USOO961 1323B2
(12) United States Patent (10) Patent No.: US 9,611,323 B2 Dennis et al. (45) Date of Patent: Apr. 4, 2017
(54) LOW AFFINITY BLOOD BRAIN BARRIER 4,447,547 A 5/1984 Allen et al.
RECEPTOR ANTIBODIES AND USES 13:was A 98. Eastrowbridge f al. ettal al. THEREFOR 4,801,575 A 1/1989 Pardridge et al. 4.956,453 A 9, 1990 B tal. (71) Applicant: Genentech, Inc., South San Francisco, 5,141,736 A 8, 1992 E. A. CA (US) 5,141,743 A 8/1992 Schryvers 5,154,924 A 10, 1992 Friden (72) Inventors: Mark Dennis, South San Francisco, CA 3:63 A SE Ele tal (US); Ryan Jefferson Watts, South San 527,713 A 6, 1993 is a Francisco, CA (US); Yunhua Joy Yu, 5,231,000 A 7/1993 Majocha et al. South San Francisco, CA (US); Yin 5,254,342 A 10/1993 Shen et al. Zhang, South San Francisco, CA (US) 5,292,869 A 3/1994 Schryvers 5,352,447 A 10, 1994 Johnson et al. (73) Assignee: Genentech, Inc., South San Francisco, 38. A 3. Yin al CA (US) 5,527,527 A 6/1996 Friden 5,601,819 A 2/1997 Wong et al. (*) Notice: Subject to any disclaimer, the term of this 5,648.469 A 7/1997 Trowbridge et al. patent is extended or adjusted under 35 5. A s 3. N.R. et al 1 U.S.C. 154(b) by 0 days. 5,672,683.ww A 9/1997 Fridenrowbridge et al. et al. 5,681,722 A 10, 1997 Newman et al. (21) Appl. No.: 14/577,006 5,693,780 A 12/1997 Newman et al. 5,708, 149 A 1/1998 Loosmore et al. (22) Filed: Dec. 19, 2014 5,728,383 A 3, 1998 Johnson et al. 5,750,105 A 5/1998 Newman et al. (65) Prior Publication Data 5,792.458 A 8, 1998 Johnson et al. 5,798,097 A 8, 1998 McKenzie et al. US 2015/0329636A1 Nov. 19, 2015 5,817,789 A 10/1998 Heartlein et al. 5,833,988 A 11/1998 Friden O O 5,912,336 A 6/1999 Sparling et al. Related U.S. Application Data 5,922,323 A 7/1999 Loosmore et al. 5,922.562 A 7/1999 Loosmore et al. (63) Continuation of application No. 13/306,524, filed on 5,922,841 A 7/1999 Loosmore et al. Nov. 29, 2011, now abandoned. 5,977.307 A 11/1999 Friden et al. 6,008,326 A 12, 1999 L tal. (60) Provisional application No. 61/418.223, filed on Nov. 6,015,555 A 1, 2000 Elore a 30, 2010. 6,015,688 A 1/2000 Loosmore et al. 6,027,921 A 2/2000 Heartlein et al. (51) Int. Cl. 6,028,049 A 2/2000 Jacobs et al. 6,060,058 A 5/2000 Schryvers A 6LX 39/395 (2006.01) 6,077,834 A 6/2000 Cheng A6 IK 47/48 (2006.01) 6,090,576 A 7/2000 Myers et al. C07K 6/28 (2006.01) 6,099,842 A 8, 2000 Pastan et al. C07K I6/46 (2006.01) 6,190,668 B1 2/2001 Yang et al. C07K 6/8 (2006.01) (Continued) C07K 6/40 (2006.01) A61 K 39/00 (2006.01) FOREIGN PATENT DOCUMENTS (52) U.S. Cl. EP O79696 B1 3, 1988 CPC ...... C07K 16/28 (2013.01); C07K 16/18 EP O592564 B1 9, 1996 (2013.01); C07K 16/2881 (2013.01); C07K I6/40 (2013.01); C07K 16/468 (2013.01); (Continued) A61 K47/483.69 (2013.01); A6 IK 2039/505 (2013.01); C07K 23.17/31 (2013.01); C07K OTHER PUBLICATIONS 2317/76 (2013.01); C07K 2317/92 (2013.01) Adams GP et al. High affinity restricts the localization and tumor (58) Field of Classification Search penetration of single-chain Fv antibody molecules. Cancer Res. CPC. C07K 16/2881; C07K 16/28: C07K 16/468; C07K 2317/31; C07K 2317/92; C07K 2001, 61:4750-4755.* 2317/77; A61K 47/48369; A61 K (Continued) 47/48376; A61K 47/48384; A61 K 47/48507; A61K 47/48561; A61 K Primary Examiner — Kimberly A. Ballard 47/48676 (74) Attorney, Agent, or Firm — McNeill Baur PLLC See application file for complete search history. (57) ABSTRACT (56) References Cited The present invention relates to antibodies that bind blood U.S. PATENT DOCUMENTS brain barrier receptors (BBB-R) and methods of using the SaC. 4,332,785 A 6, 1982 Allen et al. 4,434,156 A 2/1984 Trowbridge 17 Claims, 20 Drawing Sheets US 9,611,323 B2 Page 2
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... c. 31 Anti-iR^ -o-fi Anti-FRA + Coid (4mg/kg) rer 125 Control igG % injected Dosef Grant rai
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hiof's Post-injection 1 - 34 "fs
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Anti-frk i Anti-coilagen W
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2Orr-r------... o. Anti-FRA 100-E------Anti-FRP a k------v- - Anti-FRC --Anti-ifR 8O - - -Anti-TARE % of inhibition so
