WO2023111018A1 - Anticorps contre le facteur de coagulation x et leurs utilisations - Google Patents

Anticorps contre le facteur de coagulation x et leurs utilisations Download PDF

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WO2023111018A1
WO2023111018A1 PCT/EP2022/085850 EP2022085850W WO2023111018A1 WO 2023111018 A1 WO2023111018 A1 WO 2023111018A1 EP 2022085850 W EP2022085850 W EP 2022085850W WO 2023111018 A1 WO2023111018 A1 WO 2023111018A1
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antibody
seq
amino acid
acid sequence
anyone
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PCT/EP2022/085850
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English (en)
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Tristan W. WASLEY
Elizabeth A. BOOTH
Charles HOLZ
Robyn CASSAN
Jody Melton Witt
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F. Hoffmann-La Roche Ag
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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
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • 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

  • compositions and kits comprising said antibodies and to methods for detecting, inhibiting and/or depleting FX from samples with said antibodies, compositions or kits or uses thereof.
  • present invention also refers to compositions generated from blood-derived samples by depleting and/or inhibiting FX, or fragments thereof, and their uses as abnormal coagulation controls in in vitro assays.
  • Coagulation Factor X also called Stuart factor, is a vitamin-K dependent serine protease zymogen that plays a central role in blood coagulation cascade as its activation is the convergent step of the two coagulation initiation routes.
  • the blood coagulation cascade consists of a set of coagulation factors such that each plays a specific role in clot formation.
  • the activation of these factors is referred to as a cascade due to the sequential nature of the activation.
  • the intrinsic pathway is activated by contact with negatively charged surfaces while the extrinsic pathway is activated by tissue injury and release of tissue factor (TF or Factor III).
  • tissue factor tissue factor
  • These two pathways intersect in the common pathway with the activation of FX and the formation of the activation complexes for FX are referred to as the intrinsic or extrinsic ‘X-ase’ where the complexes consist of a substrate, enzyme and cofactor ( Figure 1 ).
  • Activated Factor X in combination with the cofactor Factor Va, convert Prothrombin to Thrombin (Factor Ila).
  • Activated thrombin cleaves fibrinogen converting it to fibrin which can polymerize to form a clot.
  • FX deficiency is a rare blood disorder, that can be either hereditary or acquired, that causes coagulation process to take longer than normal, leading people bearing this deficiency to bleed for a longer period than those bearing normal FX.
  • Hereditary FX deficiency affects both men and women and can occur in individuals of any ethnic or racial group. The disorder is estimated to affect about 1 in every 500,000-1 ,000,000 people in the general population. Rare disorders like FX deficiency often go unrecognized or misdiagnosed, making it difficult to determine the true frequency in the general population.
  • FX deficiency The signs and symptoms of FX deficiency are highly variable mostly depending on the extent of reduction in the activity of the FX. Clinical manifestations are most commonly seen as mucocutaneous bleeds (particularly epistaxis), hemarthroses (soft tissue and joint bleeding) and can include severe bleedings as bleeding within the central nervous system or the gastrointestinal tract, among others. In addition to exhibiting the general bleeding symptoms associated with FX deficiency, women and girls with this disorder may also experience heavy menstrual bleeding and complications associated with pregnancy and childbirth.
  • FX deficiency There are several diseases that may be caused by a FX deficiency including inborn deficiency in FX (Stuart- Power factor deficiency), adsorption of FX into amyloid fibrils (amyloidosis), or deficiency in vitamin K leading to production of inactive FX which could also be antagonized by warfarin therapy often to prevent thrombosis.
  • Hemophilia a disease that negatively impacts the ability of a person to form a clot, is a serious and potentially life-threatening disease and can arise from a variety of mutations to the blood factors or deficiency in blood factors involved in the clotting cascade. Hemophilia is characterized by excessive bruising and bleeding, depriving the body of nutrition and oxygen, and in more severe cases, fainting and eventually death.
  • FX deficiency The diagnosis of FX deficiency is usually suspected when both the PT and the APTT tests are abnormal and correct with a 1 :1 mix with normal plasma. However, further specific assays are required to confirm a diagnosis and differentiate FX deficiency from deficiencies in other clotting factors. Thus, FX functional activity (FX:C) must be quantified by performing serial dilutions with FX-deficient plasma.
  • Additional methods available to support diagnosis include protein characterization with different assays as the dilute Russell Viper Venom (RVV) assay (a metalloproteinase that activates FX directly and can therefore detect FX deficiency if FX-deficient plasma is used as substrate, chromogenic assays (spectrophotometric detection of a substrate sensitive to activated FX), and immunological assays (e.g. an enzyme-linked immunosorbent assay [ELISA] that measures FX antigen).
