specifically to PD-L1, PD-1, or CTLA4, respectively.
  • the treatment methods provided herein may further comprise administering radiation therapy to the subject. Radiation therapy uses high-energy radiation to shrink tumors and kill cancer cells. X-rays, gamma rays, and charged particles are types of radiation used for cancer treatment.
  • compositions comprising one or both antibodies according to the present disclosure and pharmaceutically acceptable carrier are also provided.
  • the compositions comprise a Hu8F4 or 8F4 antibody, or an antigen binding fragment thereof, or variant of any thereof, such as corresponding or synthetic antibodies (or other variations described in the present disclosure).
  • the compositions comprise any suitable molecule that blocks the CD47-SIRP ⁇ axis.
  • molecules that block the CD47-SIRP ⁇ axis are anti-CD47 or anti-CD172 antibodies, such as, but not limited to a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure, Magrolimab, TJ011133, or TTI-622, or antigen binding fragments of such anti-CD47 antibodies, or variants of any thereof, such as corresponding or synthetic antibodies (or other variations described in the present disclosure).
  • molecules that block the CD47-SIRP ⁇ axis are shown in Table 3.
  • compositions comprise a nucleic acid encoding a Hu8F4 or 8F4 antibody, or an antigen binding fragment thereof, or variant of any thereof, such as corresponding or synthetic antibodies (or other variations described in the present disclosure).
  • compositions comprise encoding any suitable molecule that blocks the CD47-SIRP ⁇ axis.
  • molecules that block the CD47-SIRP ⁇ axis are anti- CD47 or anti-CD172 antibodies, such as, but not limited to a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure, Magrolimab, TJ011133, or Attorney Docket No.: 090723-1472570 MDA24-017PCT TTI-622, or antigen binding fragments of such anti-CD47 antibodies, or variant of any thereof, such as corresponding or synthetic antibodies (or other variations described in the present disclosure).
  • Some examples of molecules that block the CD47-SIRP ⁇ axis are shown in Table 3.
  • compositions according to the embodiments of the present invention and comprising nucleic acids one or more of the nucleic acids may be incorporated into one or more vectors. In the above compositions according to the embodiments of the present invention and comprising nucleic acids, one or more of the nucleic acids may be incorporated into one or more cells.
  • the compositions may further comprise a diluent, solubilizer, emulsifier, preservative, and/or adjuvant to be used with the methods disclosed herein.
  • Such compositions can be used in a subject with a myeloid malignancy, such as AML, that would benefit from any of the myeloid malignancy antigen-specific antibodies or antigen binding fragments thereof described herein.
  • acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed.
  • the formulation material(s) are for s.c. and/or I.V. administration.
  • the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
  • suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying amino acids (such
  • the optimal pharmaceutical composition is determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Allen (2012) Remington – The Science and Practice of Pharmacy, 22d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press. In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and/or rate of in vivo clearance of the antibodies according to the present disclosure.
  • the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
  • the saline comprises isotonic phosphate-buffered saline.
  • neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • pharmaceutical compositions comprise a pH controlling buffer such phosphate-buffered saline or acetate-buffered saline.
  • a composition comprising one or both antibodies of the present disclosure can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (see Allen (2012) Remington – The Science and Practice of Pharmacy, 22d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press) in the form of a lyophilized cake or an aqueous solution.
  • a composition comprising one or both antibodies of the present disclosure can be formulated as a lyophilizate using appropriate excipients.
  • appropriate excipients may include a cryo-preservative, a bulking agent, a surfactant, or a combination of any thereof.
  • Exemplary excipients include one or more of a polyol, a disaccharide, or a polysaccharide, such as, for example, mannitol, sorbitol, sucrose, trehalose, and dextran 40.
  • the cryo-preservative may be sucrose or trehalose.
  • the bulking agent may be glycine or mannitol.
  • the surfactant may be a polysorbate such as, for example, polysorbate-20 or polysorbate-80.
  • the pharmaceutical composition can be selected for parenteral delivery.
  • the compositions can be selected for inhalation Attorney Docket No.: 090723-1472570 MDA24-017PCT or for delivery through the digestive tract, such as orally.
  • the preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art.