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% injected Dosef Gran sain
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herapeutic osing
Average % of 8.4 Gri Estain
FIG. 2C " Col.all xFFRAXTFR8xifrcxFFRExifri. 4|A|L Scenario 1: High Affinity in Ab (Ex. Anti-fr) Trace Josig - vote iptake herapestic Dosing a less iptake
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Art w Anti-frtNet N Staining
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o Anti-fr’s --Anti-FR^18ACE
. t ( ; OC O.C. (OOC F.G. 3B Antibody Concentration (nM)
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. O.O. O. t F.G. 3C Antibody Concentration (M) U.S. Patent Apr. 4, 2017 Sheet 7 of 20 US 9,611,323 B2
--125Anti-FRA -(-128; Anti-fraRACE1 5 "o 28; Anti-BACE1 % injected Dosef Gran 3rai O
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3 30 SO F.G. F Hours Post-injection U.S. Patent Apr. 4, 2017 Sheet 8 of 20 US 9,611,323 B2
U.S. Patent Apr. 4, 2017 Sheet 9 of 20 US 9,611,323 B2
g 3. s FIG. 4A g
control go Anti- Antitra EACE BAE
g g FIG. 4B .
control g3 Anti- Anti-tra, BAE SACE:
80- 25 gikg s s FIG. 4C c & i. Controi igg Anti- Anti-frif BACE BACE U.S. Patent Apr. 4, 2017 Sheet 10 of 20 US 9,611,323 B2
8. s
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Anti- Anti-TéR^ Anti- Anti-FRA to BACE1 BACE BACE1 BACE:
U.S. Patent Apr. 4, 2017 Sheet 16 of 20 US 9,611,323 B2
000- Seri i.eves
-- re-si-. va * - - - t -
Corcefiratic (ig/iii)
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CO ... o... Anti-ER^18ACE?. --Anti-frieACE - - -Anti-FREiBACE:
% of fibition 40
10-2 (-1 2 13 g4 g5 6 Competitor Concentration (iv) G. 9A
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i' is or Anti-TFRA1BACE 'o -- Anti-TFRE/SACE - 8 - Anti-Tirf/ESACE; O. O 2 4. 6 8 2 Days Post-injection F.G. 93 U.S. Patent Apr. 4, 2017 Sheet 18 of 20 US 9,611,323 B2
1GO Brai Antibody leweis
Antibody in Brain (nM)
was a ma-8----. Anti-BACE ov, Anti-TFRIBACE: --- Ati-RDiBACE r & Atti-TREiBAC:
2 4. 8 8 12 Days Post-injection G. 9C
Pasa Aisetas eyes 87.5
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O 2 4. 6 8 2 Days Post-injection F.G. 9D U.S. Patent Apr. 4, 2017 Sheet 19 of 20 US 9,611,323 B2
3rai Aiea leves 375
3OO}} Brain A49 (pgfg} 253
was a ma-8----. Anti-BACE ov, Anti-TFRIBACE: SCO --- Anti-RDiBACE r & Anti-FREBAC:
2 4. 8 8 12 Days Post-injection
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2O CO
Anti-g Anti-Abeta Anti-ifr; Anti-fRE; Abeta Aheta F.G. 10A U.S. Patent Apr. 4, 2017 Sheet 20 of 20 US 9,611,323 B2
SA ice Bfair PK gig
Brai PK 2 (agg) 8
F.G. O.3 Anti-g Anti-Abeta Anti-FRB, Anti-FRE; Abeta Axeia
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F.G. 18 Anti-g Anti-Abeta Anti-TfR; Anti-fr; Anti-fri s Abeta Abeta Abeta US 9,611,323 B2 1. 2 LOWAFFINITY BLOOD BRAIN BARRIER through brain endothelial cells to reach the parenchyma. The RECEPTOR ANTIBODIES AND USES magnitude of antibody uptake into and distribution in the THEREFOR CNS was inversely related to its binding affinity to TfR for the anti-TfR variants studied. Proof of BBB transport was RELATED APPLICATIONS achieved using a bispecific antibody that binds both TfR and the amyloid precursor protein (APP) cleavage enzyme, This application is a continuation of U.S. application Ser. B-secretase (BACE1). A single systemic dose of the bispe No. 13/306,524 having a filing date of Nov. 29, 2011; which cific anti-TfR/BACE1 antibody engineered using the meth claims priority under 35 U.S.C. S 119(e) to U.S. provisional odology of the invention not only resulted in significant 10 antibody uptake in brain, but also dramatically reduced application No. 61/418.223, filed Nov. 30, 2010, the con levels of brain Aflao compared to monospecific anti tents of which are incorporated in their entirety herein by BACE1 alone, suggesting that BBB penetrance affects the reference. potency of anti-BACE1. Similarly, a bispecific antibody that REFERENCE TO SEQUENCE LISTING binds both TfR and amyloid beta (i.e., a portion of APP that SUBMITTED AS A TEXT FILE VIA EFS-WEB 15 results from BACE1 cleavage of APP, which is one of the