ANTIBODY VARIANTS TRANSMIGRATING THE BLOOD-BRAIN BARRIER AND USES THEREOF
20200095316 · 2020-03-26
Assignee
Inventors
- Danica Stanimirovic (Ottawa, CA)
- Traian Sulea (Kirkland, CA)
- Kristin Kemmerich (Ottawa, CA)
- David Wilson (Redwood City, CA)
- Jennifer Stratton (Redwood City, CA)
- Matthew Pollard (Pullenvale, AU)
- Adam Clarke (Sydney, AU)
Cpc classification
C07K2317/90
CHEMISTRY; METALLURGY
C07K2317/32
CHEMISTRY; METALLURGY
C07K2317/569
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
C07K16/28
CHEMISTRY; METALLURGY
A61K47/6803
HUMAN NECESSITIES
C07K17/00
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/22
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates, in general, to polypeptides capable of transmigrating the blood-brain barrier, and uses thereof. More specifically, the present invention relates to polypeptides derived by site-directed mutagenesis of an existing antibody fragment and uses thereof, and methods of making such molecules. The polypeptides of the present invention show enhanced blood-brain barrier crossing and brain exposure levels in vitro and in vivo.
Claims
1. An isolated or purified antibody fragment, comprising: a complementarity determining region (CDR) 1 sequence of GFKITHYTMG (SEQ ID NO:1); a CDR2 sequence of RITWGGX.sub.1X.sub.2TX.sub.3YSNSVKG (SEQ ID NO:2), where X.sub.1 is D or K, X.sub.2 is N or D, and X.sub.3 is F, I or L; and a CDR3 sequence of GSTSTAX.sub.4PLRVDY (SEQ ID NO:3), where X.sub.4 is T or K, wherein at least one of the amino acid residues at positions X.sub.1, X.sub.2, X.sub.3 or X.sub.4 is different from the corresponding residue in CDR2 (SEQ ID NO:4) or CDR3 (SEQ ID NO:10) of SEQ ID NO:16.
2. The isolated or purified antibody fragment of claim 1, wherein CDR2 is selected from the group consisting of RITWGGDNTFYSNSVKG (SEQ ID NO:4), RITWGGKNTFYSNSVKG (SEQ ID NO:5), RITWGGDDTFYSNSVKG (SEQ ID NO:6), RITWGGDNTIYSNSVKG (SEQ ID NO:7), and RITWGGDNTLYSNSVKG (SEQ ID NO:8); with the proviso that if the CDR2 sequence is RITWGGDNTFYSNSVKG (SEQ ID NO:4), CDR3 is not GSTSTATPLRVDY (SEQ ID NO:10).
3. The isolated or purified antibody fragment of claim 1, wherein CDR3 is GSTSTAKPLRVDY (SEQ ID NO:9) or GSTSTATPLRVDY (SEQ ID NO:10); with the proviso that if CDR3 sequence is GSTSTATPLRVDY (SEQ ID NO:10), CRD2 is not RITWGGDNTFYSNSVKG (SEQ ID NO:4).
4. The isolated or purified antibody fragment of any one of claims 1 to 3, comprising: a CDR1 sequence of GFKITHYTMG (SEQ ID NO:1), a CDR2 sequence of RITWGGDNTFYSNSVKG (SEQ ID NO:4), and a CDR3 sequence of GSTSTAKPLRVDY (SEQ ID NO:9); a CDR1 sequence of GFKITHYTMG (SEQ ID NO:1), a CDR2 sequence of RITWGGKNTFYSNSVKG (SEQ ID NO:5), and a CDR3 sequence of GSTSTATPLRVDY (SEQ ID NO:10); a CDR1 sequence of GFKITHYTMG (SEQ ID NO:1), a CDR2 sequence of RITWGGDDTFYSNSVKG (SEQ ID NO:6), and a CDR3 sequence of GSTSTATPLRVDY (SEQ ID NO:10); a CDR1 sequence of GFKITHYTMG (SEQ ID NO:1), a CDR2 sequence of RITWGGDNTIYSNSVKG (SEQ ID NO:7), and a CDR3 sequence of GSTSTATPLRVDY (SEQ ID NO:10); or a CDR1 sequence of GFKITHYTMG (SEQ ID NO:1), a CDR2 sequence of RITWGGDNTLYSNSVKG (SEQ ID NO:8), and a CDR3 sequence of GSTSTATPLRVDY (SEQ ID NO:10).
5. The isolated or purified antibody fragment of any one of claims 1 to 4, comprising a sequence selected from the group consisting of: TABLE-US-00006 (SEQIDNO:11) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSR ITWGGDNTFYSNSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGS TSTAKPLRVDYWGQGTLVTVSS; (SEQIDNO:12) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSR ITWGGKNTFYSNSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGS TSTATPLRVDYWGQGTLVTVSS; (SEQIDNO:13) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSR ITWGGDDTFYSNSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGS TSTATPLRVDYWGQGTLVTVSS; (SEQIDNO:14) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSR ITWGGDNTIYSNSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGS TSTATPLRVDYWGQGTLVTVSS; (SEQIDNO:15) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSR ITWGGDNTLYSNSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGS TSTATPLRVDYWGQGTLVTVSS; and a sequence substantially identical thereto.
6. The isolated or purified antibody fragment of any one of claims 1 to 5, wherein the antibody fragment is a single-domain antibody (sdAb).
7. The isolated or purified antibody fragment of claim 6, wherein the sdAb is humanized.
8. The isolated or purified antibody fragment of any one of claims 1 to 7, wherein the antibody fragment is in a multivalent display format.
9. The isolated or purified antibody fragment of claim 8, wherein the antibody fragment is linked to a Fc fragment.
10. The isolated or purified antibody fragment of claim 9, wherein the Fc fragment is the mouse Fc2b or human Fc1.
11. The isolated or purified antibody fragment of claim 10, wherein the Fc comprises the sequence of SEQ ID NO:29.
12. The isolated or purified antibody fragment of any one of claims 1 to 11, wherein the isolated or purified antibody fragment transmigrates the blood-brain barrier.
13. A nucleic acid molecule encoding the isolated or purified antibody fragment of any one of claims 1 to 12.
14. A vector comprising the nucleic acid molecule of claim 13.
15. The isolated or purified antibody fragment of any one of claims 1 to 12, wherein the antibody or fragment thereof is immobilized onto a surface.
16. The isolated or purified antibody fragment of any one of claims 1 to 12, wherein the antibody or fragment thereof is linked to a cargo molecule.
17. The isolated or purified antibody fragment of claim 16, wherein the cargo molecule has a molecular weight in the range of about 1 kDa to about 200 kDa.
18. The isolated or purified antibody fragment of claim 16 or 17, wherein the cargo molecule is a detectable agent, a therapeutic, a drug, a peptide, a growth factor, a cytokine, a receptor trap, a chemical compound, a carbohydrate moiety, an enzyme, an antibody or fragment thereof, a DNA-based molecule, a viral vector, or a cytotoxic agent; one or more liposomes or nanocarriers loaded with a detectable agent, a therapeutic, a drug, a peptide, an enzyme, an antibody or fragment thereof, a DNA-based molecule, a viral vector, or a cytotoxic agent; or one or more nanoparticle, nanowire, nanotube, or quantum dots.
19. A composition comprising one or more than one isolated or purified antibody fragment of any one of claims 1 to 12 and 15 to 18 and a pharmaceutically-acceptable carrier, diluent, or excipient.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] These and other features of the invention will now be described by way of example, with reference to the appended drawings, wherein:
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DETAILED DESCRIPTION OF THE INVENTION
[0048] The present invention relates to isolated or purified antibody fragments that transmigrate the blood-brain barrier, and uses thereof. More specifically, the present invention relates to antibody fragments derived by point-mutation of an existing antibody fragment and uses thereof; the antibody fragments of the present invention show enhanced transmigration across the blood-brain barrier.
[0049] The present invention provides an isolated or purified antibody fragment, comprising: [0050] a complementarity determining region (CDR) 1 sequence of GFKITHYTMG (SEQ ID NO:1); [0051] a CDR2 sequence of RITWGGX.sub.1X.sub.2TX.sub.3YSNSVKG (SEQ ID NO:2), where X.sub.1 is D or K, X.sub.2 is N or D, and X.sub.3 is F, I or L; and [0052] a CDR3 sequence of GSTSTAX.sub.4PLRVDY (SEQ ID NO:3), where X.sub.4 is T or K.
[0053] The antibody fragment of the present invention is a mutated version of the FC5 antibody described in WO 2002/057445. FC5 (SEQ ID NO:16) binds to the surface of mammalian brain endothelial cells and subsequently transmigrates the blood-brain barrier (BBB). FC5 has also been shown to act as a carrier to usher molecules of various sizes across the BBB (see for example, WO 2011/127580). Without wishing to be bound by theory, the antigen to which FC5 selectively binds and that mediates FC5 transmigration has been proposed to be transmembrane domain protein 30A (TMEM30A; WO 2007/036021), which is enriched on the surface of brain endothelial cells.
