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US-12624094-B2 - Factor H potentiating antibodies and uses thereof

US12624094B2US 12624094 B2US12624094 B2US 12624094B2US-12624094-B2

Abstract

The invention relates to novel isolated, synthetic or recombinant antibodies and fragments thereof specific for factor H. The invention further relates to the use of such antibodies and fragments for inhibiting complement activation and treatment of disorders associated with complement activation.

Inventors

  • Taco Willem Kuijpers
  • Diana WOUTERS
  • Maria Clara BROUWER
  • Richard Benjamin POUW

Assignees

  • STICHTING SANQUIN BLOEDVOORZIENING

Dates

Publication Date
20260512
Application Date
20220601
Priority Date
20180115

Claims (17)

  1. 1 . A method of identifying an antibody or a fragment thereof that binds Complement Factor H (FH) and potentiates FH activity, the method comprising: (a) providing a test antibody or an antigen-binding fragment thereof that specifically binds human FH wherein the test antibody or antigen-binding fragment is obtained from immunizing an animal with an antigen protein that comprises the amino acid sequence of human FH-related 1 (FHR-1); (b) assessing competition of the test antibody or the fragment thereof with a reference antibody, or a corresponding fragment thereof, for binding CCP18 of FH in a competition binding assay; and (c) assessing an FH activity in the presence and absence of the test antibody or the fragment thereof, wherein the reference antibody comprises a light chain CDR1, a light chain CDR2, a light chain CDR3, a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 33, 34, 35, 37, 38, and 39; 17, 18, 19, 21, 22, and 23; 49, 50, 51, 53, 54, and 55; 65, 66, 67, 69, 70, and 71; or 1, 2, 3, 5, 6, and 7, respectively.
  2. 2 . The method of claim 1 , wherein the reference antibody comprises: (i) a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 36 and a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 40; (ii) a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 20 and a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 24; (iii) a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 52 and a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 56; (iv) a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 68 and a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 71; or (v) a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 4 and a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 8.
  3. 3 . The method of claim 1 , wherein the reference antibody comprises a light chain CDR1, a light chain CDR2, a light chain CDR3, a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 33, 34, 35, 37, 38, and 39; 17, 18, 19, 21, 22, and 23; 49, 50, 51, 53, 54, and 55; or 65, 66, 67, 69, 70, and 71, respectively.
  4. 4 . The method of claim 3 , wherein the reference antibody comprises: (i) a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 36 and a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 40; (ii) a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 20 and a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 24; (iii) a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 52 and a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 56; or (iv) a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 68 and a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 71.
  5. 5 . The method of claim 1 , wherein the animal is a rodent.
  6. 6 . The method of claim 5 , wherein the rodent is a mouse.
  7. 7 . The method of claim 6 , wherein the mouse is a BALB/c mouse.
  8. 8 . The method of claim 1 , further comprising producing the test antibody or the fragment thereof, wherein the test antibody or the fragment thereof has greater binding affinity for human FH than FH.07.
  9. 9 . The method of claim 1 , further comprising humanizing the test antibody or the fragment thereof and producing the humanized antibody or an antigen-binding fragment thereof, wherein the humanized antibody or the fragment thereof has greater binding affinity for human FH than FH.07.
  10. 10 . The method of claim 9 , wherein the humanized antibody or fragment thereof comprises human framework regions in the heavy and light chain variable regions.
  11. 11 . The method of claim 9 , wherein the humanized antibody or the fragment thereof binds human FH with a K D of 2.5×10 −8 M or less and/or binds a human FH fragment comprised of CCP18-20 with a K D of 1×10 −10 M or less.
  12. 12 . The method of claim 9 , wherein the humanized antibody or the fragment thereof binds human FH with a K D of 6×10 −9 M or less and/or binds a human FH fragment comprised of CCP18-20 with a K D of 6×10 −12 M or less.
  13. 13 . The method of claim 9 , wherein the humanized antibody or the fragment thereof does not compete with a second reference antibody for binding CCP18 of FH in a competition binding assay, wherein the second reference antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 84 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 88.
  14. 14 . The method of claim 9 , wherein the humanized antibody or the fragment thereof: (a) inhibits C3 deposition on LPS in vitro with an IC 50 value of 38 nM or less; (b) inhibits hemolytic activity in vitro with an IC 50 value of 150 nM or less, and/or (c) increases binding affinity of FH for C3b in vitro to a K D value of at most 2 μM and/or increases binding affinity of FH for C3b in vitro by at least 3 fold.
  15. 15 . The method of claim 9 , wherein producing the humanized antibody or the fragment thereof comprises: (i) providing a cell with a nucleic acid encoding the humanized antibody or fragment thereof, or a vector comprising the nucleic acid; and (ii) allowing the cell to translate the sequence of the nucleic acid.
  16. 16 . The method of claim 15 , wherein producing the humanized antibody or the fragment thereof further comprises harvesting and purifying the humanized antibody or fragment thereof.
  17. 17 . The method of claim 14 , wherein the humanized antibody or the fragment thereof: (a) inhibits C3 deposition on LPS in vitro with an C 50 value of 30 nM or less; and/or (b) inhibits hemolytic activity in vitro with an IC 50 value of 130 nM or less.

