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JP-2026514355-A - Antibodies against methotrexate and their uses

JP2026514355AJP 2026514355 AJP2026514355 AJP 2026514355AJP-2026514355-A

Abstract

This specification discloses antibodies that specifically bind to methotrexate analyte. Such antibodies can be used to detect methotrexate analyte in samples in homogeneous enzyme immunoassays and the like. Hapten structures for inducing such antibodies are also described. Furthermore, conjugates useful for immunoassays using such antibodies are also described.

Inventors

  • ヴァルデス，ジョニー
  • ムン，ビョンスク
  • シン，ラジェンドラ
  • グエン－チョイ，ジャクリーン

Assignees

  • アーク ダイアグノスティックス インコーポレイテッド

Dates

Publication Date
20260511
Application Date
20240318
Priority Date
20230320

Claims (20)

  1. The compound represented by the following formula (1): During the ceremony, R1 is -Y-Z, Y is a linking group, Z is selected from the group consisting of hydrogen, OH, SH, S-acyl, O-alkyl, halogen, NH2 , epoxy, maleidyl, haloacetamide, carboxyl, activated carboxyl, N3 , and alkenes, immunogenic carriers, proteins, labels, and solid supports, as well as salts thereof.
  2. The compound according to claim 1, wherein the linking group comprises 1 to 15 carbon atoms and/or 0 to 6 heteroatoms.
  3. The linking group is -(CH 2 ) n C(O)-, -C(O)(CH 2 ) n -, -C(O)(CH 2 ) n NHC(O)-, -C(O)(CH 2 ) n NHC(O)(CH 2 ) n -, -(CH 2 ) n SCH 2 C(O)-, -(CH 2 ) n C(O)NH(CH 2 ) n -, -(CH 2 ) n NHC(O)-, -(CH 2 ) n NHC(O)(CH 2 ) n -, -(CH 2 ) n NHC(O)(CH 2 ) n O(CH 2 ) n NHC(O)(CH 2 ) n −, −(CH 2 ) n The compound according to claim 1, selected from the group consisting of NHC(O)( CH2 ) n NHC(O)( CH2 ) n- , -NH( CH2 ) n C( O ) - , -( CH2 ) n- , -C(O)NH( CH2CH2O ) m (CH2) n NHC( O )(CH2) n- , and -( CH2 ) n (heterocyclyl)S( CH2 ) n C(O)-, and salts thereof, where each m is an integer from 1 to 10 and each n is an integer from 1 to 10.
  4. The compound according to claim 1, wherein Z is a protein.
  5. The compound according to claim 1, wherein the protein is an immunogenic carrier selected from the group consisting of hemocyanin, globulin, and albumin.
  6. The compound according to claim 5, wherein the immunogenic carrier is bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).
  7. The compound according to claim 1, wherein Z is an immunogenic carrier, and the immunogenic carrier is a polysaccharide.
  8. The compound according to claim 1, wherein Z is a label.
  9. The compound according to claim 8, wherein the label is an enzyme.
  10. The compound according to claim 9, wherein the enzyme is selected from the group consisting of alkaline phosphatase, β-galactosidase, and horseradish peroxidase.
  11. The compound according to claim 9, wherein the enzyme is glucose-6-phosphate dehydrogenase (G6PDH).
  12. The compound according to claim 1, wherein the linking group comprises an acyl group or alkyl group bonded to a phenyl group.
  13. The compound according to claim 1, wherein the linking group comprises an alkyl group bonded to a phenyl group or a substituted alkyl group.