[0054] In the isolated or purified antibody fragment of the present invention, at least one of X.sub.1, X.sub.2, X.sub.3, and X.sub.4 is different from the corresponding wild type residues in CDR2 or CDR3 of FC5. More specifically, in an embodiment of the present invention, at least one of X.sub.1, X.sub.2, X.sub.3 of CDR2 (SEQ ID NO: 2) and X.sub.4 of CDR3 (SEQ ID NO: 3) is different from the corresponding wild type CDR2 and CDR3 sequences of SED ID NO:16, where the wild type CDR2 (SEQ ID NO:4) and CDR3 (SEQ ID NO: 10) correspond to residues 50-66 and 99-111 of SEQ ID NO: 16, respectively.
[0055] In antibody fragment described above, the CDR2 is selected from the group consisting of RITWGGDNTFYSNSVKG (SEQ ID NO:4), RITWGGKNTFYSNSVKG (SEQ ID NO:5), RITWGGDDTFYSNSVKG (SEQ ID NO:6), RITWGGDNTIYSNSVKG (SEQ ID NO:7), and RITWGGDNTLYSNSVKG (SEQ ID NO:8); whereas CDR3 may be selected from the group consisting of is GSTSTAKPLRVDY (SEQ ID NO:9) or GSTSTATPLRVDY (SEQ ID NO:10), with the proviso that when CDR3 is GSTSTATPLRVDY (SEQ ID NO:10), CDR2 is not RITWGGDNTFYSNSVKG (SEQ ID NO:4).
[0056] The term antibody, also referred to in the art as immunoglobulin (Ig), generally refers to a protein constructed from paired heavy and light polypeptide chains; various Ig isotypes exist, including IgA, IgD, IgE, IgG, and IgM. When an antibody is correctly folded, each chain folds into a number of distinct globular domains joined by more linear polypeptide sequences. For example, the immunoglobulin light chain folds into a variable (V.sub.L) and a constant (C.sub.L) domain, while the heavy chain folds into a variable (V.sub.H) and three constant (C.sub.H, C.sub.H2, C.sub.H3) domains. Interaction of the heavy and light chain variable domains (V.sub.H and V.sub.L) results in the formation of an antigen binding region (Fv). Each domain has a well-established structure familiar to those of skill in the art.
[0057] The light and heavy chain variable regions are responsible for binding the target antigen and can therefore show significant sequence diversity between antibodies. The constant regions show less sequence diversity, and are responsible for binding a number of natural proteins to elicit important biochemical events. The variable region of an antibody contains the antigen-binding determinants of the molecule, and thus determines the specificity of an antibody for its target antigen. The majority of sequence variability occurs in six hypervariable regions, three each per variable heavy (V.sub.H) and light (V.sub.L) chain; the hypervariable regions combine to form the antigen-binding site, and contribute to binding and recognition of an antigenic determinant. The specificity and affinity of an antibody for its antigen is determined by the structure of the hypervariable regions, as well as their size, shape, and chemistry of the surface they present to the antigen. Various schemes exist for identification of the regions of hypervariability, the two most common being those of Kabat and of Chothia and Lesk. Kabat et al (1991) define the complementarity-determining regions (CDR) based on sequence variability at the antigen-binding regions of the V.sub.H and V.sub.L domains. Chothia and Lesk (1987) define the hypervariable loops (H or L) based on the location of the structural loop regions in the V.sub.H and V.sub.L domains. These individual schemes define CDR and hypervariable loop regions that are adjacent or overlapping, those of skill in the antibody art often utilize the terms CDR and hypervariable loop interchangeably, and they may be so used herein. The CDR/loops are identified herein according to the Kabat scheme.
[0058] An antibody fragment as referred to herein may include a single-domain antibody (sdAb; a fragment composed of a single V.sub.L or V.sub.H) and multivalent presentations of sdAb. Antibody fragments such as those just described may require linker sequences, disulfide bonds, or other type of covalent bond to link different portions of the fragments; those of skill in the art will be familiar with the requirements of the different types of fragments and various approaches and various approaches for their construction.
[0059] The antibody fragment is derived from an sdAb from naturally-occurring or recombinant sources. Heavy chain antibodies of camelid origin (Hamers-Casterman et al, 1993) lack light chains and thus their antigen binding sites consist of one domain, termed V.sub.HH. sdAb have also been observed in shark and are termed V.sub.NAR (Nuttall et al, 2003). Other sdAb may be engineered based on human Ig heavy or light chain sequences (Jespers et al, 2004; To et al, 2005). As used herein, the term sdAb includes those sdAb directly isolated from V.sub.H, V.sub.HH, V.sub.L, or V.sub.NAR reservoir of any origin through phage display or other technologies, sdAb derived from the aforementioned sdAb, recombinantly produced sdAb, as well as those sdAb generated through further modification of such sdAb by humanization, affinity maturation, stabilization, solubilization, camelization, or other methods of antibody engineering. Also encompassed by the present invention are homologues, derivatives, or fragments that retain the antigen-binding function and specificity of the sdAb and transmigrate the BBB with improved ability over FC5.
[0060] SdAb possess desirable properties for antibody molecules, such as high thermostability, high detergent resistance, relatively high resistance to proteases (Dumoulin et al, 2002) and high production yield (Arbabi-Ghahroudi et al, 1997); they can also be engineered to have very high affinity by isolation from an immune library (Li et al, 2009) or by in vitro affinity maturation (Davies & Riechmann, 1996). Further modifications to increase stability, such as the introduction of non-canonical disulfide bonds (Hussack et al, 2011; Kim et al, 2012), may also be brought to the sdAb.
[0061] A person of skill in the art would be well-acquainted with the structure of a single-domain antibody (see, for example, 3DVVT, 2P42 in Protein Data Bank). An sdAb comprises a single immunoglobulin domain that retains the immunoglobulin fold; most notably, only three CDR/hypervariable loops form the antigen-binding site. However, and as would be understood by those of skill in the art, not all CDR may be required for binding the antigen. For example, and without wishing to be limiting, one, two, or three of the CDR may contribute to binding and recognition of the antigen by the sdAb of the present invention. The CDR of the sdAb or variable domain are referred to herein as CDR1, CDR2, and CDR3.
[0062] In one non-limiting example, the antibody fragment of the present invention may comprise: [0063] a CDR1 sequence of GFKITHYTMG (SEQ ID NO:1), a CDR2 sequence of RITWGGDNTFYSNSVKG (SEQ ID NO:4), and a CDR3 sequence of GSTSTAKPLRVDY (SEQ ID NO:9); [0064] a CDR1 sequence of GFKITHYTMG (SEQ ID NO:1), a CDR2 sequence of RITWGGKNTFYSNSVKG (SEQ ID NO:5), and a CDR3 sequence of GSTSTATPLRVDY (SEQ ID NO:10); [0065] a CDR1 sequence of GFKITHYTMG (SEQ ID NO:1), a CDR2 sequence of RITWGGDDTFYSNSVKG (SEQ ID NO:6), and a CDR3 sequence of GSTSTATPLRVDY (SEQ ID NO:10); [0066] a CDR1 sequence of GFKITHYTMG (SEQ ID NO:1), a CDR2 sequence of RITWGGDNTIYSNSVKG (SEQ ID NO:7), and a CDR3 sequence of GSTSTATPLRVDY (SEQ ID NO:10); or [0067] a CDR1 sequence of GFKITHYTMG (SEQ ID NO:1), a CDR2 sequence of RITWGGDNTLYSNSVKG (SEQ ID NO:8), and a CDR3 sequence of GSTSTATPLRVDY (SEQ ID NO:10).
[0068] As previously stated, the antibody fragment may be an sdAb of camelid origin or derived from a camelid V.sub.HH, and thus may be based on camelid framework regions. The present invention further encompasses an antibody fragment that is chimeric (or chimerized), veneered, or humanized. Chimeric antibody fragments encompass constructs in which the native variable domain (of camelid origin) is linked to human constant domain(s) (see Gonzales et al 2005). Veneering or re-surfacing of antibodies involves replacing exposed residues in the framework region of the native antibody fragment with the amino acid residues in their human counterpart (Padlan, 1991; Gonzales et al 2005). Humanization of an antibody comprises replacing an amino acid in the sequence with its human counterpart, as found in the human consensus sequence, without loss of antigen-binding ability or specificity; this approach reduces immunogenicity of the antibody fragment when introduced into human subjects. In this process, one or more than one of the CDR defined herein may be fused or grafted to a human variable region (V.sub.H, or V.sub.L), to other human antibody (IgA, IgD, IgE, IgG, and IgM), to human antibody fragment framework regions (Fv, scFv, Fab), or to human proteins of similar size and nature onto which CDR can be grafted (Nicaise et al, 2004). In such a case, the conformation of said one or more than one hypervariable loop is likely preserved, and the affinity and specificity of the sdAb for its target (i.e., brain endothelial cells) is likely minimally affected. As is known by those of skill in the art, it may be necessary to incorporate certain native amino acid residues into the human framework in order to retain binding and specificity. Humanization by CDR grafting is known in the art (for example, see Tsurushita et al, 2005; Jones et al, 1986; Tempest et al, 1991; Riechmann et al, 1988; Queen et al, 1989; reviewed in Gonzales et al, 2005see also references cited therein), and thus persons of skill would be amply familiar with methods of preparing such humanized antibody or fragments thereof.