  • RVV dilute Russell Viper Venom
  • chromogenic assays spectrophotometric detection of a substrate sensitive to activated FX
  • immunological assays e.g. an enzyme-linked immunosorbent assay [ELISA] that measures FX antigen.
  • Blood factor depleted plasma and in particular FX-depleted plasma, are essential reagents in all these assays and play a critical role in the diagnosis of hemostatic disorders, such as FX-deficiency.
  • the target blood factor needs to be >99% depleted from the plasma while the other blood factors are retained at >50% in the plasma in order to ensure that the activity of the depleted factor is the only one affecting the result of the assay.
  • >1 g/L fibrinogen must be retained, and all coagulation inhibitors must be absent.
  • These depleted plasma reagents are then used in a variety of test formats including but not limited to ELISAs, benchtop coagulation assays, and automated hemostasis analyzers. With FX-depleted blood plasma, it is possible to identify patients with this specific deficiency. Identification and understanding of the specific deficiency that a patient suffers supports providing better treatment and control of the disease.
  • a first aspect of the present invention relates to an antibody that binds to an epitope of FX comprising the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24, and in a preferred embodiment to an antibody that binds to an epitope of FX with the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24.
  • FX or “Factor X” are used interchangeably and refer to a protein consisting of the nonactivated form of the coagulation Factor X, also known as Stuart factor. "FX”.
  • FX zymogen consists of an N-terminal light chain of 139 amino acids (SEQ ID NO: 28) and a C-terminal heavy chain of 306 amino acids (SEQ ID NO:26) which are covalently linked via a disulfide bridge.
  • FX within the above definition includes proteins that have the amino acid sequence of native human FX in its different stages of processing as explained above and also proteins with a slightly modified amino acid sequence, for instance, a modified N-terminal end including N-terminal amino acid deletions or additions.
  • FX within the above definition also includes natural allelic variations that may exist and occur from one individual to another.
  • FX within the above definition further includes variants of FX. Such variants differ in one or more amino acid residues from the wild type sequence. Examples of such differences may include truncation of the N-and/or C-terminus by one or more amino acid residues (e.g.
  • amino acid residues 1 to 10 amino acid residues
  • addition of one or more extra residues at the N- and/or C-terminus e.g. addition of a methionine residue at the N-terminus
  • conservative amino acid substitutions i.e. substitutions performed within groups of amino acids with similar characteristics, e.g. (1 ) small amino acids, (2) acidic amino acids, (3) polar amino acids, (4) basic amino acids, (5) hydrophobic amino acids, (6) aromatic amino acids.
  • antibody includes polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies, recombinant antibodies, chimeric antibodies, and antibody fragments.
  • antibody fragments include but are not limited to variable fragments (Fv), single-chain Fvs (scFv), bispecific antibodies (sc(Fv)2), single chain antibodies, single domain antibodies, Fab fragments, F(ab')2 fragments, Fab’ fragments, disulfide-linked Fv (dsFv), chemically conjugated Fv (ccFv), diabodies and anti-idiotypic (anti-ld) antibodies, and functionally active epitope-binding fragments of any of the above.
  • antibodies also include affibodies, nanobodies, and unibodies.
  • particular antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an antigen binding site.
  • Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA and IgY), class (for example, lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass.
  • the terms “monoclonal antibody” or “mAb” refer to an antibody composition having a homogeneous antibody population that bind to the same epitope.
  • the term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made.
  • the term encompasses antibodies obtained from murine hybridomas, as well as human monoclonal antibodies obtained using human rather than murine hybridomas.
  • recombinant antibody refers to antibodies or fragments thereof that are non-naturally occurring and can be associated with a polypeptide or fragment thereof that is not found in nature. Recombinant antibodies can be produced by any of the recombinant techniques well known by the skilled person.
  • Fab antigen-binding fragment
  • an antibody comprises a fragment crystallizable region (Fc) and two antigen-binding fragments (Fab).
  • Fc fragment crystallizable region
  • Fab antigen-binding fragments
  • the Fab fragments can be separated from the Fc region resulting in two Fab fragments, which is also known as F(ab')2 fragment or dimeric fragment antigen binding.
  • epitope refers to the portion of an antigen to which an antibody specifically binds.
  • epitopope includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor.
  • said antibody binds to FX and does not bind to FXa and in another embodiment, said antibody binds to the activation peptide of FX.
  • FXa or “Factor Xa” are used interchangeably and refer to a protein consisting of the activated form of the coagulation Factor X.
  • FXa differs from FX in that the activation peptide (SEQ ID NO: 29) is cleaved off from the heavy chain, resulting in a different C-terminal heavy chain of 254 amino acids (SEQ ID NO: 27) covalently linked via a disulfide bridge to the N-terminal light chain of 139 amino acids (SEQ ID NO: 28).