  14. An antibody that specifically binds to the compound represented by the following formula (1): During the ceremony, R1 is -Y-Z, Y is a linking group, Z is selected from the group consisting of hydrogen, OH, SH, S-acyl, O-alkyl, halogen, NH2 , epoxy, maleidyl, haloacetamide, carboxyl, activated carboxyl, N3 , and alkenes, immunogenic carriers, proteins, labels, and solid supports, as well as salts thereof.
  15. The aforementioned antibody V H CDR1 containing the amino acid sequence (SEQ ID NO: 2), A heavy chain variable region ( VH ) polypeptide comprising VHCDR2 containing the amino acid sequence (SEQ ID NO: 3) and VHCDR3 containing the amino acid sequence (SEQ ID NO: 4), V L CDR1 containing the amino acid sequence (SEQ ID NO: 6), A light chain variable region ( VL ) polypeptide comprising VLCDR2 containing the amino acid sequence (SEQ ID NO: 7) and VLCDR3 containing the amino acid sequence (SEQ ID NO: 8), The antibody according to claim 14, comprising:
  16. The aforementioned antibody A heavy chain variable region ( VH ) polypeptide containing an amino acid sequence having 95% or more identity with the amino acid sequence described in Sequence ID No. 1, A light chain variable region ( VL ) polypeptide containing an amino acid sequence having 95% or more identity with the amino acid sequence described in Sequence ID No. 5, The antibody according to claim 15, comprising:
  17. The aforementioned antibody V H CDR1 containing the amino acid sequence (SEQ ID NO: 10), A heavy chain variable region ( VH ) polypeptide comprising VHCDR2 containing the amino acid sequence (SEQ ID NO: 11) and VHCDR3 containing the amino acid sequence (SEQ ID NO: 12), V L CDR1 containing the amino acid sequence (SEQ ID NO: 14), A light chain variable region ( VL ) polypeptide comprising VLCDR2 containing the amino acid sequence (SEQ ID NO: 15) and VLCDR3 containing the amino acid sequence (SEQ ID NO: 16), The antibody according to claim 14, comprising:
  18. The aforementioned antibody A heavy chain variable region ( VH ) polypeptide containing an amino acid sequence having 95% or more identity with the amino acid sequence described in Sequence ID No. 9, A light chain variable region ( VL ) polypeptide containing an amino acid sequence having 95% or more identity with the amino acid sequence described in SEQ ID NO: 13, The antibody according to claim 17, comprising:
  19. The aforementioned antibody V H CDR1 containing the amino acid sequence (SEQ ID NO: 18), A heavy chain variable region ( VH ) polypeptide comprising VHCDR2 containing the amino acid sequence (SEQ ID NO: 19) and VHCDR3 containing the amino acid sequence (SEQ ID NO: 20), V L CDR1 containing the amino acid sequence (SEQ ID NO: 22), A light chain variable region ( VL ) polypeptide comprising VLCDR2 containing the amino acid sequence (SEQ ID NO: 23) and VLCDR3 containing the amino acid sequence (SEQ ID NO: 24), The antibody according to claim 14, which includes an antibody that competes with the compound for binding.
  20. The aforementioned antibody A heavy chain variable region ( VH ) polypeptide containing an amino acid sequence having 95% or more identity with the amino acid sequence described in Sequence ID No. 17, A light chain variable region ( VL ) polypeptide containing an amino acid sequence having 95% or more identity with the amino acid sequence described in SEQ ID NO: 21, The antibody according to claim 19, comprising:

Description

[Reference to related applications] This application claims priority under U.S. Patent Application No. 18/186,899, filed on 20 March 2023, the disclosures of which are incorporated herein by reference. [Technical field to which the review belongs] The present invention relates to a method and system for detecting methotrexate (MTX) using an immunoassay. In particular, it relates to antibodies and haptens used in an immunoassay for detecting and quantifying MTX in a biological sample. [Inclusion via array listing reference] The Sequence Listing was created on March 20, 2023, and is provided to this application as a Sequence Listing XML file named "ARKD-009_SEQ_LIST.xml" with a size of 35,274 bytes. The contents of the Sequence Listing XML are incorporated into this application by reference. MTX [N-[4[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid, abbreviated as MTX], formerly known as ametopterin, is an antimetabolite used in the treatment of certain neoplastic diseases, severe psoriasis, and adult rheumatoid arthritis. MTX has a unique mechanism of action for use in chemotherapy and immunosuppression in autoimmune diseases. In cancer treatment, MTX acts as an anti-folate anti-metabolite. MTX is taken up into cells by a carrier called the human reduced folate carrier, forming MTX-polyglutamate. Both MTX and MTX-polyglutamate inhibit dihydrofolate reductase, an enzyme that catalyzes the conversion of dihydrofolate to tetrahydrofolate, the active form of folate. Tetrahydrofolate is essential for the synthesis of both DNA and RNA nucleotides. MTX-polyglutamate further inhibits the de novo synthesis of purines by both purine synthase and thymidylate synthase, thereby inhibiting DNA synthesis. This mechanism of action is utilized in cancer treatment due to its cytotoxic effects. In autoimmune diseases, different mechanisms of action are involved when MTX is selected as a drug. MTX inhibits the enzyme transformylase, resulting in impaired adenosine and guanine metabolism. The accumulation of adenosine is converted into its anti-inflammatory effects, manifesting as suppression of T cell activation, downregulation of B cells, and increased sensitivity of activated CD-95 T cells. Furthermore, it also manifests as suppression of methyltransferase activity and inhibition of interleukin-β1 binding to cell surface receptors. MTX may have serious toxicity. Patients receiving MTX therapy should be closely monitored to ensure rapid detection of toxic effects. Moderate to high doses of MTX (approximately 35 mg/ m² to 12 g/ m² ) in combination with leucovorin (citroborum factor) rescue have shown favorable results in the treatment of osteosarcoma, leukemia, non-Hodgkin lymphoma, lung cancer, and breast cancer. High-dose MTX (HDMTX) refers to doses exceeding 500 mg/mL. Patients may experience nausea, mucosal ulcers, hair loss, fatigue, fever, increased risk of infection, leukopenia, gastrointestinal bleeding, pancreatitis, cirrhosis, aplastic anemia, malignant tumors (lymphoproliferative disorders), infections, interstitial pneumonia, renal dysfunction, and teratogenicity. Accurate monitoring of MTX blood levels is crucial to ensure safe and effective treatment and to avoid toxicity from excessive exposure. The three antidotes used for MTX toxicity are leucovorin, thymidine, and glucarpidase. Leucovorin is the reduced active form of folic acid and rescues normal cells from the toxic effects caused by MTX inhibition of reduced folic acid. Leucovorin is particularly effective in preventing myelosuppression, gastrointestinal toxicity, and neurotoxicity during MTX treatment. Thymidine rescues cells from the cytotoxic effects of MTX, but its use is still in the research stage and is always used in combination with other drugs. Glucarpidase converts MTX into non-toxic metabolites, 4-(((2,4-diaminopteridine-6-yl)methyl)(methyl)amino)benzoic acid (DAMPA) and glutamic acid, and rapidly removes MTX from patients with renal impairment. Glucarpidase, in combination with leucovorin, is a common treatment for MTX toxicity. A single dose of glucarpidase reduces plasma MTX concentration by more than 97% within 15 minutes. For patients with renal impairment, hydration and urinary alkalinization are recommended. Serum concentrations of MTX depend on the indication for use, dosage, route of administration, treatment regimen, individual pharmacokinetics, metabolism, and other clinical factors. For example, in breast cancer treatment, serum concentrations usually reach 10–100 μmol/L, but in high-dose therapy for osteosarcoma, they can exceed 1000 μmol/L, and in some cases, pediatric osteosarcoma patients have reached 3100 μmol/L after 4 hours of intravenous administration. The decay curve of MTX in osteosarcoma treatment shows wide variation, ranging from 30–300 μmol/L at 24 hours after the start of administration, to 3–30 μmol/L at 48 hours, and less than 0.3 μmol/L at 72 hours. Typically, 10 mg of leucovorin is administe