WO2023108101A1 - Antibodies that inhibit glycoprotein ib-ix mediated platelet signaling and uses in managing bleeding conditions - Google Patents

Abstract

This disclosure relates to antibodies that inhibit glycoprotein Ib-IX-mediated platelet signaling and clearance. In certain embodiments, this disclosure relates to recombinant chimeric antibodies or antibody fragments comprising one or more complementarity determining regions (CDRs) of antibodies disclosed herein. In certain embodiments, this disclosure relates to methods of treating or preventing glycoprotein Ib-IX-mediated bleeding disorders or conditions using antibodies or fragments disclosed herein.

Description

ANTIBODIES THAT INHIBIT GLYCOPROTEIN IB-IX MEDIATED PLATELET

SIGNALING AND USES IN MANAGING BLEEDING CONDITIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/287,818 filed December 9, 2021. The entirety of this application is hereby incorporated by reference for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under HL082808 awarded by the National Institutes of Health. The government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN XML FILE VIA THE OFFICE ELECTRONIC FILING SYSTEM

The Sequence Listing associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is 21153PCT.xml. The XML file is 19 KB, was created on December 9, 2022, and is being submitted electronically via the USPTO patent electronic filing system.

BACKGROUND

The interaction of platelets with von Willebrand Factor (VWF) is a mechanism that facilitates blood clotting. Lack of a sufficient number of platelets in blood, referred to as thrombocytopenia, can cause uncontrolled bleeding and is sometimes fatal. For instance, mechanical circulatory support systems, including extracorporeal membrane oxygenation (ECMO), have been increasingly used to support patients suffering from respiratory and/or cardiac failure. ECMO is frequently used in the intensive care unit (ICU) in the hospital, for both pediatric and adult populations. ECMO, with multiple additional components and long periods of support, has a high occurrence of thrombotic and bleeding complications that, when they occur, sometimes leads to death. Thus, there is a need to identify improved strategies for managing bleeding complications. Deng et al. report platelet clearance via shear-induced unfolding of a membrane mechanoreceptor. Nat Commun, 2016, 7: 12863.

Liang et al. report dimerization of glycoprotein Iba is not sufficient to induce platelet clearance. J Thromb Haemost, 2016, 14(2):381-6.

Perrault et al. report a monoclonal antibody against the extracellular domain of GPIbp modulates vWF mediated platelet adhesion. Thromb Haemost, 2001, 86(5): 1238-48.

Ojima-Kato et al. report ‘Zipbody’ leucine zipper-fused Fab in E. coli in vitro and in vivo expression systems. Protein Engineering, Design & Selection, 2016, 29(4): 149-157.

References cited herein are not an admission of prior art.

SUMMARY

This disclosure relates to antibodies that inhibit glycoprotein Ib-IX-mediated platelet signaling and clearance. In certain embodiments, this disclosure relates to recombinant chimeric antibodies or antibody fragments comprising one or more complementarity determining regions (CDRs) of antibodies disclosed herein. In certain embodiments, this disclosure relates to pharmaceutical compositions comprising antibodies or fragments disclosed herein. In certain embodiments, this disclosure relates to nucleic acids and vectors encoding antibodies or fragments disclosed herein and cells and expression system for producing said antibodies or fragments. In certain embodiments, this disclosure relates to methods of treating or preventing bleeding conditions or disorders using antibodies or fragments disclosed herein.

In certain embodiments, this disclosure relates to recombinant chimeric antibodies or antibody fragments comprising six complementarity determining regions (CDRs) of antibody RAM.l wherein the CDRs comprise three heavy chain CDRs, wherein heavy chain CDR 1 comprises the amino acid sequence of GFNVNNDWMG (SEQ ID NO: 1); CDR 2 comprises the amino acid sequence of EINKDSSTINYNPSLKG (SEQ ID NO: 2), and CDR 3 comprises the amino acid sequence of ALTMGIDY (SEQ ID NO: 3), and, wherein the CDRs comprise three light chain CDRs, wherein light chain CDR 1 comprises the amino acid sequence of KASQNVGNNIA (SEQ ID NO: 4); light chain CDR2 comprises the amino acid sequence of FASSRYT (SEQ ID NO: 5); and light chain CDR3 comprises the amino acid sequence of QRVYNSP (SEQ ID NO: 6); and, wherein the antibody comprises a heavy chain having a variable and constant region and a light chain having a variable and constant region, and wherein the antibody or fragment specifically binds GPIb-beta extracellular domain, or specifically binds GPIb-IX complex and does not bind GPIb-alpha subunit.

In certain embodiments, the heavy chain comprises a leucine containing/zipper/repeat sequence C-terminal to a heavy chain constant region. In certain embodiments, the leucine containing sequence comprises the amino acid sequence

LX¹X¹X²X¹X¹X¹LX¹X¹X²X¹X¹X¹LX¹X¹X²X¹X¹X¹LX¹X¹X²X¹X¹X¹L (SEQ ID NO: 7) or LX¹X¹X²X¹X¹X¹LX¹X¹X²X¹X¹X¹LX¹X¹X²X¹X¹X¹L, (SEQ ID NO: 8) wherein X¹ is individually and independently at each occurrence any amino acid, and X² is individually and independently at each occurrence an amino acid selected from K, D, E, R, S, Q, T, and L or any amino acid. In certain embodiments, the leucine containing sequence is capable of forming dimers or multimers. In certain embodiments, the leucine containing sequence comprises the amino acid sequence of RMKQLEDKVEELLSKNYHLENEVARLKKLVGER (SEQ ID NO: 9, GCN4).

In certain embodiments, the heavy chain comprises the amino acid sequence of

EVKLVESGGGLVQPGRSLKLSCAASGFNVNNDWMGWVRQAPGKGLEWIGEIN KDSSTINYNPSLKGKLTISRDNAQNTLYLQMSKLGSEDTAIYYCARALTMGIDYWGPG VMVTVSS (SEQ ID NO: 10) or variants thereof with greater than 60% 70%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98 %, 99% identity thereto.

In certain embodiments, the heavy chain comprises the amino acid sequence of

EVKLVESGGGLVQPGRSLKLSCAASGFNVNNDWMGWVRQAPGKGLEWIGEIN KDSSTINYNPSLKGKLTISRDNAQNTLYLQMSKLGSEDTAIYYCARALTMGIDYWGPG VMVTVS S S AKTTPPS VYPL APGS AAQTNSMVTLGCLVKGYFPEP VTVTWNSGSLS SGVH TFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTG GGRMKQLEDKVEELLSKNYHLENEVARLKKLVGER (SEQ ID NO: 11) or variants thereof with greater than 60% 70%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98 %, 99% identity thereto.

In certain embodiments, the light chain comprises the amino acid sequence of

NNIVMTQSPKSMSISVGDRVTMNCKASQNVGNNIAWYQQKPGQSPKLLIYFASS RYTGVPDRFTGGGYGTDFTLTINGVQVEDAAFYYCQRVYNSPWTFGGGTNLELK (SEQ ID NO: 12) or variants thereof with greater than 60% 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% identity thereto. In certain embodiments, this disclosure relates to pharmaceutical composition comprising the recombinant chimeric antibody disclosed herein and a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition is in the form of an aqueous buffered solution. In certain embodiments, the pharmaceutical composition is in the form of an aqueous buffered saline solution. In certain embodiments, the pharmaceutical composition is in the form of an aqueous solution comprising a saccharide or polysaccharide.

In certain embodiments, this disclosure relates to kits comprising an antibody disclosed herein. In certain embodiments, an antibody disclosed herein is within a container, e.g., box, vial, syringe, optionally containing instructions for administration on or in the container.

In certain embodiments, this disclosure relates to nucleic acids or vectors encoding the heavy chain and/or light chain of an antibody or fragment disclosed herein in operable combination with a heterologous promoter.

In certain embodiments, this disclosure relates to cells or other expression systems comprising a nucleic acid or vector encoding the heavy chain and/or light chain of an antibody fragment thereof in operable combination with a heterologous promoter.

In certain embodiments, this disclosure relates to method treating or preventing a bleeding disorder comprising administering an effective amount of an antibody or fragment disclosed herein to a subject in need thereof. In certain embodiments, the bleeding disorder is thrombocytopenia, spontaneous or excessive bleeding. In certain embodiments, the bleeding disorder is von Willebrand disease or acquired von Willebrand syndrome (A VWS). In certain embodiments, the bleeding disorder is a GPIb-IX mediated disorder.

In certain embodiments, this disclosure relates to method treating, reducing, or preventing thrombocytopenia, such as inflammation-linked thrombocytopenia or thrombotic thrombocytopenia (e.g. VWF binding induced thrombocytopenia). In certain embodiments, this disclosure relates to methods of treating, reducing, or preventing thrombocytopenia using RAM.1- GCN4.

In certain embodiments, this disclosure relates to uses of an antibody disclosed herein for treating or preventing a bleeding disorder. In certain embodiments, this disclosure relates to uses of an antibody disclosed herein in the production of a medicament for treating or preventing a bleeding disorder or thrombocytopenia. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Figure 1 illustrates unfolding of the mechanosensory domain (MSD) in the GPIb-IX complex leads to platelet signaling and its clearance.

Figure 2A illustrates that VWF interacts with GPIb-alpha causing filopodia formation which can be inhibited with RAM.1.

Figure 2B illustrates that RAM.l inhibits signaling of a mutant GPIb-IX complex that mimics the unfolded state of MSD and induces ligand-free filopodia formation.