main constituents of amyloid plaques) was shown to be A sequence listing is Submitted concurrently with the readily taken up into the brain using the methodology of the specification as an ASCII formatted text file via EFS-Web, invention. The data and experiments described herein high with a file name of “P4499R1C1US.txt, a creation date of light several causative mechanisms behind increasing Dec. 18, 2014, and a size of 19,714 bytes. The sequence uptake of an antibody into the CNS using a lower-affinity listing filed via EFS-Web is part of the specification and is antibody approach. First, high affinity anti-BBB receptor hereby incorporated by reference in its entirety. (BBB-R) antibodies (e.g., anti-TfR) limit brain uptake by quickly saturating the BBB-R in the brain vasculature, thus FIELD OF THE INVENTION reducing the total amount of antibody taken up into the brain 25 and also restricting its distribution to the vasculature. Strik The present invention relates to antibodies that bind blood ingly, lowering affinity for the BBB-R improves brain brain barrier receptors (BBB-R) and methods of using the uptake and distribution, with a robust shift observed in SaC. localization from the vasculature to neurons and associated neuropil distributed within the CNS. Second, the lower BACKGROUND 30 affinity of the antibody for the BBB-R is proposed to impair the ability of the antibody to return to the vascular side of the Brain penetration of large molecule drugs is severely BBB via the BBB-R from the CNS side of the membrane limited by the largely impermeable blood-brain barrier because the overall affinity of the antibody for the BBB-R is (BBB). Among the many strategies to overcome this low and the local concentration of the antibody on the CNS obstacle is to utilize transcytosis trafficking pathways of 35 side of the BBB is non-saturating due to the rapid dispersal endogenous receptors expressed at the brain capillary of the antibody into the CNS compartment. Third, in vivo, endothelium. Recombinant proteins such as monoclonal and as observed for the TfR system, antibodies with less antibodies have been designed against these receptors to affinity for the BBB-R are not cleared from the system as enable receptor-mediated delivery of large molecules to the efficiently as those with greater affinity for the BBB-R, and brain. However, strategies to maximize brain uptake while 40 thus remain at higher circulating concentrations than their minimizing reverse transcytosis back to the blood, and the higher-affinity counterparts. This is advantageous because extent of accumulation after therapeutic dosing, remain the circulating antibody levels of the lower-affinity antibody unexplored. Furthermore, whether antibodies that cross the are Sustained at therapeutic levels for a longer period of time BBB are pharmacodynamically functional is unknown. than the higher-affinity antibody, which consequently 45 improves uptake of antibody in brain for a longer period of SUMMARY time. Furthermore, this improvement in both plasma and brain exposure may reduce the frequency of dosing in the Monoclonal antibodies have vast therapeutic potential for clinic, which would have potential benefit not only for treatment of neurological or central nervous system (CNS) patient compliance and convenience but also in lessening diseases, but their passage into the brain is restricted by the 50 any potential side effects or off-target effects of the antibody blood-brain barrier (BBB). Past studies have shown that a and/or of a therapeutic compound coupled thereto. Anti very Small percentage (approximately 0.1%) of an IgG TfR/BACE1 and anti-TfR/Abeta are each promising and circulating in the bloodstream crosses through the BBB into novel therapeutic candidates for the treatment of Alzheim the CNS (Felgenhauer, Klin. Wschr. 