  • the formulation components are present in concentrations that are acceptable to the site of administration.
  • buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
  • the pH may be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8.6.9, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5.
  • the pH of the pharmaceutical composition may be in the range of 6.6-8.5 such as, for example, 7.0-8.5, 6.6-7.2, 6.8-7.2, 6.8-7.4, 7.2-7.8, 7.0-7.5, 7.5-8.0, 7.2-8.2, 7.6-8.5, or 7.8-8.3.
  • the pH of the pharmaceutical composition may be in the range of 5.5-7.5 such as, for example, 5.5-5.8, 5.5- 6.0, 5.7-6.2, 5.8-6.5, 6.0-6.5, 6.2-6.8, 6.5-7.0, 6.8-7.2, or 6.8-7.5.
  • the pH of the pharmaceutical composition may be in the range of 4.0-5.5 such as, for example, 4.0-4.3, 4.0-4.5, 4.2-4.8, 4.5-4.8, 4.5-5.0, 4.8-5.2, or 5.0-5.5.
  • the pH is 7.2.
  • a therapeutic composition when parenteral administration is contemplated, can be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising one or both antibodies of the present disclosure in a pharmaceutically acceptable vehicle.
  • a vehicle for parenteral injection is sterile distilled water in which one or both antibodies of the present disclosure is formulated as a sterile, isotonic solution and properly preserved.
  • the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection.
  • an agent such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection.
  • hyaluronic acid can also be used, and can have the effect of promoting sustained duration in the circulation.
  • implantable drug delivery devices can be used to introduce the desired molecule.
  • a pharmaceutical composition can be formulated for inhalation.
  • one or both antibodies of the present disclosure can be formulated as a dry powder for inhalation.
  • an inhalation solution comprising one or both antibodies of the present disclosure can be formulated with a propellant for aerosol delivery.
  • solutions can be nebulized.
  • Pulmonary administration is further described in International Application Publication No. WO/1994/020069, which describes pulmonary delivery of chemically modified proteins.
  • formulations can be administered orally.
  • one or both antibodies of the present disclosure that is administered in this fashion can be formulated with or without carriers customarily used in compounding solid dosage forms, such as tablets and capsules.
  • a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized, and pre-systemic degradation is minimized.
  • at least one additional agent can be included to facilitate absorption of one or both antibodies of the present disclosure.
  • diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.
  • a pharmaceutical composition can involve an effective quantity of one or both antibodies of the present disclosure in a mixture with non-toxic excipients suitable for the manufacture of tablets.
  • solutions can be prepared in unit-dose form.
  • suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
  • Additional pharmaceutical compositions can be selected by one skilled in the art, including formulations one or both antibodies of the present disclosure in sustained- or controlled-delivery formulations.
  • sustained-release preparations can include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules.
  • Sustained release matrices can include polyesters, hydrogels, polylactides (see, e.g., U.S. Patent No.3,773,919; U.S.
  • PHAs polyhydroxyalkanoates
  • sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. (See, e.g., Eppstein et al.
  • the pharmaceutical composition to be used for in vivo administration typically is sterile. In certain embodiments, sterilization is accomplished by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization using this method can be conducted either prior to or following lyophilization and reconstitution. In certain embodiments, the composition for parenteral administration can be stored in lyophilized form or in a solution.
  • parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • a sterile access port for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the pharmaceutical composition once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
  • such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.
  • kits are provided for producing a single-dose administration unit.
  • the kit can contain both a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments, kits containing single and multi-chambered pre-filled syringes are included.
  • the effective amount of a pharmaceutical composition comprising one or both antibodies of the present disclosure to be employed therapeutically depends, for example, upon the therapeutic context and objectives.
  • the appropriate dosage levels for treatment vary depending, in part, upon the molecule delivered, the indication for which the antibodies of the present disclosure are used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient.
  • the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
  • Attorney Docket No.: 090723-1472570 MDA24-017PCT [0154]
  • the clinician also selects the frequency of dosing, taking into account the pharmacokinetic parameters of the one or both antibodies of the present disclosure in the formulation used.
  • a clinician administers the composition until a dosage is reached that achieves the desired effect.
