EP3958898A1 - Formulations et anticorps anti-cd38 - Google Patents

Formulations et anticorps anti-cd38

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Publication number
EP3958898A1
EP3958898A1 EP20726267.6A EP20726267A EP3958898A1 EP 3958898 A1 EP3958898 A1 EP 3958898A1 EP 20726267 A EP20726267 A EP 20726267A EP 3958898 A1 EP3958898 A1 EP 3958898A1
Authority
EP
European Patent Office
Prior art keywords
amino acid
antibody
seq
acid sequence
heavy chain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20726267.6A
Other languages
German (de)
English (en)
Inventor
Béatrice Cameron
Marielle CHIRON BLONDEL
Jacques Dumas
Alain Fournier
Jonathan KINGSBURY
Cendrine Lemoine
Brian Murray
Nathan OSTBERG
Sanket PATKE
Angela VIRONE-ODDOS
Zichuan ZHANG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sanofi SA
Original Assignee
Sanofi SA
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Filing date
Publication date
Application filed by Sanofi SA filed Critical Sanofi SA
Publication of EP3958898A1 publication Critical patent/EP3958898A1/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • anti-CD38 antibodies having improved cytotoxic activity, and stable formulations thereof.
  • CD38 is a Type II glycosylated 45 kilodalton (kDa) membrane protein that was identified as a lymphocyte marker.
  • CD38 has a role in leukocyte homeostasis through modulation of hematopoietic cell survival and differentiation (Richards JO, et al., Mol Cancer Ther. 2008; 7(8):2517-27).
  • CD38 functions as a receptor binding to CD31 and is involved in cell adhesion and signal transduction.
  • the function of CD38 in signal transduction appears to be versatile depending on the cell lineage, the differentiation stage, and, possibly, the association with different co-receptors (Richards JO, et al., 2008).
  • CD38 is also an ectoenzyme catalyzing the synthesis and hydrolysis of cyclic adenosine-diphosphate-ribose (cADPR) from nicotinamide adenine dinucleotide (NAD+) to ADP-ribose (DiLillo DJ, Ravetch JV., Cell. 2015; 161(5): 1035-45).
  • cADPR cyclic adenosine-diphosphate-ribose
  • NAD+ nicotinamide adenine dinucleotide
  • ADP-ribose DiLillo DJ, Ravetch JV., Cell. 2015; 161(5): 1035-45.
  • CD38 The expression of CD38 in healthy humans can be detected on NK cells, monocytes, dendritic cells, macrophages, granulocytes, activated T and B cells, and plasma cells. In contrast, expression has not been detected in hematopoietic stem cells, resting T and B cells, or tissue macrophages. Furthermore, several hematological malignancies express CD38, such as plasma cell dyscrasias (e.g., multiple myeloma (MM), amyloidosis) and other cancers of hematopoietic origin including, including for example, Waldenstrom’s disease, non-Hodgkin’s lymphoma (NHL), acute lymphocytic leukemia (ALL), and acute
  • AML myelogenous leukemia
  • CD38 is especially notable in MM as >98% of patients are positive for this protein (Reinherz EL, et ak, Proc Natl Acad Sci USA. 1980; 77(3): 1588-92, Lin P, et al, Am J Clin Pathol. 2004; 121(4):482-8).
  • the strong and uniform expression of CD38 on malignant clonal MM cells contrasts with the restricted expression pattern on normal cells suggesting this antigen may be useful for specific targeting of tumor cells.
  • MM is a malignant plasma cell disease that is characterized by CD38 expression on the cell surface, clonal proliferation of plasma cells in the bone marrow (BM) and the production of excessive amounts of a monoclonal immunoglobulin (usually of the IgG or IgA type or free urinary light chain (also referred to as paraprotein, M-protein or M-component)). It is a disease predominantly associated with advancing age with more than 80% of patients aged 60 years or older.
  • BM bone marrow
  • Induction treatment regimens include alkylating agents, dexamethasone alone, thalidomide plus dexamethasone, and vincristine, Adriamycin® (doxorubicin), and dexamethasone (VAD; or modifications to this regimen); however, the latter two regimens are associated with higher toxicity (Richardson PG, et al, Blood. 2010; 116(5):679-86,
  • Additional approved drugs include pomalidomide (belonging to the same IMiD ® class as lenalidomide) and carfilzomib and ixazomib (belonging to the same proteasome inhibitor class as bortezomib).
  • monoclonal antibodies particularly anti-CD38 antibodies, have begun to play a major role in the treatment of myeloma patients.
  • Daratumumab an anti-CD38 antibody, has been approved for treatment of MM as a single agent and in combination with other treatments for MM
  • Isatuximab an anti-CD38 antibody, has been reported to induce 25-29% response rate as a single agent and 60% response rate in combination therapy in relapsed or refractory multiple myeloma (Martin T, et al, Blood.
  • dexamethasone for treatment of adult patients with MM who have received one to three prior lines of therapy, and in combination with pomalidomide and dexamethasone for the treatment of adult patients with MM who have received at least two prior therapies including lenalidomide and a proteasome inhibitor (Bristol-Myers Squibb Company. EMPLICITI® (elotuzumab) [package insert] U.S. Food and Drug Administration website www-dot- accessdata-dot-fda.gov/drugsatfda_docs/label/2018/761035s0081bl.pdf. Revised November 2018.
  • the antibodies, uses, and methods of treatment provided herein may provide a superior clinical response or treatment of the disease.
  • antibodies that specifically bind human CD38 and mediate superior killing of cells that express CD38 formulations and unit dosage forms comprising the antibodies, methods of preparing the antibodies, and methods of using the antibodies.
  • antibodies that specifically bind human CD38 are provided.
  • the antibodies provided herein comprise a light chain (LC) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, and 9 and a heavy chain (HC) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, and 6.
  • LC light chain
  • HC heavy chain
  • the antibodies provided herein comprise an LC having an amino acid sequence of SEQ ID NO: 7 and an HC having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.
  • the antibodies provided herein comprise an LC having an amino acid sequence of SEQ ID NO: 7 and an HC having an amino acid sequence of SEQ ID NO: 2.
  • the antibodies provided herein comprise an LC having an amino acid sequence of SEQ ID NO: 7 and an HC having an amino acid sequence of SEQ ID NO: 3.
  • the antibodies provided herein comprise an LC having an amino acid sequence of SEQ ID NO: 7 and an HC having an amino acid sequence of SEQ ID NO: 4.
  • the antibodies provided herein comprise an LC having an amino acid sequence of SEQ ID NO: 8 and an HC having an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 6. In some embodiments, the antibodies provided herein comprise and LC having an amino acid sequence of SEQ ID NO:
  • the antibodies provided herein comprise and LC having an amino acid sequence of SEQ ID NO:
  • the antibodies provided herein comprise an LC having an amino acid sequence of SEQ ID NO: 9 and an HC having an amino acid sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 7. In some embodiments, the antibodies provided herein comprise and LC having an amino acid sequence of SEQ ID NO:
  • the antibodies provided herein comprise and LC having an amino acid sequence of SEQ ID NO:
  • compositions comprising a formulated antibody are provided herein.