  • FXa within the above definition includes proteins that have the amino acid sequence of native human FXa as explained above and also proteins with a slightly modified amino acid sequence and also proteins with a slightly modified amino acid sequence, for instance, a modified N-terminal end including N-terminal amino acid deletions or additions, as long as they retain the activity of FXa.
  • FXa within the above definition also includes natural allelic variations that may exist and occur from one individual to another.
  • FXa” within the above definition further includes variants of FXa. Such variants differ in one or more amino acid residues from the wild type sequence. Examples of such differences may include truncation of the N-and/or C- terminus by one or more amino acid residues (e.g.
  • amino acid residues 1 to 10 amino acid residues
  • addition of one or more extra residues at the N- and/or C-terminus e.g. addition of a methionine residue at the N-terminus
  • conservative amino acid substitutions i.e. substitutions performed within groups of amino acids with similar characteristics, e.g. (1 ) small amino acids, (2) acidic amino acids, (3) polar amino acids, (4) basic amino acids, (5) hydrophobic amino acids, (6) aromatic amino acids.
  • said antibody can inhibit the enzymatic activity of FX or the enzymatic activity of FX and FXa.
  • said antibody comprises a light chain comprising complementary determining regions L-CDR1 comprising the amino acid sequence of SEQ ID NO:14 or SEQ NO: 20, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 21 , and L-CDR3 comprising the amino acid sequence of SEQ ID. NO 16 or SEQ ID NO: 22.
  • each heavy chain is linked to a light chain by a disulfide bond.
  • Each chain contains distinct sequence domains.
  • the light chain includes two domains, a variable domain (VL) and a constant domain (CL).
  • the heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1 , CH2 and CH3, collectively referred to as CH).
  • variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen.
  • the constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR).
  • the Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain.
  • the specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant.
  • Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs).
  • Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site.
  • the light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1 , L-CDR2, L-CDR3 and H-CDR1 , H-CDR2, H-CDR3, respectively.
  • An antigen-binding site therefore, normally includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
  • Framework Regions (FRs) refer to amino acid sequences interposed between CDRs.
  • CDRs can be identified in accordance with the definitions of the Kabat, Chothia, the accumulation of both Kabat and Chothia, AbM, contact, IMGT unique numbering, and/or conformational definitions or any method of CDR determination well known in the art.
  • Antibody CDRs may be identified as the hypervariable regions originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.
  • the positions of the CDRs may also be identified as the structural loop structures originally described by Chothia and others (See, e.g., Chothia et al., Nature 342:877-883, 1989).
  • Other approaches to CDR identification include the "AbM definition,” which is a compromise between Kabat and Chothia and is derived using Oxford Molecular's AbM antibody modeling software (now AccelrysO), the "contact definition" of CDRs based on observed antigen contacts, set forth in MacCallum et al., J. Mol. Biol., 262:732-745, 1996, or “IMGT unique numbering”, which relies on the high conservation of the structure of the variable region (see Lefranc, M.-P. Nucl.
  • CDRs In another approach, referred to herein as the "conformational definition" of CDRs, the positions of the CDRs may be identified as the residues that make enthalpic contributions to antigen binding. See, e.g., Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008. Still other CDR boundary definitions may not strictly follow one of the above approaches, but will nonetheless overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding.
  • a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods used herein may utilize CDRs defined according to any of these approaches. For any given embodiment containing more than one CDR, each CDR may be defined in accordance with any one of Kabat, Chothia, extended, AbM, contact, IMGT unique numbering, or conformational definitions.
  • sequences are analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT and the Protein Data Bank (PDB) with structural data from the PDB. Unless otherwise indicated, all CDRs set forth herein are derived according to the Abysis database website as per the numbering scheme indicated.
  • said antibody comprises a heavy chain comprising complementary determining regions H-CDR1 comprising the amino acid sequence of SEQ ID NO:11 or SEQ NO: 17, a H-CDR2 comprising the amino acid sequence of SEQ ID NO: 12 or SEQ ID NOU 8, and H-CDR3 comprising the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 19.
  • said antibody comprises a light chain comprising complementary determining regions L-CDR1 comprising the amino acid sequence of SEQ ID NO:14 or SEQ NO: 20, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 21 , and L-CDR3 comprising the amino acid sequence of SEQ ID.
  • H-CDR1 comprising the amino acid sequence of SEQ ID NO:11 or SEQ NO: 17, a H-CDR2 comprising the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO:18, and H-CDR3 comprising the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 19.
  • said antibody comprises a light chain comprising a light chain variable region comprising an amino acid sequence at least about 85%, 90%, 91% ,92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identical to the sequence set forth in SEQ ID NO: 7 or in SEQ 9.
  • said the light chain variable region comprises the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9.
  • said antibody comprises a heavy chain comprising a heavy chain variable region comprising an amino acid sequence at least about 85%, 90%, 91% ,92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identical to the sequence set forth in SEQ ID NO: 6 or in SEQ 8 or SEQ ID. NO: 10.
