FUSION PROTEIN OF SINGLE DOMAIN ANTIBODY AND PROCOAGULANT
20220041716 · 2022-02-10
Inventors
Cpc classification
C07K2317/569
CHEMISTRY; METALLURGY
C07K2319/33
CHEMISTRY; METALLURGY
C07K2317/51
CHEMISTRY; METALLURGY
A61P7/00
HUMAN NECESSITIES
C07K2317/33
CHEMISTRY; METALLURGY
A61P7/04
HUMAN NECESSITIES
C07K14/70596
CHEMISTRY; METALLURGY
C07K2317/34
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/22
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to single domain antibodies (sdAbs) against TREM (triggering receptors expressed on myeloid cells) like transcript-1 (TLT-1) molecules that are present on activated platelets at the site of an injury, and especially on a subset of activated platelets, coated platelets. Furthermore, the present invention relates to fusion proteins comprising sdAbs against TLT-1 and an extracellular (soluble) domain of tissue factor (sTF), to direct targeting of such fusion proteins to activated platelets at the site of injury through binding of the sdAbs to TLT-1, a membrane protein receptor that is only present on activated platelets. Specific interaction of sdAbs with the TLT-1 receptor positions the sTF domain of the fusion to interact with, and activate, FVII. As a result, a targeted procoagulant effect is achieved at the site of injury via activated platelets. The fusion proteins are useful to treat individuals that have a bleeding disorder, such as hemophilia A, hemophilia B, or acute bleeding due to traumatic injury.
Claims
1. A single domain antibody against TLT-1, comprising: (a) CDR1 being SEQ ID NO: 6, CDR2 being SEQ ID NO: 32, CDR3 being SEQ ID NO: 43; (b) CDR1 being SEQ ID NO: 8, CDR2 being SEQ ID NO: 31, CDR3 being SEQ ID NO: 44; (c) CDR1 being SEQ ID NO: 3, CDR2 being SEQ ID NO: 31, CDR3 being SEQ ID NO: 41; (d) CDR1 being SEQ ID NO: 3, CDR2 being SEQ ID NO: 31, CDR3 being SEQ ID NO: 42; (e) CDR1 being SEQ ID NO: 1, CDR2 being SEQ ID NO: 31, CDR3 being SEQ ID NO: 40; (f) CDR1 being SEQ ID NO: 25, CDR2 being SEQ ID NO: 35, CDR3 being SEQ ID NO: 45; (g) CDR1 being SEQ ID NO: 3, CDR2 being SEQ ID NO: 31, CDR3 being SEQ ID NO: 46; (h) CDR1 being SEQ ID NO: 11, CDR2 being SEQ ID NO: 31, CDR3 being SEQ ID NO: 47; (i) CDR1 being SEQ ID NO: 16, CDR2 being SEQ ID NO: 31, CDR3 being SEQ ID NO: 50; or (j) CDR1 being SEQ ID NO: 25, CDR2 being SEQ ID NO: 35, CDR3 being SEQ ID NO: 59.
2. The single domain antibody according to claim 1, comprising the sequence selected from the group consisting of SEQ ID NOs: 68, 70, 64, 65, 62, 71, 74, 76, 80, and 95, or a sequence having at least 95% identity thereof, provided that the sequence variation is in the non-CDR framework region.
3. A fusion protein comprising (i) an extracellular domain of a tissue factor protein having the amino acid sequence of 1-208, 1-209, 1-210, 1-211, 1-212, 1-213, 1-214, 1-215, 1-216, 1-217, 1-218, 1-219, 1-220, or 1-221 amino acid residues of SEQ ID NO: 110, (ii) a single domain antibody according claim 1, and (iii) a linker.
4. The fusion protein according to claim 3, wherein the linker has a length of 15-30 amino acids.
5. The fusion protein according to claim 3, further comprises a protease cleavage site.
6. The fusion protein according to claim 5, wherein the protease cleavage site is a thrombin cleavage site or a FXa cleavage site.
7. A pharmaceutical composition comprising the fusion protein of claim 3 and a pharmaceutically acceptable carrier.
8. A single domain antibody against TLT-1, comprising: CDR1 selected from the group consisting of: SEQ ID NOs: 1-30, CDR2 selected from the group consisting of: SEQ ID NOs: 31-39, and CDR3 selected from the group consisting of: SEQ ID NOs: 40-61.
9. The single domain antibody according to claim 8, comprising the sequence selected from the group consisting of SEQ ID NOs: 62-99, or a sequence having 95% identity thereof, provided that the sequence variation is in the non-CDR framework region.
10. A fusion protein comprising (i) an extracellular domain of a tissue factor protein having the amino acid sequence of 1-208, 1-209, 1-210, 1-211, 1-212, 1-213, 1-214, 1-215, 1-216, 1-217, 1-218, 1-219, 1-220, or 1-221 amino acid residues of SEQ ID NO: 110 (ii) a single domain antibody according to claim 8, and (iii) a linker.
11. The fusion protein according to claim 10, wherein the linker has a length of 15-30 amino acids.
12. The fusion protein according to claim 10, further comprises a protease cleavage site.
13. The fusion protein according to claim 12, wherein the protease cleavage site is a thrombin cleavage site or a FXa cleavage site.