[0069] The CDR sequences as described above may be incorporated into a suitable antibody fragment scaffold. For example and without wishing to be limiting, the CDR sequences may be incorporated into the FC5 scaffold (SEQ ID NO:16); or into a humanized version of the FC5 scaffold as described in U.S. Provisional patent application No. 62/358,777 filed on Jul. 6, 2016, for example FC5-H7 (SEQ ID NO:17). The CDRs in the FC5 or humanized scaffolds correspond to residues 26-35 (CDR1), residues 50-66 (CDR2), and residues 99-111 (CDR3).
[0070] For example, and without wishing to be limiting in any manner, the isolated or purified antibody fragment as described above may be selected from the group consisting of:
TABLE-US-00002 (SEQIDNO:11) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSR ITWGGDNTFYSNSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGS TSTAKPLRVDYWGQGTLVTVSS; (SEQIDNO:12) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSR ITWGGKNTFYSNSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGS TSTATPLRVDYWGQGTLVTVSS; (SEQIDNO:13) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSR ITWGGDDTFYSNSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGS TSTATPLRVDYWGQGTLVTVSS; (SEQIDNO:14) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSR ITWGGDNTIYSNSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGS TSTATPLRVDYWGQGTLVTVSS; (SEQIDNO:15) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSR ITWGGDNTLYSNSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGS TSTATPLRVDYWGQGTLVTVSS;
and [0071] a sequence substantially identical thereto.
[0072] A substantially identical sequence may comprise one or more conservative amino acid mutations. It is known in the art that one or more conservative amino acid mutations to a reference sequence may yield a mutant peptide with no substantial change in physiological, chemical, physico-chemical or functional properties compared to the reference sequence; in such a case, the reference and mutant sequences would be considered substantially identical polypeptides.
[0073] In a non-limiting example, a conservative mutation may be a conservative amino acid substitution. A conservative amino acid substitution is defined herein as the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g. size, charge, or polarity). These conservative amino acid mutations are made to the framework regions of the sdAb while maintaining the CDR sequences listed above and the overall structure of the CDR of the antibody fragment; thus the specificity and binding of the antibody are maintained. Such a conservative amino acid substitution may substitute a basic, neutral, hydrophobic, or acidic amino acid for another of the same group. By the term basic amino acid it is meant hydrophilic amino acids having a side chain pK value of greater than 7, which are typically positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). By the term neutral amino acid (also polar amino acid), it is meant hydrophilic amino acids having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gln or Q). The term hydrophobic amino acid (also non-polar amino acid) is meant to include amino acids exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (Ile or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G). Acidic amino acid refers to hydrophilic amino acids having a side chain pK value of less than 7, which are typically negatively charged at physiological pH. Acidic amino acids include glutamate (Glu or E), and aspartate (Asp or D).
[0074] Sequence identity is used to evaluate the similarity of two sequences; it is determined by calculating the percent of residues that are the same when the two sequences are aligned for maximum correspondence between residue positions. Any known method may be used to calculate sequence identity; for example, computer software is available to calculate sequence identity. Without wishing to be limiting, sequence identity can be calculated by software such as NCBI BLAST2 service maintained by the Swiss Institute of Bioinformatics (and as found at ca.expasy.org/tools/blast/), BLAST-P, Blast-N, or FASTA-N, or any other appropriate software that is known in the art.
[0075] The substantially identical sequences of the present invention may be at least 90% identical; in another example, the substantially identical sequences may be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical, or any percentage therebetween, at the amino acid level to sequences described herein. This corresponds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 conservative amino acid mutations. Importantly, the substantially identical sequences retain the activity and specificity of the reference sequence (that is, the ability to transmigrate the BBB). In a non-limiting embodiment, the difference in sequence identity may be due to conservative amino acid mutation(s). In a non-limiting example, the present invention may be directed to an antibody fragment comprising a sequence at least 95% (corresponding to 6 conservative amino acid mutations), 98% (corresponding to 2 conservative amino acid mutations), or 99% (corresponding to 1 conservative amino acid mutation) identical to that of the antibodies described herein. It is additionally understood that calculating a percentage sequence identity involves a comparison with the complete sequence of the antibody fragment.
[0076] The antibody fragment of the present invention may also comprise additional sequences to aid in expression, detection or purification of a recombinant antibody or fragment thereof. Any such sequences or tags known to those of skill in the art may be used. For example, and without wishing to be limiting, the antibody fragment may comprise a targeting or signal sequence (for example, but not limited to ompA), a detection/purification tag (for example, but not limited to c-Myc, His.sub.5, or His.sub.6), or a combination thereof. In another example, the additional sequence may be a biotin recognition site such as that described by Cronan et al in WO 95/04069 or Voges et al in WO/2004/076670. As is also known to those of skill in the art, linker sequences may be used in conjunction with the additional sequences or tags, or may serve as a detection/purification tag.
[0077] The antibody fragment of the present invention may also be in a multivalent display format, also referred to herein as multivalent presentation. Multimerization may be achieved by any suitable method of known in the art. For example, and without wishing to be limiting in any manner, multimerization may be achieved using self-assembly molecules such as those described in WO2003/046560, where pentabodies are produced by expressing a fusion protein comprising an antibody fragment and the pentamerization domain of the B-subunit of an AB.sub.5 toxin family (Merritt & Hol, 1995). A multimer may also be formed using the multimerization domains described by Zhu et al. (2010); this form, referred to herein as a combody form, is a fusion of the antibody fragment with a coiled-coil peptide resulting in a multimeric molecule. Other forms of multivalent display are also encompassed by the present invention. For example, and without wishing to be limiting, the antibody fragment may be presented as a dimer, a trimer, or any other suitable oligomer. This may be achieved by methods known in the art, for example direct linking connection (Nielson et al, 2000), c-jun/Fos interaction (de Kruif & Logtenberg, 1996), Knob into holes interaction (Ridgway et al, 1996).
[0078] Another method known in the art for multimerization is to dimerize the antibody fragment using an Fc domain, for example, but not limited to, human Fc domains. The Fc domains may be selected from various classes including, but not limited to, IgG, IgM, or various subclasses including, but not limited to IgG1, IgG2, IgG3, and IgG4. In this approach, the Fc encoding polynucleotide is inserted into a vector along with the sdAb encoding polynucleotide to generate a sdAb-Fc fusion protein (Bell et al, 2010; Iqbal et al, 2010); the fusion protein is recombinantly expressed then purified. For example, and without wishing to be limiting in any manner, multivalent display formats may encompass chimeric formats of FC5-H7 mutational variants linked to an Fc domain. Such chimeric format molecules are readily engineered and produced, can greatly extend the serum half-life of sdAb, and may be excellent tumor imaging reagents (Bell et al., 2010).
[0079] The Fc domain in the multimeric complex as just described may be any suitable Fc fragment known in the art. The Fc fragment may be from any suitable source; for example, the Fc may be of mouse or human origin. In a specific, non-limiting example, the Fc may be the mouse IgG2b Fc fragment or human IgG1, IgG2 or IgG4 Fc fragment (Bell et al, 2010; Iqbal et al, 2010). In a specific, non-limiting example, the multimerized construct may comprise the isolated or purified antibody fragment as described herein and an Fc comprising the sequence of SEQ ID NO:29.
[0080] Each subunit of the multimers described above may comprise the same or different antibody fragments of the present invention, which may have the same or different specificity. Additionally, the multimerization domains may be linked to the antibody fragment using a linker, as required; such a linker should be of sufficient length and appropriate composition to provide flexible attachment of the two molecules, but should not hamper the antigen-binding properties of the antibody.