  • the antibody comprises HCs and LCs having the amino acid sequences as described above, in combination with sucrose, L-histidine, and polysorbate 80.
  • the antibody is present at a concentration of 50 mg/mL
  • the sucrose is present at a concentration of 8 % (w/v)
  • the L- histidine is present at a concentration of 10 mM
  • the polysorbate 80 (PS80) is present at a concentration of 0.05 % (v/v)
  • the formulation has a pH of 6.2.
  • the pharmaceutical composition is lyophilized.
  • unit dosage forms comprising a formulated antibody are provided herein.
  • the antibody comprises HCs and LCs having the amino acid sequences as described above and the unit dosage form comprises 215 mg of the antibody, 6.21 mg L-histidine, 344 mg sucrose, and 2.15 mg polysorbate 80.
  • the unit dosage form of the antibody is lyophilized.
  • the antibody comprises HCs and LCs having the amino acid sequences as described above, wherein the process comprises expressing the antibody in a cell culture, subjecting the antibody to at least one of a chromatography purification step and an ultrafiltration step to produce a purified antibody solution; and adjusting the purified antibody solution to produce an antibody formulation.
  • the antibody formulation comprises the purified antibody, sucrose, L-histidine, and polysorbate 80.
  • antibody formulation comprises the antibody at a concentration of 50 mg/mL, sucrose at a concentration of 8 % w/v, L-histidine at a concentration of 10 mM; and polysorbate 80 (PS80) at a concentration of 0.05 % v/v.
  • the antibody formulation is prepared such that it has a pH of 6.2. In some embodiments of the process for preparing the antibody formulation, the antibody formulation is lyophilized.
  • the lyophilized antibody formulation comprises sucrose, L- histidine, PS80, and an antibody that specifically binds human CD38.
  • the antibody comprises HCs and LCs having the amino acid sequences as described above.
  • the lyophilized antibody formulation is reconstituted in a diluent, thereby preparing the reconstituted antibody formulation.
  • the reconstituted antibody formulation comprises the antibody at a concentration of 50 mg/mL, sucrose at a concentration of 8 % w/v, L-histidine at a concentration of 10 mM; and PS80 at a concentration of 0.05 % v/v.
  • the reconstituted antibody formulation has a pH of 6.2.
  • methods of treating a patient having a Multiple Myeloma are provided.
  • the method comprises administering to the patient one or more doses of an antibody that specifically binds human CD38, wherein the antibody comprises HCs and LCs having the amino acid sequences as described above.
  • antibodies for use in a method of treating Multiple Myeloma are provided.
  • the antibody specifically binds human CD38, wherein the antibody comprises HCs and LCs having the amino acid sequences as described above.
  • FIG. 1 is a graph showing the onset of unfolding for mAbs 1, 2, 3, 5, 6, 7, and 8.
  • FIG. 2 is a graph showing isoelectric point (pi) for mAbs 1, 3, 5, 6, 7, and 8.
  • anti-CD38 antibodies that have superior antibody-dependent cellular cytotoxicity (ADCC) against cells expressing high, medium, and low levels of CD38 on their cell surfaces.
  • the anti-CD38 antibodies provided herein also have superior antibody-dependent cellular phagocytosis (ADCP) activity against cells expressing CD38 on their cell surfaces.
  • the antibodies provided herein have a lower than expected isoelectric point (pi). Despite the lower than expected pi and onset of protein unfolding at a lower temperature, which could negatively impact the stability of the antibody, especially in commercial production processes, the antibodies provided herein are provided in stable suitable form for administration to human patients.
  • Antibodies are provided in stable suitable form for administration to human patients.
  • anti-CD38 antibodies that specifically bind human CD38.
  • the anti-CD38 antibodies provided herein may be produced using recombinant methods.
  • nucleic acid encoding the antibody is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression.
  • DNA encoding the antibody may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • Many vectors are available.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • the vector is typically transformed into a host cell suitable for expression of the nucleic acid.
  • the host cell is a eukaryotic cell or a prokaryotic cell.
  • the eukaryotic host cell is a mammalian cell. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al, J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod.
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • CHO Chinese hamster ovary
  • DHFR-CHO cells Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)
  • myeloma cell lines such as NS0 and Sp2/0.
  • Yazaki and Wu Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 255-268.
  • the anti-CD38 antibody prepared from the cells can be purified using, for example, hydroxylapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being among one of the typically preferred purification steps.
  • affinity chromatography being among one of the typically preferred purification steps.
  • the antibody is expressed by the CHO 8D6 host cell line (DXB11 derivative) using a 500 L single-use bioreactor, operated in fed-batch mode.
  • the cell culture process is initiated by thawing a master cell bank vial, followed by a series of seed train cell expansion steps.
  • the cells are then transferred into a bioreactor and grown using a serum free, chemically-defined cell culture medium.
  • the culture is terminated for harvest of the antibodies after 10-14 days.
  • the cells and cell culture debris can be removed from the harvested cell culture by depth filtration.
  • the purified antibody is formulated in a liquid solution and is stored at ⁇ -30° C.
  • the liquid solution Prior to dispensing the formulated antibody into suitable vials, the liquid solution is thawed and sterile filtered using a 0.2 pm filtration device.
  • the formulated antibody is dispensed into a suitable container, such as a USP Type 1 glass vial, 4.3 mL/vial.
  • the filled vial includes a 0.3 mL overage of the formulated antibody.
  • the formulated antibody is lyophilized in the vial.
  • the term“antibody” typically refers to a tetrameric protein comprising two heavy chains (HC) and two light chains (LC). Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one LC (typically having a molecular weight of about 25 kDa) and one HC (typically having a molecular weight of about 50-70 kDa).
  • the terms“HC” and“LC” as used herein refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen.
  • the amino-terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids that typically is responsible for antigen recognition.
  • a full-length HC immunoglobulin polypeptide includes a variable domain (VH) and three constant domains (CHI, CH2, and CH3), wherein the VH domain is at the amino-terminus of the polypeptide and the CH3 domain is at the carboxyl-terminus, and a full-length LC immunoglobulin polypeptide includes a variable domain (VL) and a constant domain (CL), wherein the VL domain is at the amino-terminus of the polypeptide and the CL domain is at the carboxyl-terminus.
  • antibody is used herein in the broadest sense and specifically covers typical antibodies, as described above, including monoclonal antibodies, and multispecific antibodies (e.g., bi and tri-specific antibodies so long as they exhibit the desired biological activity including specific binding to the CD38 target and ability to trigger ADCC and ADCP).
  • Fc refers to a molecule comprising the sequence of a non-antigen-binding fragment resulting from digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region.
  • Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association.
  • the number of intermolecular disulfide bonds between monomeric subunits of typical Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgGl, IgG2, IgG3, IgAl, IgGA2, and IgG4).
  • class e.g., IgG, IgA, and IgE
  • subclass e.g., IgGl, IgG2, IgG3, IgAl, IgGA2, and IgG4
  • Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG.
  • the antibodies described herein may be isolated.