14. A pharmaceutical composition comprising the fusion protein of claim 8 and a pharmaceutically acceptable carrier.
15. A fusion protein comprising the amino acid sequence of SEQ ID NO: 106, 107, 108, 109, 111, or 112.
16. A pharmaceutical composition comprising the fusion protein of claim 15 and a pharmaceutically acceptable carrier.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0030] “CDR”s are complementary-determining Regions of VH or VL chains of antibody which are critical for binding with antigen.
[0031] A “domain” means one region in a polypeptide which is folded into a particular structure independently of other regions.
[0032] A “single domain antibody” (sdAb), or a “variable domain of heavy chain of heavy-chain antibody” (VHH), also known as a nanobody, is an antibody fragment consisting of a single monomeric variable antibody, i.e., a variable domain of a heavy chain of an antibody. A single domain antibody is typically derived from the Camelidae family. VHH and sdAb are used interchangeably in this application.
[0033] A “tissue factor” (TF), also called platelet tissue factor, factor III, or CD142, is a membrane-bound protein encoded by the F3 gene, present in subendothelial tissue and leukocytes.
[0034] Its role in the clotting process is the initiation of thrombin formation from the zymogen prothrombin.
[0035] “TREM (triggering receptors expressed on myeloid cells) like transcript-1” (TLT-1), as used herein, is a membrane protein receptor found only in alpha-granules of platelets and megakaryocytes. TLT-1 contains an extracellular V-set Ig domain, a proline-rich region, and an immune receptor tyrosine-based inhibitory motif in its cytoplasmic tail. Upon platelet activation, TLT-1 is rapidly brought to the surface of platelets where it can enhance Ca.sup.++ influx and promote platelet aggregation.
[0036] The present invention is directed to high-affinity single-domain antibodies (sdAb) that specifically bind both mouse and human TLT-1 proteins on activated, but not resting, platelets. Due to their smaller size, elevated stability, larger number of accessible epitopes, relatively low production costs and improved robustness, the inventors selected sdAb as targeting agents to prepare fusion proteins.
[0037] The present invention is also directed to fusion proteins comprising an extracellular (soluble) domain of tissue factor (sTF) linked to these single-domain antibodies for efficiently targeting sTF to sites of vascular injury. The targeting is through binding of the sdAbs to TLT-1, a membrane protein receptor confined exclusively to the alpha-granules of resting platelets and megakaryocytes that then translocates to the surface of platelets upon their activation; positioning of sTF for interaction with FVII is achieved on activated platelet membranes to promote procoagulant activity. This targeting maximizes TF's ability to function as a strong hemostatic agent, while minimizing the chance of inducing disseminated intravascular coagulation (DIC) by excess thrombin formation. The fusion proteins of present invention fulfill the needs to treating patients with severe bleeding disorders.
Single-Domain Antibodies (sdAb) against TLT-1 (TREM-Like Transcript 1)
[0038] TLT-1 (TREM-like transcript 1) protein is expressed selectively on the surface of activated platelets and contains a number of described grooves on its surface (Gattis et al., 2006, Proc. Natl. Acad. Sci. USA, volume 281, pp. 13396-13403). The inventors discovered that such characteristics making TLT-1 ideally suited for interacting with the single-domain antibodies. These surface grooves appear to contain amino acid residues with both negatively-charged and uncharged electrostatic properties that allow interaction with selected amino acids distinctly- and conformationally-displayed on sdAbs.
[0039] The inventors have prepared high-affinity single domain antibodies, that target TLT-1 protein. The inventors have generated a total of 103 sdAb, in which 38 sdAb sequences were identified.
[0040] The present invention is directed to a single-domain antibody against TLT-1, comprising CDR1 selected from the group consisting of: SEQ ID NOs: 1-30, CDR2 selected from the group consisting of: SEQ ID NOs: 31-39, and CDR3 selected from the group consisting of: SEQ ID NOs: 40-61.