[0081] The antibody fragment as described herein transmigrates the blood-brain barrier. The brain is separated from the rest of the body by a specialized endothelial tissue known as the blood-brain barrier (BBB). The endothelial cells of the BBB are connected by tight junctions and efficiently prevent many therapeutic compounds from entering the brain. In addition to low rates of vesicular transport, one specific feature of the BBB is the existence of enzymatic barrier(s) and high level(s) of expression of ATP-dependent transporters on the abluminal (brain) side of the BBB, including P-glycoprotein (Gottesman et al., 1993; Watanabe, 1995), which actively transport various molecules from the brain into the blood stream (Samuels, 1993). Only small (<500 Daltons) and hydrophobic (Pardridge, 1995) molecules can more readily cross the BBB. Thus, the ability of the antibody fragment as described above to specifically bind the surface receptor, internalize into brain endothelial cells, and undergo transcytosis across the BBB by evading lysosomal degradation is useful in the neurological field.
[0082] The present invention also encompasses nucleic acid sequences encoding the molecules as described herein. Given the degeneracy of the genetic code, a number of nucleotide sequences would have the effect of encoding the polypeptide, as would be readily understood by a skilled artisan. The nucleic acid sequence may be codon-optimized for expression in various micro-organisms. The present invention also encompasses vectors comprising the nucleic acids as just described. Furthermore, the invention encompasses cells comprising the nucleic acid and/or vector as described.
[0083] The present invention further encompasses the isolated or purified antibody fragments immobilized onto a surface using various methodologies; for example, and without wishing to be limiting, the antibody fragment may be linked or coupled to the surface via His-tag coupling, biotin binding, covalent binding, adsorption, and the like. Immobilization of the antibody fragment of the present invention may be useful in various applications for capturing, purifying or isolating proteins. The solid surface may be any suitable surface, for example, but not limited to the well surface of a microtiter plate, channels of surface plasmon resonance (SPR) sensorchips, membranes, beads (such as magnetic-based or sepharose-based beads or other chromatography resin), glass, plastic, stainless steel, a film, or any other useful surface such as nanoparticles, nanowires and cantilever surfaces.
[0084] The invention also encompasses one or more antibody fragments as described above linked to a cargo molecule. The cargo molecule may be any suitable molecule, which is delivered across the BBB by the antibody fragment. The cargo molecule may have a molecular weight in the range of about 1 kD to about 200 kDa; for example, the cargo molecule may have a molecular weight of about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 kDa, or any weight therebetween, or any range of weights defined by any two aforementioned weights. In specific, non-limiting examples, the cargo molecule may have a molecular weight of about 80 kDa (for example, but not limited to a Fc fragment, toxin, growth factor, cytokine, enzyme, protein, antibody, single-domain antibody, or antibody fragment, etc), or about 200 kDa (for example, but not limited to a monoclonal antibody).
[0085] For example, and without wishing to be limiting in any manner, the cargo molecule may be a detectable agent, a therapeutic agent, a drug, a peptide, an enzyme, a growth factor, a cytokine, a receptor trap, a cargo antibody or a fragment of a cargo antibody (e.g., IgG, scFv, Fab, F(ab).sub.2, V.sub.HH, etc) a chemical compound, a carbohydrate moiety, DNA-based molecules (anti-sense oligonucleotide, microRNA, siRNA, plasmid), a cytotoxic agent, viral vector (adeno-, lenti-, retro), one or more liposomes loaded with any of the previously recited types of cargo molecules, or one or more nanoparticle, nanowire, nanotube, or quantum dots. The cargo molecule as described above may be a detectable agent. For example, the FC5 antibody variant fragment may be linked to a radioisotope, a paramagnetic label, a fluorophore, a fluorescent agent, Near Infra-Red (NIR; for example Cy5.5) fluorochrome or dye, an echogenic microbubble, an affinity label, a detectable protein-based molecule, nucleotide, quantum dot, nanoparticle, nanowire, or nanotube or any other suitable agent that may be detected by imaging methods. The antibody fragment may be linked to the cargo molecule using any method known in the art (recombinant technology, chemical conjugation, etc.).
[0086] The cargo molecule as described herein may be linked, also referred to herein as conjugated, to the antibody fragment by any suitable method known in the art. For example, and without wishing to be limiting, the cargo molecule may be linked to the antibody fragment by a covalent bond, by a peptide bond, or by ionic interaction. For example, and without wishing to be limiting, the antibody fragment linked to a cargo molecule may be expressed as a fusion protein. The linkage may be achieved through a chemical cross-linking reaction, or through fusion using recombinant DNA methodology combined with any peptide expression system, such as bacteria, yeast or mammalian cell-based systems. When conjugating the cargo molecule to the antibody fragment, a suitable linker may be used. Methods for linking an antibody fragment to a cargo molecule such as a therapeutic or detectable agent would be well-known to a person of skill in the art.
[0087] In one non-limiting example, the cargo molecule may be a detectable label, a radioisotope, a paramagnetic label such as gadolinium or iron oxide, a fluorophore, Near Infra-Red (NIR) fluorochrome or dye, an echogenic microbubble, an affinity label (for example biotin, avidin, etc), enzymes, or any other suitable agent that may be detected by diagnostic imaging methods. In a specific, non-limiting example, the antibody fragment may be linked to a near infrared fluorescence (NIRF) imaging dye, for example and not wishing to be limiting Cy5.5, Alexa680, Dylight680, or Dylight800.
[0088] The in vivo detection step in the methods described above may be whole body imaging for diagnostic purposes or local imaging at specific sites, such as but not limited to brain vessels or brain tumor vessels, in a quantitative manner to assess the progression of disease or host response to a treatment regimen. The detection step in the methods as described above may be immunohistochemistry, or a non-invasive (molecular) diagnostic imaging technology including, but not limited to: [0089] Optical imaging; [0090] Positron emission tomography (PET), wherein the detectable agent is an isotopes such as .sup.11C, .sup.13N, .sup.15O, .sup.18F, .sup.64Cu, .sup.62Cu, 1.sup.24I, .sup.76Br, .sup.82Rb and .sup.68 Ga, with .sup.18F being the most clinically utilized; [0091] Single photon emission computed tomography (SPECT), wherein the detectable agent is a radiotracer such as .sup.99mTc, .sup.111In, .sup.123I, .sup.201Tl, .sup.133Xe, depending on the specific application; [0092] Magnetic resonance imaging (MRI), wherein the detectable agent may be, for example and not limited to gadolinium, iron oxide nanoparticles and carbon-coated iron-cobalt nanoparticles thereby increasing the sensitivity of MRI for the detection of plaques. [0093] Contrast-Enhanced Ultrasonography (CEUS) or ultrasound, wherein the detectable agent is at least one acoustically active and gas-filled microbubble. Ultrasound is a widespread technology for the screening and early detection of human diseases. It is less expensive than MRI or scintigraphy and safer than molecular imaging modalities such as radionuclide imaging because it does not involve radiation.
[0094] The present invention further provides a method of transporting a molecule of interest across the blood-brain barrier. The method comprises administering the molecule linked to an antibody fragment as described herein to a subject. The molecule may be any desired molecule, including the cargo molecules, as previously described; the molecule may be linked to the antibody fragment using any suitable method, including, but not limited to conjugation or expression in a fusion protein. The administration may be by any suitable method, for example parenteral administration, including but not limited to intravenous (iv), subcutaneous (sc), and intramuscular (im) administration. In this method, the antibody fragment of the present invention ferries the molecule of interest across the BBB to its brain target.
[0095] The present invention also encompasses a composition comprising one or more than one isolated or purified antibody fragment as described herein. The composition may comprise a single antibody fragment as described above, or may be a mixture of antibody fragments. Furthermore, in a composition comprising a mixture of antibody fragments of the present invention, the antibodies may have the same specificity, or may differ in their specificities;
[0096] The composition may also comprise a pharmaceutically acceptable diluent, excipient, or carrier. The diluent, excipient, or carrier may be any suitable diluent, excipient, or carrier known in the art, and must be compatible with other ingredients in the composition, with the method of delivery of the composition, and is not deleterious to the recipient of the composition. The composition may be in any suitable form; for example, the composition may be provided in suspension form, powder form (for example, but not necessarily limited to lyophilised or encapsulated), capsule or tablet form. For example, and without wishing to be limiting, when the composition is provided in suspension form, the carrier may comprise water, saline, a suitable buffer, or additives to improve solubility and/or stability; reconstitution to produce the suspension is effected in a buffer at a suitable pH to ensure the viability of the antibody fragment. Dry powders may also include additives to improve stability and/or carriers to increase bulk/volume; for example, and without wishing to be limiting, the dry powder composition may comprise sucrose or trehalose. In a specific, non-limiting example, the composition may be so formulated as to deliver the antibody fragment to the gastrointestinal tract of the subject. Thus, the composition may comprise encapsulation, time-release, or other suitable technologies for delivery of the antibody fragment. It would be within the competency of a person of skill in the art to prepare suitable compositions comprising the present compounds.