52: 1158-1164 (1974)), er's disease. Furthermore, receptor mediated transport where the CNS concentration of the antibody may be 55 (RMT)-based bispecific targeting technology opens the door insufficient to permit a robust effect. The methods and for a wide range of potential therapeutics for CNS diseases. compositions of the invention provide a way to improve the The invention provides methods of engineering BBB-pen percentage of the antibody that distributes into the CNS and etrant therapeutics that greatly improve transport across the thus more readily attain therapeutic antibody concentrations BBB and CNS distribution of the therapeutic. in the CNS. 60 Accordingly, in a first embodiment, the invention pro Herein is described a group of antibodies against the vides a method of transporting a compound across the transferrin receptor (TfR) that can deliver therapeutics blood-brain barrier comprising exposing an antibody which including antibodies and small molecules across the BBB at binds with low affinity to a blood-brain barrier receptor both trace and therapeutically relevant doses after a single (BBB-R) coupled to a compound to the blood-brain barrier systemic injection in mice. Distribution of antibody changed 65 Such that the antibody transports the compound coupled from vascular to neuronal 24 hours after injection, indicating thereto across the blood-brain barrier. In one aspect, the that a significant amount of antibody had transcytosed compound is a neurological disorder drug. In another aspect, US 9,611,323 B2 3 4 the compound is an imaging agent. In another aspect, the the antibody by a linker. In one such aspect, the linker is compound is labeled. In another aspect, the antibody is cleavable. In another such aspect, the linker is not cleavable. labeled. In another aspect, the antibody does not impair the In another Such aspect, the compound is directly linked to binding of the BBB-R to one or more of its native ligands. the antibody. In one such aspect, the antibody is a multi In another such aspect, the antibody specifically binds to specific antibody and the compound forms one portion of the TfR in such a manner that it does not inhibit binding of the multispecific antibody. In another Such aspect, the multispe TfR to transferrin. In another aspect, the BBB is in a cific antibody comprises a first antigen binding site which mammal. In another Such aspect, the mammal is a human. In binds the BBB-R and a second antigen binding site which another Such aspect, the mammal has a neurological disor binds a brain antigen. In another Such aspect, the brain der. In another such aspect, the neurological disorder is 10 antigen is selected from the group consisting of beta selected from the group consisting of Alzheimer's disease secretase 1 (BACE1), Abeta, epidermal growth factor recep (AD), stroke, dementia, muscular dystrophy (MD), multiple tor (EGFR), human epidermal growth factor receptor 2 sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic (HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, fibrosis, Angelman’s syndrome, Liddle syndrome, Parkin CD20, huntingtin, prion protein (PrP), leucine rich repeat son's disease, Pick's disease, Paget’s disease, cancer, and 15 kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma traumatic brain injury. In another aspect, the BBB is in a secretase, death receptor 6 (DR6), amyloid precursor protein human. (APP), p75 neurotrophin receptor (p75NTR), and caspase 6. In another aspect, the antibody has an IC50 for the BBB-R In another such aspect, the multispecific antibody binds both from about 1 nM to about 100 uM. In another such aspect, TfR and BACE1. In another such aspect, the multispecific the IC50 is from about 5 nM to about 100 uM. In another antibody binds both TfR and Abeta. In another such aspect, such aspect, the IC50 is from about 50 nM to about 100 uM. the multispecific antibody is labeled. In another aspect, the In another such aspect, the IC50 is from about 100 nM to compound is reversibly coupled to the antibody such that the about 100 LM. In another aspect, the antibody has an affinity compound is released from the antibody concurrent with or for the BBB-R from about 5 nM to about 10 uM. In another after BBB transport. Such aspect, the antibody, when coupled to a compound, has 25 In another embodiment, the invention provides a method an affinity for the BBB-R