[0041] The present invention is also directed to a single domain antibody against TLT-1, comprising: (a) CDR1 being SEQ ID NO: 6, CDR2 being SEQ ID NO: 32, CDR3 being SEQ ID NO: 43; (b) CDR1 being SEQ ID NO: 8, CDR2 being SEQ ID NO: 31, CDR3 being SEQ ID NO: 44; (c) CDR1 being SEQ ID NO: 3, CDR2 being SEQ ID NO: 31, CDR3 being SEQ ID NO: 41; (d) CDR1 being SEQ ID NO: 3, CDR2 being SEQ ID NO: 31, CDR3 being SEQ ID NO: 42; (e) CDR1 being SEQ ID NO: 1, CDR2 being SEQ ID NO: 31, CDR3 being SEQ ID NO: 40; (f) CDR1 being SEQ ID NO: 25, CDR2 being SEQ ID NO: 35, CDR3 being SEQ ID NO: 45; (g) CDR1 being SEQ ID NO: 3, CDR2 being SEQ ID NO: 31, CDR3 being SEQ ID NO: 46; (h) CDR1 being SEQ ID NO: 11, CDR2 being SEQ ID NO: 31, CDR3 being SEQ ID NO: 47; (i) CDR1 being SEQ ID NO: 16, CDR2 being SEQ ID NO: 31, CDR3 being SEQ ID NO: 50; or (j) CDR1 being SEQ ID NO: 25, CDR2 being SEQ ID NO: 35, CDR3 being SEQ ID NO: 59. (
[0042] The present invention is further directed to a single domain antibody comprising the sequence selected from the group consisting of SEQ ID NOs: 62-99, or a sequence having at least 95%, or 96%, or 97%, or 98%, or 99% sequence identify thereof, provided that the sequence variations are in the non-CDR framework regions. Preferred single domain antibodies include those comprising the sequence selected from the group consisting of SEQ ID NOs: 62, 64, 65, 68, 70, 71, 74, 76, 80, and 95, preferably SEQ ID NOs. 68 and 70, or a sequence having at least 95%, or 96%, or 97%, or 98%, or 99% sequence identify thereof, provided that the sequence variations are in the non-CDR framework regions. The sequence variation, i.e., the amino acid changes are preferably of a minor amino acid change such as a conservative amino acid substitution. A conservative amino acid substitution is well known to a person skilled in the art.
[0043] The present invention provides single domain antibodies that interact both with human and mouse forms of the TLT-1 protein. Such antibodies are suitable for testing in both human and mouse models of bleeding, such as in transgenic mouse models of hemophilia or in acquired bleeding though inhibitors of coagulation pathways.
Fusion Proteins
[0044] The second aspect of the invention is directed to a fusion protein comprising (a) an extracellular domain of tissue factor, (b) a single domain antibody against TLT-1, and (c) a linker.
[0045] Activated platelets, and in particular, “coated” platelets, are substrates for numerous coagulation cascade components that, in combination with fibrinogen, are able to generate a fibrin-based clot needed to seal a vascular injury. By fusing sdAbs with the soluble domain of human tissue factor (sTF), the inventors have demonstrated the targeting of these protein fusions to activated platelets directly and specifically. This specific targeting thus “bypasses” the normal coagulation cascade much in the way of a recombinant FVIIa. Mechanistically, however, the two ‘bypass agents’ are very different. For the chimeric sTF-sdAb fusions, the extracellular portion of TF becomes anchored to activated platelets through the insertion of a high-affinity sdAb fusion partner into relevant epitope folds of the TLT-1 protein; in the correct surface orientation, the sTF domain is thermodynamically-favored to bind to circulating plasma FVII, and activates it in situ to FVIIa; factor VIIa in turns activates FX to FXa, and further stimulates and promotes the common coagulation cascade. This mechanism is considerably different than the mechanics of recombinant FVIIa activation of coagulation factors and direct platelet binding.
[0046] The amino acid sequence of full length of human tissue factor protein is shown in
[0047] SEQ ID NO: 110. For example, sTF is 1-208, 1-209, 1-210, 1-211, 1-212, 1-213, 1-214, 1-215, 1-216, 1-217, 1-218, 1-219, 1-220, or 1-221 of SEQ ID NO: 110. A preferred sTF is 1-209 of SEQ ID NO: 110.
[0048] In the fusion protein of the present invention, the sdAb is any sdAb described above in the preceding sections.
[0049] In the fusion protein of the present invention, the sdAb may be C-terminal or N-terminal to the sTF, and a flexible linker is used to connect the sdAb with the soluble tissue factor. A flexible linker can be any length that links the two proteins, spaces the two protein properly, and does not affect the functionality of the two proteins. The length of linker sequence can be optimized in order to allow ideal positioning sTF of the fusion molecule on the surface of the platelet, as a function of its insertion into the TLT-1 molecule, to efficiently bind FVII, which is the first step in propagating the extrinsic coagulation pathway. The length of the linker sequence is in general 5-40, 10-30, or 15-30 amino acids, preferably the length of the linker is 18-26 amino acids.
[0050] A flexible linker may contain a variety of amino acids. In one embodiment, a flexible linker comprises various combinations of glycine and serine, as well as other amino acids, such as threonine. For example, a flexible linker can be a natural amino acid sequence derived from a human transthyretin protein such as GSGGGTGGGSGGSGGGTGGGSG (SEQ ID NO: 113). For example, the flexible linker can be an artificial sequence such as GGGGSGGGGSGGGGS (SEQ ID NO: 114).
[0051] In one embodiment, the fusion protein of the present invention may further comprise a protease cleavage site. In this embodiment, the fusion protein comprises: (a) an extracellular domain of tissue factor, (b) a single domain antibody against TLT-1, (c) a linker, and (d) a polypeptide sequence that can be proteolytically-cleaved by a protease. The polypeptide sequence of (d) includes, but not limited to, a thrombin cleavage site, a FXa cleavage site, or a FXIa cleavage site, to allow auto-regulation of thrombin production (
[0052] In one embodiment, the present invention provides nucleotide sequences encoding the fusion proteins of the present invention. The nucleotide sequences allow inclusion as part of a prokaryotic, fungal, or eukaryotic expression vector for expression in bacterial cells (like Escherichia coli), yeast (like Saccharomyces cerevisiae), insect cells (like Sf9, Sf21 and High Five), or mammalian cells (like CHO, HEK, BHK, for example), respectively. Due to the small size of the sdAb, the fusion protein can be expressed in bacteria, yeast, insect cells or other eukaryotic cells, such as mammalian cells.
