Search

US-12617863-B2 - Humanized anti-glycoprotein Ib alpha (GPIbalpha) antibodies

US12617863B2US 12617863 B2US12617863 B2US 12617863B2US-12617863-B2

Abstract

Multivalent anti-platelet glycoprotein I(b)alpha antibodies can cause severe side effects. The present disclosure provides humanized antibodies specifically recognizing glycoprotein I(b)alpha and lacking a Fc portion, therefore Bleeding Time do not interact with Fc receptor. The humanized antibodies are capable of preventing platelet activation and aggregation, and reducing thrombus size/growth and prevent vessel occlusion. They can be also very useful to decrease platelet-tumor cell interaction and decrease tumor metastasis. At therapeutic doses, the humanized antibodies lack the ability to induce platelet activation, induce thrombocytopenia; and/or prolong bleeding time.

Inventors

  • Heyu Ni

Assignees

  • CCOA Therapeutics Inc.

Dates

Publication Date
20260505
Application Date
20201210

Claims (20)

  1. 1 . A humanized antibody or fragment thereof specifically recognizing platelet glycoprotein I(b)α (GPIbα), wherein a Fc moiety is present or absent, wherein the humanized antibody or fragment thereof: is capable of preventing platelet activation, aggregation, and/or thrombus growth; lacks the ability to activate platelets; lacks the ability to induce thrombocytopenia; and/or at a therapeutic dose, lacks the ability to prolong bleeding time; wherein the humanized antibody or fragment thereof has a heavy chain and a light chain comprising: i) the heavy chain variable region having an amino acid sequence of QVQLVESGGGLVKPGGSLRLSCAASGFTFSSFAMSWIRQAPGKGLEWVSSITSAG TPYYPDSVLGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSRGYEDYFDYW GQGTLVTVSS (SEQ ID NO: 93); and the light chain variable region having an amino acid of sequence DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSRNOKNYLAWYQQKPGKAPKLLIY FTSTRESGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCQQHYSSPWTFGQGTKVE IK (SEQ ID NO: 95); or ii) the heavy chain variable region having an amino acid sequence of QVQLVESGGGLVKPGGSLRLSCAASGFTFSSFAMSWIRQAPGKGLEWVSSITSAG TPYYPDSVLGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSRGYEDYFDYW GQGTLVTVSS (SEQ ID NO: 93); and the light chain variable region having an amino acid of sequence DIVMTQSPLSLPVTPGEPASISCKSSQSLLNSRNQKNYLAWYLOKPGQSPOLLIYF TSTRESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSSPWTFGQGTKVE IK (SEQ ID NO: 96); or iii) the heavy chain variable region having an amino acid sequence of QVOLVESGGGVVQPGRSLRLSCAASGFTFSSFAMSWVRQAPGKGLEWVASITSA GTPYYPDSVLGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSRGYEDYFDY WGQGTLVTVSS (SEQ ID NO: 94); and the light chain variable region having an amino sequence acid of DIVMTQSPLSLPVTPGEPASISCKSSQSLLNSRNQKNYLAWYLOKPGOSPQLLIYF TSTRESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSSPWTFGQGTKVE IK (SEQ ID NO: 96); and wherein the humanize antibody or fragment thereof comprises: a first heavy chain CDR having the amino acid sequence of SEQ ID NO: 37; a second heavy chain CDR having the amino acid sequence of SEQ ID NO: 38; a third heavy chain CDR having the amino acid sequence of SEQ ID NO: 39; a first light chain CDR having the amino acid sequence of SEQ ID NO: 65; a second light chain CDR having the amino acid sequence of SEQ ID NO: 66; and a third light chain CDR having the amino acid sequence of SEQ ID NO: 67.
  2. 2 . The humanized antibody or fragment thereof of claim 1 being capable of recognizing a human GPIbα, a mouse GPIbα, a dog GPIbα, a rat GPIbα, a rabbit GPIbα and/or a monkey GPIbα.
  3. 3 . The humanized antibody or fragment thereof of claim 1 being an antibody fragment.
  4. 4 . The humanized antibody or fragment thereof of claim 3 being a F(ab) 2 fragment.
  5. 5 . The humanized antibody or fragment thereof of claim 3 , wherein the antibody is a monovalent antibody.
  6. 6 . The humanized antibody or fragment thereof of claim 5 being a Fab antibody fragment.
  7. 7 . The humanized antibody or fragment thereof of claim 5 being a single chain variable fragment (scFv).
  8. 8 . The humanized antibody or fragment thereof of claim 1 , wherein the heavy chain further comprises a CH1 region of a human IgG 1 antibody, wherein the CH1 region of the human IgG 1 antibody has the amino acid sequence of SEQ ID NO: 40 or 61.
  9. 9 . The humanized antibody or fragment thereof of claim 8 , wherein the heavy chain has the amino acid sequence of SEQ ID NO: 36 or 57.
  10. 10 . The humanized antibody or fragment thereof of claim 1 , wherein the light chain further comprises a kappa chain C region of a human IgG 1 antibody, wherein the kappa chain C region has the amino acid sequence of SEQ ID NO: 75 or 82.
  11. 11 . The humanized antibody or fragment thereof of claim 10 , wherein the light chain has the amino acid sequence of SEQ ID NO: 71 or 78.
  12. 12 . The humanized antibody or fragment thereof of claim 1 having: the heavy chain of SEQ ID NO: 36 and the light chain of SEQ ID NO: 71; the heavy chain of SEQ ID NO: 36 and the light chain of SEQ ID NO: 78; or the heavy chain of SEQ ID NO: 57 and the light chain of SEQ ID NO: 78.
  13. 13 . A chimeric protein comprising the humanized antibody or fragment thereof of claim 1 and a carrier protein.
  14. 14 . A pharmaceutical composition comprising (i) the humanized antibody or fragment thereof of claim 1 or the chimeric protein of claim 13 and (ii) a pharmaceutical excipient.
  15. 15 . A method of preventing or limiting the interaction between glycoprotein I(b)α (GPIbα) present on a platelet and a GPIbα ligand, the method comprising contacting the humanized antibody or fragment thereof of claim 1 , the chimeric protein of claim 13 or the pharmaceutical composition of claim 14 with the platelet.