  • the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via, for example, an implantation device or catheter.
  • the route of administration of the pharmaceutical composition is in accordance with known methods, such as the following: orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebral, intraventricular, intramuscular, subcutaneously, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices.
  • the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.
  • individual elements of a combination therapy may be administered by different routes.
  • the composition can be administered locally, e.g., during surgery or topically.
  • local administration is via implantation of a membrane, sponge, or another appropriate material onto which the desired molecule has been absorbed or encapsulated.
  • the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.
  • one or both antibodies of the present disclosure can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptides.
  • such cells can be animal or human cells, and can be autologous, heterologous, or xenogeneic.
  • the cells can be immortalized.
  • the cells in order to decrease the chance of an immunological response, the cells can be encapsulated to avoid infiltration of surrounding tissues.
  • the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the Attorney Docket No.: 090723-1472570 MDA24-017PCT cells by a subject’s immune system or by other detrimental factors from the surrounding tissues.
  • KITS KITS
  • the therapeutics according to the present disclosure are ideally suited for the preparation of kits, which can be used for carrying out the therapeutic methods according to the present disclosure.
  • the kits of this disclosure may comprise a carrier container being compartmentalized to receive in close confinement one or more containers such as vials, tubes, and the like, each of the containers comprising one of the separate elements to be used in the method.
  • one of the containers may comprise an Hu8F4 or 8F4 antibody (or their fragments or variants, such as corresponding or synthetic antibodies, or other variations described in the present disclosure), or a nucleic acid encoding the foregoing.
  • Another one of the containers may comprise a suitable molecule that blocks the CD47-SIRP ⁇ axis.
  • molecules that block the CD47-SIRP ⁇ axis are anti-CD47 or anti- CD172 antibodies, such as, but not limited to a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure, Magrolimab, TJ011133, or TTI-622, or antigen binding fragments of such anti-CD47 antibodies, or variant of any thereof, such as corresponding or synthetic antibodies (or other variations described in the present disclosure).
  • molecules that block the CD47-SIRP ⁇ axis are shown in Table 3.
  • another one of the containers may comprise a nucleic acid encoding.
  • a suitable molecule that blocks the CD47-SIRP ⁇ axis may comprise a nucleic acid encoding.
  • kits may include a pharmaceutical package for delivery to doctors and cancer patients. Such packaging is intended to improve patient convenience and compliance with the treatment plan. Typically, the packaging comprises paper (cardboard) or plastic. In some embodiments, the kit may include instructions for use (e.g., for administering according to a method as described herein). [0161] In some embodiments, a kit comprises unit dose forms of antibodies according to the present disclosure. In some embodiments, a kit further comprises unit dose forms of one or more of a chemotherapeutic agent, a cytotoxic agent, a radiotherapeutic agent, or an immunotherapeutic agent.
  • a kit comprises antibodies according to the present disclosure in defined, therapeutically effective dose in a single unit dosage forms or as separate unit Attorney Docket No.: 090723-1472570 MDA24-017PCT doses.
  • the dose and form of the unit dose e.g., tablet, capsule, immediate release, delayed release, etc.
  • the kit or pharmaceutical package includes doses suitable for multiple days of administration, such as one week, one month, or three months.
  • kits are provided for producing a single-dose administration unit.
  • kits containing single or multi-chambered pre-filled syringes are included.
  • kits containing one or more containers of a formulation described in this disclosure are included.
  • Example 1 Loss of CD47 expression delays the growth of AML.
  • BLI was accessed weekly. Each data point shown in FIG.4 represents Total Flux Signal of one mouse at a timepoint. Survival was calculated using Log-rank (Mantel-Cox) test (FIG.5).
  • Treatment with Hu8F4-2 for 7 weeks (1 mg/kg three times per week; started on day 2), significantly reduced THP1 leukemia growth and fully eliminated CD47 KO THP1 cells, confirming the role of CD47 in the activity of Hu8F4 antibody.
  • FIG.7 illustrates the summary of BLI data.
  • the survival was calculated using Log-rank (Mantel-Cox) test (FIG.8).