[0053] In a further aspect, the present invention provides a pharmaceutical composition comprising the fusion protein of the present invention and a pharmaceutically acceptable carrier. In a further aspect, the present invention provides a method for treating bleeding disorders, such as those of congenital or acquired coagulopathies, traumatic bleeding due to injury, or other uses where bleeding cannot easily be controlled. The method comprises the step of administering an effective amount of the fusion protein of the present invention to a patient in need thereof with. The fusion protein, for example, can be administered by injection or other parenteral administration, or by oral administration.
[0054] The fusion protein of the present invention avidly, and specifically, binds to TLT-1 molecules on activated platelets. This binding to TLT-1 then conformationally-promotes interaction of sTF to FVII, the molecule that, when activated, further facilitates the downstream common coagulation cascade leading to thrombin formation. The resulting fusion protein exhibits the desired properties of a functional procoagulant: high-affinity binding to activated platelets, high-affinity binding to FVII and conversion to FVIIa, conversion of factor X to factor Xa, and incorporation of a proteolytic (thrombin) cleavage site to self-limit excess thrombin formation. Cleavage allows selective dissociation of the sTF domain (domain responsible for FVII activation but only when bound as a fusion) from the sdAb antibody domain that binds TLT-1 on the activated platelet (these domains do not promote coagulation or platelet aggregation in any case). The fusion proteins optionally have a hexanucleotide His tag incorporated at their C-terminus to facilitate purification and detection.
[0055] The inventors have demonstrated two high-affinity sdAb domains, sdAb 2-33.sub.TLT (SEQ ID: 68) and sdAb 2-90.sub.TLT (SEQ ID: 70), to act as fusion partners with the extracellular domain of tissue factor (amino acid 1-209 of SEQ ID NO: 100). The resulting preferred fusion molecules, named sTF.sub.209-PC1-sdAb 2-33.sub.TLT (SEQ ID: 106) and sTF.sub.209-PC1-sdAb 2-90.sub.TLT SEQ ID: 107), bind efficiently to both mouse and human platelets through the interaction with the platelet TLT-1 receptor. They effectively bind to FVIIa to promote the generation of FXa from FX, to generate formation of thrombin (FIIa) from prothrombin, and to reduce blood loss in a mouse model of bleeding. On the other hand, neither the sTF domain alone, nor the sdAb antibody domain alone, nor sTF-sdAb fusion protein is able to mediate platelet aggregation or activation at the tested dose.
[0056] The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting.
EXAMPLES
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TABLE-US-00001 TABLE 1 Abbreviations Abbreviation Name Description TLT-1 TREM-like transcript-1 sdAb Single domain antibody sTF Tissue factor extracellular (soluble) domain VHH Variable domain of the heavy chain immunoglobulin FVIIa Activated factor VII CHO Chinese hamster ovary BHK Baby Hamster Kidney HEK Human embryonic kidney SEQ Sequence CDR Complementary determining regions PBMC Peripheral blood mononuclear cell GLY Glycine SER Serine HIS Histidine PC1 Thrombin cleavage site PC2 Factor Xa cleavage site
Example 1: Human TLT-1 Amino Acid Sequence
[0058] The amino acid sequence of human TLT-1, an abundant platelet type I. transmembrane receptor with an immunoglobulin-like structure, is presented below; the underlined sequence is the signal sequence at the N-terminus of the protein and the highlighted sequence is the TLT-1 transmembrane domain. The extracellular domain of human ILT-1 that was used in generation of the anti-TLT-1 single domain antibodies (sdAbs) is a 147 amino acid protein between the end. of the signal sequence and the beginning of the transmembrane domain (underlined), UniProt sequence Q86YW5:
TABLE-US-00002 (SEQ ID NO: 118) MGLTLLLLLLLGLEGQGIVGSLPEVLQAPVGSSILVQCHYRLQDVKAQKV WCRFLPEGCQPLVSSAVDRRAPAGRRTFLTDLGGGLLQVEMVTLQEEDAG EYGCMVDGARGPQILHRVSLNILPPEEEEETHKIGSLAENAFSDPAGSAN PLEPSQDEKSIPLIWGAVLLVGLLVAAVVLFAVMAKRKQGNRLGVCGRFL SSRVSGMNPSSVVHHVSDSGPAAELPLDVPHIRLDSPPSFDNTTYTSLPL DSPSGKPSLPAPSSLPPLPPKVLVCSKPVTYATVIFPGGNKGGGTSCGPA QNPPNNQTPSS
Example 2. Generation of Anti-Human TLT41 Single Domain Antibodies (sdAbs)
[0059] In order to create coagulation co-factors targeting specific proteins on platelets, the inventors first identified sdAb antibodies specific for human TLT-1 by immunizing llamas five times with recombinant, soluble human TLT-1 protein and recovering the mRNAs coding for the heavy-chain antibody fragments from the B-cells of the immunized llamas. Messenger RNAs were converted into complementary DNA (cDNA) and cloned into a major coat protein gene (pIII) of bacteriophage M13 for expression. VHH domains of interest were selected by phage display methods (e.g., Kushwaha et al., 2014, J. Vis. Exp., volume 84, e50685; Saw and Song, 2019, Protein Cell, volume 10, pp. 787-807). The recovered sdAbs were selected from the pool of antibody fragments by binding repeatedly to immobilized human TLT-1 protein, as well as to unrelated proteins; repeated selection by this method identified only those that were true and high-affinity binders to TLT-1, while non-specific binders were discarded. DNA sequencing and sequence alignment were used to validate the structure and sequence of the resulting specific sdAb. Further characterization of potential candidates was made by testing of binding of individual sdAb expressed in, and purified from, bacterial cells to full-length, membrane-bound human recombinant TLT-1 that was transfected into, and expressed on the surface of, Chinese hamster ovary (CHO) cells and on both resting and activated platelets using Flow cytometry to demonstrate specificity and functional binding of sdAbs. Finally, select sdAbs were tested as fusions with sTF to determine their ability to reduce blood loss in animal models of bleeding.