  • isolated protein isolated polypeptide or“isolated antibody” refers to a protein, polypeptide or antibody that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other proteins from the same species, (3) is expressed by a cell from a different species, and/or (4) does not occur in nature.
  • a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be“isolated” from its naturally associated components.
  • a protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
  • the antibodies provided herein comprise HC and LC amino acid sequences as provided in Table 1.
  • the term“affinity” refers to a measure of the attraction between two polypeptides, such as receptor/ligand or antigen/antibody, for example.
  • the intrinsic attractiveness between two polypeptides can be expressed as the binding affinity equilibrium constant (KD) of a particular interaction.
  • KD binding affinity equilibrium constant
  • An antibody is said to specifically bind to an antigen when the KD is ⁇ 1 mM, preferably ⁇ 100 nM.
  • a KD binding affinity constant can be measured, e.g., by surface plasmon resonance (SPR) (BIAcore®) or Bio-Layer Interferometry.
  • SPR surface plasmon resonance
  • koff refers to the dissociation rate constant of a particular antibody- antigen interaction. A koff dissociation rate constant can be measured, e.g., by Bio-Layer Interferometry.
  • the antibodies provided herein comprise a tetrameric protein comprising two HCs and two LCs and binds to human CD38 with an affinity or KD of about 1.91 x 10 10 M to about 6.7 x 10 10 M when measured by Surface Plasmon
  • the HC and LC have the amino acid sequences of SEQ ID NOs: 2 and 7 (mAb 2), 3 and 7 (mAb 3), 4 and 7 (mAb 4), 5 and 8 (mAb 5), 5 and 9 (mAb 6), 6 and 8 (mAb 7), or 6 and 9 (mAb 8), respectively.
  • the antibody comprises HC and LC having the amino acid sequences of SEQ ID NOs: 3 and 7, respectively, and the antibody binds to human CD38 with an affinity or KD of about 2 x 10 -10 M.
  • the antibody comprises HC and LC having the amino acid sequences of SEQ ID NOs: 5 and 8, respectively, and the antibody binds to human CD38 with an affinity or KD of about 6.7 x 10 -10 M.
  • the antibody comprises HC and LC having the amino acid sequences of SEQ ID NOs: 5 and 9, respectively, and the antibody binds to human CD38 with an affinity or KD of about 4.15 x 10 -10 M.
  • the antibody comprises HC and LC having the amino acid sequences of SEQ ID NOs: 6 and 8, respectively, and the antibody binds to human CD38 with an affinity or KD of about 3.85 x 10 -10 M. In some embodiments, the antibody comprises HC and LC having the amino acid sequences of SEQ ID NOs: 6 and 9, respectively, and the antibody binds to human CD38 with an affinity or KD of about 1.91 x 10 -10 M.
  • the antibodies provided herein are also capable of binding to the lower affinity FcyRIIIa (CD 16a) variant having phenylalanine (F) at amino acid position 158 (158F) and are capable of binding to the higher affinity FcyRIIIa (CD 16a) variant having valine (V) at amino acid position 158 (158V) with KDs within less than an order of magnitude, respectively, and with KDs of less than 100 nM as measured by SPR.
  • the antibodies provided herein bind to the 158F variant and to the 158V variant of FcyRIIIa (CD16a) with KDs of less than 100 nM as measured by SPR and where in the binding to the 158F and 158V variants differ by less than 2-fold. In some embodiments, the antibodies provided herein bind to FcyRIIIa (CD16a) variant (158F) with a KD of about 59 nM or less when measured, for example, by SPR.
  • the antibody comprises an HC having an amino acid sequence of SEQ ID NO: 2 and an LC having an amino acid sequence of SEQ ID NO: 7 and is capable of binding to FcyRIIIa (158F) with a KD of about 53 nM and of binding to FcyRIIIa (158V) with a KD or about 47 nM as measured, for example, by SPR.
  • the antibody comprises an HC having an amino acid sequence of SEQ ID NO: 3 and an LC having an amino acid sequence of SEQ ID NO: 7 and is capable of binding to FcyRIIIa (158F) with a KD of about 59 nM and is capable of binding to FcyRIIIa (158V) with a KD of about 75 nM as measured, for example, by SPR.
  • the antibody comprises an HC having an amino acid sequence of SEQ ID NO: 4 and an LC having an amino acid sequence of SEQ ID NO: 7 and is capable of binding to FcyRIIIa (158F) with a KD of about 51 nM and is capable of binding to FcyRIIIa (158V) with a KD of about 47 nM as measured, for example, by SPR.
  • the antibodies provided herein are also capable of binding to the lower affinity FcyRIIa (CD32a) variant having arginine at amino acid position 131 (131R) with a KD of ⁇ 690 nM and are capable of binding to the higher affinity FcyRIIa (CD32a) variant having a histidine at position 131 (131H) with a KD of less than about 270 nM as measured, for example, by SPR.
  • the antibody comprises an HC having an amino acid sequence of SEQ ID NO: 2 and an LC having an amino acid sequence of SEQ ID NO: 7 and is capable of binding to FcyRIIa (CD32a) (131R) with a KD of about 690 nM and of binding to FcyRIIa (CD32a) (131H) with a KD or about 120 nM as measured, for example, by SPR.
  • the antibody comprises an HC having an amino acid sequence of SEQ ID NO: 3 and an LC having an amino acid sequence of SEQ ID NO: 7 and is capable of binding to FcyRIIa (CD32a) (131R) with a KD of ⁇ about 125 nM or ⁇ 100 nM and is capable of binding to FcyRIIa (CD32a) (131H) with a KD of about 220 nm as measured, for example, by SPR.
  • the antibody comprises an HC having an amino acid sequence of SEQ ID NO: 4 and an LC having an amino acid sequence of SEQ ID NO: 7 and is capable of binding to FcyRIIa (CD32a) (131R) with a KD of about 510 nM and is capable of binding to FcyRIIa (CD32a) (131H) with a KD of about 60 nM as measured, for example, by SPR.
  • the antibody is also capable of binding to FcyRIIIa (CD16a) 158F with apparent KD of about 70 nM or less and to FcyRIIIa (CD16a) 158V with apparent KD of about 32 nM or less as measured by binding to FcyRIIIa (CD16a) 158F- or 158V-expressing HEK cells, respectively.
  • the antibody comprising an HC having an amino acid sequence of SEQ ID NO: 2 and an LC having an amino acid sequence of SEQ ID NO: 7 is also capable of binding to FcyRIIIa (CD16a) 158F with apparent KD of about 70 nM and is capable of binding FcyRIIIa (CD 16a) 158V with apparent KD of about 18 nM as measured by binding to FcyRIIIa (CD16a) 158F- or 158V-expressing HEK cells, respectively.