DETAILED DISCUSSION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims. Although the function of certain compositions disclosed herein are believed to operate by particular mechanisms, it is not intended that embodiments of this disclosure be limited by any specific mechanism.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. An "embodiment" of this disclosure refers to an example and infers that the example is not necessarily limited to the example. Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

As used in this disclosure and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") have the meaning ascribed to them in U.S. Patent law in that they are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

"Consisting essentially of' or "consists of' or the like, have the meaning ascribed to them in U.S. Patent law in that when applied to methods and compositions encompassed by the present disclosure refers to the idea of excluding certain prior art element(s) as an inventive feature of a claim, but which may contain additional composition components or method steps, etc., that do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein.

"Subject" refers to any animal, preferably a human patient, livestock, rodent, monkey, or domestic pet.

As used herein, the terms "prevent" and "preventing" include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease is reduced.

As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments, of the present disclosure also contemplate treatment that merely reduces symptoms, and/or delays disease progression. As used herein, the term "combination with" when used to describe administration with an additional treatment means that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof.

The term "effective amount" refers to that amount of a compound or pharmaceutical composition described herein that is sufficient to effect the intended application including, but not limited to, disease treatment as illustrated below. The therapeutically effective amount can vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The specific dose will vary depending on, for example, the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other agents, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

In certain contexts, an “antibody” refers to a protein-based molecule that is naturally produced by animals in response to the presence of a protein or other molecule or that is not recognized by the animal’s immune system to be a “self’ molecule, i.e., recognized by the animal to be a foreign molecule, i.e., an antigen to the antibody. The immune system of the animal will create an antibody to specifically bind the antigen, and thereby targeting the antigen for degradation or elimination, or any cell or organism attached to the antigen. It is well recognized by skilled artisans that the molecular structure of a natural antibody can be synthesized and altered by laboratory techniques. Recombinant engineering can be used to generate fully synthetic antibodies or fragments thereof providing control over variations of the amino acid sequences of the antibody. Thus, the term “antibody” is intended to include natural antibodies, monoclonal antibody, or non-naturally produced synthetic antibodies, such as specific binding single chain antibodies, bispecific antibodies, or fragments thereof. These antibodies may have chemical modifications. The term "monoclonal antibodies" refers to a collection of antibodies encoded by the same nucleic acid molecule that are optionally produced by a single hybridoma (or clone thereof) or other cell line, or by a transgenic mammal such that each monoclonal antibody will typically recognize the same antigen. The term "monoclonal" is not limited to any particular method for making the antibody, nor is the term limited to antibodies produced in a particular species, e.g., mouse, rat, etc. From a structural standpoint, an antibody is a combination of proteins: two heavy chain proteins and two light chain proteins. The heavy chains are longer than the light chains. The two heavy chains typically have the same amino acid sequence. Similarly, the two light chains typically have the same amino acid sequence. Each of the heavy and light chains contain a variable segment that contains amino acid sequences which participate in binding to the antigen. The variable segments of the heavy chain do not have the same amino acid sequences as the light chains. The variable segments are often referred to as the antigen binding domains. The antigen and the variable regions of the antibody may physically interact with each other at specific smaller segments of an antigen often referred to as the "epitope." Epitopes usually consist of surface groupings of molecules, for example, amino acids or carbohydrates. The terms “variable region,” "antigen binding domain," and "antigen binding region" refer to that portion of the antibody molecule which contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. Small binding regions within the antigenbinding domain that typically interact with the epitope are also commonly alternatively referred to as the "complementarity-determining regions, or CDRs."

With regard to variable chain immunoglobulins, the location of binding complementaritydetermining regions (CDRs) sometimes varies depending on the specific sequence context and animal. The CDRs can be determined through epitope studies and sequence alignment comparisons of the constant and framework regions for the specific animal. As is well-known in the art that there are multiple conventions to define and describe the CDRs of a VH or VHH fragment, such as the Kabat definition (which is based on sequence variability) and the Chothia definition (which is based on the location of the structural loop regions).

In general, identifying CDRs can be accomplished utilized the following rules using Kabat or Chothia antibody sequence criteria. Note that there are examples where these constant features do not occur; however, the Cys residues are the most common conserved feature.

For CDR-L1, the start residue is approximately 24 to 30 after the first amino acid and typically after a Cys. The residue after is typically a Trp such as Trp-Tyr-Gln, Trp-Leu-Gln, Trp- Phe-Gln, or Trp-Tyr-Leu. Length is typically 10 to 17 residues. CDR-L2 starts about 16 residues after the end of LI. The residues before are typically Ile-Tyr, Val-Tyr, Ile-Lys, Ile-Phe, with a length of about 4-7 residues. CDR-L3 starts at about 33 residues after end of L2 before a Cys residue with a length of about 7 to 11 residues typically ending before a Phe-Gly. For CDR-H1, the start residue is approximately 26 to 30 after the first amino acid and typically 4 amino acids after a Cys and typically ends with Trp, e.g., Trp-Val, but also, Trp-Ile, Trp-Ala. The length is typically about 6 to 12 residues. CDR-H2 typically starts at about 4-15 residues after the end of CDR-H1. Residues before the start are typically Trp-Ile-Gly but can be a number of variations, and residues after typical ends with Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala- Thr/Ser/Ile/Ala. The length can vary from about 8 to 20 amino acids; CDR-H3 is typically about 30-33 residues after the end of CDR-H2, and often identified 3 amino acids after a Cys, such as in the example Cys-Ala-Arg. The end is sometimes identified before residues such as Trp-Gly. The length can vary widely, e.g., 4-25 or more depending on the animal.

A "chimeric antibody" is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such that the entire molecule is not naturally occurring. Examples of chimeric antibodies include those having a variable region derived from a non-human antibody and a human immunoglobulin constant region. The term is also intended to include antibodies having a variable region derived from one human antibody grafted to an immunoglobulin constant region of a predetermined sequences or the constant region from another human for which there are allotypic differences residing in the constant regions of any naturally occurring antibody having the variable regions, e.g., CDRs 1, 2, and 3 of the light and heavy chain. Human heavy chain genes exhibit structural polymorphism (allotypes) that are inherited as a haplotype. The serologically defined allotypes differ within and between population groups. See Jefferis et al. mAb, 1 (2009), pp. 332-338. In certain embodiments, the antibody, antigen binding fragment, the light chain, or the heavy chain comprises a non-naturally occurring chimeric amino acid sequence such that there is at least one mutation that is not present in naturally occurring antibodies comprising one or all of the six CDRs.

The term "antibody fragment" refers to an antibody which comprises less than a complete, intact antibody. Complete antibodies comprise two functionally independent parts or fragments: an antigen binding fragment known as "Fab," and a carboxy terminal crystallizable fragment known as the "Fc" fragment. The Fab fragment includes the first constant domain from both the heavy and light chain (CHI and CL1) together with the variable regions from both the heavy and light chains that bind the specific antigen. Each of the heavy and light chain variable regions includes three complementarity determining regions (CDRs) and framework amino acid residues which separate the individual CDRs. The Fc region comprises the second and third heavy chain constant regions (CH2 and CH3) and is involved in effector functions such as complement activation and attack by phagocytic cells. In some antibodies, the Fc and Fab regions are separated by an antibody "hinge region," and depending on how the full-length antibody is proteolytically cleaved, the hinge region may be associated with either the Fab or Fc fragment. For example, cleavage of an antibody with the protease papain results in the hinge region being associated with the resulting Fc fragment, while cleavage with the protease pepsin provides a fragment wherein the hinge is associated with both Fab fragments simultaneously. Because the two Fab fragments are covalently linked following pepsin cleavage, the resulting fragment is termed the F(ab')2 fragment.

The term, "humanized" refers to an antibody containing one or more amino acid mutations so that immunogenicity upon administration in human patients, e.g., due to "pre-existing antibodies", is reduced, made highly unlikely, or nonexistent. Anaphylaxis is a severe allergic reaction to an allergen, e.g., polypeptide. Non-human proteins contain amino acid residues that may be immunogenic when targeted by preexisting antibodies circulating in a human patient. Thus, it is desirable to mutate residues within a therapeutic antibody so that the peptide sequences are similar to peptide sequences that commonly occurs in human antibodies/proteins, provided that the desirable therapeutic properties are retained, thereby reducing the risk of undesirable allergic reactions. In antibodies, this is typically accomplished by transfer of complementaritydetermining regions (CDRs) of a non-human antibody to a human framework sequence, yielding a human like antibody with reduced immunogenicity. Another method entails comparing sequences, preferably framework sequences, and identifying amino acid substitutions providing "humanized" sequences frequently found within human antibody sequence repertoire. These humanized sequences reduce the risk of undesirable immune reactions providing an antibody that is substantially non-immunogenic in humans and retain the affinity and activity of the original polypeptide. In one example humanization, e.g., framework region humanization, one screens the available human consensus sequences for existing known sequences that are most similar to the original sequence. Using computer databases to compare human consensus frameworks for humanizing antibodies is well-known. See e.g., A universal combinatorial design of antibody framework to graft distinct CDR sequences: a bioinformatics approach, Proteins, 2012, 80(3):896- 912 and Clavero- Alvarez et al. Humanization of Antibodies using a Statistical Inference Approach, Scientific Reports, 2018, volume 8, Article number: 14820. European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, veneering or resurfacing (European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al., 1994, Proc. Natl. Acad. Sci. 91 :969-973), chain shuffling (U.S. Pat. No. 5,565,332), and techniques disclosed in, e.g., U.S. Pat. Nos. 6,407,213, 5,766,886, 5,585,089, International Publication No. WO 9317105, Tan et al., 2002, J. Immunol. 169:1119- 25, Caldas et al., 2000, Protein Eng. 13:353-60, Morea et al., 2000, Methods 20:267-79, Baca et al., 1997, J. Biol. Chem. 272: 10678-84, Roguska et al., 1996, Protein Eng. 9:895-904, Couto et al., 1995, Cancer Res. 55 (23 Supp):5973s-5977s, Couto et al., 1995, Cancer Res. 55: 1717-22, Sandhu, 1994, Gene 150:409-10, Pedersen et al., 1994, J. Mol. Biol. 235:959-73, Jones et al., 1986, Nature 321 :522-525, Riechmann et al., 1988, Nature 332:323, and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596.

The framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor CDR or the consensus framework may be mutagenized by substitution, insertion, or deletion of at least one residue so that the CDR or framework residue at that site does not correspond exactly to either the consensus or the donor antibody. Such mutations, however, are preferably not extensive. Usually, at least 75% of the humanized antibody residues will correspond to those of the parental framework region (FR) and CDR sequences, more often 90%, and most preferably greater than 95%.

In certain instances, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; U.S. Publication Nos. 2004/0049014 and 2003/0229208; U.S. Pat. Nos. 6,350,861; 6,180,370; 5,693,762; 5,693,761; 5,585,089; and 5,530,101 and Riechmann et al., 1988, Nature 332:323).

The terms "protein," "peptide," and "polypeptide" refer to polymers comprising amino acids joined via peptide bonds and are used interchangeably. Amino acids may be naturally or non-naturally occurring. A "chimeric protein" or "fusion protein" is a molecule in which different portions of the protein are derived from different origins such that the entire molecule is not naturally occurring. A chimeric protein may contain amino acid sequences from the same species or different species as long as they are not arranged together in the same way that they exist in a natural state. Examples of a chimeric protein include sequences disclosed herein that contain one, two or more amino acids attached to the C-terminal or N-terminal end that are not identical to any naturally occurring protein, such as in the case of adding an amino acid containing an amine side chain group, e.g., lysine, an amino acid containing a carboxylic acid side chain group such as aspartic acid or glutamic acid, a polyhistidine (HIS) tag, e.g., typically four or more histidine amino acids, a human influenza hemagglutinin (HA) tag, a TAT polypeptide, GST peptide, or a selfcleaving peptide P2A-GSG.

The term “comprising” in reference to a protein having an amino acid sequence refers a peptide that may contain additional N-terminal (amine end) or C-terminal (carboxylic acid end) amino acids, i.e., the term is intended to include the amino acid sequence within a larger peptide. The term “consisting of’ in reference to a peptide having an amino acid sequence refers a peptide having the exact number of amino acids in the sequence and not more or having not more than a rage of amino acids expressly specified in the claim. In certain embodiments, the disclosure contemplates that the “N-terminus of a peptide consists of an amino acid sequence,” which refers to the N-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a rage of amino acids specified in the claim however the C-terminus may be connected to additional amino acids, e.g., as part of a larger peptide. Similarly, the disclosure contemplates that the “C-terminus of a peptide consists of an amino acid sequence,” which refers to the C-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a rage of amino acids specified in the claim however the N-terminus may be connected to additional amino acids, e.g., as part of a larger peptide.

A "variant" refers to a chemically similar sequence because of amino acid changes. In certain embodiments, a variant contains one or two, or more amino acid deletions or substitutions. In certain embodiments, the substitutions are conserved substitutions. In certain embodiments, a variant contains one, two, or ten or more, or ten or less amino acid additions. In certain embodiments, the additions may be to the N-terminus or the C-terminus. The variant may be substituted with one or more chemical substituents. A conservative amino acid substitution refers to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. A variant may have "non-conservative" changes (e.g., replacement of a glycine with a tryptophan). Similar minor variations may also include amino acid deletions or insertions (in other words, additions), or both. Guidance in determining which and how many amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art. Variants can be tested in functional assays. Certain variants have less than 10%, and preferably less than 5%, and still more preferably less than 2% changes (whether substitutions, deletions, and so on). Variants can be prepared for testing by mutating a vector to produce appropriate codon alternatives for peptide translation.

The term "derivative" additionally encompasses non-amino acid modifications, for example, amino acids that may be glycosylated (e.g., have altered mannose, 2-N- acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5- glycolneuraminic acid, etc.), acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytic cleavage, linked to a cellular ligand or other protein, etc. In some embodiments, the altered carbohydrate modifications modulate one or more of the following: solubilization of the antibody, facilitation of subcellular transport and secretion of the antibody, promotion of antibody assembly, conformational integrity, and antibody-mediated effector function.

Derivatized antibodies may be used to alter the half-lives (e.g., serum half-lives) of parental antibodies in a mammal, preferably a human. Preferably such alteration will result in a half-life of greater than 15 days, preferably greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The increased half-lives of the humanized antibodies of the present disclosure or fragments thereof in a mammal, preferably a human, results in a higher serum titer of said antibodies or antibody fragments in the mammal, and thus, reduces the frequency of the administration of said antibodies or antibody fragments and/or reduces the concentration of said antibodies or antibody fragments to be administered. Antibodies or fragments thereof having increased in vivo half-lives can be generated by techniques known to those of skill in the art. For example, antibodies or fragments thereof with increased in vivo halflives can be generated by modifying (e.g., substituting, deleting, or adding) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor. Antibodies can be engineered to increase biological half-lives (see, e.g. U.S. Pat. No. 6,277,375).

Antibodies or fragments thereof with increased in vivo half-lives can be generated by attaching to said antibodies or antibody fragments polymer molecules such as high molecular weight polyethylene glycol (PEG). PEG can be attached to said antibodies or antibody fragments with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C-terminus of said antibodies or antibody fragments or via epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity will be used. The degree of conjugation will be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies. Unreacted PEG can be separated from antibody-PEG conjugates by, e.g., size exclusion or ion-exchange chromatography.

The term “recombinant” as used herein refers to an antibody produced by recombinant means, such as isolation of immunoglobulin sequences from human cells and their expression in transfected cells.

In certain embodiments, the antibodies disclosed herein have at least one non-naturally occurring molecular modification, such as the attachment of polyethylene glycol, the attachment of a chimeric peptide, the attachment of a fluorescent dye comprising aromatic groups, fluorescent peptide, a chelating agent capable of binding a radionuclide, e.g., ¹⁸F. In certain embodiments, the peptides contain an N-terminal acetyl, propionyl group, myristoyl and palmitoyl, group or N- terminal mono- or di-methylation, or a C-terminal alkyl ester or amide. In certain embodiments, this disclosure contemplates antibodies disclosed herein labeled using commercially available biotinylation reagents. Biotinylated antibodies can be used in streptavidin/avidin affinity binding, purification, and detection. In certain embodiments, the disclosure contemplates an antibody disclose herein containing azide-derivatives of naturally occurring monosaccharides such as N- azidoacetylglucosamine, N-azidoacetylmannosamine, and N-azidoacetylgalactosamine.

In certain embodiments, this disclosure contemplates derivatives of antibodies disclose herein wherein one or more amino acids are substituted with chemical groups to improve pharmacokinetic properties such as solubility and serum half-life, optionally connected through a linker. In certain embodiments, such a derivative may be a prodrug wherein the substituent or linker is biodegradable, or the substituent or linker is not biodegradable. In certain embodiments, contemplated substituents include a saccharide, polysaccharide, acetyl, fatty acid, lipid, and/or polyethylene glycol. The substituent may be covalently bonded through the formation of amide bonds on the C-terminus or N-terminus of the antibody optionally connected through a linker. In certain embodiments, it is contemplated that the substituent may be covalently bonded through an amino acid within the antibody, e.g., through an amine side chain group such as lysine or an amino acid containing a carboxylic acid side chain group such as aspartic acid or glutamic acid, within the antibody comprising a sequence disclosed herein. In certain embodiments, it is contemplated that the substituent may be covalently bonded through a cysteine in a sequence disclosed herein optionally connected through a linker. In certain embodiments, a substituent is connected through a linker that forms a disulfide with a cysteine amino acid side group.

As used herein, a "lipid" group refers to a hydrophobic group that is naturally or non- naturally occurring that is highly insoluble in water. As used herein a lipid group is considered highly insoluble in water when the point of connection on the lipid is replaced with a hydrogen and the resulting compound has a solubility of less than 0.63 x 10'⁴ % w/w (at 25 °C) in water, which is the percent solubility of octane in water by weight. See Solvent Recovery Handbook, 2^nd Ed, Smallwood, 2002 by Blackwell Science, page 195. Examples of naturally occurring lipids include saturated or unsaturated hydrocarbon chains found in fatty acids, glycerolipids, cholesterol, steroids, polyketides, and derivatives. Non-naturally occurring lipids include derivatives of naturally occurring lipids, acrylic polymers, aromatic, and alkylated compounds and derivatives thereof.

As used herein, the term “conjugated” refers to linking molecular entities through covalent bonds, linking groups, or by other specific binding interactions, such as due to hydrogen bonding or other van der Walls forces. The force to break a covalent bond is high, e.g., about 1500 pN for a carbon-to-carbon bond. The force to break a combination of strong protein interactions is typically a magnitude less, e.g., biotin to streptavidin is about 150 pN. Thus, a skilled artisan would understand that conjugation must be strong enough to bind molecular entities in order to implement the intended results.