Animal Immunization
[0060] One llama was immunized subcutaneously at 3-week intervals and at multiple site of injection. Over the course of 5 rounds of injections with 0.5-1.0 mg of human TLT-1-His antigen (encompassing the TLT-1 extracellular domain shown in
Immune Library Construction and Screening
[0061] After the immunization protocol was completed, whole blood was collected from the immunized llama for PBMC isolation. RNA was extracted and tested by gel electrophoresis to be intact. The VHH genes of immunoglobulin RNA were amplified by two rounds of PCR after reverse transcription using unique primers to camelid variable and constant region domain sequences. The PCR products and the phagemid DNA were digested with Sfi I restriction site endonuclease and ligated together with T4 DNA ligase. The ligation mix was transformed into E. coli TG1 cells. The final constructed library consisted of 5.2×10.sup.8 independent members. Three rounds of bio-panning for single-domain binders against TLT-1-His protein was then performed and an enriching factor of about a thousand-fold was achieved.
Binder Validation
[0062] Based on the bio-panning strategy, a total of 300 clones were validated against TLT-l1 using ELISA and, of these, 147 clones were identified as positive. DNA sequence indicated that 145 of 147 clones were correctly identified as authentic camelid antibodies. A total of 103 unique clones have been identified at the amino acid level. As some groups of the identified unique clones present the same CDR3 region but have differences in their CDR1 and/or CDR2 regions, these unique sequences were further analyzed based on their CDR3 regions (the CDR regions are predicted via IMGT database). A total of 38 unique sdAb sequences with different CDR1, CDR2, and/or CDR3 were identified (
ELISA Ligand Binding Assay
[0063] The top 10 clones were then confirmed in the final soluble ELISA validation. Soluble TLT-1 extracellular domain His tag (sTLT-1-His) protein was coated (0.1 μg/well) onto a 96-well plate and incubated overnight at 4° C. An irrelevant protein with His tag and an no coating group were used in the assay as negative controls. On the next day, the coated plate was washed 3 times with 200 μL PBST buffer per well and blocked with 300 μL blocking buffer per well for 1 h at 37° C. The blocking buffer was then removed and the plate was washed 3 times with the washing buffer. After washing, 100 μL of HRP-anti-TLT-1 sdAb antibody in blocking buffer was added to each well and incubated at 37° C. for 1 h. The plate was washed three times with the washing buffer and then 100 μL of TMB substrate solution was added per well and incubated at room temperature for 15 minutes; 100 μL of 2M H.sub.2SO.sub.4 were then added to stop the reaction and the plate was analyzed using a microplate reader at 490 nm. According to the results, consistent results were obtained. In the meantime, the negative control groups present expected low signal, which indicated all the Top 10 clones did not cross-react with His tag and can bind to the target specifically.