[0097] Typically, only a small fraction (<0.01%) of injected antibody or antibody fragment dose crosses the BBB. The FC5 V.sub.HH (WO 2002/057445) was previously identified for its ability to transmigrate the BBB. Various antibody or protein fusion constructs with FC5 have shown 10-15 fold improvement of brain uptake compared to control antibody without an FC5 component. The present inventors have now identified specific mutations within the complementarity-determining regions of FC5 that lead to improved binding of the antibody to brain endothelial cells. Specifically, mutants D56K, N57D, F591, F59L, and T105K demonstrated improved binding in both SV-ARBEC and HBEC. The same mutants showed improvements in the range of 33-448% increase in P.sub.app values compared to FC5-H7 (a humanized FC5), indicating improvement in their rate of transport across the in vitro BBB model. When fused to a Fc, most mutants maintained improved binding to SV-ARBEC compared to FC5-Fc and showed higher P.sub.app values compared to FC5-H7-Fc fusion. The results suggest that specific mutations introduced into CDRs resulted in both the improvement in affinity and in enhanced BBB crossing in vitro. Furthermore, selected mutants tested for delivery of the antibody fragments to the brain showed 1.5 to 3-fold increase in brain levels, demonstrating better brain penetration and higher brain levels compared to the FC5-H7-Fc construct.
[0098] The present invention will be further illustrated in the following examples. However, it is to be understood that these examples are for illustrative purposes only and should not be used to limit the scope of the present invention in any manner.
Example 1: Design of CDR Mutation Library
[0099] Based on the sequence of FC5 (SEQ ID NO:16), single point-mutations within the antibody's CDR were designed to improve performance. The modeled 3D-structure of the CDR conformation of FC5 was used to inform the construction of the sdAb mutants.
[0100] 3D-Structure Modeling of Camelid V.sub.HH.
[0101] Template structures similar to FC5 V.sub.HH were identified using BLAST searches against the Protein Data Bank (PDB). The 3D structure of the FC5 V.sub.HH was approximated using homology modeling based on the 2X1O|A (PDB code|Chain ID) structure as template. The FC5 V.sub.HH structure was then built by mutating the template structure to the FC5 sequence; this included 32 mutations at various positions (26 in the CDR and 6 in the framework region). The FC5 V.sub.HH model was then refined by energy minimization with the AMBER force-field and a stepwise release of constraints, ranging from the CDR loops, which were relaxed first, to the backbone heavy atoms of the framework region, which were fully relaxed only in the last stage. The CDR-H3 loop of the V.sub.HH model was then refined by Monte-Carlo-minimization (MCM) conformational sampling, in which dihedral angles in the CDR-H3 region were sampled followed by energy minimization.
[0102] The selected humanized framework for this work was that of FC5-H7 (SEQ ID NO:17), which demonstrated an optimal balance of low predicted immunogenicity, high production yield, and elevated thermal stability. Humanized FC5-H7 was constructed and produced as described in PCT application PCT/IB2017/054036, the contents of which are incorporated herein by reference.
[0103] The FC5 CDR contain 29 residues: 7 in CDR1 (residues 26-35, SEQ ID NO:16), 10 in CDR2 (residues 50-66, SEQ ID NO:16) and 12 in CDR3 (residues 99-111, SEQ ID NO:16). Mutations were focused on solvent-accessible residues that did not appear to play a structural role in the CDR. The following mutations were selected for introducing into the FC5-H7: D56K, N57D, F591, F59L, and T105K.
Example 2: Library Construction and Plasmid Production
[0104] The single point-mutations identified in Example 1 were introduced into the FC5-H7 sequence.
[0105] To generate the cDNA sequences for the single-point mutants, the respective preferred E. coli codon was used for the point mutant and embedded within the cDNA sequence of the FC5-H7. All variants were expressed in fusion with His5 and c-myc tags to allow for purification by immobilized metal affinity chromatography using HiTrap Chelating column and for detection by immunochemistry, respectively.
[0106] Briefly, DNA encoding sdAb FC5-H7 (SEQ ID NO:17) or point-mutants was cloned into the BbsI/BamHI sites of plasmid pSJF2H to generate expression vector for FC5 (Muruganandam et al, 2002). The DNA constructs were confirmed by nucleotide sequencing on 373A DNA Sequencer Stretch (PE Applied Biosystems) using primers fdTGIII, 5-GTGAAAAAATTATTATTATTCGCAATTCCT-3 (SEQ ID NO:18) and 96GIII, 5-CCCTCATAGTTAGCGTAACG-3 (SEQ ID NO:19).
Example 3: Very Small Scale Expression and Purification of 318 FC5-H7 CDR Mutants
[0107] The plasmids produced in Example 2 were cloned into E. coli cells, expressed, and purified on a small scale to assess the performance of each clone.
[0108] Protein Expression:
[0109] The CDR-mutated variants were synthesized and directly cloned into pSJF2H as described in Example 2. Subsequently, 50 ng of DNA was transformed into 5 l of Zymo Research Mix and Go electro-competent TG1 E. coli. (Cedarlane) for 10 min at 4 C. then 100 l of 2YT media was added (tryptone 16 g, yeast extract 10 g and NaCl 5 g in 1 L). Clones were selected on 6 well 2YT agar plates+100 g/ml ampicillin and grown overnight at 32 C. followed by 3-4 hours of growth at 37 C. Cultures were inoculated into 5 ml of 2YT/glu/amp (2YT media supplemented with 100 g/ml ampicillin and 0.1% glucose) and grown at 37 C., 250 rpm in Kingfisher 24 well deep plates. Protein expression was induced at an OD.sub.600 of 0.4-0.5 with addition of IPTG to a final concentration of 1 mM. The cultures were grown at 250 rpm overnight at 37 C. The bacteria were pelleted by plate centrifugation, 3000 rpm for 15 min. The supernatant was discarded and the pellets were frozen at 80 C. for 20 min. The partially thawed pellets were re-suspended in 1 ml of lysis buffer (1PBS supplemented with 0.1 M Hepes pH 7.5, 10 ml of FastBreak (Fisher), 1EDTA protease inhibitor tablet (Roche), 200 l of DNase (Sigma), in 100 ml) and lysed for 30 min at 250 rpm. The plates were centrifuged at 3000 rpm for 15 min and the supernatant was transferred into new 24 well deep plates for Kingfisher purification. Pure Proteome nickel magnetic beads (Millipore) were centrifuged at 400g for 1 min then washed in 40 ml of buffer A (500 mM NaCl; 10 mM Hepes pH 7.5) centrifuged again then re-suspended in a final volume of 10 ml. A 200 l volume of prepared beads was added to the Kingfisher 24 well deep plates containing the expressed c-myc/His-dual tagged V.sub.HH single domain antibody variants and incubated for 30 min at 270 rpm.
[0110] KingFisher Flex Magnetic Particle Processor Purification:
[0111] A washing buffer plate (500 mM NaCl, 10 mM Hepes pH7.5 and 10 mM imidazole, 2 ml/well), an elution buffer plate (500 mM NaCl, 10 mM Hepes pH 7.5 and 300 mM imidazole, 300 l/well), a collection buffer plate (50 mM EDTA, 0.5 ml/well), a 24 well tip comb (VWR), and the Kingfisher 24 well deep plates containing the c-myc/His-dual tagged V.sub.HH single domain antibody/nickel magnetic beads were inserted into the KingFisher Flex Magnetic Particle Processor. The purification protocol (Hiba_KF optimized) was initiated with the following steps: collection of beads from sample platewash 1 min at medium speedbead collectionelution by mixing 5 min at medium speedbead collectionrelease of beads into EDTA and drive home. EDTA at a 2 mM final concentration was added to each protein elution plate prior to storage at 4 C. In preparation for cell binding analysis and affinity ranking by Mirrorball High Sensitivity Microplate Cytometry the samples were transferred to 96-well Sephadex G-25 desalting plates (GE Healthcare) to remove the imidazole and to buffer exchange the purified V.sub.HH single domain antibodies into 250 l PBS+EDTA 0.5 mM. Protein purity was assessed by Mini-Protein TGX 4-20% stain free SDS-Page gels and band visualization with a Bio-Rad Gel Dock EZ System. Protein concentration was measured by Nanodrop.
Example 4. Initial Selection for Affinity-Improved FC5 Variants
[0112] The selection strategy for the FC5 mutants was based on screening of all variants expressed in small quantity using Mirrorball binding assay (see below). The first screening round is performed in rat brain endothelial cells SV-ARBEC. The clones with best binding affinity to SV-ARBEC were then re-tested for binding in both SV-ARBEC and human brain endothelial cells (HBEC) using the same binding assay.