A "linking group" refers to any variety of molecular arrangements that can be used to bridge two molecular moieties together. An example formula may be -Rm- wherein R is selected individually and independently at each occurrence as: -CRmRm-, -CHRm-, -CH-, -C-, -CH2-, -C(OH)R_m, -C(OH)(OH)-, -C(OH)H, -C(Hal)R_m-, -C(Hal)(Hal)-, -C(Hal)H-, -C(N₃)Rm-, -C(CN)R_m-, -C(CN)(CN)-, -C(CN)H-, -C(N₃)(N₃)-, -C(N₃)H-, -O-, -S-, -N-, -NH-, -NRm-, -(C=O)-, -(C=NH)-, -(C=S)-, -(C=CH2)-, which may contain single, double, or triple bonds individually and independently between the R groups. If an R is branched with an Rm it may be terminated with a group such as -CH₃, -H, -CH=CH2, -CCH, -OH, -SH, -NH2, -N₃, -CN, or -Hal, or two branched Rs may form a cyclic structure. It is contemplated that in certain instances, the total Rs or “m” may be less than 100, or 50, or 25, or 10. Examples of linking groups include bridging alkyl groups and alkoxyalkyl groups. Linking groups may be substituted with one or more substituents.

As used herein, the term "biodegradable" in reference to a substituent or linker refers to a molecular arrangement in a peptide derivative that when administered to a subject, e.g., human, will be broken down by biological mechanism such that a metabolite will be formed and the molecular arrangement will not persist for over a long period of time, e.g., the molecular arrangement will be broken down by the body after a several hours or days. In certain embodiments, the disclosure contemplates that the biodegradable linker or substituent will not exist after a week or a month.

Antibodies and the peptide sequences that make up antibodies can be produced by any commonly used method. Typical examples include the recombinant expression in suitable host systems, e.g., bacteria or yeast. In general, the peptides may be produced by living host cells that have been genetically engineered to produce the peptide. Methods of genetically engineering cells to produce proteins are well known in the art. See e.g., Ausubel et al., eds. (1990), Current Protocols in Molecular Biology (Wiley, New York). Such methods include introducing nucleic acids that encode and allow expression of the antibody/peptide into host cells. These host cells can be bacterial cells, fungal cells, or animal cells grown in culture. In one embodiment, the antibodies or peptides are produced in mammalian cells. Typical mammalian host cells for expressing the clone antibodies or antigen-binding fragments thereof include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216- 4220(1980), used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, Mol. Biol. 159:601-621 (1982)), lymphocytic cell lines, e.g., NS0 myeloma cells and SP2 cells, COS cells, and a cell from a transgenic animal, e.g., a transgenic mammal. For example, the cell is a mammary epithelial cell.

In addition to the nucleic acid sequences encoding the peptide, recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216; 4,634,665; and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on a host cell into which the vector has been introduced.

Standard molecular biology techniques can be used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the peptides from the culture medium. For example, the polypeptides can be isolated by affinity chromatography.

In certain embodiments, this disclosure relates to nucleotide sequences or nucleic acids that encode the antibodies and the peptides contained therein in genetic constructs that include the foregoing nucleotide sequences or nucleic acids and one or more elements for genetic constructs known per se. In certain embodiments, this disclosure relates to hosts or host cells that contain such nucleotide sequences or nucleic acids, and/or that express (or are capable of expressing), the antibodies and the peptides contained therein.

In certain embodiments, this disclosure relates to methods for preparing antibodies and the peptides contained therein, which method comprises cultivating or maintaining a host cell as described herein under conditions such that said host cell produces or expresses the antibodies and the peptides contained therein.

The term "nucleic acid" refers to a polymer of nucleotides, or a polynucleotide, e.g., RNA, DNA, or a combination thereof. The term is used to designate a single molecule, or a collection of molecules. Nucleic acids may be single stranded or double stranded and may include coding regions and regions of various control elements. A "heterologous" nucleic acid sequence or peptide sequence refers to a nucleic acid sequence or a peptide sequence that does not naturally occur, e.g., because the whole sequence contains a segment from other plants, bacteria, viruses, other organisms, or joinder of two sequences that occur the same organism but are joined together in a manner that does not naturally occur in the same organism or any natural state.

The term "recombinant" when made in reference to a nucleic acid molecule refers to a nucleic acid molecule which is comprised of segments of nucleic acid joined together by means of molecular biological techniques provided that the entire nucleic acid sequence does not occurring in nature, i.e., there is at least one mutation in the overall sequence such that the entire sequence is not naturally occurring even though separately segments may occur in nature. The segments may be joined in an altered arrangement such that the entire nucleic acid sequence from start to finish does not naturally occur. The term "recombinant" when made in reference to a protein or a peptide refers to a protein molecule that is expressed using a recombinant nucleic acid molecule.

The terms "vector" or " expression vector " refer to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism or expression system, e.g., cellular or cell-free expression system. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. In certain embodiments, this disclosure contemplates a vector encoding a peptide disclosed herein in operable combination with a heterologous promoter.

Protein "expression systems" refer to in vivo and in vitro (cell free) systems. Systems for recombinant protein expression typically utilize somatic cells transfected with a DNA expression vector that contains the template. The cells are cultured under conditions such that they translate the desired protein. Expressed proteins are extracted for subsequent purification. In vivo protein expression systems using prokaryotic and eukaryotic cells are well known. Proteins may be recovered using denaturants and protein-refolding procedures. In vitro (cell-free) protein expression systems typically use translation-compatible extracts of whole cells or compositions that contain components sufficient for transcription, translation, and optionally post-translational modifications such as RNA polymerase, regulatory protein factors, transcription factors, ribosomes, tRNA cofactors, amino acids, and nucleotides. In the presence of an expression vectors, these extracts and components can synthesize proteins of interest. Cell-free systems typically do not contain proteases and enable labeling of the protein with modified amino acids. See, e.g., Shimizu et al., Cell-free translation reconstituted with purified components, 2001, Nat. Biotechnol., 19, 751-755 and Asahara & Chong, Nucleic Acids Research, 2010, 38(13): el41, both hereby incorporated by reference in their entirety.

A "selectable marker" is a nucleic acid introduced into a recombinant vector that encodes a peptide that confers a trait suitable for artificial selection or identification (report gene), e.g., beta-lactamase confers antibiotic resistance, which allows an organism expressing beta-lactamase to survive in the presence antibiotic in a growth medium. Another example is thymidine kinase, which makes the host sensitive to ganciclovir selection. It may be a screenable marker that allows one to distinguish between wanted and unwanted cells based on the presence or absence of an expected color. For example, the lac-z-gene produces a beta-galactosidase enzyme that confers a blue color in the presence of X-gal (5-bromo-4-chloro-3-indolyl-P-D-galactoside). If recombinant insertion inactivates the lac-z-gene, then the resulting colonies are colorless. There may be one or more selectable markers, e.g., an enzyme that can complement the inability of an organism to synthesize a particular compound required for its growth (auxotrophic) and one able to convert a compound to another that is toxic for growth. Additional contemplated selectable markers include any genes that impart antibacterial resistance or express a fluorescent protein. Examples include, but are not limited to, the following genes: amp^r, cam^r, tet^r, blasticidin¹) neo^r, hyg^r, abx^r, neomycin phosphotransferase type II gene (nptll), p-glucuronidase (gus), green fluorescent protein (gfp), egfp, yfp, mCherry, p-galactosidase (lacZ), lacZa, lacZAM15, chloramphenicol acetyltransferase (cat), alkaline phosphatase (phoA), bacterial luciferase (luxAB), bialaphos resistance gene (bar), phosphomannose isomerase (pmi), xylose isomerase (xylA), arabitol dehydrogenase (atlD), UDP- glucose:galactose-l -phosphate uridyltransferasel (galT), feedback-insensitive a subunit of anthranilate synthase (OASA1D), 2-deoxy glucose (2-DOGR), benzyladenine-N-3 -glucuronide, E. coli threonine deaminase, glutamate 1 -semialdehyde aminotransferase (GSA-AT), D-amino acidoxidase (DAAO), salt-tolerance gene (rstB), ferredoxin-like protein (pflp), trehalose-6-P synthase gene (AtTPSl), lysine racemase (lyr), dihydrodipicolinate synthase (dapA), tryptophan synthase beta 1 (AtTSBl), dehalogenase (dhlA), mannose-6-phosphate reductase gene (M6PR), hygromycin phosphotransferase (HPT), and D-serine ammonialyase (dsdA). A "label" refers to a detectable moiety that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, nonlimiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. In one example, a "label receptor" refers to incorporation of a heterologous peptide in the receptor. A label includes the incorporation of a radiolabeled amino acid or the covalent attachment of biotinyl moieties to a peptide that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling peptides and glycoproteins are known in the art and may be used. Examples of labels for peptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as ³⁵S or ¹³¹I), fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined peptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, labels are attached by spacer arms (linking groups) of various lengths to reduce potential steric hindrance.

In certain embodiments, the disclosure relates to recombinant antibodies and the peptides contained therein comprising sequences disclosed herein or variants or fusions thereof wherein the amino terminal end or the carbon terminal end of the amino acid sequence are optionally attached to a heterologous amino acid sequence, label, or reporter molecule.

In certain embodiments, the disclosure relates to the recombinant vectors comprising a nucleic acid encoding a peptide disclosed herein or chimeric protein thereof.

In certain embodiments, the recombinant vector optionally comprises a mammalian, human, insect, viral, bacterial, bacterial plasmid, yeast associated origin of replication or gene such as a gene or retroviral gene or lentiviral LTR, TAR, RRE, PE, SLIP, CRS, and INS nucleotide segment or gene selected from tat, rev, nef, vif, vpr, vpu, and vpx or structural genes selected from gag, pol, and env.