Description

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS This application is a Continuation Application of U.S. application Ser. No. 16/961,737, filed on Jul. 13, 2020, which is a U.S. National Stage Application under 35 U.S.C. § 371 of International Patent Application No. PCT/NL2019/050018 filed Jan. 15, 2019, which claims the benefit of priority of European Patent Application No. EP 18151726.9 filed Jan. 15, 2018, each of which are incorporated by reference in their entireties. FIELD OF THE INVENTION The invention relates to the field of immunology and medicine. In particular, the invention relates to factor H specific antibodies and uses thereof. BACKGROUND The complement system is an important element of innate immunity that contributes to the protection of many organisms such as mammals against invading pathogens. The complement system consists of over 30 different components which are mainly synthesized in the liver. Activation of the complement system occurs by three different pathways, the classical pathway, the lectin pathway and the alternative pathway. The three pathways converge at the formation of a C3 convertase, which are different for each pathway but have similar activity. In the classical complement pathway, activation of the complement component (C) 1 complex, consisting of C1q, C1r and C1s, occurs upon binding to antibody-antigen complexes. The C1 complex cleaves C4 and C2 leading to the formation of a C3 convertase consisting of C4bC2a. The C3 convertase cleaves C3 into the active components C3a and C3b. In the lectin pathway, mannose binding lectin binds to mannose residues on pathogenic surfaces which activates serine proteases MASP-1 and MASP-2 that are able to cleave C4 and C2. As in the classical pathway, this leads to the formation of the C4bC2a C3 convertase. This C3 convertase can bind C3b to form a C5 convertase. Contrary to the classical and lectin pathways, the alternative pathway has a low level of continuous activity due to spontaneous hydrolysis of C3 to C3(H2O) in plasma. This C3b-like C3(H2O) can form a fluid phase C3 convertase by binding factor B (FB) which in turn is activated into Bb by factor D. Similarly, when C3b binds to a surface, it may bind FB to form C3bB. This complex is cleaved by factor D to C3bBb which is the C3 convertase of the alternative pathway that can be stabilized by properdin (factor P) to C3bBbP. This C3 convertase is able to cleave C3 into C3a and C3b. In addition to this process the alternative pathway acts as amplification loop for the classical and lectin activation pathways as C3b generated in these pathways may act as starting point for the alternative pathway. Thereby, the amplification loop results in a reinforcement of the classical and lectin pathway. The C3 convertase formed in one of the three activation pathways can bind C3b to form a C5 convertase. The C5 convertases of all three complement pathways activate C5 into C5a and C5b which initiates the terminal pathway of the complement system. C5b binds C6, C7, C8 and C9 to form the membrane attack complex (MAC) which forms a transmembrane channel and causes cell lysis. Next to forming a pore in the membrane of pathogens, complement helps clearing pathogens or altered self-cells by opsonisation with C3b molecules and production of pro-inflammatory peptides such as C3a and C5a that attract and activate immune cells to the site of infection. Because of the strong pro-inflammatory nature of complement, host cells are well protected by several membrane and soluble complement-regulating proteins. The alternative pathway contributes for 80-90% to total complement activity. Regulation of this pathway is therefore crucial. C3(H2O) that is formed by spontaneous hydrolysis of C3, and C3b are generally, if not bound by a pathogen, rapidly inactivated by factor H (FH), factor I (FI) and host cell surface molecules thereby inhibiting the formation of the C3 convertases. CD55 (also termed decay accelerating factor or DAF) binds C3b at the host cell surface. FI cleaves C3b to an inactive form but is dependent on co-factors either expressed on cell surfaces (CD46, MCP) or circulating in plasma (FH). FH is a plasma glycoprotein that is essential for controlling the alternative pathway of complement both in solution and on cell surfaces. FH binds C3b at the same position as FB, thereby preventing the formation of C3 convertases. FH also has decay accelerating activity, i.e. it promotes the dissociation of alternative pathway C3 convertases once they have been formed. Whether FH binds to C3b is determined by the carbohydrates present on the cell surface. Sialic acid, glycosaminoglycans and heparin present on the host cell surface promotes binding of FH to C3b, whereas binding of C3b to molecules expressed on the surface of pathogens results in binding of FB. FH thus exerts its complement inhibitory activity on host cells but not on the surface of pathogenic cells because the cell surface molecules that bind FH are