  • the antibody comprising an HC having an amino acid sequence of SEQ ID NO: 3 and an LC having an amino acid sequence of SEQ ID NO: 7 is also capable of binding to FcyRIIIa (CD16a) 158F with apparent KD of about 60 nM and is capable of binding FcyRIIIa (CD16a) 158V with apparent KD of about 32 nM as measured by binding to FcyRIIIa (CD16a) 158F- or 158V-expressing HEK cells,
  • the antibody comprising an HC having an amino acid sequence of SEQ ID NO: 4 and an LC having an amino acid sequence of SEQ ID NO: 7 is also capable of binding to FcyRIIIa (CD 16a) 158F with apparent KD of about 44 nM and is capable of binding FcyRIIIa (CD16a) 158V with apparent KD of about 28 nM as measured by binding to FcyRIIIa (CD16a) 158F- or 158V-expressing HEK cells, respectively.
  • the antibody is also capable of binding to FcyRIIa (CD32a) variant having arginine at amino acid position 131 (131 R) with apparent KD of about 890 nM or less and to FcyRIIa (CD32a) variant 131H with apparent KD of about 840 nM or less as measured by binding to FcyRIIa (CD32a) 131R- or 131H-expressing HEK cells, respectively.
  • the antibody comprising an HC having an amino acid sequence of SEQ ID NO: 2 and an LC having an amino acid sequence of SEQ ID NO: 7 is also capable of binding to FcyRIIa (CD32a) 131R with apparent KD of about 890 nM and is capable of binding FcyRIIa (CD32a) 131H with apparent KD of about 840 nM as measured by binding to FcyRIIa (CD32a) 131R- or 131H-expressing HEK cells, respectively.
  • the antibody comprising an HC having an amino acid sequence of SEQ ID NO: 3 and an LC having an amino acid sequence of SEQ ID NO: 7 is also capable of binding to FcyRIIa (CD32a) 131R with apparent KD of about 87 nM and is capable of binding FcyRIIa (CD32a) 131H with apparent KD of about 222 nM as measured by binding to FcyRIIa (CD32a) 131R- or 131H-expressing HEK cells, respectively.
  • the antibody comprising an HC having an amino acid sequence of SEQ ID NO: 4 and an LC having an amino acid sequence of SEQ ID NO: 7 is also capable of binding to FcyRIIa (CD32a) 131R with apparent KD of about 467 nM and is capable of binding FcyRIIa (CD32a) 131H with apparent KD of about 544 nM as measured by binding to FcyRIIa (CD32a) 131R- or 131H-expressing HEK cells, respectively.
  • the antibodies provided herein are capable of killing a CD38-expressing cell by Antibody Dependent Cellular Cytotoxicity (ADCC) of cells expressing high (-400, 000/cell), medium ( ⁇ 100,000/cell), and low (-13, 000/cell) levels of CD38 molecules on the cell surface.
  • ADCC Antibody Dependent Cellular Cytotoxicity
  • the antibodies provided herein trigger such ADCC in the presence of Natural Killer (NK) cells that express the lower affinity Fcyllla (CD16a) receptor variant (158F) and/or express the higher affinity Fcyllla (CD16a) receptor variant (158V).
  • ADCC can be measured using methods known in the art, for example by measuring target cell lysis in the presence of the antibody and effector cells in a cell-based potency assay.
  • the target cell can be, for example, a CD38 expressing cell such as KMS12-BM, RPMI-8226, or MOLP-8.
  • the effector cells can be, for example, any cell that can be induced to kill target cells by ADCC.
  • the effector cells are primary cells isolated from human blood such as peripheral blood mononuclear cells (PBMC) or human NK cells purified from PBMC.
  • the cells are a Natural Killer cell line such as NK-92 cells that express FcyRIIIa (158V) or FcyRIIIa (158F) on the surface of the cells (see WO
  • the NK cell line is a cell line such as a Jurkat cell line that has been engineered to express Fcyllla (CD 16a) 158F or 158V (see for example Promega, G701A).
  • the antibodies provided herein are capable of killing cells expressing a high level of CD38 on the cell surface by Antibody Dependent Cellular Phagocytosis (ADCP).
  • the antibodies provided herein trigger such ADCP in the presence of human PBMC.
  • the antibodies provided herein trigger about 41% phagocytosis of CD38- expressing cells by human PMBCs that express FcyRIIIa (158V) with a relative EC50 of 212.6 pM.
  • composition refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations typically are sterile. “Pharmaceutically acceptable” excipients (vehicles, additives) are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed.
  • compositions and formulations of the antibodies provided herein can be prepared by mixing the antibody, having the desired degree of purity, with one or more optional pharmaceutically acceptable carriers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), and can be provided in the form of lyophilized formulations or aqueous solutions.
  • the pharmaceutical compositions provided herein comprise a formulated antibody comprising an antibody and one or more of the following excipients: sucrose, L-histidine, and polysorbate 80 (PS80).
  • the antibodies comprise HC and LC amino acid sequences of any one of the monoclonal antibodies provided in Table 1.
  • the antibody is present in the pharmaceutical composition at a concentration of 5 mg/mL to 50 mg/mL.
  • the antibody is present in the pharmaceutical composition at a concentration of 5 mg/mL.
  • the antibody is present in the pharmaceutical composition at a concentration of 10 mg/mL.
  • the antibody is present in the pharmaceutical composition at a concentration of 20 mg/mL.
  • the antibody is present in the pharmaceutical composition at a concentration of 50 mg/mL.
  • the concentration of each excipient is chosen such that the pharmaceutical composition can be diluted for
  • the sucrose is present at a concentration of 8 % (w/v) to 10 % (w/v). In some embodiments, the sucrose is present at a concentration of 8 % (w/v). In other embodiments, the sucrose is present at a concentration of 10% (w/v).
  • the L-histidine is present at a concentration of 10 mM to 20 mM. In some embodiments, the L-histidine is present at a concentration of 10 mM. In some embodiments, the L-histidine is present at a concentration of 20 mM.
  • the PS80 is present at a concentration of 0.005 % (v/v) to 0.05 % (v/v). In some embodiments, the PS80 is present at a concentration of 0.005 % (v/v). In some embodiments, the PS80 is present at a concentration of 0.02 % (v/v). In some embodiments, the PS80 is present at a concentration of 0.05 % (v/v).
  • the pH of the pharmaceutical composition is optimized to maintain stability of the antibody for example when the pharmaceutical composition is in liquid form. In some embodiments, the pH of the formulated antibody has a pH of about 6.0 to about 6.5. In some embodiments, the formulated antibody has a pH of about 6.0.
  • the formulated antibody has a pH of about 6.2. In some embodiments, the formulated antibody has a pH of about 6.5. In some embodiments, the formulated antibody has a pH of 6.0. In some embodiments, the formulated antibody has a pH of 6.2. In some embodiments, the formulated antibody has a pH of 6.5.
  • the antibody of the pharmaceutical composition and/or formulated antibodies provided herein comprise one or more charged isoforms.
  • Charged isoforms are typically described as main isoform, acidic isoform(s), and basic isoform(s).
  • the main isoform refers to the most prevalent isoform in a given batch of antibody. Acidic isoform(s) have lower isoelectric point (pi) compared to the main isoform, and basic isoforms have higher pH compared to the main isoform.