[0113] Mirrorball High Sensitivity Microplate Cytometry (TTP Labtech):
[0114] All buffers and reagents were pre-chilled to 4 C. Each sdAb mutant was diluted to a starting concentration of 1000 nM in a 1:1 buffer mix of 0.5PBS/2.5 mM EDTA and Mirrorball assay bufferLive Cell Imaging Solution, LCIB (Invitrogen, 140 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl.sub.2, 1.0 mM MgCl.sub.2, 20 mM Hepes, pH 7.4, mOsm=300). A 20 l volume of a 1:1 mix of LCIB and 0.5PBS/2.5 mM EDTA was added to all wells of each 384 well Mirrorball assay plate (Corning 3712); with the exception of row A which received 40 l of 1000 nM test V.sub.HH antibody. Serial dilutions were prepared for each test variant within the Mirrorball 384 well assay plate. A 16-channel Finn pipette (Thermo Scientific) was used to transfer 20 l of V.sub.HH antibody from row A-columns 1-24 into row B-columns 1-24 mixing 8, then transferring 20 l of V.sub.HH antibody from row B-columns 1-24 into row C-columns 1-24 mixing 8. Dilutions were repeated until row G-columns 1-24 to create 7 point curve for each mutant. A second set of test V.sub.HH antibody variants (1000 nM) were added to row I-columns 1-24 and the dilution protocol was repeated until row O-columns 1-24. Row H-columns 1-24 were reserved on each plate for the reference FC5-H7 V.sub.HH single domain antibody. Row P-columns 1-24 received no antibody; this was background control for non-specific binding of the secondary to the cells of interest; thus 48 variants could be tested on each 384 well Mirrorball assay plate. Immortalized adult rat brain microvascular endothelial cells (SV-ARBEC) and/or human microvascular brain endothelial cells (HBEC-D3) were dissociated in Accutase solution (Sigma Aldrich) to generate single cell populations. Cells were washed in LCIB then centrifuged at 200g, 5 min to pellet. Wash buffer was removed and the cell pellet was re-suspended into 1 mL of LCIB. Cell number was calculated using a Bio-Rad TC20 automated cell counter with Trypan Blue dye to assess viability. The cells were diluted to 350,000 live cells/ml in LCIB. A fluorescent conjugate c-myc Alexa 488 detection antibody (1600 ng/ml, Santa Cruz Biotechnology) supplemented with Draq 5 nuclear stain (2 uM, Cell Signaling) was prepared in LCIB assay buffer. The cells and the detection secondary/Drag 5 solution were mixed 1:1 and 20 l of solution containing 3500 cells was added into each well of the Mirrorball 384 well assay plate; which already contained each V.sub.HH antibody variant in a 7 point dilution series resulting in a final concentration of 500, 250, 125, 62.5, 31.25, 15.63 and 7.81 nM. All plates were incubated at 4 C. for 2 h and 20 h. Readings were taken at each time point using Mirrorball High Sensitivity Microplate Cytometry with the following settings: [0115] Laser Settings: 488 and 640 enabled, 6.0 mW [0116] Channel Settings: FL-2 (488-540 nm) voltage 600, sensitivity 4, Tiff files saved and FL-4 (650-690 nm) voltage 600, sensitivity 4, trigger 4, Tiff files saved [0117] Object Characteristics: FL-2 (peak intensity, mean intensity, total intensity, and baseline) and FL-4 (peak intensity, mean intensity, total intensity and baseline) [0118] Population Definition: ObjectsCells Filters (FL-4 perimeter range 0-500 nm and FL-2 mean intensity range 0-15000) [0119] Population Statistics: Objects: number of objects, Objects: mean (FL-2 peak, mean, total intensities and perimeter) and Objects: mean FL-2 baseline. Objects: median (FL-2 peak, mean, and total intensities) Objects: mean (FL-4 peak, mean, total intensities and perimeter) and Objects: mean FL-4 baseline. Objects: median (FL-4 peak, mean, and total intensities) Cells: number of objects, Cells: mean (FL-2 peak, mean, and total intensities) and Cells: mean FL-2 baseline. Cells: median (FL-2 peak, mean, and total intensities) Cells: mean (FL-4 peak, mean, and total intensities) and Cells: mean FL-4 baseline. Cells: median (FL-4 peak, mean, and total intensities)
[0120] The remaining live cell material was incubated at 4 C. adjacent to the assay plate so cell viability could be monitored at both 2 h and 20 h time points. The Mirrorball assay procedure was repeated for all V.sub.HH single domain antibodies until all 318 variants were screened in both SV-ARBEC and HBEC-D3 cell lines of interest. The data was analysed with Cellista software (TTP Labtech) and GraphPad Prism 6 software programs.
[0121] Some clones showed no binding or lower binding to SV-ARBEC compared to FC5. The clones showing improved binding were selected for further studies in both SV-ARBEC and HBEC cells, as described above.
Example 5: Transport of FC5-H7 CDR Variants Across In Vitro BBB Model
[0122] The top candidates of the 48 clones identified in Example 4 demonstrating improved binding in both SV-ARBEC and HBEC cells were screened in in vitro BBB permeability assay, using a single-time point for Papp determination. The quantification of variants was done using by MRM-ILIS.
[0123] SV40-immortalized Adult Rat Brain Endothelial Cells (SV-ARBEC) were used to generate an in vitro blood-brain barrier (BBB) model as described (Garberg et al., 2005; Haqqani et al., 2012). Sv-ARBEC (80,000 cells/membrane) were seeded on a 0.1 mg/mL rat tail collagen type I-coated tissue culture inserts (pore size-1 m; surface area 0.9 cm.sup.2, Falcon) in 1 ml of growth medium. The bottom chamber of the insert assembly contained 2 ml of growth medium supplemented with the immortalized neonatal rat astrocytes-conditioned medium in a 1:1 (v/v) ratio. Equimolar amounts (5.6 M) of positive (FC5) or negative controls (A20.1, a Clostridium difficile toxin A binding V.sub.HH; and EG2, an EGFR binding V.sub.HH), FC5, FC5-H7 and 29 point-mutants from Example 4 were tested for their ability to cross the rat in vitro BBB model. Following exposure of equimolar amounts of the sdAb to the luminal side of the BBB, samples were taken after 15, 30 and 60 min from the abluminal side. The sdAb content of each sample was then quantified by mass spectrometry (multiple reaction monitoring-isotype labeled internal standards; MRM-ILIS).
[0124] MRM-ILIS:
[0125] The methods are all as described in Haqqani et al. (2012). Briefly, to develop the SRM (selected reaction monitoring also known as multiple reaction monitoring (MRM)) assay for V.sub.HH, each V.sub.HH was first analyzed by nanoLC-MS/MS using data-dependent acquisition to identify all ionizible peptides. For each peptide, the 3 to 5 most intense fragment ions were chosen. An initial SRM assay was developed to monitor these fragments at attomole amounts of the digest (about 100-300 amol). Fragments that showed reproducible intensity ratios at low amounts (i.e., had Pearson r20.95 compared to higher amounts) were considered stable and were chosen for the final SRM assay. To further optimize the assay, elution times for each peptide were also included, with care taken to not choose peptides that have close m/z (mass-to-charge ratio) and elution times.
[0126] A typical multiplexed SRM analysis of V.sub.HH in cell media or body fluids (serum or cerebrospinal fluid (CSF)) involved spiking known amount of ILIS (0.1-10 nM) followed by injecting 100-400 ng of CSF or cultured media proteins (0.3-1 L) or about 50-100 ng of serum proteins (1-3 nanoliters) into the nanoLC-MS system. The precursor m/z of each target peptide ion was selected in the ion trap (and the remaining unrelated ions were discarded) at the specified elution time for the target, followed by collision induced dissociation (CID) fragmentation, and selection of only the desired fragment ions in the ion trap for monitoring by the detector. For quantification analysis, raw files generated by the LTQ (ThermoFisher) were converted to the standard mass spectrometry data format mzXML and intensities were extracted using an in-house software called Q-MRM (Quantitative-MRM; see Haqqani et al. 2012), which is a modified version of MatchRx software. For each V.sub.HH, extracted-ion chromatograms were generated for each of its fragment ion that consisted of combined intensities within 0.25 Da of the fragment m/z over the entire elution time. To obtain a final intensity value for each fragment, all intensities within 0.5 min of the expected retention times were summed. A V.sub.HH was defined as detectable in a sample if the fragments of at least one of its peptides showed the expected intensity ratios, i.e., the final intensity values showed a strong Pearson correlation r0.95 and p<0.05 compared with the final intensities values of its corresponding pure V.sub.HH.