from about 30 nM to about 1 uM. of increasing exposure of the CNS to a compound, wherein In another Such aspect, the antibody, when coupled to a the compound is coupled to an antibody which binds with compound, has an affinity for the BBB-R from about 50 nM low affinity to a BBB-R, thereby increasing the exposure of to about 1 LM. In another Such aspect, the compound the CNS to the compound. In one aspect, the compound is coupled antibody specifically binds to TfR and has an 30 a neurological disorder drug. In another aspect, the com affinity for TfR between those affinities observed for the pound is an imaging agent. In another aspect, the compound anti-TfR/BACE1 antibody and the anti-TfR/BACE1 anti is labeled. In another aspect, the antibody is labeled. In body. In another such aspect, the compound-coupled anti another aspect, the antibody does not impair the binding of body specifically binds to TfR and has an affinity for TfR the BBB-R to one or more of its native ligands. In another between those affinities observed for the anti-TfRP/BACE1 35 such aspect, the antibody specifically binds to TfR in such a antibody and the anti-TfR'/BACE1 antibody. In another manner that it does not inhibit binding of the TfR to Such aspect, the compound-coupled antibody specifically transferrin. In another aspect, the antibody-coupled com binds to TfR and has an IC50 for TfR between those IC50s pound is administered to a mammal. In another Such aspect, observed for the anti-TfR"/BACE1 antibody and the anti the mammal is a human. In another Such aspect, the mammal TfR/BACE1 antibody. In another such aspect, the com 40 has a neurological disorder. In another such aspect, the pound-coupled antibody specifically binds to TfR and has an neurological disorder is selected from the group consisting IC50 for TfR between those IC50s observed for the anti of Alzheimer's disease (AD), stroke, dementia, muscular TfRP/BACE1 antibody and the anti-TfR/BACE1 antibody. dystrophy (MD), multiple sclerosis (MS), amyotrophic lat In one aspect, the affinity of the anti-BBB-R or anti-BBB eral Sclerosis (ALS), cystic fibrosis, Angelman's syndrome, R/compound for the BBB-R is measured using scatchard 45 Liddle syndrome, Parkinson's disease, Pick's disease, Pag analysis. In another aspect, the affinity of the anti-BBB-R or et's disease, cancer, and traumatic brain injury. anti-BBB-R/compound for the BBB-R is measured using In another aspect, the increase in CNS exposure to the BIACORE analysis. In another aspect, the affinity of the compound is measured relative to the CNS exposure of a anti-BBB-R or anti-BBB-R/compound for the BBB-R is compound coupled with a typical antibody not having measured using a competition ELISA. 50 lowered affinity for the BBB-R. In another aspect, the In another aspect, the BBB-R is selected from the group increase in CNS exposure to the compound is measured as consisting of transferrin receptor (TfR), insulin receptor, a ratio of the amount of the compound found in the CNS insulin-like growth factor receptor (IGF receptor), low den relative to the amount found in the serum after administra sity lipoprotein receptor-related protein 8 (LRP8), low den tion. In another Such aspect, the increase in CNS exposure sity lipoprotein receptor-related protein 1 (LRP1), and hepa 55 results in a ratio of greater than 0.1%. In another aspect, the rin-binding epidermal growth factor-like growth factor (HB increase in CNS exposure to the compound is measured EGF). In another such aspect, the BBB-R is a human relative to the CNS exposure of a compound in the absence BBB-R. In one such aspect, the BBB-R is TfR. In another of a coupled antibody. In another aspect, the increase in CNS such aspect, the BBB-R is TfR and the antibody does not exposure to the compound is measured by imaging. In inhibit TfR activity. In another such aspect, the BBB-R is 60 another aspect, the increase in CNS exposure to the com TfR and the antibody does not inhibit the binding of TfR to pound is measured by an indirect readout such as a modi transferrin. In another aspect, the compound-coupled anti fication of one or more physiological symptoms. body is administered at a therapeutic dose. In one Such In another aspect, the antibody has an IC50 for the BBB-R aspect, the therapeutic dose is a dose that Saturates the from about 1 nM to about 100 uM. In another such aspect, BBB-R to which the antibody specifically binds. 