  16. 16 . The method of claim 15 for preventing or limiting platelet activation.
  17. 17 . The method of claim 15 , wherein the GPIbα ligand is von Willebrand factor (VWF), P-selectin, kininogen, thrombospondin and/or thrombin.
  18. 18 . The method of claim 15 , wherein the contacting occurs under high or low shear rates.
  19. 19 . The method of any claim 15 , wherein the humanized antibody, the chimeric protein or the pharmaceutical composition is contacted with the platelet prior to, at the same time or after the GPIbα ligand is contacted with the platelet.
  20. 20 . The method of claim 15 for preventing or limiting the interaction in vivo in a subject in need thereof, wherein the subject is at risk of experiencing or has experienced a pathological thrombosis.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS AND SEQUENCE LISTING STATEMENT This application claims priority from U.S. provisional application Ser. No. 62/946,086 filed on Dec. 10, 2019 and incorporated herewith in its entirety. The sequence listing associated with this application is provided in text format and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is PCT_-_Sequence_listing_as_filed. The text file is 81.2 Ko, was created on Dec. 9, 2020 and is being submitted electronically. TECHNOLOGICAL FIELD The present disclosure concerns humanized antibodies which specifically recognize and bind to the platelet glycoprotein I(b)α (GPIbα) as well as protein construct comprising same, and therapeutic uses associated thereto. BACKGROUND Platelet adhesion and aggregation at sites of atherosclerotic rupture in coronary or cerebral arteries are usually the critical events in acute thrombosis. Therefore, anti-platelet therapy represents one of the key treatment regimens in reducing cardiovascular deaths, including (i) cyclooxygenase inhibitors such as aspirin; (ii) platelet P2Y12 receptor antagonists such as clopidogrel, prasugrel and ticagrelor; (iii) aIIbβ3 antagonists such as abciximab, eptifibatide and tirofiban; and (iv) PAR1 antagonists such as vorapaxar, etc. However, limitations of current anti-platelet therapies, such as a slow onset/weak/poor inhibition of platelet function, excessive bleeding complications, thrombocytopenia and unexpected platelet activation are major concerns that drive therapeutic advances. Notably, concerning acute ischemic stroke, since the risk for intracranial hemorrhage and/or potential neurotoxicity (e.g. recombinant tissue plasminogen activator (tPA)) of current anti-thrombotic/thrombolytic drugs, as well as for patients who have passed the window for intravenous thrombolysis, treatments are very limited if it is not available. Platelet GPIb-IX-V complex has emerged as a promising anti-platelet target. GPIb-IX-V complex is a key platelet receptor in initiating platelet adhesion and translocation to the injured vessel wall, particularly at high shear. Platelet adhesion/translocation onto the subendothelium is mediated by the binding of GPIbα subunit to von Willebrand Factor (VWF) that anchored/immobilized on the injured vessel wall. VWF is a multimeric adhesive blood protein secreted from activated endothelial cells and platelets. GPIbα-VWF binding can then trigger a signal transduction process that leads to the release of platelet agonists, such as thromboxane A2 and ADP, and the activation of platelet αIIbβ3 integrin that results in platelet aggregation mediated by αIIbβ3 binding to fibrinogen, VWF, etc. Under high shear conditions, the GPIbα-VWF interaction are required for pathologic growth of occlusive thrombi (both platelet adhesion and platelet aggregation/agglutination) at sites of arterial stenosis where blood flows with wall shear rates that may exceed 10,000-40,000 s−1, while under low shear conditions (such as in the most cases of hemostasis), platelet adhesion can be directly mediated by αIIbβ3-fibrinogen/fibrin and α2β1/GPVI-collagen interactions, etc. Therefore, pharmacological inhibition of GPIbα may result in a lower risk of systemic bleeding and improved safety compared to other antiplatelet drugs that are not specifically target thrombosis at high shear. It has also been shown that GPIbα is important for leukocyte recruitment under thrombo-inflammatory conditions, such as in acute ischemic stroke. Moreover, ischemia—reperfusion (e.g. by thrombolysis or thrombectomy) of the previously hypoxic brain areas can increase the pro-inflammatory function of platelets via GPIbα, which may further promote thrombo-inflammatory neuronal damage and infarct growth. Furthermore, GPIbα has been considered exclusively expressed on platelets and megakaryocytes. Thus, direct platelet GPIbα antagonists have a great potential to be developed as effective and safer anti-platelet drugs for the treatment of acute thrombotic events, such as heart attack and stroke. Notably, novel anti-platelet strategies targeting GPIbα-VWF interaction has been demonstrated as an effective therapy to treat acquired thrombotic thrombocytopenic purpura (aTTP), a thrombotic microangiopathy and a life-threatening condition with a high mortality rate if untreated. Autoantibodies against ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13), the VWF-cleaving protease that cleaves/reduces the multimeric size of VWF, results in the severe deficiency of ADAMTS13 activity. These ultra-large VWF are hyper-adhesive that cause the formation of platelet (GPIbα)-VWF microthrombi in blood vessels, leading to the end-organ ischemia and infarction, low platelet counts and destruction of red blood cells. Therefore, blocking the interaction between VWF and platelet GPIbα can prevent the development of acute TTP by achievin