  • Charged isoforms can result from post- translational modifications to the amino acid sequence of the HC and/or LC. For example, increases in deamidation, glycation and sialylation, may cause a decrease in the pi of an antibody. Conversion of an N-terminal glutamic acid to pyroGlu (or pyroQ) results in the loss of one positive charge, which may cause a decrease in the pi. In addition, the presence of a C-terminal lysine may cause an increase the pi of an antibody. Furthermore, amidation of a C-terminal proline may cause an increase in pi of the antibody.
  • the amino acid at position 1 of the HC amino acid sequence is pyroglutamine.
  • the HC has a C-terminal amino acid consisting of glycine.
  • Charged isoforms of the antibodies provided herein can be separated and quantified using methods known in the art for separating polypeptides based on charge.
  • a weak cation exchange column can be used in a high performance liquid chromatography (HPLC) system with a phosphate/sodium chloride gradient buffer.
  • HPLC high performance liquid chromatography
  • the acidic isoforms of the antibody would elute first, followed by the main isoform and then the basic isoforms of the antibody.
  • capillary isoelectric focusing cIEF
  • Capillary isoelectric focusing is a method that separates proteins by their isoelectric point (pi) values.
  • cIEF the antibody sample migrates to its isoelectric points on a pH gradient within the capillary, thereby resolving the different charge isoforms along the length of the capillary.
  • charge isoforms can be isolated by strong cation exchange chromatography (SCX) and analyzed by cIEF. An image of the resolved charged isoforms in the capillary is taken using a charge coupled device camera using whole column detection and measuring absorbance at 280 nm. The charge isoform distribution of the test sample is compared to a reference standard using cIEF to identify the charge isoforms of the antibody.
  • SCX strong cation exchange chromatography
  • cIEF is used as a quality control measure to confirm the identity of the antibody during commercial production, and by determining the charge isoform distribution of the antibody in comparison to a reference standard, e.g., a test antibody having a charge isoform distribution pattern within a predefined distribution pattern or compared to the distribution pattern of a reference standard.
  • the antibodies (including antibodies in the pharmaceutical composition, and formulated antibodies) comprise HC and LC amino acid sequences of any one of the monoclonal antibodies provided in Table 1, and one or more charged isoform parameters selected from the group consisting of: at least about 70% main isoform, no more than about 30% acidic isoform, and less than 4% basic isoform.
  • the antibody comprises 71% main isoform, 28% acidic isoform, and less than 4% basic isoform.
  • the antibody contains charge isoforms with isoelectric points ranging from about 5.8 to about 9.0.
  • the antibody contains charge isoforms with isoelectric points ranging from about 5.85 to about 8.97.
  • the antibodies are lyophilized.
  • the antibodies can be in a container (e.g., a vial) such that a prescribed dose of the antibody can be removed from the container for administration to a patient.
  • Unit dosage forms of formulated antibodies that specifically bind CD38 are provided herein.
  • the antibodies comprise HC and LC amino acid sequences of any one of the monoclonal antibodies provided in Table 1, and the unit dosage form comprises the antibody and one or more excipients selected from the group of sucrose, L-histidine, and polysorbate 80.
  • the unit dosage form comprises about 215 mg of the antibody, about 6.21 mg L-histidine, about 344 mg sucrose, and about 2.15 mg polysorbate 80.
  • the unit dosage form comprises 215 mg of the antibody, 6.21 mg L-histidine, 344 mg sucrose, and 2.15 mg polysorbate 80.
  • the unit dosage form is lyophilized.
  • the unit dosage form can be in a container (e.g., a vial) such that a prescribed dosage of the antibody can be removed from the container for administration to a patient.
  • the antibody, pharmaceutical formulation, and/or formulated antibody are in a glass vial fitted with elastomeric closure.
  • the vial contains about 215 mg of antibody. In some embodiments, the vial contains 215 mg of antibody.
  • the fill volume of the vial has been established to enable removal of about 4 mL. In some embodiments, the fill volume of the vial has been established to enable removal of 4 mL.
  • the antibodies comprise HC and LC amino acid sequences of any one of the monoclonal antibodies provided in Table 1.
  • the process comprises expressing the antibody in a suitable cell culture.
  • the antibody is subjected to at least one of a chromatography step and at least one ultrafiltration step, thereby producing a purified antibody solution.
  • the purified antibody solution is adjusted to concentrate the antibody and to add excipients and adjust pH.
  • the concentration of the antibody is adjusted to be about 50 mg/ml; sucrose, L- histidine, and polysorbate 80 are added, and the pH is adjusted to be at least about 6.0.
  • the concentration of the antibody is adjusted to be 50 mg/ml; sucrose, L- histidine, and polysorbate 80 are added, and the pH is adjusted to be at least 6.0.
  • the pH is about 6.2. In some embodiments, the pH is 6.2.
  • the sucrose is at a concentration of about 8 % w/v
  • the L-histidine is at a concentration of about 10 mM
  • the polysorbate 80 is at a concentration of about 0.05 % v/v.
  • the sucrose is at a concentration of 8 % w/v
  • the L-histidine is at a concentration of 10 mM
  • the polysorbate 80 is at a concentration of 0.05 % v/v.
  • the antibody, pharmaceutical composition and/or formulated antibody are lyophilized.
  • methods of preparing reconstituted formulated antibody are provided wherein the formulated antibody is provided in lyophilized form and comprises HC and LC amino acid sequences of any one of the monoclonal antibodies provided in Table 1, sucrose at a concentration of about 8 % w/v, L-histidine at a concentration of about 10 mM, and polysorbate 80 at a concentration of about 0.05 % v/v.
  • methods of preparing reconstituted formulated antibody are provided wherein the formulated antibody is provided in lyophilized form and comprises HC and LC amino acid sequences of any one of the monoclonal antibodies provided in Table 1, sucrose at a concentration of 8 % w/v, L- histidine at a concentration of 10 mM, and polysorbate 80 at a concentration of 0.05 % v/v.
  • the formulated antibody prior to use is reconstituted in a suitable volume of water for injection to produce a solution comprising the antibody at a
  • the formulated antibody prior to use is reconstituted in a suitable volume of water for injection to produce a solution comprising the antibody at a concentration of 50 mg/mL, sucrose at a concentration of about 8 % w/v, L-Histidine at a concentration of about 10 mM, and polysorbate 80 at a concentration of about 0.05 % v/v, wherein the pH of the reconstituted antibody is about 6.2.
  • the formulated antibody prior to use the formulated antibody is reconstituted in a suitable volume of water for injection to produce a solution comprising the antibody at a concentration of 50 mg/mL, sucrose at a
  • treatment refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis.
  • an individual is successfully“treated” if one or more symptoms associated with cancer are mitigated or eliminated, including, but not limited to, reducing the proliferation of cancerous cells, destroying cancerous cells, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, and/or prolonging survival of individuals.
  • kits for treating or delaying progression of multiple myeloma comprising administering to a subject in need thereof an effective amount of an anti-CD38 antibody provided herein.
  • the antibodies comprise HC and LC amino acid sequences of any one of the monoclonal antibodies provided in Table 1.
  • the antibody is capable of improving survival of mice in a murine MM model, wherein mice in the model express the low affinity variant of human CD16a (158F), and wherein the mice have been injected with 500,000 EL4 cells expressing human CD38.