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO:8 or SEQ ID. NO: 10.
  • said antibody comprises a light chain comprising a light chain variable region comprising an amino acid sequence at least about 85%, 90%, 91% ,92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identical to the sequence set forth in SEQ ID NO: 7 and a heavy chain comprising a heavy chain variable region comprising an amino acid sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identical to the sequence set forth in SEQ ID NO: 6.
  • said the light chain variable region comprises the amino acid sequence of SEQ ID NO:7 and said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 6.
  • the terms “identical” “identity, or “percent identity,” in the context of two or more nucleic acids or amino acid sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence. To determine the percent identity, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the two sequences that are compared are the same length after gaps are introduced within the sequences, as appropriate (e.g., excluding additional sequence extending beyond the sequences being compared).
  • a "corresponding" CDR refers to a CDR in the same location in both sequences (e.g., CDR-H1 of each sequence).
  • the determination of percent identity or percent similarity between two sequences can be accomplished using a mathematical algorithm.
  • a preferred, nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877.
  • Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410.
  • Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
  • a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
  • said antibody comprises a light chain comprising a light chain variable region comprising an amino acid sequence at least about 85%, 90%, 91% ,92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identical to the sequence set forth in SEQ ID NO: 9 and a heavy chain comprising a heavy chain variable region comprising an amino acid sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identical to the sequence set forth in SEQ ID NO: 8 or SEQ ID. NO: 10 .
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:9 and said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8. In another preferred embodiment said the light chain variable region comprises the amino acid sequence of SEQ ID NO:9 and said heavy chain variable region comprises the amino acid sequence of SEQ ID. NO: 10.
  • said antibody comprises a light chain comprising complementary determining regions L-CDR1 , L-CDR2 and L-CDR3, each of them comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, respectively, or the amino acid sequence of SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, respectively.
  • said antibody comprises a heavy chain comprising complementary determining regions H-CDR1 , H-CDR2 and H-CDR3, each of them comprising the amino acid sequence of SEQ ID NO:11 , SEQ ID NO:12 and SEQ ID NO:13, respectively, or the amino acid sequence of SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, respectively.
  • said antibody comprises a light chain comprising complementary determining regions L-CDR1 , L-CDR2 and L-CDR3, each of them comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, respectively, or the amino acid sequence of SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, respectively and a heavy chain comprising complementary determining regions H-CDR1 , H-CDR2 and H-CDR3, each of them comprising the amino acid sequence of SEQ ID NO:11 , SEQ ID NO:12 and SEQ ID NO:13, respectively, or the amino acid sequence of SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, respectively.
  • L-CDR1 , L-CDR2 and L-CDR3 each of them comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, respectively, or the amino acid sequence of SEQ ID NO:20, SEQ ID NO
  • said antibody comprises a light chain comprising complementary determining regions L-CDR1 , L-CDR2 and L-CDR3, each of them comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, respectively, and a heavy chain comprising complementary determining regions H-CDR1 , H-CDR2 and H-CDR3, each of them comprising the amino acid sequence of SEQ ID NO:11 , SEQ ID NO:12 and SEQ ID NO:13, respectively.
  • said antibody comprises a light chain comprising complementary determining regions L-CDR1 , L-CDR2 and L-CDR3, each of them comprising the amino acid sequence of SEQ ID NQ:20, SEQ ID NO:21 and SEQ ID NO:22, respectively, and a heavy chain comprising complementary determining regions H-CDR1 , H-CDR2 and H-CDR3, each of them comprising the amino acid sequence of SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, respectively.
  • said antibody comprises a light chain comprising the amino acid sequences of SEQ ID NO: 2 or SEQ ID NO: 4.
  • said antibody comprises a heavy chain comprising the amino acid sequences of SEQ ID NO:1 , or SEQ ID NO: 3 or SEQ ID NO: 5.
  • said antibody comprises a light chain comprising the amino acid sequences of SEQ ID NO: 2 or SEQ ID NO: 4 and a heavy light chain comprising the amino acid sequences of SEQ ID NO:1 , or SEQ ID NO: 3 or SEQ ID NO: 5.
  • said antibody comprises a the light chain comprising the amino acid sequence of SEQ ID NO: 2 and a heavy chain comprising the amino acid sequence of SEQ 1 .
  • said antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 4 and a heavy chain comprising the amino acid sequence of SEQ 3.
  • said antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 4 and a heavy chain comprising the amino acid sequence of SEQ 5.
  • the antibody of the present invention is a recombinant antibody, or a monoclonal antibody or a fragment of said recombinant or monoclonal antibody.