TABLE-US-00003 TABLE 2 [OD 490 nm] Coating: Coating: No Clone TLT-1 protein (3 μg/mL) Irrelevant protein (3 μg/mL) Coating 2-2 0.796 0.103 0.096 2-25 0.512 0.073 0.104 2-33 1.501 0.070 0.095 2-64 0.911 0.066 0.104 2-69 0.494 0.076 0.099 2-90 0.762 0.081 0.132 2-127 0.760 0.076 0.102 2-132 0.981 0.114 0.079 3-32 0.858 0.097 0.106 3-38 0.818 0.087 0.099
Example 3. Development of pNT-sdAb 2-33.SUB.TLT.-His, pNT-sdAb 2-132.SUB.TLT.His, pNT-sdAb 2-25.SUB.TLT.-His, pNT-sdAb 2-64.SUB.TLT.-His, pNT-sdAb 2-90.SUB.TLT.-His, pNT-sdAb 2-127.SUB.TLT.-His, pNT-sdAb 2-2.SUB.TLT.-His, pNT-sdAb 3-32.SUB.TLT.-His, pNT-sdAb 3-38.SUB.TLT.-His and pNT-sdAb 2-69.SUB.ILT.-His Expression Constructs
[0064] In order to evaluate the utility of these novel antibodies, DNAs corresponding to ten selected single-domain antibodies identified in TABLE 1 were synthesized and codon-optimized for bacterial expression (GenScript, Piscataway N.J.); corresponding amino acid sequences and SEQ ID numbers are shown in
Example 4. Development of pNT-sTF.SUB.209.-His Expression Construct
[0065] DNA corresponding to the extracellular domain of tissue factor (sTF) amino acid 1-209 was synthesized as previously described and codon-optimized for expression in bacteria. A Nhe I restriction enzyme site at the 5′-end and a Bam HI restriction enzyme site at the 3′-end were included for cloning purposes. To facilitate recombinant sTF purification, a sequence encoding six histidine amino acids (His) was also incorporated at the 3′-end of the synthesized genes upstream of the Bam HI site. The synthesized sTF209-His was inserted into Nhe I and Bam HI restriction enzyme sites of a pNT-based expression vector and the resulting vector was designated as pNT-sTF.sub.209-His (TABLE 2). A representative illustration of the plasmid expression vector for the extracellular domain of soluble tissue factor (sTF) is shown in
Example 5. Development of pNT-sTF.SUB.209.-PC1-sdAb 2-33.SUB.TLT .and pNT-sTF.SUB.209.-PC1-sdAb 2-90.SUB.TLT .Expression Constructs
[0066] The expression cassettes encoding sTF.sub.209-PC1-sdAb 2-33.sub.TLT-His and sTF.sub.209-PC1-sdAb 2-90.sub.TLTl -His were synthesized (GenScript, Piscataway N.J.) and codon-optimized for bacterial expression. A Nhe I restriction enzyme site at the 5′-end and a Bam HI restriction enzyme site at the 3′-end were included for cloning purposes. To facilitate purification of the recombinant fusion proteins, a sequence encoding six histidine amino acids (His) was also incorporated at the 3′-end of the synthesized genes upstream of the Bam HI site. To properly position sTF on the surface of the cell surface and to limit thrombin overexpression, a Gly-Ser linker sequence from human transthyretin (encoding 22 amino acids) and a thrombin cleavage site from human factor VIII (‘PCI’), respectively, were inserted between the sTF and sdAb sequences. The synthesized genes were inserted into Nhe I and Bam HI restriction enzyme sites of a pNT expression vector, itself based on the pET9d plasmid vector. The resulting vectors were designated as pNT-sTF.sub.209-PC1-sdAb 2-33.sub.TLT-His and pNT-sTF209-PC1-sdAb 2-90.sub.TLT-His, respectively (Table 3). A representative illustration of the plasmid expression vector for the sTF-sdAb fusions is shown in
TABLE-US-00004 TABLE 3 Expression Construct Name Coding Protein Description 1 pNT-sdAb 2-33.sub.TLT-His sdAb 2-33.sub.TLT-His 2 pNT-sdAb 2-132.sub.TLT-His sdAb 2-132.sub.TLT-His 3 pNT-sdAb 2-25.sub.TLT-His sdAb 2-25.sub.TLT-His 4 pNT-sdAb 2-64.sub.TLT-His sdAb 2-64.sub.TLT-His 5 pNT-sdAb 2-90.sub.TLT-His sdAb 2-90.sub.TLT-His 6 pNT-sdAb 2-127.sub.TLT-His sdAb 2-127.sub.TLT-His 7 pNT-sdAb 2-2.sub.TLT-His sdAb 2-2.sub.TLT-His 8 pNT-sdAb 3-32.sub.TLT-His sdAb 2-32.sub.TLT-His 9 pNT-sdAb 3-38.sub.TLT-His dAb 2-38.sub.TLT-His 10 pNT-sdAb 2-69.sub.TLT-His sdAb 2-69.sub.TLT-His 11 pNT-sTF.sub.209-His sTF.sub.209-His 12 pNT- sTF.sub.209-PC1-sdAb sTF.sub.209-PC1-sdAb 2-33.sub.TLT-His 2-33.sub.TLT-His fusion 13 pNT- sTF.sub.209-PC1-sdAb sTF.sub.209-PC1-sdAb 2-90.sub.TLT-His 2-90.sub.TLT-His fusion
Example 6. Expression and Purification of Recombinant sTF.SUB.209., TLT-1 sdAbs and sTF.SUB.209.-sdAb Fusion Proteins Expressed in Bacteria
[0067] All ten sdAbs, as well as sTF.sub.209, and the two sTF.sub.209-sdAb fusion protein DNA sequences described in TABLE 2 were chemically-transformed into an E. coli BL21-based bacteria strain and expressed in LB medium. The bacteria were harvested after protein expression and sonicated in lysis buffer (20 mM HEPES pH 8.0, 300 mM KCl and 10% glycerol). The supernatants were then collected by high-speed centrifugation and applied to a His-Trap HP column (GE) for His-tag protein purification using GE AKTA chromatography system. After washing with 20 column volumes of washing buffer (20 mM HEPES pH 8.0, 20 mM imidazole, 300 mM KCl and 10% glycerol), the absorbed proteins were eluted by using gradient elution buffer (20 mM HEPES pH 8.0, 40-300 mM imidazole, 300 mM KCl and 10% glycerol). Fluted proteins were then concentrated and buffer exchanged into PBS buffer. The purified proteins were analyzed using 10% SDS-PAGE method and confirmed with Western blot.