In certain embodiments, the recombinant vector optionally comprises a gene vector element (nucleic acid) such as a selectable marker region, lac operon, a CMV promoter, a hybrid chicken B-actin/CMV enhancer (CAG) promoter, tac promoter, T7 RNA polymerase promoter, SP6 RNA polymerase promoter, SV40 promoter, internal ribosome entry site (IRES) sequence, cis-acting woodchuck post regulatory element (WPRE), scaffold-attachment region (SAR), inverted terminal repeats (ITR), c-myc tag coding region, metal affinity tag coding region, streptavidin binding peptide tag coding region, polyHis tag coding region, HA tag coding region, MBP tag coding region, GST tag coding region, polyadenylation coding region, SV40 polyadenylation signal, SV40 origin of replication, Col El origin of replication, fl origin, pBR322 origin, or pUC origin, TEV protease recognition site, loxP site, Cre recombinase coding region, or a multiple cloning site such as having 5, 6, or 7 or more restriction sites within a continuous segment of less than 50 or 60 nucleotides or having 3 or 4 or more restriction sites with a continuous segment of less than 20 or 30 nucleotides.

Sequence "identity" refers to the number of exactly matching amino acids (expressed as a percentage) in a sequence alignment between two sequences of the alignment calculated using the number of identical positions divided by the greater of the shortest sequence or the number of equivalent positions excluding overhangs wherein internal gaps are counted as an equivalent position. In certain embodiments, any recitation of sequence identity expressed herein may be substituted for sequence similarity. Percent “similarity” is used to quantify the similarity between two sequences of the alignment. This method is identical to determining the identity except that certain amino acids do not have to be identical to have a match. Amino acids are classified as matches if they are among a group with similar properties according to the following amino acid groups: Aromatic - F Y W; hydrophobic-A V I L; Charged positive: R K H; Charged negative - D E; Polar - S T N Q.

Antibodies that inhibit glycoprotein Ib-IX-mediated platelet signaling and clearance

This disclosure relates to antibodies that inhibit glycoprotein Ib-IX-mediated platelet signaling and clearance. In certain embodiments, this disclosure relates to recombinant chimeric antibodies or antibody fragments comprising one or more or all of the six complementarity determining regions (CDRs) of antibody RAM.1.

In certain embodiments, this disclosure relates to recombinant chimeric antibodies or antibody fragments comprising one or more or all of the six complementarity determining regions (CDRs) of antibody RAM.1 wherein the CDRs comprise three heavy chain CDRs, wherein heavy chain CDR 1 comprises the amino acid sequence of GFNVNNDWMG (SEQ ID NO: 1); CDR 2 comprises the amino acid sequence of EINKDSSTINYNPSLKG (SEQ ID NO: 2), and CDR 3 comprises the amino acid sequence of ALTMGIDY (SEQ ID NO: 3), and, wherein the CDRs comprise three light chain CDRs, wherein light chain CDR 1 comprises the amino acid sequence of KASQNVGNNIA (SEQ ID NO: 4); light chain CDR2 comprises the amino acid sequence of FASSRYT (SEQ ID NO: 5); and light chain CDR3 comprises the amino acid sequence of QRVYNSP (SEQ ID NO: 6); and, wherein the antibody comprises a heavy chain having a variable and constant region and a light chain having a variable and constant region, and wherein the antibody or fragment specifically binds GPIb-beta extracellular domain, or specifically binds GPIb-IX complex and does not bind GPIb-alpha domain.

In certain embodiments, the heavy chain comprises a leucine containing sequence C- terminal to a heavy chain constant region. In certain embodiments, the leucine containing sequence comprises the amino acid sequence of

RMKQLEDKVEELLSKNYHLENEVARLKKLVGER (SEQ ID NO: 9, GCN4) or variant thereof. In certain embodiments, the variant comprises conservative and/or nonconservative amino acid substitutions. Tropsha et al. report physical modeling (CPK) and in vacuo molecular mechanics calculations of the stability of the GCN4 and other leucine zipper proteins that dimerize. Proc. Nall. Acad. Sci. USA, 1991, Vol. 88, pp. 9488-9492. Ojima-Kato et al. report ‘Zipbody’ leucine zipper-fused Fab in E. coli in vitro and in vivo expression systems. Protein Engineering, Design & Selection, 2016, 29(4): 149-157.

In certain embodiments, the leucine zipper sequence comprises the amino acid sequence LX¹X¹X²X¹X¹X¹LX¹X¹X²X¹X¹X¹LX¹X¹X²X¹X¹X¹LX¹X¹X²X¹X¹X¹L (SEQ ID NO: 7) or LX¹X¹X²X¹X¹X¹LX¹X¹X²X¹X¹X¹LX¹X¹X²X¹X¹X¹L, (SEQ ID NO: 8) wherein X¹ is individually and independently at each occurrence any amino acid, and X² is individually and independently at each occurrence an amino acid selected from K, D, E, R, S, Q, T, and L or any amino acid.

In certain embodiments, the heavy chain comprises the amino acid sequence of

EVKLVESGGGLVQPGRSLKLSCAASGFNVNNDWMGWVRQAPGKGLEWIGEIN KDSSTINYNPSLKGKLTISRDNAQNTLYLQMSKLGSEDTAIYYCARALTMGIDYWGPG VMVTVSS (SEQ ID NO: 10) or variants thereof with greater than 60% 70%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98 %, 99% identity thereto. In certain embodiments, the variant comprises conservative amino acid substitutions. In certain embodiments, the variant comprises nonconservative amino acid substitutions. In certain embodiments, the conservative and/or nonconservative amino acid substitutions are within the framework regions. In certain embodiments, the variant does not contain substitutions within the heavy chain CDR1, CDR2, or CDR3. In certain embodiments, the variant has one conservative amino acid substitutions within the heavy chain CDR1, CDR2, or CDR3. In certain embodiments, the variant has two conservative amino acid substitutions within the heavy chain CDR1, CDR2, or CDR3.

In certain embodiments, the heavy chain comprises the amino acid sequence of

EVKLVESGGGLVQPGRSLKLSCAASGFNVNNDWMGWVRQAPGKGLEWIGEIN KDSSTINYNPSLKGKLTISRDNAQNTLYLQMSKLGSEDTAIYYCARALTMGIDYWGPG VMVTVS S S AKTTPPS VYPL APGS AAQTNSMVTLGCLVKGYFPEP VTVTWNSGSLS SGVH TFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTG GGRMKQLEDKVEELLSKNYHLENEVARLKKLVGER (SEQ ID NO: 11) or variants thereof with greater than 60% 70%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98 %, 99% identity thereto.

In certain embodiments, the variant comprises conservative amino acid substitutions. In certain embodiments, the variant comprises nonconservative amino acid substitutions. In certain embodiments, the conservative and/or nonconservative amino acid substitutions are within the framework regions. In certain embodiments, the variant does not contain substitutions within the heavy chain CDR1, CDR2, or CDR3. In certain embodiments, the variant has one conservative amino acid substitutions within the heavy chain CDR1, CDR2, or CDR3. In certain embodiments, the variant has two conservative amino acid substitutions within the heavy chain CDR1, CDR2, or CDR3.

In certain embodiments, the light chain comprises the amino acid sequence of

NNIVMTQSPKSMSISVGDRVTMNCKASQNVGNNIAWYQQKPGQSPKLLIYFASS RYTGVPDRFTGGGYGTDFTLTINGVQVEDAAFYYCQRVYNSPWTFGGGTNLELK (SEQ ID NO: 12) or variants thereof with greater than 60% 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% identity thereto.

In certain embodiments, the variant comprises conservative amino acid substitutions. In certain embodiments, the variant comprises nonconservative amino acid substitutions. In certain embodiments, the conservative and/or nonconservative amino acid substitutions are within the framework regions. In certain embodiments, the variant does not contain substitutions within the light chain CDR1, CDR2, or CDR3. In certain embodiments, the variant has one conservative amino acid substitutions within the light chain CDR1, CDR2, or CDR3. In certain embodiments, the variant has two conservative amino acid substitutions within the light chain CDR1, CDR2, or CDR3.

In certain embodiments, the antibody, antigen binding fragment, or heavy chain, comprises a human constant domain from an immunoglobulin constant region (Fc) or fragment thereof having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more of the following mutations G236A, S239D, A330L, I332E, S267E, L328F, P238D, H268F, S324T, S228P, G236R, L328R, L234A, L235A, M252Y, S254T, T256E, M428L, N434S, P329G, D265A, N297A, N297G, N297Q, F243L, R292P, Y300L, V305I, P396L, S298A, E333A, K334A, L234Y, L235Q, G236W, S239M, H268D, D270E, K326D, A330M, K334E, K326W, E333S, E345R, E430G, S440Y, L235E, N325S. With regard to IgGFc mutations reported herein, the sequences are in reference to following, amino acid sequence (SEQ ID NO: 18) starting at amino acid 119:

STKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS

HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC

LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW

QQGNVFSCSV MHEALHNHYT QKSLSLSPG (SEQ ID NO: 18).

It is noted that in reference to SEQ ID NO: 18, it is specifically for IgGl. IgG2, IgG3, and IgG4 will have some alternative amino acids as the same positions. For example, IgG2 contains a V at position 309 instead of L at position 309 for IgGl. IgG4 contains a F at position 234 instead of L at position 234 for IgGl.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation that activates immune responses, enhance ADC by increasing FcyRIIIa binding or decreasing FcyRIIb binding, enhance ADCP by increasing FcyRIIa binding or increased FcyRIIIa binding, enhance CDC by increasing Clq binding or hexamerization, reduce effector functions by aglycosylation, reducing FcyR and Clq binding, increasing co-engagement by increasing FcyRIIb binding, increasing FcyRIIa binding, or decreasing FcyRIIIa binding, and/or increases half-life.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from L234A and L235A, or both.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from L234A, L235A, and P329G, or all or combinations thereof.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from D265A and N297A, or both.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from D265A and N297G, or both.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from D265A and N297Q, or both.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from M252Y, S254T, T256E, or all or combinations thereof.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from F243L, R292P, Y300L, V305I, P396L, or combinations thereof.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from S239D, I332E or both.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from S239D, I332E, A330L, or all or combinations thereof. In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from S239D, I332E, G236A, A330L, or all or combinations thereof.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from S298A, E333A, K334A, or all or combinations thereof.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from S298A, E333A, K334A, or all or combinations thereof.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from L234Y, L235Q, G236W, S239M, H268D, D270E, S298A, or all or combinations thereof.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from D270E, K326D, A330M, K334E, or all or combinations thereof.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from G236A, S239D, I332E, or all or combinations thereof.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from K326W, E333S or both.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from E345R, E430G, S440Y, or all or combinations thereof.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from E345R, E430G, S440Y, or all or combinations thereof.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from F234A, L235A or both of IgG4. In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from H268Q, V309L, A330S, P331S or all or combinations thereof of IgG2.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from V234A, G237A, P238S, H268A, V309L, A330S, P331S, or all or combinations thereof of IgG2.