  • said antibody fragment is selected from variable fragments (Fv), single-chain Fvs (scFv), bispecific antibodies (sc(Fv) 2 ), single chain antibodies, single domain antibodies, Fab fragments, F(ab') 2 fragments, Fab’ fragments, disulfide-linked Fv (dsFv), chemically conjugated Fv (ccFv), diabodies, anti-idiotypic (anti-ld) antibodies, affibodies, nanobodies, and unibodies.
  • the antibody of the present invention comprises a constant region of the murine IgG 1 class or the murine lgG3 class.
  • NUCLEIC ACIDS ECODING THE ANTIBODIES OF THE PRESNET INVENTION AND CELLS COMPRISING AND EXPRESING SAID NUCLEIC ACIDS.
  • the present invention also refers to a nucleic acid comprising a nucleotide sequence encoding the antibody of the present invention, a promoter operably linked to the nucleotide sequence and a selectable marker.
  • nucleic acid refers to any materials comprised of DNA or RNA. Nucleic acids can be made synthetically or by living cells.
  • the present invention refers to a cell comprising the antibody of the present invention or the nucleic acid comprising encoding the antibody of the present invention.
  • the term “cell” refers to any kind of cellular system which can be engineered to generate proteins, protein fragments, or peptides of interest, including antibodies, antibody fragments, and fusion proteins.
  • cells that have been manipulated to express a particular protein or fragment of protein or peptide include cultured cells, e.g., mammalian cultured cells, such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, plant cells or bacterial cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.
  • mammalian cultured cells such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6
  • the present invention relates to a composition
  • a composition comprising at least a first antibody according to the invention and a solid support to which the antibody is covalently or non-covalently bound.
  • the composition further comprises a second antibody according to the invention that binds to an epitope of FX different than the first antibody, the second antibody being covalently or non-covalently bound to a solid support.
  • both antibodies can be bound to the same or to different support.
  • the solid support comprises a particle, a bead, a membrane, a surface, a polypeptide chip, a microtiter plate, or the solid-phase of a chromatography column.
  • the present invention relates to a kit for detecting the presence of, depleting or inhibiting FX, FXa, or a fragment thereof, in a sample, said kit comprising at least one antibody or at least one composition according to the invention.
  • fragment thereof when referred to FX or FXa, means any amino acid sequence comprised within the amino acid sequence of the FX or FXa protein that retain the ability to bind to anyone of the antibodies of the invention.
  • amino acid sequences may include truncation of the N- and/or C-terminus by one or more amino acid residues wherein the resulting amino acid sequence after truncation still bind to the antibodies of the invention.
  • the present invention relates to a method for detecting the presence of FX, FXa, or a fragment thereof, in a sample, comprising the steps of i) contacting said sample with at least one antibody, or at least one composition or the kit according to the invention and ii) identifying the FX, FXa, or the fragment thereof, bound in step i).
  • the sample is a liquid sample.
  • the liquid sample is a blood sample or a plasma sample.
  • sample refers to any biological material obtained from a subject or patient.
  • a sample can comprise blood, peritoneal fluid, CSF, saliva or urine.
  • a sample can comprise whole blood, blood plasma, blood serum, B cells enriched from blood samples, and cultured cells (e.g., B cells from a subject).
  • a sample can also include a biopsy or tissue sample including neural tissue.
  • a sample can comprise whole cells and/or a lysate of the cells.
  • the present invention related to a method for depleting and/or inhibiting FX, FXa, or a fragment thereof, comprising the step of contacting said sample with at least one antibody, or at least one composition or the kit according to the invention.
  • the sample is a liquid sample.
  • the liquid sample is a blood sample or a plasma sample.
  • the term “deplete”, with respect to Factor X, Factor Xa or a fragment thereof, are used as refers to the physical or virtual removal of whole or part of the Factor X, Factor Xa or a fragment thereof present in a sample from the remaining content of said sample.
  • Physical removal refers to the separation or the extraction of a component from a sample such that said component is not present within said sample.
  • Virtual removal means making unavailable or nonfunctional a component in a sample without separating or extracting said component from said sample.
  • the invention related to the use of an antibody according to the invention to determine the presence of FX, FXa, or a fragment thereof, in a sample.
  • the sample is a liquid sample.
  • the liquid sample is a blood sample or a plasma sample.
  • the invention related to the use of an antibody according to the invention to deplete and/or inhibit FX, FXa, or a fragment thereof, from a sample.
  • the sample is a liquid sample.
  • the liquid sample is a blood sample or a plasma sample.
  • the invention relates to a composition obtainable by the method for depleting and/or inhibiting FX, FXa, or a fragment thereof, according to the invention.
  • the composition is obtainable from a human blood-derived sample.
  • such human blood-derived sample is one that has a normal coagulation time value.
  • the human blood-derived sample is a sample from a donor and in a preferred embodiment, the human blood-derived sample is a pool sample from two or more human donors. In another particular embodiment the human-derived sample is a blood sample or a plasma sample.