Example 7. Binding Affinity (Kd) Determination of TLT-1 sdAbs and sTF.SUB.209.-sdAb Fusion Proteins to Extracellular Domain of TLT-1 Receptor
[0068] The binding of sdAb 2-33.sub.TLT-His, sdAb 2-132.sub.TLT-His, sdAb 2-25.sub.TLT-His, sdAb 2-64 .sub.TLT-His, sdAb 2-90.sub.TLT-His, sdAb 2-127.sub.TLT-His, sdAb 2-2 .sub.TLT-His, sdAb 3-32.sub.TLTcur-His; sdAb 3-38.sub.TLT-His and sdAb 2-69.sub.TLT-His, sTF.sub.209-PC1-sdAb 2-33.sub.TT-His and s TF.sub.209-PC1-sdAb 2-90.sub.TLT-His proteins to the human extracellular domain of TLT-1-Fc tagged protein (sTLT-1-Fc) was analyzed using ELISA, sTLT-1-Fc (3 μg/ml) was immobilized onto a 96-well plate for 24 hours at 4° C. and each well immobilized with SILT-1-Fc was blocked with 2% BSA PBST (PBS plus 0.1% Tween 20) for 2 hours at room temperature (RT). Serial dilution (1000 nM to 0.001 nM) of TLT-sdAbs and sTF-sdAb fusion proteins was performed and diluted proteins were then added to the coated 96-well platelet and incubated for 1 hour. After 3 times of washing with PBST, anti-His HRP antibody was added and incubated for 1 hour at RT. The plate was then washed for 3 times to remove the excess HRP conjugate and 100 μL TMB substrate was then added and incubated for 10-15 mins. To stop the reaction of color development, 2M sulfuric acid was added to the well. The binding affinity (Kd) was calculated based on OD.sub.450 nm measurement using GraphPad Prism 8.0 (
Example 8. Binding to Activated Human and Mouse Platelets
[0069] The binding capability of sdAb 2-33.sub.TLT-His, sdAb sTF.sub.209-PC1-sdAb 2-33.sub.TLT-His and sTF.sub.209-PC1-sdAb 2-90.sub.TLT-His proteins to both human and mouse resting and activated platelets was tested by FACS assay. Citrated human (3 donors) and mouse (12 mice) whole blood were collected at room temperature (RT) and 10 μL of whole blood was used for each sample. To activate human platelets, ADP (5 and 20 μM) was used, and incubated with whole blood for 10 min at room temperature (RT). To activate mouse platelet, Type I fibrillary collagen (5 and 10 ug/ml) was used and incubated with whole blood for 10 min at room temperature (RT). Both ADP and collagen used were from Helena Laboratory, Beaumont Tex., Then, for each sample, 10 μg/ml of test article (i.e, sdAb 2-33.sub.TLT-His, sdAb 2-90.sub.TLT-His, sTF.sub.209-PC1-sdAb 2-33.sub.TLT-His or sTF.sub.209-PC1-sdAb 2-90.sub.TLT-His proteins) was added, followed by one or the other labeled antibodies, either APC-anti-CD41a antibody that was used in gating platelet population, or FITC anti-His antibody, that was used in detecting TLT-1 binding of TLT-sdAbs or sTF.sub.209-sdAb fusion proteins on activated platelets. APC-anti-CD62P antibody was used as an activated platelet binding control antibody in the assay. After incubation for 30 min at room temperature, all samples were fixed with 500 μL of 5% paraformaldehyde for 10 min at RT and. analyzed by FACS (LSR II, Beckon Dickinson, San Jose, Calif.). Data in
[0070] These observations demonstrate the novelty of these sdAbs in their ability to bind both mouse and human TLT-1 on activated platelets. This observation further indicates that testing of sdAbs and their fusion counterparts can proceed directly in mouse bleeding models without resorting to the use of transfused human platelets to facilitate binding (Example 13).