FcgRIIb has immunosuppressive function. In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from S267E, L328F or both.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from S267E, L328F, P238D, or combinations thereof.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from N325S and L328F or both.

Antibodies interact with the complement cascade through Clq binding enabling antibodies to activate complement-dependent cytotoxicity (CDC). In certain embodiments, this disclosure relates to antibodies reported wherein the constant region or fragment thereof comprises a mutation that effectively active complement-dependent cytotoxicity such as those selected from S267E, H268F, S324T, or all or combinations thereof.

In certain embodiments, this disclosure relates to antibodies reported wherein the constant region comprises a mutation in the Fc domain or fragment thereof selected from M428L, N434S or both.

In certain embodiments, is may be desirable to have antibodies wherein constant region of the Fc has been to increase or decrease antibody half-life. In certain embodiments, this disclosure relates to antibodies reported wherein the constant region or fragment thereof comprises a mutation that increases or decreases the antibodies half-life such as those selected from M252Y, S254T, T256E, M428L, N434S or all or combinations thereof.

In certain embodiments, this disclosure contemplates that a heavy chain contains at least one mutation wherein both heavy chains are not identical. In certain embodiments, this disclosure contemplates that one heavy chain may have alternative mutations than the opposite heavy chain, i.e., one of the two heavy chains contain a mutation that the other sequence does not, or one of the two heavy chains contain one or more mutations, and the other heavy chain contains different mutations.

Methods of Use

GPIb-IX-mediated platelet signaling leads to accelerated platelet clearance and thrombocytopenia in a number of thrombotic thrombocytopenic disorders. They include thrombotic microangiopathy, bacterial infection, viral infection, sepsis, certain kinds of immune thrombocytopenia, and platelet storage lesions. In addition, many mechanical devices induce thrombotic thrombocytopenia in patients, which involve pathological activation of von Willebrand factor (VWF) and subsequent GPIb-IX activation. Inhibitors of GPIb-IX-mediated signaling may be used to treat thrombocytopenia and related symptoms in these conditions, optionally in combination with other drugs.

In certain embodiments, this disclosure relates to method treating or preventing a bleeding disorder comprising administering an effective amount of an antibody disclosed herein to a subject in need thereof. In certain embodiments, the bleeding disorder is thrombocytopenia, spontaneous or excessive bleeding. In certain embodiments, the bleeding disorder is von Willebrand disease or acquired von Willebrand syndrome (A VWS).

In certain embodiments, this disclosure relates to methods of treating or preventing blood clotting or thrombosis comprising administering an effective amount of an antibody disclosed herein to a subject in need thereof. In certain embodiments the subject is exposed to a mechanical circulatory support system, e.g., a device that provides extracorporeal membrane oxygenation (ECMO).

Therapeutic plasma exchange (TPE) is a procedure in which the blood of a subject is filtered by apheresis removing plasma and donor plasma is reinfused. Plasma exchange with plasma is used as therapy for thrombotic thrombocytopenic purpura (TTP). Depending on the condition of the patient, plasma exchanges may be instituted multiple times. In certain embodiments, this disclosure relates to methods of treating or preventing thrombotic thrombocytopenic purpura comprising administering an effective amount of an antibody disclosed herein or fragment thereof, to a subject in need thereof. In certain embodiments, administering an effective amount of an antibody disclosed herein to a subject in need thereof is before, during, or after a plasma exchange.

In certain embodiments, this disclosure relates to methods of treating or alleviating a blood clotting or excessive bleeding diseases or conditions in a subject by administering to the subject an antibody disclosed herein or fragment thereof, wherein the amount of the antibody or fragment administered is effective to reduce the time-to-response, to reduce exacerbations, to reduce hospitalization, to reduce ischemia, to reduce the death toll and/or to reduce the number of required Plasma Exchanges (PE).

In certain embodiments, this disclosure relates to methods of using an antibody or fragment as disclosed herein wherein said disease is chosen from acute coronary syndrome (ACS), transient cerebral ischemic attack, unstable or stable angina pectoris, stroke, myocardial infarction, or thrombotic thrombocytopenic purpura (TTP).

In certain embodiments, this disclosure relates to methods of using an antibody or fragment disclosed herein to increase platelet concentrations or reduce the risk of organ damage, ischemic damage and/or microthrombi formation.

In certain embodiments, this disclosure relates to methods of using an antibody or fragment as disclosed herein comprising administering an antibody or fragment as disclosed herein; measuring the platelet number; and if said platelet number is lower than 150,000 platelets per microliter of blood, then repeating said antibody or fragment administration.

In certain embodiments, this disclosure relates to methods of using an antibody or fragment as disclosed herein wherein administering the antibody or fragment is repeated until said platelet number is at least 150,000 platelets per microliter of blood.

In certain embodiments, this disclosure relates to methods of treating or preventing spontaneous bleeding in a subject comprising administering an effective amount of an antibody or fragment as disclosed herein. In certain embodiments, this disclosure relates to methods of treating or preventing spontaneous bleeding comprising administering an effective amount of an antibody or fragment as disclosed herein to a subject in need thereof. In certain embodiments, the subject is exhibiting symptoms of, at risk of, or diagnosed with von Willebrand Disease (VWD), or thrombocytopenia.

In certain embodiments, thrombocytopenia due to a hematological disorder or malignancy such as chronic immune thrombocytopenia, aplastic anemia, or a hematological malignancy such as leukemia, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL), lymphoma, Hodgkin's lymphoma, or NonHodgkin's lymphoma.

In certain embodiments, this disclosure relates to uses of an antibody or fragment disclosed herein for treating or preventing a bleeding disorder. In certain embodiments, this disclosure relates to uses of an antibody or fragment disclosed herein in the production of a medicament for treating or preventing a bleeding disorder. In certain embodiments, this disclosure relates to method treating or preventing a bleeding disorder comprising administering an effective amount of an antibody or fragment as disclosed herein to a subject in need thereof in combination with an anticoagulant such as heparin, unfractionated heparin (UFH), low molecular weight heparin (LMWH), and ultra-low-molecular weight heparin (ULMWH), fondaparinux, idraparinux, idrabiotaparinux, warfarin, dabigatran, rivaroxaban, apixaban, betrixaban, edoxaban, hirudin, bivalirudin, acenocoumarol, phenprocoumon, and phenindione or in combination with reversal agents such as idarucizumab and andexanet alfa.

Methods of administering antibodies and antigen binding fragments include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In a specific embodiment, the antibodies or fragments are administered intramuscularly, intravenously, or subcutaneously. The compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968; 5,985, 20; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903. In a specific embodiment, it may be desirable to administer an antibody or fragment or pharmaceutical composition comprising the same locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as silastic membranes, or fibers.

The dosages and frequencies of administration provided herein are encompassed by the terms therapeutically effective and prophylactically effective. The dosage and frequency further will typically vary according to factors specific for each patient depending on the specific therapeutic or prophylactic agents administered, the severity, the route of administration, as well as age, body weight, response, and the past medical history of the patient. Suitable regimens can be selected by one skilled in the art by considering such factors and by following, for example, dosages reported in the literature and recommended in the Physician's Desk Reference (56th Ed., 2002).

Various delivery systems are known and can be used to administer the therapeutic or prophylactic compositions, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or fragment, construction of a nucleic acid as part of a retroviral or other vector, etc.

Pharmaceutical compositions

In certain embodiments, this disclosure contemplates a pharmaceutical unit dosage form suitable for parenteral administration to a patient, preferably a human patient, comprising an antibody or fragment as described herein or a formulation as described herein.

The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient (the antibody or fragment) to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

The term "excipient" as used herein refers to an inert substance which is commonly used as a diluent, vehicle, preservative, surfactant, binder, carrier, or stabilizing agent for compounds which impart a beneficial physical property to a formulation. The skilled person is familiar with excipients suitable for pharmaceutical purposes, which may merely be a filler or may have particular functions in the formulation, such as stabilization, preservation, etc.

A "sterile" formulation is aseptic or free or essentially free from all living microorganisms and their spores. This is readily accomplished by filtration through sterile filtration membranes. A "stable" formulation is one in which the antibody or fragment therein essentially retains its physical stability and/or chemical stability and/or biological activity upon storage. The storage period is generally selected based on the intended shelf-life of the formulation. In certain embodiments, the formulation comprises an aqueous carrier. In certain embodiments, the aqueous carrier is in particular a buffer.