  • the invention relates to the use of a composition obtainable by the method for depleting and/or inhibiting FX, FXa, or a fragment thereof, according to the invention as an abnormal coagulation control in an in vitro assay.
  • the composition is obtainable from a human blood-derived sample.
  • such human blood-derived sample is one that has a normal coagulation time value.
  • the human blood-derived sample is a sample from a donor and in a preferred embodiment, the human blood-derived sample is a pool sample from two or more human donors.
  • the human-derived sample is a blood sample or a plasma sample.
  • the term “obtainable by” does not limit the method used for obtaining a composition but the features of said composition. Then the term “obtainable by” comprises any method for obtaining a composition as long as the features of the obtained composition are the same.
  • FIG. 1 shows an illustration of the Clotting Cascade.
  • FX is an integral protein in the clotting cascade. Both the Intrinsic and Extrinsic pathways require FX to then activate Prothrombin and then form fibrin clots. Common Pathway. This view is a modern take on the clotting cascade. Shown are the complexes formed throughout the course of the cascade.
  • Figure 2 shows SDS-PAGE Gel analysis of anti-FX monoclonal antibody purifications. It shows relative purity of expression. Description of lanes in gel are located above each image. Molecular weights on the left of each image are in kDa. Each antibody, A) 3E12.1 , B) 5C7.1 , and C) 4D6.1 , was run non-reduced (left image) and reduced (right image). Resulting Heavy Chain and Light Chain from reducing are identified alongside their respective mAb.
  • Figure 3 shows results from printed peptide microarray assays for 3E12.1 antibody. It shows that 3E12.1 antibody recognizes SMKTRGLPKAK.
  • Columns 2-4 represent increasing concentrations of exposed 3E12.1 to the microarray. Values represent fluorescence intensity measured per peptide.
  • SMKTRGLPKAK was identified out of the entire FX sequence by the epitope sequence search and is underlined in Column 1 . It is located on the Heavy Chain of FX, i.e. the serine protease.
  • B Intensity of black color denotes increased preference. More intense color denotes preferred amino acids and lower intense color denotes less preferred amino acids.
  • Figure 4 shows results from printed peptide microarray assays for 5C7.1 antibody. It shows that 5C7.1 antibody recognizes EAPDSIT.
  • Columns 2-4 represent increasing concentrations of exposed 5C7.1 to the microarray. Values represent fluorescence intensity measured per peptide. EAPDSIT was identified out of the entire sequence of FX by the epitope sequence search and is underlined in Column 1 . There were no other peaks of noteworthy intensity. The epitope is located on the heavy chain of Factor X. Specifically, it is located on the activation peptide. When this peptide is cleaved, 5C7.1 can no longer bind.
  • B Intensity of black color denotes increased preference. More intense color denotes preferred amino acids and lower intense color denotes less preferred amino acids.
  • FIG. 5 shows Western Blots for 3E12.1 and 4D6.1/5C7.1 antibodies.
  • FX/FXa were loaded at 200 ng/well.
  • 3E12.1 was used as a primary antibody at a dilution of 6.25 pg/mL.
  • 3E12.1 binds the heavy chain of FX (-34.2 kDa) and FXa (-28.5 kDa) under non-reducing and reducing conditions.
  • B FX/FXa were loaded at 50 ng/well.
  • 4D6.1 was used as a primary antibody at a dilution of 0.25 pg/mL.
  • 4D6.1 binds FX and not FXa.
  • FX/FXa were loaded at 200 ng/well.
  • HX-1 a control antibody, was used as a primary antibody at a dilution of 6.25 pg/mL.
  • HX-1 binds non- reduced FX and FXa.
  • Figure 6 shows an In vitro Clotting Assays to identify varying degrees of inhibitory effects of anti-FX antibodies on the Intrinsic Clotting Cascade. Clotting was measured by change in absorbance at 405 nm over time. Increased absorbance at 405 nm is proportional to increased clotting. Inflection point times were used to rank degree of inhibition.
  • the Negative control (a) contains no antibody and all proteins required for clotting. This control shows the clotting cascade, uninhibited.
  • the Positive control (b) contains FX-deficient plasma. Theoretically, this sample should clot very slowly, if at all, and is used as the practical “maximum” inhibition of the system.
  • control antibody HX-1 (d) is incubated with FX prior to initiation of clotting in the assay. Inhibited clotting shown, as compared to the Negative Control.
  • C) 5C7.1 antibody (c) is incubated with FX prior to initiation of the clotting cascade.
  • FIG. 7 shows Binning Study for 3E12.1 , 4D6.1 and 5C7.1 antibodies.