Example 9. Binding of sTF-sdAb Fusion Proteins to FVIIa
[0071] sTF.sub.209-PC1-sdAb 2-33.sub.TLT-His and sTF.sub.209-PC1-sdAb 2-90.sub.TLT-His fusion proteins are designed to target sTF to the surface of activated platelets through sdAb/TLT-1 receptor interaction. To verify whether fusing TLT-1 sdAb to sTF would affect its binding to FVIIa, are FVIIa amidolytic activity assay was performed. Various concentrations (0-100 nM) of sTF.sub.209 and sTF.sub.209-sdAb fusion proteins were incubated with factor FVIIa (5 nM) in a butler containing 100 nM NaCl, 50 mM HEPES, pH 7.4, 5 mM CaCl.sub.2, 0.1% BSA at 37° C. for 5 minutes. FVIIa amidolytic activity was assayed with the addition of a 5 mM Chromozym tPA substrate and the absorbance were measured at 405 nm at room temperature. Both sdAb 2-33.sub.TLT-His and sdAb 2-90.sub.TLT-His were included in the assay as negative controls. The data (
Example 10. Procoagulant Effect of Targeted sTF.SUB.209 .in a One-Stage Clotting Assay
[0072] Targeting sTF.sub.209to TLT-1 receptor is expected to promote coagulant activity. To confirm the hypothesis, the procoagulant activity of sTF.sub.209-PC1-sdAb 2-33.sub.TLT and sTF.sub.209-PC1-sdAb 2-90.sub.TLT fusion proteins were evaluated in a modified one-stage activated partial thromboplastin time (APTT) clot assay. The APTT clotting times were measured using a STar 4 Hemostasis Analyzer (Diagnostica Stago). Fifty microliters of hemophilia A patient plasma (George King Bio-Medical, Overland Park, Kans.), 50 μL containing 0.5×10.sup.6 CHO-K1 cells expressing human TLT-1 protein and 1 nM of test article (sTF.sub.209-PC1-sdAb 2-33.sub.TLT-His or sTF.sub.209-PC1-sdAb 2-90.sub.TLT-His) were added to the sample cuvette with total volume of 100 μL. After 200 seconds incubation at 37° C., 50 μL calcium chloride (20 mM) was added to initiate the clot formation. The data (
Example 11. Targeted sTF to the Surface of Activated Platelets Promotes Thrombin Generation
[0073] Human platelet-rich plasma (PRP) was prepared by centrifugation of human whole blood containing 0.32% Sodium Citrate at 150×g for 20 min. Thrombin generation assay was performed by adding 20 μL of PRP reagent (Diagnostica Stago), 80 μL of PRP and 25 nM of testing samples. The reaction was started by the addition of 20 μL FluCa substrate (Diagnostica Stago) to U-bottom 96-well plates (ThermoFisher) and the fluorescent signal from the substrate was detected in a Fluoroskan Ascent plate reader (ThermoFisher). The results showed that sTF.sub.209-PC1-sdAb 2-33.sub.TLT-His and sTF.sub.209-PC1-sdAb-290.sub.TLT-His have an increased potency in thrombin generation compared to sTF.sub.209-His, sdAb 2-33.sub.TLT-His and sdAb-290.sub.TLT-His (
Example 13. sTF.SUB.209.-sdAb Fusion Proteins Reduced Tail-bleeding in Enoxaparin Treated Mice
[0074] The procoagulant effect of sTF.sub.209-PC1-sdAb 2-33.sub.TLT-His and sTF.sub.209-PC1-sdAb 2-90.sub.FLT-His fusion proteins were tested in an enoxaparin-induced tail-bleeding model in mice (Washington Biotechnology Inc, Baltimore Md.). Mice (4 per group) were injected subcutaneously with sodium enoxaparin (30 mg/kg) and two hours later were anaesthetized by intraperitoneal injection of ketamine/xylazine (10 mg/kg). The baseline bleeding time and blood loss were determined by transecting the mouse tail at a point 10 mm from tail tip. The time required for bleeding to stop was recorded, and blood loss was determined by collecting blood in a warmed (37° C.) normal saline solution. An intravenous injection of sTF.sub.209-PC1-sdAb 2-33.sub.TLT-His and sTF.sub.209-PC1-sdAb 2-90 .sub.FLT-His fusion proteins (90 μg/kg) were performed immediately after the first bleeding time determination. A second bleeding time was then measured 5 minutes after the injection of the above proteins, and bleeding time and blood loss was determined in a similar manner as described. The results show that administration of the sTF.sub.209-PC1-sdAb 2-33.sub.TLT-His and sTF.sub.209-PC1-sdAb 2-90.sub.TLT-His fusion proteins completely normalize the bleeding time to that of control animals in this bleeding model (
Example 14. Targeted sTF to the Surface of Activated Platelets Promotes Fibrin Clot Formation in Human Whole Blood (Prophetic Example)
[0075] Citrated human whole blood (HWB) is drawn from normal donors. Clot formation is measured by thrombelastography (TEG5000) analyzer (Haemonetics, Boston, Mass.). The final concentrations (0-100 nM) of sdAb 2-33.sub.TLT-His, sdAb 2-90.sub.TLT-His, sTF.sub.209-His, sTF.sub.209-PC1-sdAb 2-33.sub.TLT-His and sTF.sub.209-PC1-sdAb 2-90.sub.TLT-His are added to 340 μL of whole blood containing the kaolin activator. Clotting formation measurement is initiated with addition of 20 μL of 0.2 M CaCl.sub.2. The TEG trace is followed continuously for up to 60 min. The R-time (clotting time) is recorded for potency comparison of testing samples. The data are expected to demonstrate that sTF.sub.209-sdAb fusion proteins shortened R-time (clotting time) in a concentration dependent manner compared to sTF.sub.209-His and sdAb 2-33.sub.TLT-His and sdAb 2-90.sub.TLT-His proteins. The results are expected to further demonstrate that the enhanced thrombin generation seen in Example 11 generates bona fide fibrin formation necessary to generate a functional clot.