As used herein, "buffer" refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components. In certain embodiments, the formulation comprises a buffer selected from at least one of citrate or phosphate buffer, preferably a citrate buffer. Buffers enhance the stability of the antibodies or fragments. The pH of the formulation is typically in the range 5.0 to 7.5, wherein each value is understood to encompass a range of plus or minus 0.2. The most advantageous pH will depend on the buffer comprised in the formulation. Hence, a formulation comprising a phosphate buffer, which preferably has a pH in the range of 6.5 to 7.5, preferably 6.9, 7.0, 7.1, e.g., 7.1. A formulation comprising a citrate buffer is suitable for storage and use. A formulation comprising a citrate buffer, which preferably has a pH between 6.0 and 7.0, more preferably 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8 or 6.9, e.g., 6.5.

In certain embodiments, this disclosure contemplates pharmaceutical compositions comprising antibodies disclosed herein, and optionally at least one pharmaceutically acceptable carrier, diluent, or excipient. Any pharmaceutical product or composition comprising an antibody disclosed herein may also comprise one or more further components known per se for use in pharmaceutical products or compositions depending on the intended pharmaceutical form and/or for example one or more other compounds or active principles intended for therapeutic use to provide a combination product. The pharmaceutical compositions can be administered in any suitable manner that allows the compound or polypeptide to enter the circulation, such as intravenously, via injection or infusion, or in any other suitable manner (including oral administration, subcutaneous administration, intramuscular administration, administration through the skin, intranasal administration, administration via the lungs, etc.).

In certain embodiments, this disclosure contemplates an antibody or fragment formulation as described herein, wherein said formulation comprises a citrate or phosphate buffer with a pH in the range of 5.0 to 7.5. In certain embodiments, this disclosure contemplates an antibody or fragment formulation as described herein, wherein said formulation is in liquid, lyophilized, spray-dried, reconstituted lyophilized or frozen form.

In certain embodiments, formulations comprise an antibody or fragment disclosed herein at a concentration that is suitable for clinical purposes, which includes concentrations used in stock solutions for dilution prior to use on the patient. Typical concentrations comprise the non-limiting examples of concentrations in the range of 0.1 to 150 mg/mL, such as 1-100 mg/mL, 5-80 mg/mL, or 10-40 mg/mL, preferably 10 mg/mL, wherein each value is understood to optionally encompass a range (e.g. a value of 10 optionally encompasses a range of 8 to 12 mg/mL).

In a further embodiment, the formulation may further comprise a detergent or surfactant. A "surfactant" refers to a surface-active agent, preferably a nonionic surfactant. Examples of surfactants herein include polysorbate; poloxamer; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol etc.

The formulation may further comprise excipients such as preservatives. A "preservative" is a compound which can be optionally included in the formulation to essentially reduce bacterial action therein, thus facilitating the production of a multi-use formulation. Examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3 -pentanol, and m-cresol.

The formulation may further comprise stabilizing agents, such as a polyols. A "polyol" is a substance with multiple hydroxyl groups, and includes sugars (reducing and nonreducing sugars), sugar alcohols and sugar acids. A polyol may optionally be included in the formulation, for instance to improve stability. In certain embodiments, polyols herein have a molecular weight which is less than about 600 kD (e.g. in the range from about 120 to about 400 kD). A "reducing sugar" is one which contains a hemi-acetal group that can reduce metal ions or react covalently with lysine and other amino groups in proteins and a "nonreducing sugar" is one which does not have these properties of a reducing sugar. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose and glucose. Nonreducing sugars include sucrose, trehalose, sorbose, and raffinose. Mannitol, xylitol, erythritol, threitol, sorbitol and glycerol are examples of sugar alcohols. As to sugar acids, these include L-gluconate and metallic salts thereof. Where it desired that the formulation is freeze-thaw stable, the polyol is preferably one which does not crystallize at freezing temperatures (e.g. -20 degrees C) such that it destabilizes the antibody or fragment in the formulation. In certain embodiments, nonreducing sugars such as sucrose and trehalose are examples of polyols, with sucrose being preferred.

Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, hydrophilic polymers such as polyvinyl pyrrolidone, cellulose based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, gelatin, polyethylene polyoxypropylene block polymers, polyethylene glycol and antioxidants including ascorbic acid and methionine; low molecular weight (less than about 10 residues) polypeptides; and amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine.

The disclosure also encompasses products obtainable by further processing of a liquid formulation, such as a frozen, lyophilized or spray-dried product. Upon reconstitution, these solid products can become liquid formulations as described herein. In its broadest sense, therefore, the term "formulation" encompasses both liquid and solid formulations.

In certain embodiment, the formulations are isotonic in relation to human blood. Isotonic solutions possess the same osmotic pressure as blood plasma, and so can be intravenously infused into a subject without changing the osmotic pressure of the blood plasma.

In some embodiments, the antibodies or antigen binding fragments are formulated in liposomes for targeted delivery of the antibodies or fragments. Liposomes are vesicles comprised of concentrically ordered phospholipid bilayers which encapsulate an aqueous phase. Liposomes typically comprise various types of lipids, phospholipids, and/or surfactants. The components of liposomes are arranged in a bilayer configuration, similar to the lipid arrangement of biological membranes.

The antibodies, or antigen binding fragments may also be formulated as immunoliposomes. Immunoliposomes refer to a liposomal composition, wherein an antibody or a fragment thereof is linked, covalently or non-covalently to the liposomal surface. The chemistry of linking an antibody to the liposomal surface is known in the art and encompassed within the disclosure, see, e.g., U.S. Pat. No. 6,787,153; Allen et al., 1995, Stealth Liposomes, BocaRotan: CRC Press, 233-44; Hansen et al., 1995, Biochim. Biophys. Acta, 1239: 133-144. In certain embodiments, the antibodies or antigen binding fragments are linked covalently or non-covalently to a hydrophobic anchor, which is stably rooted in the lipid bilayer of the liposome. Examples of hydrophobic anchors include, but are not limited to, phospholipids, e.g., phosphatidylethanolamine (PE), phosphatidylinositol (PI).

The antibodies and antigen binding fragments can be packaged in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity of antibody or fragment. In one embodiment, the antibodies or fragments are supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline to the appropriate concentration for administration to a subject. Preferably, the antibodies or fragment are supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg.

In certain embodiments, an antibody or fragment is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the antibody or fragment. Preferably, the liquid form of the antibodies or fusion proteins are supplied in a hermetically sealed container at least 1 mg/ml, more preferably at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, at least 100 mg/ml, at least 150 mg/ml, at least 200 mg/ml of the antibody or fragment.

In certain embodiments, the therapeutic or prophylactic composition is a nucleic acid encoding an antibody or an antigen-binding fragment thereof as disclosed herein. The nucleic acid can be administered in vivo to promote expression of its encoded antibody or fragment, by constructing it as part of an appropriate nucleic acid expression vector and administering by use of a retroviral vector (See U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cellsurface receptors or transfecting agents.

Kits

In certain embodiments, this disclosure relates to kits containing antibodies and fragments disclosed herein useful for the treatment of a disease as described. In certain embodiments, the kit comprises a container, a product label and a package insert. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be of a variety of materials such as glass or plastic. The container holds the composition which is effective in treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a injection needle). At least one active agent in the composition is the antibody or fragment disclosed herein. The product label on, or associated with, the container indicates that the composition is used for treating the condition of choice. In certain embodiments, the kit may further comprise a second container comprising a pharmaceutically acceptable buffer, such as a phosphate buffer saline or a citrate buffered saline. It may further include other materials desirable from a user or commercial standpoint, including other buffers, diluents, filters, needles, and syringes. In certain embodiments, a dosage unit form can be e.g., in the format of a prefilled syringe, an ampoule, cartridge or a vial.

In certain embodiments, the kit may further comprise instructions for use and/or a clinical package leaflet. In any embodiment of the products as defined herein, this disclosure also encompasses the presence of packaging material, instructions for use, and/or clinical package leaflets, e.g., as required by regulatory aspects.

EXAMPLES

An antibody derivative was developed to inhibit activation and signaling of platelet glycoprotein (GP) Ib-IX receptor complex, and subsequently inhibit GPIb-IX-mediated platelet clearance. Uses to treat thrombocytopenia, bleeding, and related symptoms in a number of thrombotic thrombocytopenic disease settings are contemplated. Antibody RAM.l is a rat monoclonal antibody. See Perrault et al. A novel monoclonal antibody against the extracellular domain of GPIbp modulates vWF mediated platelet adhesion. Thromb Haemost. 2001, 86(5): 1238-48. It has been discovered that antibody RAM.l binds human GPIbp subunit in the GPIb-IX complex and inhibits GPIb-IX-mediated signaling.

The RAM.l gene was sequenced. The CDR loops were grafted onto a mouse IgGl framework. The recombinant mouse RAM.1 antibody retains the inhibitory function of the original antibody. However, the Fab fragment of RAM.l or RAM. l-derived scFv does not express well. Thus, a chimeric protein in which a portion the Fc of the RAM.1 heavy chain was replaced with a GCN4 leucine zipper. The RAM.1-GCN4 protein is a disulfide-linked homodimer of RAM.1 Fab. Experiments indicate that the RAM.1 -derived protein complex will inhibitor platelet clearance and ameliorating thrombocytopenia in animal models.

Plasma protein VWF often becomes activated in inflammatory and/or elevated shear flow conditions. Activated VWF binds to the ligand-binding domain (LBD) of platelet GPIb-IX complex and activates the latter by inducing unfolding of the mechanosensory domain (MSD) in GPIba, which in turn induces a conformational change in the juxtaposed GPIbp/GPIX extracellular domains, thereby triggering certain signals into the platelet and inducing its rapid clearance by the body (See Fig. 1).