  • Octet assay was run by loading AMC tips with each antibody: 3E12.1 , 4D6.1 , 5C7.1 , and the control antibody HX-1 . Loaded tips were then exposed to A) FX or B) FXa, forming initial Ab:Ag complexes. These complexes were then all introduced to each antibody one at a time. Binding response (nm) was recorded during this step and entered into cells corresponding to their respective antibody conditions. Columns show initially loaded antibody. Rows show antibody associated to mAb:Ag complex. Cells where antibody is matched to itself act as controls for identical bins.
  • Figure 8 shows an Octet Kinetics Assay to estimate KD values of 3E12.1 and 5C7.1 , when interacting with FX. Curves from -60 to 0 seconds are used to baseline so that association and dissociation curves can be compared. Association with FX occurs from 0 to 160 seconds. Dissociation into buffer occurs from 160 to 2660 seconds. Legend shown below each graph are varying nanomolar concentrations of each antibody, A) 3E12.1 , or B) 5C7.1. Descending order of the curves in the figure correspond to values in the legend, from left to right (i.e. larger binding curve response correlates with greater concentration). Y-axis is the binding response of FX to each antibody, in nanometers.
  • Figure 9 shows modeling of 3E12.1 bound to epitope region of truncated FX.
  • the Schrodinger Suite was used to homology build 3E12.1 from sequence and calculate docked states with FX (PDBID:1 HCG). Two different views of the same docked state of 3E12.1 and a portion of FX. Portion of FX (A), Light chain of 3E12.1 (B) and Heavy Chain of 3E12.1 (C). One-letter residue code and position shown for clarity of interacting residues.
  • Figure 10 shows modeling of 5C7.1 bound to epitope region of truncated FX.
  • the Schrodinger Suite was used to homology build 5C7.1 from sequence and calculate docked states with FX. Two different views of the same docked state of 5C7.1 and a portion of FX. Portion of FX (A), Light chain of 5C7.1 (B) and Heavy Chain of 5C7.1 (C). One-letter residue code and position shown for clarity of interacting residues. Note: due to recognition of unstructured region of FX, a linear peptide was built in pyMOL and imported into Schrodinger for the docking simulation.
  • mice Six female BALB/c mice were immunized with human Factor X purified from plasma. Four of those mice were immunized via a rapid prime method. After a 19-day immunization protocol, lymph nodes were harvested for fusion and hybridoma generation. The two remaining mice were immunized via a slower, standard prime method where the initial antigen immunization was followed by boosts all with CpG adjuvant. Titers were checked and, following a final boost, spleens were harvested for fusion. Two methods were utilized to provide ample opportunity for identification of antibodies of interest.
  • Lymphocytes from all four rapid prime method mice and both standard prime method mice were isolated and fused with murine SP2/0 myeloma cells in the presence of poly-ethylene glycol. Fused cells were cultured using a single-step cloning method, with HAT selection. Approximately 10 days after the fusion event, clones were picked from semi-solid media and placed in 96-well format across 10 plates.
  • Example 2 Sequencing [0080] Next Generation sequencing was used to sequence the hybridomas of the top three performing mAbs. Variable regions of each mAb was returned and CDR regions were identified via KABAT schema. These CDR regions were then compared to identify similarities or differences. Sequencing showed that two of the antibodies, 4D6.1 and 5C7.1 , had identical CDRs, despite an isotype switch. Sequences obtained are listed in Table 1 .
  • Table 1 Description of the sequences obtained from sequencing the hybridomas of the top three performing mAbs.
  • Example 3 Epitope specificity. Epitope sequence identification.
  • Epitope specificity is an important property of monoclonal antibodies and particularly relevant when select blood factor depleting antibodies due to similarity across blood factors. Every antibody has a unique way in which it binds its antigen, which are further differentiated by the strength, or affinity, with which it binds. These properties are dictated by the unique sequence of the antibody.
  • 3E12.1 , 4D6.1 , and 5C7.1 many techniques were applied to experimentally determine epitope sequences and their functional importance. We utilized western blot, clotting analysis, printed peptide microarray, 3D modelling and simulated docking, BLI, and 12-mer peptide phage display assays to narrow down potential binding sites.
  • the printed peptide microarray assay explores the entirety of the antigenic sequence in a microplate format.
  • a library of 15-mer peptides with a frame shift of 1 amino acid along the sequence was generated and then used to coat each wells of a microplate with a unique peptide.
  • this plate is exposed to a set concentration of antibody (3E12.1 or 5C7.1 in this case) and an IgG-Fc specific secondary antibody then quantitates the binding occurring in each well, i.e. to each peptide.
  • An intensity map is generated from the data and specific peptide sequences are typically identified.