[0127] Samples containing mixtures of V.sub.HH (media, serum, CSF) were reduced, alkylated and trypsin-digested as previously described (Haqqani et al., 2012; Gergov et al., 2003). The digests (tryptic peptides) were acidified with acetic acid (5% final concentration) and analyzed on a reversed-phase nanoAcquity UPLC (Waters, Milford, Mass.) coupled to LTQ XL ETD or LTQ Orbitrap ETD mass spectrometer (ThermoFisher, Waltham, Mass.). The desired aliquot of the sample was injected and loaded onto a 300 m I.D.0.5 mm 3 m PepMaps C18 trap (ThermoFisher) then eluted onto a 100 m I.D.10 cm 1.7 m BEH130C18 nanoLC column (Waters) using a gradient from 0%-20% acetonitrile (in 0.1% formic) in 1 minute, 20%-46% in 16 min, and 46%-95% in 1 min at a flow rate of 400 nL/min. The eluted peptides were ionized into the mass spectrometer by electrospray ionization (ESI) for MS/MS and SRM analysis using CID for fragmentation of the peptide ions. The CID was performed with helium as collision gas at normalized collision energy of 35% and 30 ms of activation time. Ion injection times into linear ion trap were adjusted by the instrument using an automatic gain control (AGC) target value of 610.sup.3 and a maximum accumulation time of 200 ms.
[0128] The specific peptides used for detection and quantification of FC5-H7 CDR mutational variants are shown in Table 1.
TABLE-US-00003 TABLE1 PeptidesusedinnanoLC-SRMdetectionofselected FC5-H7CDRmutationvariantsInvariousstudies described,assaysweremultiplexedindifferent combinationsforsimultaneousmonitoringinthe samesample.Limitsofdetectionand quantificationoftheSRMassayforeachpeptide rangedfrom1.5-2.5ng/ml.1ng/mLcorrespondsto about60-70pMofV.sub.HH.A20-1asdescribedin Hussacketal,2011b). SEQ ID Protein Signatures NO: Unique FC5 ITWGGDNTFYSNSVK 20 Yes FC5-ILIS ITWGGDNTFYSNSVK.sup.(a) 20 Yes A20.1 TTYYADSVK 21 Yes EFVAAGSSTGR 22 Yes TFSMDPMAWFR 23 Yes DEYAYWGQGTQVTVSSGQAGQGSEQK 24 Yes FC5-H7 LSCAASGFK 25 Yes variants NTLYLQMNSLR 26 Yes EVQLVESGGGLVQPGGSLR 27 Yes .sup.(a)Heavy-labeled peptide.
[0129] Determination of the Apparent Permeability Coefficient:
[0130] Quantified values can be directly plotted or the P.sub.app (apparent permeability coefficient) values can be determined with the given formula [Qr/dt=cumulative amount in the receiver compartment versus time; A=area of the cell monolayer; C0=initial concentration of the dosing solution] and plotted. The P.sub.app value is commonly used to determine the ability of a molecule to cross the BBB. P.sub.app values are a measure of the specific permeability of the compound across brain endothelial monolayer.
[0131] The screening results for the variants with similar or higher P.sub.app values compared to FC5-H7 are shown in
Example 6: Expression and Purification of FC5 Variant-Fc Constructs
[0132] Constructs comprising FC5 variants FC5, FC5-H7, D56K, N57D, F591, F59L, and T105K V.sub.HH (
[0133] The FC5 variant cDNA was cloned into mammalian expression vector pTT5 (Durocher 2002) containing the human Fc fragment. Polyplexes of the resulting vector were pre-formed by mixing 25 ml of plasmid DNA solution containing 187.5 g pTT5-IR5mFc2b, 56.25 g pTT-AKTdd (activated mutant of Protein Kinase B), 18.75 g pTTo-GFP (to monitor transfection efficiency), and 112.5 g of salmon testis DNA (Sigma-Aldrich); and 25 ml of PEI solution containing 1.125 mg of PEIpro (PolyPlus Transfection), both made in F17 medium. The mixture was incubated for 10 minutes prior to addition to the cell culture. A 450 ml culture of CHO cells stably expressing a truncated EBNA1 protein (CHO-3E7) and grown in F17 medium (Invitrogen) was transfected with 50 ml of polyplexes. Twenty four hours post-transfection, the culture was fed with 12.5 ml of 40% (w/v) tryptone N1 (Organotechnie) solution and 1.25 ml of 200 mM valproic acid solution. The culture was harvested 8 days post-transfection and clarified by centrifugation. Clarified medium was filtered through a 0.22 m membrane prior to its application on a column packed with 5 ml of protein-A MabSelect SuRe resin (GE Healthcare). After loading, the column was washed with 5 volumes of phosphate-buffered saline pH 7.1 (PBS) and the antibody was eluted with 100 mM sodium citrate buffer pH 3.0. Fractions containing the eluted antibody were pooled and a buffer exchange was performed by loading on a desalting Econo-Pac column (BioRad) equilibrated in PBS. Desalted antibody was then sterile-filtered by passing through a Millex GP (Millipore) filter unit (0.22 m) and aliquoted.
Example 7: Binding of Fc-Fused FC5-H7 CDR Variants to Brain Endothelial Cells
[0134] The binding of Fc-fused FC5 CDR variants (FC5, FC5-H7, D56K, N57D, F591, F59L, and T105K; Example 6) to rat (SV-ARBEC), human (HBEC-D3) and non-human primate (CynoBEC) brain endothelial cells was evaluated using Mirrorball High Sensitivity Microplate Cytometry (TTP Labtech) as described in Example 4.
[0135] The results are shown in
Example 8: Transport of Fc-Fused FC5-H7 CDR Variant Across In Vitro BBB Model
[0136] To evaluate whether Fc-fused FC5-H7 CDR mutational variants (FC5, FC5-H7, D56K, N57D, F591, F59L, and T105K) from Example 6 transmigrate the blood-brain barrier, the in vitro assay and quantification method as described in Example 5 was used.
[0137] As shown in
Example 9: Pharmacokinetics of FC5 CDR Variants-Fc Fusions in CSF and Plasma
[0138] An in vivo assay was carried out to determine whether Fc fusions with FC5-H7 mutated variants F591 and T105K are able to cross into the brain, and specifically into the cerebrospinal fluid (CSF), as well as to quantify its presence in CSF and serum.
[0139] The technique used for multiple sampling of cisterna magna CSF was developed at NRC by modification of previously described methods (Huang et al., 1995; Kornhuber et al., 1986). All animals were purchased from Charles River Laboratories International, Inc. (Wilmington, Mass., USA). Animals were housed in groups of three in a 12 h light/dark cycle at a temperature of 24 C., a relative humidity of 505%, and were allowed free access to food and water. All animal procedures were approved by the NRC's Animal Care Committee and were in compliance with the Canadian Council of Animal Care guidelines. Male Wistar rats aged 8-10 weeks (weight range, 230-250 g) were used in all studies.
[0140] In all experiments, test antibodies (FC5-H7 mutant Fc-fusions) were administered i.v. into tail vein in equimolar doses (7 mg/kg). CSF sample collections were made from cisterna magna by needle puncture up to five times over 96 hours. For sample collection rats were briefly and lightly anesthetized with 3% isoflurane, placed in a stereotaxic frame with the head rotated downward at a 45 angle. A 2-cm midline incision between the ears beginning at the occipital crest was made and muscles separated to expose dura mater covering cisternae magna. A 27G butterfly needle (QuickMedical, Cat # SV27EL) with tubing attached to 1 ml syringe was used to puncture dura and aspirate the 20 l of CSF. The CSF was then transferred into the sample glass vial (Waters, Cat #186000384c) and placed in 80 C. freezer until further analysis.
[0141] Blood samples were collected from the tail vein in a commercially available tube (BD microtainer, Cat #365956). After clotting at room temperature for 15-30 minutes, the clot was removed by centrifuging at 1100 rcf (3422 rpm) for 10 min; serum was then transferred into a clean glass vial (Waters, Cat #186000384c), frozen on dry ice and stored at 80 C. until further analysis. At the end of collection, rats were sacrificed by cardiac puncture. Blood and CSF PK analyses were performed using WinLin 6.0 program.
[0142] Serum and CSF samples were analyzed by mass spectrometry and nanoLC-SRM based quantification as described in Example 5 using peptide signatures shown in Table 1.
[0143] CSF collection is a delicate procedure during which CSF can be easily contaminated with blood. Since the amounts of V.sub.HH s were expected to be much smaller in the CSF (<0.1%) than blood, even a slight contamination with blood could seriously compromise the value of an individual CSF sample. It was therefore necessary to develop stringent exclusion criteria for blood-contaminated CSF samples. To evaluate blood-CSF albumin ratio, a nanoLC-SRM method was developed for quantifying albumin levels in plasma and CSF. An albumin peptide APQVSTPTLVEAAR (SEQ ID NO:28) was selected based on its unique retention time and m/z value (Mol Pharm) in order to have minimum interference with other peptide peaks in the multiplex assay. The intensity of the peptide was quantified in both CSF and plasma samples using SRM as described above. The albumin ratio was calculated as follows for each rat:
Albumin Ratio=Intensity per nL of plasma analyzed/Intensity per nL of CSF analyzed
A ratio of 1500 and below was considered as blood contaminated.