  • one or more doses of an anti-CD38 antibody is provided as a lyophilized formulation, wherein prior to administration, the lyophilized formulation is reconstituted forming a reconstituted antibody formulation such that the reconstituted antibody formulation has a pH of 6.2 and comprises the antibody at a concentration of about 50 mg/mL in a liquid comprising about 10 mM L-histidine, about 8 % w/v sucrose, and about 0.05 % v/v polysorbate 80.
  • one or more doses of an anti-CD38 antibody is provided as a lyophilized formulation, wherein prior to administration, the lyophilized formulation is reconstituted forming a reconstituted antibody formulation such that the reconstituted antibody formulation has a pH of 6.2 and comprises the antibody at a concentration of 50 mg/mL in a liquid comprising 10 mM L-histidine, 8 % w/v sucrose, and 0.05 % v/v polysorbate 80.
  • one or more doses of antibody is administered to the patent.
  • the dose can be about 0.1, about 0.2, about 0.3, about 0.5, about 1.0, about 2.5, about 5, about 10, or about 20 mg/kg of the antibody.
  • the dose can be 0.1, 0.2, 0.3, 0.5, 1.0, 2.5, 5, 10, and 20 mg/kg of the antibody.
  • the antibody is administered intravenously. In some embodiments, the antibody is administered subcutaneously.
  • the term“about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field.
  • Reference to“about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.
  • the term“about” signifies a stated value plus or minus 10% of said value; for example,“about 10 mg/kg” can encompass 9 to 11 mg/kg.
  • the term“about” signifies a stated value plus or minus 5% of said value; for example,“about 20 mg/kg” can encompass 19 to 21 mg/kg.
  • A“subject” or an“individual” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.
  • the mammal is human.
  • Fc-engineered anti-CD38 antibodies were generated having mutations in the Fc part of the antibody to enhance the affinity for the activating FcyRIIIa and FcyRIIa receptors on effector cells.
  • Two humanized heavy chains (HC) (SEQ ID NO: 5 and SEQ ID NO: 6) carry 14 and 10 mutations in the framework regions (compared to SEQ ID NO: 1) which result in an increase in Homo sapiens germinality score from 74.49 % to 88.78 % and 84.69 %, respectively.
  • LC humanized light chains
  • SEQ ID NO: 8 and SEQ ID NO: 9 carry 17 and 15 mutations in the framework regions (compared to SEQ ID NO: 7) which result in an increase in Homo sapiens germinality score from 64.36 % to 81.19 % and 79.21 %, respectively.
  • methionine at position 11 was substituted with leucine to avoid a potential problematic methionine oxidation.
  • Table 2 The HC and LC sequences are provided in Table 2, and the mAh HC and LC pairings are provided in Table 3.
  • binding affinity and kinetic constants of mAbs 1-8 for human CD38 was measured by SPR using a Biacore® instrument. Briefly, anti-CD38 antibodies were captured to a CM5 chip to which anti-Fc antibody had been covalently bound. Multiple concentrations of huCD38 were injected over the captured antibodies being tested. Binding curves (sensorgrams) were generated and kinetic analyses were performed using the Biacore Evaluation software.
  • binding affinity of mAbs 1 and 3 to human CD38 was measured by SPR using a Biacore® instrument as described above.
  • mAb 1 has a Kd of 0.22 nM and mAb 3 had a Kd of 0.20 nM.
  • affinity of the mAbs for FcyRlIIa 158V receptors or FcyRlIIa 158F receptors was measured using Surface Plasmon Resonance (SPR).
  • the mAbs were produced in HEK293 cells by transient transfection according to supplier (ThermoFisher Scientific), and purified by protein A affinity chromatography according to supplier (GE Healthcare).
  • FcyRlIIa (CD16a) extracellular domains with V158 or F158 were produced with a C-terminal histag in HEK293 cells by transient expression and purified by
  • Immobilized Metal Affinity Chromatography according to the supplier (Ni- Sepharose GE, Healthcare). Affinity by SPR on BIACore 2000 (GE Healthcare) was performed by capture assay using immobilized anti -His IgG on CM5 chip. FcyRIIIa-histag was then added to the running buffer, followed afterwards by the anti-CD38 mAh at concentrations ranging from 8 to 256 nM. Analysis was performed using the two-state reaction with acceptable c 2 values of less than 5 (meaning 2% of the highest tested concentration) and a percentage of maximal theoretical resonance unit greater than 25 (meaning more than 1 ⁇ 4 productive interaction), using BIAevaluation Software 4 1 (GE Healthcare).
  • mAbs 2-4 have a 9-fold higher affinity for the FcyRlIIa 158V than mAh 1 and more than 27-fold higher affinity for the FcyRlIIa 158F receptor than mAh 1.
  • FcyRIIa extracellular domains with R131 or HI 31 were provided by R&D System (batch 1330-CD) or Biorbyt (batch ORB138408), respectively.
  • the assay was a bead based proximity assay described by supplier (Perkin Elmer), permitting a ranking of the anti-CD38 Fc variants by competition to anti-CD38 with native IgGl. Biotinylated-anti-CD38 with native IgGl was captured on the donor beads, and FcyRIIa histag-protein was captured on the acceptor beads. When anti-CD38 and FcyRIIa interact, beads are brought into proximity.
  • Excitation of the donor beads at 680 nm causes the release of singlet oxygen, which diffuses and triggers the emission of light at 520-620 nm from the acceptor beads, when in proximity.
  • the amount of light is directly proportional to the degree of interaction and it is measured with the EnVision multimode plate reader (Perkin Elmer).
  • the untagged competitive mAbs anti-CD38 Fc-variants DE S239D/I332E, Eu numbering
  • ADE G236 A/S239D/I332E, Eu numbering
  • ADLE G236A/S239D/F243L/I332E, Eu numbering
  • the untagged competitive mAbs were used at varying levels such that increasing concentration of untagged mAbs can displace the biotinylated anti-CD38 and decreases the signal. Data were normalized for maximal signal. Two experiments were run and IC50 evaluated by BIOST@T-SPEED-LTS 2.1.0.
  • HEK293 T-F cyRIIa- 131 R, HEK293T-FcyRI or HEK293T-FcyRIIb were seeded in 96-well microplates at 10 5 cells/well.
  • the plates were centrifuged at 800 g for 1 minute and resuspended in 50 mL PBS containing the antibodies at different concentrations for 30 minutes at 4°C. 200 mL PBS 1% FBS was then added to wash the cells, followed by a 800 g centrifugation for 1 minute.
  • the washing step was repeated three times in total before staining the cells with the secondary antibody conjugated with FITC (Fragment Goat Anti- human IgG (H+L) FITC, ref 109-546-088 Jackson ImmunoResearch, 10 pg/mL final) for 30 minutes at 4°C.
  • FITC Frignment Goat Anti- human IgG (H+L) FITC, ref 109-546-088 Jackson ImmunoResearch, 10 pg/mL final
  • Cells were washed with 200 mL PBS 1% FBS three times and resuspended in 100 mL PBS before acquisition on MACSVYB (B1 channel).