  • sequence “SMKTRGLPKAK” was identified for 3E12.1 ( Figure 3), and “EAPDSIT” for 5C7.1 ( Figure 4). These sequences are located on the heavy chain and activation peptide of the heavy chain of FX, respectively. Similar to how the epitope scan was run, libraries of peptides were generated for 3E12.1 and 5C7.1. Each library consisted of the respective antibody’s epitope with point mutations at each position with every other possible residue. Wells of a microplate were coated, and respective antibodies were exposed to each microarray. Intensity maps were generated, and analysis looked at the relative binding intensities of each point mutation. A colorimetric chart was then constructed to visualize how drastically each mutation affects binding.
  • a substitutional scan of the epitope revealed that there was a strong dependence on certain residues for the interaction to be successful.
  • the conserved core motif is “KTRGLPKAK.” Highly conserved residues are R6, K10, K12, where substitutions cause a 70% decrease in binding. K10 showed tolerance for substitution by Arginine.
  • Well conserved residues are K4 and L8, with a 48% and 44% decrease, respectively, due to substitution (Figure 3).
  • the conserved core motif is “EAPDSIT.” Highly conserved residues are P and I, where substitutions cause a 73% decrease in binding.
  • Well conserved residue is T, with a 50% decrease due to substitution (Figure 4).
  • Example 4 Epitope specificity. Western blot.
  • a clotting assay was designed to identify any inhibition of the Intrinsic pathway caused by the binding interaction between FX and mAb.
  • Various positive and negative controls were designed to aid in this analysis.
  • Positive control contained 50 pL 40x diluted FX-depleted plasma, 50 pL FX-depleted plasma, and 50 pL HEPES Buffered Solution (HBS).
  • Negative control contained 50 pL 40x diluted Normal Human Plasma, 50 pL FX-depleted plasma, and 50 pL HBS.
  • mAb samples contained 50 pL 40x diluted Normal Human Plasma, 50 pL FX-depleted plasma, and 50 pL of 40x excess mAb in HBS.
  • This assay incorporates FX deficient plasma, normal human plasma, and incubation with 3E12.1 , 4D6.1 , or 5C7.1 , and control antibodies to identify any inhibition caused by the interaction between them and FX. Inhibition of the clotting cascade would reduce or cease the rate of coagulation, measured at 405nm, and then be compared against positive and negative controls. The decreased rates can be quantified, and antibodies can then be compared. Results from this assay indicate that 3E12.1 , 4D6.1 and 5C7.1 are all inhibitory. With the addition of 3E12.1 to normal human plasma, the rate of coagulation was slowed by roughly 3.3x as compared to no antibody.
  • 96-well plates were prepared with two-fold serial dilutions of FX from 403.5 nM to 6.305 nM and each anti-FX antibody set at 20 pg/mL.
  • Anti-mlgG Fc Capture (AMC) tips were loaded with 3E12.1 , 4D6.1 , and 5C7.1 for 200 seconds, 1200 seconds, and 200 seconds, respectively, and then introduced to the varying concentrations of FX for 160 seconds. These complexes were then dissociated in buffer for 2500 seconds. Changes in binding on the surface of each tip was recorded throughout the experiment for later analysis. Using analysis tools on the association and dissociation of the anti-FX:FX complexes, kon, koff, and KD could be calculated and used to rank antibodies by affinity.
  • Depletion studies involved comparing antibodies 3E12.1 , 4D6.1 , and 5C7.1 against commercially available sources. Depletion studies incorporated a series of experiments including inhibition, magnetic-particle, and column (Poly-prep) based FX- depletion. Small-scale depletion or inhibition was assayed first.
  • Table 2 Commercial antibodies tested during the development of FX deficient plasma. It is shown commercial reference, type of antibody (M: monoclonal; P: polyclonal), clone name (monoclonal mAbs), isotype, manufacturer/distributor, recognized epitope in FX structure. NA: Not applicable.
  • Antibodies ab61362 and 5051 are the same clone distributed by two different companies Abeam and Haemtech. Ill; international units ND; not determined.
  • SMKTRGLPKAK SEQ ID NO: 24 Epitope of 5C7.1/4D6.1, 193-199 FX

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Abstract

La présente invention concerne des anticorps qui se lient de manière spécifique au facteur de coagulation X (FX), ou des fragments de ceux-ci, inhibent son activité enzymatique et peuvent l'appauvrir dans des échantillons dérivés du sang humain. La présente invention concerne également des compositions et des kits comprenant lesdits anticorps et des procédés de détection, d'inhibition et/ou de déplétion de FX à partir d'échantillons avec lesdits anticorps, compositions ou kits ou leurs utilisations. La présente invention concerne en outre des compositions produites à partir d'échantillons dérivés du sang par déplétion et/ou inhibition du FX, ou des fragments de celui-ci, et leurs utilisations en tant que témoins de coagulation anormaux dans des dosages in vitro.
PCT/EP2022/085850 2021-12-17 2022-12-14 Anticorps contre le facteur de coagulation x et leurs utilisations WO2023111018A1 (fr)

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