[0144] Results are shown in
TABLE-US-00004 TABLE 2 Serum and CSF exposure (area under Curve-AUC) of systemically injected FC5-H7 CDR mutant variant-Fc fusions and A20.1Fc (from FIG. 9). Variant H7 T105K D56K N57D F59L A20.1 Serum 1127000 1180000 410855 975072 761068 1740399 CSF 5246 9122 3395 7438 2969 941 CSF/Serum 0.465 0.773 0.826 0.762 0.390 0.022 AUC ratio
Example 10. Brain Levels of Fc-Fused FC5-H7 CDR Mutants after Systemic Administration
[0145] In separate experiments, FC5-H7-Fc, F591-Fc, T105K-Fc, D56K-Fc, N57D-Fc, F59L-Fc and A20.1-Fc were administered by i.v. injection via tail vein, each at 7 mg/kg, and circulated for 24 h. Rats were then thoroughly perfused with 10 ml of heparinized (100U/ml) saline at a rate of 1 ml/min via the left common carotid artery to facilitate specific perfusion of the brain. Brains were then removed and homogenized in ice-cold homogenization buffer containing 50 mM Tris-HCl pH 8, 150 mM NaCl and protease inhibitor cocktail (Sigma-Aldrich, Oakville, ON) using Dounce homogenizer (10-12 stroke at 4 C.). Samples were then sonicated three times for 10 s each at 4 C. and insoluble material was removed (10,000g for 10 min at 4 C.). The supernatant was analyzed for protein content, and about 0.5 g of protein was used for SRM analysis using methods described in Example 5 and peptide signatures shown in Table 1.
[0146] The results are shown in
[0147] The embodiments and examples described herein are illustrative and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments, including alternatives, modifications and equivalents, are intended by the inventors to be encompassed by the claims. Furthermore, the discussed combination of features might not be necessary for the inventive solution.
TABLE-US-00005 SEQUENCES SEQ ID NO: Sequence Description 1 GFKITHYTMG CDR1FC5 2 RITWGGX.sub.1X.sub.2TX.sub.3YSNSVKG CDR2 consensus 3 GSTSTAX.sub.4PLRVDY CDR3 consensus 4 RITWGGDNTFYSNSVKG CDR2FC5 5 RITWGGKNTFYSNSVKG CDR2D56K 6 RITWGGDDTFYSNSVKG CDR2N57D 7 RITWGGDNTIYSNSVKG CDR2F59I 8 RITWGGDNTLYSNSVKG CDR2F59L 9 GSTSTAKPLRVDY CDR3T105K 10 GSTSTATPLRVDY CDR3FC5 11 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPG FC5-H7-T105K KEREFVSRITWGGDNTFYSNSVKGRFTISRDNSKNTLYLQMNS LRAEDTAVYYCAAGSTSTAKPLRVDYWGQGTLVTVSS 12 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPG FC5-H7-D56K KEREFVSRITWGGKNTFYSNSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCAAGSTSTATPLRVDYWGQGTLVTVSS 13 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPG FC5-H7-N57D KEREFVSRITWGGDDTFYSNSVKGRFTISRDNSKNTLYLQMNS LRAEDTAVYYCAAGSTSTATPLRVDYWGQGTLVTVSS 14 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPG FC5-H7-F59I KEREFVSRITWGGDNTIYSNSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCAAGSTSTATPLRVDYWGQGTLVTVSS 15 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPG FC5-H7-F59L KEREFVSRITWGGDNTLYSNSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCAAGSTSTATPLRVDYWGQGTLVTVSS 16 DVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMGWFRQAPG FCS KEREFVSRITWGGDNTFYSNSVKGRFTISRDNAKNTVYLQMNS LKPEDTADYYCAAGSTSTATPLRVDYWGKGTQVTVSS 17 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPG FC5-H7 KEREFVSRITWGGDNTFYSNSVKGRFTISRDNSKNTLYLQMNS LRAEDTAVYYCAAGSTSTATPLRVDYWGQGTLVTVSS 18 GTGAAAAAATTATTATTATTCGCAATTCCT fdTGIIIprimer 19 CCCTCATAGTTAGCGTAACG 96GIIIprimer 20 ITWGGDNTFYSNSVK FCSpeptide 21 TTYYADSVK A20.1peptide 22 EFVAAGSSTGR A20.1peptide 23 TFSMDPMAWFR A20.1peptide 24 DEYAYWGQGTQVTVSSGQAGQGSEQK A20.1peptide 25 LSCAASGFK FC5-H7 variantpeptide 26 NTLYLQMNSLR FC5-H7 variantpeptide 27 EVQLVESGGGLVQPGGSLR FC5-H7 variantpeptide 28 APQVSTPTLVEAAR Albumin peptide 29 AEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE IgG1Fc VTCVVVDVSHEGPEVKFNWVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EGLHNHYTQKSLSLSPG 30 GAGGTCCAGCTGGTGGAATCTGGAGGAGGATTGGTGCAGCC GGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCA FCS-H7-T105K AAATCACTCACTATACCATGGGCTGGTTCCGCCAGGCTCCAG GGAAGGAGCGTGAATTTGTATCACGTATTACTTGGGGTGGTG ATAACACCTTCTATTCAAACTCCGTGAAGGGCCGATTCACCA TTTCCAGAGACAACAGCAAGAACACGCTGTATCTGCAAATGA ACAGCCTGCGTGCGGAGGACACGGCCGTGTATTACTGTGCA GCAGGTTCGACGTCGACTGCGAAACCACTTAGGGTGGACTA CTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA 31 GAGGTCCAGCTGGTGGAATCTGGAGGAGGATTGGTGCAGCC FCS-H7-D56K GGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCA AAATCACTCACTATACCATGGGCTGGTTCCGCCAGGCTCCAG GGAAGGAGCGTGAATTTGTATCACGTATTACTTGGGGTGGTA AAAACACCTTCTATTCAAACTCCGTGAAGGGCCGATTCACCA TTTCCAGAGACAACAGCAAGAACACGCTGTATCTGCAAATGA ACAGCCTGCGTGCGGAGGACACGGCCGTGTATTACTGTGCA GCAGGTTCGACGTCGACTGCGACGCCACTTAGGGTGGACTA CTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA 32 GAGGTCCAGCTGGTGGAATCTGGAGGAGGATTGGTGCAGCC FCS-H7-N57D GGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCA AAATCACTCACTATACCATGGGCTGGTTCCGCCAGGCTCCAG GGAAGGAGCGTGAATTTGTATCACGTATTACTTGGGGTGGTG ATGATACCTTCTATTCAAACTCCGTGAAGGGCCGATTCACCA TTTCCAGAGACAACAGCAAGAACACGCTGTATCTGCAAATGA ACAGCCTGCGTGCGGAGGACACGGCCGTGTATTACTGTGCA GCAGGTTCGACGTCGACTGCGACGCCACTTAGGGTGGACTA CTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA 33 GAGGTCCAGCTGGTGGAATCTGGAGGAGGATTGGTGCAGCC FCS-H7-F59I GGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCA AAATCACTCACTATACCATGGGCTGGTTCCGCCAGGCTCCAG GGAAGGAGCGTGAATTTGTATCACGTATTACTTGGGGTGGTG ATAACACCATCTATTCAAACTCCGTGAAGGGCCGATTCACCA TTTCCAGAGACAACAGCAAGAACACGCTGTATCTGCAAATGA ACAGCCTGCGTGCGGAGGACACGGCCGTGTATTACTGTGCA GCAGGTTCGACGTCGACTGCGACGCCACTTAGGGTGGACTA CTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA 34 GAGGTCCAGCTGGTGGAATCTGGAGGAGGATTGGTGCAGCC FCS-H7-F59L GGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCA AAATCACTCACTATACCATGGGCTGGTTCCGCCAGGCTCCAG GGAAGGAGCGTGAATTTGTATCACGTATTACTTGGGGTGGTG ATAACACCCTGTATTCAAACTCCGTGAAGGGCCGATTCACCA TTTCCAGAGACAACAGCAAGAACACGCTGTATCTGCAAATGA ACAGCCTGCGTGCGGAGGACACGGCCGTGTATTACTGTGCA GCAGGTTCGACGTCGACTGCGACGCCACTTAGGGTGGACTA CTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA 35 GAGGTCCAGCTGGTGGAATCTGGAGGAGGATTGGTGCAGCC FC5-H7 GGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCA AAATCACTCACTATACCATGGGCTGGTTCCGCCAGGCTCCAG GGAAGGAGCGTGAATTTGTATCACGTATTACTTGGGGTGGTG ATAACACCTTCTATTCAAACTCCGTGAAGGGCCGATTCACCA TTTCCAGAGACAACAGCAAGAACACGCTGTATCTGCAAATGA ACAGCCTGCGTGCGGAGGACACGGCCGTGTATTACTGTGCA GCAGGTTCGACGTCGACTGCGACGCCACTTAGGGTGGACTA CTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA
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