The interaction of VWF with GPIba is required for primary hemostasis. Although it is not intended that certain embodiments of this disclosure be limited by any particular mechanism, it is believed that direct inhibition of this interaction results in bleeding including sometimes fatal consequences such as intracranial hemorrhage. This is primarily why no direct inhibitors of the VWF-GPIba interaction has been developed. However, elucidation of the activation mechanism of GPIb-IX suggests an approach that indirectly inhibit GPIb-IX without interfering with the VWF-GPIba interaction, i.e., by inhibit GPIb-IX-mediated signaling.

The rat antibody RAM.l specifically binds to the GPIbp extracellular domain. GPIbp is highly conserved across species. RAM.l can recognize both human and murine GPIbp. RAM.l does not interfere with the binding of ligands to the N-terminal LBD. RAM.l inhibits GPIb-IX- mediated filopodia formation in platelets and transfected CHO cells expressing GPIb-IX (Illustrated in Fig. 2A) indicating that RAM.1 stabilizes GPIbp to a specific conformation, i.e., the “off’ state of GPIb-IX.

RAM. 1 inhibits signaling of a mutant GPIb-IX complex that mimics the unfolded state of MSD and induces a ligand-free filopodia formation (Fig. 2B). This observation supports the model that RAM.1 inhibits GPIb-IX-mediated signaling by blocking effect of MSD unfolding to the nearby GPIbp extracellular domains. This indicates that RAM. l can inhibit GPIb-IX-mediated platelet clearance and alleviate thrombocytopenia. Thus, this disclosure contemplates that certain antibodies and other agents that bind the GPIbp extracellular domains have potential as therapeutic agents to treat thrombocytopenia, reduce thrombo-inflammation, and down-regulate related thrombotic thrombocytopenic symptoms.

RAM.l is unique because it is the only molecule reported to inhibit GPIb-IX-mediated signaling in platelets. Other inhibitors have been developed to target the LBD of GPIba. Unlike RAM.l, they inhibit GPIb-IX function by directly blocking VWF binding to the LBD, and they have the potential of causing bleeding. In contrast, RAM.1 does not target the LBD of GPIba and interfere with this hemostatic process in vivo; thus, may prevent undesirable internal bleeding. The sequences and CDRs of agents used in experiments are provided below.

Rat RAM.l heavy chain sequence (CDRs bold):

MMGLGVILFLVALLKGVQCEVKLVESGGGLVQPGRSLKLSCAASGFNVNNDWMGWV RQAPGKGLEWIGEINKDSSTINYNPSLKGKLTISRDNAQNTLYLQMSKLGSEDTAIYYC ARALTMGIDYWGPGVMVTVSS (SEQ ID NO: 13)

Rat RAM.l light chain sequence (CDRs bold):

MESQTQVVIFVLLWLSGANGNIVMTQSPKSMSISVGDRVTMNCKASQNVGNNIAWYQ QKPGQSPKLLIYFASSRYTGVPDRFTGGGYGTDFTLTINGVQVEDAAFYYCQRVYNSP WTFGGGTNLELK (SEQ ID NO: 14)

Recombinant mouse RAM.l gamma 1 heavy chain sequence (CDRs bold):

MYRMQLLSCIALSLALVTNEVKLVESGGGLVQPGRSLKLSCAASGFNVNNDWMGWV RQAPGKGLEWIGEINKDSSTINYNPSLKGKLTISRDNAQNTLYLQMSKLGSEDTAIYYC ARALTMGIDYWGPGVMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEP VTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDK KIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFV DDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKT KGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIM DTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 15)

Complete recombinant mouse RAM.1 kappa light chain sequence (CDRs bold):

MYRMQLLSCIALSLALVTNNIVMTQSPKSMSISVGDRVTMNCKASQNVGNNIAWYQQ KPGQSPKLLIYFASSRYTGVPDRFTGGGYGTDFTLTINGVQVEDAAFYYCQRVYNSPW TFGGGTNLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 16)

Mouse RAM.1 heavy chain-GCN4 fusion protein sequence (CDRs and GCN4 domain bold): MYRMQLLSCIALSLALVTNSEVKLVESGGGLVQPGRSLKLSCAASGFNVNNDWMGWV RQAPGKGLEWIGEINKDSSTINYNPSLKGKLTISRDNAQNTLYLQMSKLGSEDTAIYYC ARALTMGIDYWGPGVMVTVSSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEP VTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDK KIVPRDCGCKPCICTGGGRMKQLEDKVEELLSKNYHLENEVARLKKLVGERHHHHH H (SEQ ID NO: 17)

In certain embodiments, this disclosure relates to antibodies or nucleic acids encoding the antibodies/peptide sequences provided above or fragments or variants thereof with greater than 60% 70%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98 %, 99% identity thereto. In certain embodiments, the variant comprises conservative or nonconservative amino acid substitutions. In certain embodiments, the variant does not contain substitutions within the heavy chain or light chain CDR1, CDR2, or CDR3. In certain embodiments, the variant has one conservative amino acid substitutions within the heavy chain or light chain CDR1, CDR2, or CDR3. In certain embodiments, the variant has two conservative amino acid substitutions within the heavy chain or light chain CDR1, CDR2, or CDR3.

Claims

1. A recombinant chimeric antibody or antibody fragment comprising six complementarity determining regions (CDRs) of antibody RAM. l, wherein the CDRs comprise three heavy chain CDRs, wherein heavy chain CDR 1 comprises the amino acid sequence of GFNVNNDWMG (SEQ ID NO: 1); CDR 2 comprises the amino acid sequence of EINKDSSTINYNPSLKG (SEQ ID NO: 2), and CDR 3 comprises the amino acid sequence of ALTMGIDY (SEQ ID NO: 3), and, wherein the CDRs comprise three light chain CDRs, wherein light chain CDR 1 comprises the amino acid sequence of KASQNVGNNIA (SEQ ID NO: 4); light chain CDR2 comprises the amino acid sequence of FASSRYT (SEQ ID NO: 5); and light chain CDR3 comprises the amino acid sequence of QRVYNSP (SEQ ID NO: 6); and, wherein the antibody comprises a heavy chain having a variable and constant region and a light chain having a variable and constant region, and wherein the antibody or fragment specifically binds GPIb-beta extracellular domain, or specifically binds GPIb-IX complex and does not bind GPIb-alpha subunit.

2. The recombinant chimeric antibody of claim 1, wherein the heavy chain comprises a heterologous leucine containing sequence C-terminal to the heavy chain constant region.

3. The recombinant chimeric antibody or claim 2, wherein leucine containing sequence comprises the amino acid sequence LX¹X¹X²X¹X¹X¹LX¹X¹X²X¹X¹X¹LX¹X¹X²X¹X¹X¹LX¹X¹X² X^X^ (SEQ ID NO: 7) or LX¹X¹X²X¹X¹X¹LX¹X¹X²X¹X¹X¹LX¹X¹X²X¹X¹X¹L, (SEQ ID NO: 8) wherein X¹ is individually and independently at each occurrence any amino acid, and X² is individually and independently at each occurrence an amino acid selected from K, D, E, R, S, Q, T, and L.

4. The recombinant chimeric antibody or claim 2, wherein leucine containing sequence comprises the amino acid sequence of RMKQLEDKVEELLSKNYHLENEVARLKKLVGER (SEQ ID NO: 9, GCN4).

5. The recombinant chimeric antibody of claim 1, wherein the heavy chain comprises the amino acid sequence of

EVKLVESGGGLVQPGRSLKLSCAASGFNVNNDWMGWVRQAPGKGLEWIGEIN KDSSTINYNPSLKGKLTISRDNAQNTLYLQMSKLGSEDTAIYYCARALTMGIDYWGPG VMVTVSS (SEQ ID NO: 10) or variants thereof

6. The recombinant chimeric antibody of claim 1, wherein the heavy chain comprises the amino acid sequence of

EVKLVESGGGLVQPGRSLKLSCAASGFNVNNDWMGWVRQAPGKGLEWIGEIN KDSSTINYNPSLKGKLTISRDNAQNTLYLQMSKLGSEDTAIYYCARALTMGIDYWGPG VMVTVS S S AKTTPPS VYPL APGS AAQTNSMVTLGCLVKGYFPEP VTVTWNSGSLS SGVH TFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTG GGRMKQLEDKVEELLSKNYHLENEVARLKKLVGER (SEQ ID NO: 11) or variants thereof

7. The recombinant chimeric antibody of any of claims 1-6, wherein the light chain comprises the amino acid sequence of

NNIVMTQSPKSMSISVGDRVTMNCKASQNVGNNIAWYQQKPGQSPKLLIYFASS RYTGVPDRFTGGGYGTDFTLTINGVQVEDAAFYYCQRVYNSPWTFGGGTNLELK (SEQ ID NO: 12) or variants thereof.

8. A pharmaceutical composition comprising the recombinant chimeric antibody of any of claims 1-7 and a pharmaceutically acceptable excipient.

9. A nucleic acid or vector encoding the heavy chain and/or light chain of the recombinant chimeric antibody of any of claims 1-7 in operable combination with a heterologous promoter.

10. A cell or other expression system comprising a nucleic acid or vector of claim 9.

11. A method treating or preventing a bleeding disorder comprising administering an effective amount of a recombinant chimeric antibody of any of claims 1-7 to a subject in need thereof.

12. The method of claim 11, wherein the bleeding disorder is thrombocytopenia, spontaneous or excessive bleeding, or GPIb-IX mediated disorder.

13. The method of claim 11, wherein the bleeding disorder is von Willebrand disease or acquired von Willebrand syndrome (A VWS).

14. Use of a recombinant chimeric antibody of any of claims 1-7 for treating or preventing a bleeding disorder or GPIb-IX mediated disorder.

15. Use of a recombinant chimeric antibody of any of claims 1-7 in the production of a medicament for treating or preventing a bleeding disorder or GPIb-IX mediated disorder.