  • Both Hu8F4 and anti-CD47 F(ab)’ 2 single-agent treatment slowed leukemia growth, while the combination of Hu8F4 and anti-CD47 F(ab)’2 completely prevented outgrowth of the aggressive U937-A2 cells, indicating that blocking CD47 significantly increased the activity of Hu8F4 in vivo.
  • THP1 (FIG.9D) cells were incubated with and without Hu8F4 and a-CD47 F(ab’)2 antibodies. Fluorescent and bright field images were taken at various time points The bar show object area of target cells at 96 hours, analyzed and calculated by using Gen5 software. Each bar shows medium calculated object area of green (target) cells in 4 images, captured from corresponding cells using x4 objective by Cytation 3, error bar shows SEM.
  • human macrophages were generated from CD14+ monocytes from healthy donors.
  • Cells were stained with anti-CD11b antibody and analyzed by flowcytometry. Phagocytosis was calculated as percent of phagocyted CD11b+ CFSE+ target cells out of total CFSE+ target cells.
  • A-CD47 antibody F(ab’)2 increased ADCP, induced buy Hu8F4, but had no effect on control antibody Herceptin.
  • Anti-CD47 F(ab)’ 2 alone and in the presence of control with Herceptin did not induce phagocytosis.
  • Example 6 Hu8F4 combined with either anti-SIRP ⁇ or anti-CD47 mAb blockage mediates synergistic ADCP activity against HLA-A2+ THP1 human AML cells.
  • Hu8F4 combined with either anti- SIRP ⁇ or anti-CD47 mAb blockage mediated synergistic ADCP activity against HLA-A2+ Attorney Docket No.: 090723-1472570 MDA24-017PCT THP1 human AML cells. The experimental results are illustrated in FIG.11.
  • GFP-transfected THP1 target cells were treated with CD47- blocking antibodies +/- Hu8F4 antibodies for 30 minutes and added to BMDM at 1:1 ratio. Fluorescent images were taken at Day 7, the number of green fluorescent target cells weas analyzed by Gen5 software. (7 days time point shown). Bar graphs show medium number of target cells in images, captured from corresponding cells using x20 objective by Cytation 3, error bar shows SEM. One representative experiment out of three shown.
  • the experiment showed H8F4- induced ADCP of target cells in presence of differentiated NSG mouse bone marrow-derived macrophages (BMDM) required inactivation of CD47.
  • BMDM differentiated NSG mouse bone marrow-derived macrophages
  • the experiments were conducted demonstrating that blockage with either anti- CD47 or anti-SIRP ⁇ showe synergistic ADCP activity with h8F4.
  • the experimental results are illustrated in FIG.12.
  • Target AML cells, transfected with GFP, were mixed 1:1 with NSG mouse macrophages, labelled with pkH26, in presence of antibodies. On Day 5 fluorescent images were assessed, and target AML cells were counted.
  • the bar graphs in FIG.12 show medium number of target cells in images, captured from corresponding cells using x20 (for MV411) and x4 (for THP1) objectives (2way ANOVA comparison, adjusted p values: * p ⁇ 0.05; ** p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001).
  • FIG.12. [0174] The experiments were conducted demonstrating that Anti-SIRP ⁇ (anti-CD172a) mediated anti-AML activity in vivo only in combination with Hu8F4.
  • the experimental design is illustrated in FIG.13.
  • the experimental results are illustrated in FIG.14.
  • Adult female NSG mice were injected intravenously with 1 million THP-1 Luc/GFP leukemia cells via tail vein.
  • In vivo bioluminescence imaging was performed on the day of inoculation and weekly thereafter to monitor leukemia burden.
  • the total signal in the region of interest (ROI) measured in photons per second, was quantified using Living Image software. The same ROI was applied to all sequential images captured during a single imaging session for each mouse.
  • the leukemia burden was plotted against the number of days post-inoculation.

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Abstract

L'invention concerne des méthodes de traitement d'une malignité myéloïde chez un sujet à l'aide d'anticorps. L'invention concerne également des compositions et des kits pour traiter une malignité myéloïde chez un sujet à l'aide d'anticorps.
PCT/US2024/054203 2023-11-01 2024-11-01 Thérapies pour le traitement de malignités myéloïdes Pending WO2025097011A2 (fr)

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