  • mAh 2, 3 and 4 had a 32-, 38-, and 51 -fold higher affinity for FcyRIIIa 158F expressed on HEK cells compared to mAh 1, respectively, and mAh 2, 3 and 4 had a 9-, 5-, and 6-fold higher affinity for FcyRIIIa 158V expressed on HEK cells compared to mAh 1, respectively.
  • Table 8
  • mAh 3 had a ⁇ 5-fold higher affinity for FcyRIIa 131H expressed on HEK cells compared to mAh 1
  • mAh 3 had a ⁇ 16-fold higher affinity for FcyRIIa 131R expressed on HEK cells compared to mAh 1.
  • mAh 2, 3 and 4 had a similar level of binding affinity for FcyRI compared to mAh 1 and a 1.8- to 3.9-fold lower affinity for FcyRIIb expressed on HEK cells compared to mAh 1.
  • ADCC ANTIBODY DEPENDENT CELLULAR CYTOTOXICITY
  • ADCC Antibody Dependent Cell Cytotoxic assays using NK92 cell line engineered to over-express the FcyRIIIa receptor as effector cells were examined using a calcein-acetyoxymethyl (Calcein-AM; Invitrogen) release assay.
  • Calcein-AM calcein-acetyoxymethyl
  • the non- fluorescent calcein AM is converted to a green-fluorescent calcein.
  • the labeled target cells are incubated with SAR442085 antibody and the NK-92 effector cells. Lysis of the target cells through ADCC leads to release of the fluorescent calcein.
  • the intensity of fluorescence is proportional to the level of ADCC activity present.
  • the multiple myeloma target cells (MOLP-8, RPMI 8226, MM1R or KMS12-
  • BM BM
  • Calcein-AM 50 mg diluted in 25 mL DMSO, then diluted 10 mL calcein in 4 mL RPMI 1640 + 1% FBS + 1% Probenecid for staining of 4 x 10 6 cells
  • NK cells expressing 158F or 158V were added as effector cells at an E:T ratio of 5: 1 (1 x 10 5 NK cells for 2 x 10 4 target cells).
  • the plates were then incubated for 1 h at 37 °C in a humidified incubator with 5% CO2, and 100 mL of supernatants were harvested and transferred into opaque 96-well microplates for analysis using fluorometry on Tecan Infinite Ml 000 to measure calcein release (excitation filter: 492 nm; emission: 515 nm).
  • the cells were lysed with 2% Triton X-100.
  • the fluorescence value of the culture medium background was subtracted from that of the experimental release (A), the target cell spontaneous release (B), and the target cell maximal release (C).
  • the cytotoxicity and ADCC percentages for each plate were calculated using the following formula:
  • ADCC of multiple myeloma cell line MOLP-8 was assessed in vitro as described above. MOLP-8 cells exhibit a high CD38 receptor density on the cell surface
  • Table 11 shows EC50 of each tested antibody against MOLP-8 cells in the presence of NK cells expressing 158V. As shown in Table 11, in the presence of NK cells expressing the high affinity variant of FcyRIIIa (158V), mAbs 2, 3, and 4 triggered ADCC of MOLP-8 cells with an EC50 of 24- to 33-fold lower than mAb 1.
  • Table 12 shows EC50 of each tested antibody against MOLP-8 cells in the presence of NK cells expressing 158F.
  • mAh 4 showed the strongest ADCC activity against MOLP-8 cells in the presence of NK cells expressing the low affinity FcyRIIIa variant (158F), with and EC50 of about 97-fold lower than mAh 1.
  • mAbs 2 and 3 triggered ADCC of MOLP-8 cells with an EC50 of about 59- to 69-fold lower than mAh 1 in the presence of NK cells expressing the low affinity FcyRIIIa variant (158F).
  • ADCC of multiple myeloma cell line RPMI-8226 was assessed in vitro as described above. RPMI-8226 cells exhibit a medium CD38 receptor density on the cell surface ( ⁇ 70, 000/cell).
  • Table 13 shows EC50 of each tested antibody against RPMI-8226 cells in the presence of NK cells expressing 158V. As shown in Table 13, mAbs 2-4 demonstrated similar levels of enhanced ability with an EC50 of about 27-fold lower compared to mAh 1 to trigger ADCC against MM cells expressing a medium CD38 receptor density in the presence of NK cells expressing the higher affinity variant of FcyRIIIa (158V).
  • Table 14 shows EC50 of each tested antibody against RPMI-8226 cells in the presence of NK cells expressing 158F.
  • mAh 4 demonstrated about a 73-fold enhanced ability compared to mAh 1 to trigger ADCC against MM cells expressing a medium CD38 receptor density in the presence of NK cells expressing the low affinity variant of FcyRIIIa.
  • mAh 3 demonstrated about a 65 -fold
  • mAh 2 demonstrated about a 49-fold enhanced ability to trigger ADCC against MM cells expressing a medium CD38 receptor density in the presence of NK cells expressing the low affinity variant of FcyRIIIa compared to mAh 1.
  • ADCC of multiple myeloma cell line KMS-12BM was assessed in vitro as described above.
  • KMS-12BM cells exhibit a low CD38 receptor density on the cell surface ( ⁇ 13,000/cell).
  • Table 15 shows EC50 of each tested antibody against KMS-12BM cells in the presence of NK cells expressing the higher affinity variant of FcyRIIIa (158V).
  • mAbs 2, 3, and 4 demonstrated enhanced ability compared to mAh 1 to trigger ADCC against MM cells expressing a low CD38 receptor density in the presence of NK cells expressing the higher affinity variant of FcyRIIIa (158V).
  • mAh 3 had an EC50 of ⁇ 69-fold lower than mAh 1
  • mAh 4 had an EC50 of ⁇ 91-fold lower than mAh 1.
  • Table 16 shows EC50 of each tested antibody against KMS-12BM cells in the presence of NK cells expressing the lower affinity variant of FcyRIIIa (158F).
  • mAbs 2, 3, and 4 demonstrated enhanced ability to trigger ADCC compared to mAh 1 against MM cells expressing a low CD38 receptor density in the presence of NK cells expressing the lower affinity variant of FcyRIIIa (158F).
  • mAh 3 had an EC50 of ⁇ 145-fold lower than mAh 1
  • mAh 4 had an EC50 of ⁇ 269-fold lower than mAh
  • ADCP ANTIBODY DEPENDENT CELLULAR PHAGOCYTOSIS
  • FCGR2A genotype (FcyRIIIa- 158V/V and FcyRIIa- 131H/H).
  • Blood was first collected in a 50 mL Falcon tube. 15 mL of Ficoll-PlaqueTM PLUS 96% was added very gently at the bohom of a Sepmate tube, then blood added slowly in the tube before centrifugation at 1,300 g for 10 minutes. The PBMC ring was then collected and washed with 50 mL PBS and another round of centrifugation at 300 g for 5 minutes. The washing process was repeated two times before PBMC staining with anti-CD 14 magnetic beads according to
  • macrophages To collect macrophages, accutase (ThermoFisher, ref: A1110501) was put in flasks for 15 minutes at 37° C to detach cells, then 20 mL RPMI 10% FBS 1% Glutamine was added to stop accutase activity. Collected cells were centrifuged at 350 g for 10 minutes followed by a wash in 20 mL PBS and another round of centrifugation at 300 g for 10 minutes. For two experiments, the macrophages used were from a macrophage batch frozen in liquid nitrogen.
  • Macrophages were resuspended in diluent C at 20x10 6 cells/mL (provided in manufacturer’s staining kit # PKH26GL-1KT from Sigma Aldrich). For 1 volume of macrophages, 1 volume of PKH26 (20 mL diluted in 1 mL diluent C) was added to the cells for 5 minutes incubation in the dark.
  • MOLP-8 cells were stained with PKH67 fluorochrome and macrophages derived from purified monocytes were stained with PKH26, making them distinguishable by flow cytometry. Then MOLP-8 cells and macrophages were put in culture in the presence of either mAh 1 or mAh 3 overnight before analysis of the double positive population
  • mAh 3 exhibited an enhanced ADCP activity as compared to mAh 1 against MOLP-8 cell line as demonstrated by the higher percentage (%) of total phagocytosis achieved in all experiments and the lower EC50 (relative) value (EC50rel).
  • the geometric mean of the % of total phagocytosis was 40.85% for mAh 3 versus 18.57% for mAh 1 and the geometric mean of EC50rel was 31.87 ng/mL (or 212.57 pM) for mAh 3 versus mAh 1 for which the value was over 64.86 ng/mL (or over 432.62 pM).
  • mAh 3 appeared to be at least 2.035 times better than mAh 1 for the ADCP activity.
  • Humanized FcyR C57BL/6 mice recapitulate huFcyR expression patterns and expression levels and are functional in a variety of huIgG-mediated models of inflammation, cytotoxicity, and tumor clearance.
  • the human variants inserted in the mouse model were, respectively, huFcyRIIIa 158F and huFcyRIIa 131R (the low affinity variants of the receptors).
  • the profile of human FcyR expression pattern and expression levels were recapitulated in the mouse. [0093] Starting on day 1 post tumor cell injection, the test or control mAbs were administered intraperitoneally on days 1, 4, 7, and 14.
  • the isotype control mAh was administered at 10 mg/kg.
  • mAbs 1, 3, and 10 were administered at 10 and 1.25 mg/kg.
  • the primary efficacy end points were Median Survival Time (MST), the percent Increased Lifespan (%ILS), and the long term survivors (defined as mice with survival duration superior or equal to 2 times the MST of control group).
  • MST Median Survival Time
  • %ILS percent Increased Lifespan
  • the survival differences between groups were evaluated with a Cox model.
  • the isotype control group had an MST of 39 days. Twenty percent of the isotype control group exhibited long term survival with this model. The group treated with mAh 1 at 10 mg/kg had a MST of more than 82.5 days, an increased lifespan greater than 112%. Fifty percent of the group treated with 10 mg/kg of mAh 1 was long term survivors. Mice treated with 1.25 mg/kg of mAh 1 had an MST greater than 46.5 days, and an increased lifespan greater than 19%. Thirty percent of the group treated with 1.25 mg/kg of mAh 1 was long term survivors.
  • the group treated with mAh 10 at 10 mg/kg had a MST of more than 70 days, an increased lifespan of 70%. Fifty percent of the group treated with 10 mg/kg of mAh 10 was long term survivors. Mice treated with 1.25 mg/kg of mAh 10 had an MST greater than 42 days, and an increased lifespan of 8%. Only 10 % of the group treated with 1.25 mg/kg of mAh 10 was long term survivors.
  • the group treated with mAh 3 at 10 mg/kg had a MST of more than 90 days, an increased lifespan greater than 131%.
  • 90 % of the group treated with 10 mg/kg of mAh 3 was long term survivors.
  • mice treated with 1.25 mg/kg of mAh 3 also had an MST greater than 90 days, and an increased lifespan greater than 131%.
  • 90% of the group treated with 1.25 mg/kg of mAh 3 was long term survivors.
  • thermostability of mAbs 1, 3, 5, 6, 7, and 8 was measured by differential scanning calorimetry (DSC) using a Malvern MicroCal VP calorimeter.
  • the samples were tested in a buffer comprising 10 mM histidine HC1 at pH 6.0.
  • the prepared samples were loaded into wells of a Wheaton 96-well round bottom plate, along with buffer as reference, in triplicate. Using a temperature ramp rate of 1° C/min and a range of 20-120° C, thermal scans were collected and processed using origin software 2 0
  • mAbs 3, 5, 6, 7, and 8 showed an onset of protein conformation change at about 40° C, at a temperature of about 17 degrees lower than the onset temperature of mAb 1 and 15-20° C lower than is typical for mAbs (FIG. 1).
  • the isoelectric point (pi) of each of mAbs 1, 3, 5, 6, 7, and 8 was measured by capillary isoelectric focusing (cIEF).
  • a lyophilized formulation was developed to ensure adequate stability of mAh 3 that was suitable for storage of the mAh at ⁇ -30° C and for storage of the lyophilized mAh at 2-8° C.
  • mAh 3 formulations using different buffering systems and at different pHs were tested in stability studies comprised of freeze-thaw cycling, agitation studies, and short-term (12 weeks) incubation at stress, accelerated and intended storage conditions of 40° C, 25° C, 5° C, -30° C, and -80 ° C.
  • aggregation and chemical degradation of the protein were evaluated in addition to particle formation (visual inspection, light obscuration-based particle counting, and micro-flow imaging).
  • a lyophilization process was developed for mAh 3 to ensure that the formulated antibody had acceptable stability and cake attributes following lyophilization.
  • Process parameters of the lyophilization cycle drying temperatures, chamber pressure, etc.
  • the robustness of the cycle was established by conducting a series of lyophilization runs with controlled excursions in freezing rate, drying temperatures and chamber vacuum pressure.
  • the stability studies on lyophilized formulated antibody comprised of short-term (12 weeks) incubation at stress, accelerated and intended storage conditions of 40° C, 25° C, and 5° C.
  • lyophilized powder attributes were evaluated in addition to aggregation, chemical degradation, and particle formation.
  • the composition of mAh 3 was selected as 50 mg/mL mAh 3, 10 mM L-histidine-HCl, 8% w/v sucrose, 0.05% w/v polysorbate 80, and at a pH 6.2.
  • the formulated antibody is stored at ⁇ -30° C and lyophilized formulated antibody is stored at 2 - 8° C.

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Abstract

L'invention concerne des anticorps qui se lient spécifiquement au CD38 humain, des formulations et des formes posologiques unitaires comprenant les anticorps, des procédés de préparation de ces anticorps et des procédés d'utilisation de ces anticorps.
EP20726267.6A 2019-04-23 2020-04-23 Formulations et anticorps anti-cd38 Pending EP3958898A1 (fr)

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KR20220003562A (ko) 2022-01-10
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SG11202111602YA (en) 2021-11-29
CA3137365A1 (fr) 2020-10-29
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