65 the IC50 is from about 5 nM to about 100 uM. In another In another aspect, the compound is covalently coupled to such aspect, the IC50 is from about 50 nM to about 100 uM. the antibody. In one Such aspect, the compound is joined to In another such aspect, the IC50 is from about 100 nM to US 9,611,323 B2 5 6 about 100 LM. In another aspect, the antibody has an affinity compound is reversibly coupled to the antibody such that the for the BBB-R from about 5 nM to about 10 uM. In another compound is released from the antibody concurrent with or Such aspect, the antibody, when coupled to a compound, has after BBB transport. an affinity for the BBB-R from about 30 nM to about 1 uM. In another embodiment, the invention provides a method In another Such aspect, the antibody, when coupled to a of decreasing clearance of a compound administered to a compound, has an affinity for the BBB-R from about 50 nM Subject, wherein the compound is coupled to an antibody to about 1 LM. In another Such aspect, the compound which binds with low affinity to a BBB-R, such that the coupled antibody specifically binds to TfR and has an clearance of the compound is decreased. In one aspect, the affinity for TfR between those affinities observed for the compound is a neurological disorder drug. In another aspect, 10 the compound is an imaging agent. In another aspect, the anti-TfR/BACE1 antibody and the anti-TfR/BACE1 anti compound is labeled. In another aspect, the antibody is body. In another such aspect, the compound-coupled anti labeled. In another aspect, the antibody does not impair the body specifically binds to TfR and has an affinity for TfR binding of the BBB-R to one or more of its native ligands. between those affinities observed for the anti-TfRP/BACE1 In another Such aspect, the antibody specifically binds to antibody and the anti-TfR/BACE1 antibody. In another 15 TfR in such a manner that it does not inhibit binding of the Such aspect, the compound-coupled antibody specifically TfR to transferrin. In another aspect, the Subject is a mam binds to TfR and has an IC50 for TfR between those IC50s mal. In another such aspect, the mammal is a human. In observed for the anti-TfR/BACE1 antibody and the anti another Such aspect, the mammal has a neurological disor TfR'/BACE1 antibody. In another such aspect, the com der. In another Such aspect, the neurological disorder is pound-coupled antibody specifically binds to TfR and has an selected from the group consisting of Alzheimer's disease IC50 for TfR between those IC50s observed for the anti (AD), stroke, dementia, muscular dystrophy (MD), multiple TfRP/BACE1 antibody and the anti-TfR/BACE1 antibody. sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic In one aspect, the affinity of the anti-BBB-R or anti-BBB fibrosis, Angelman’s syndrome, Liddle syndrome, Parkin R/compound for the BBB-R is measured using scatchard son's disease, Pick's disease, Paget’s disease, cancer, and analysis. In another aspect, the affinity of the anti-BBB-R or 25 traumatic brain injury. anti-BBB-R/compound for the BBB-R is measured using In another aspect, the decrease in clearance of the com BIACORE analysis. In another aspect, the affinity of the pound is measured relative to the clearance of a compound anti-BBB-R or anti-BBB-R/compound for the BBB-R is coupled with a typical antibody not having lowered affinity measured using a competition ELISA. for the BBB-R. In another aspect, the decrease in clearance In another aspect, the BBB-R is selected from the group 30 of the compound is measured relative to the clearance of the consisting of transferrin receptor (TfR), insulin receptor, compound in the absence of a coupled antibody. insulin-like growth factor receptor (IGF receptor), low den In another aspect, the antibody has an IC50 for the BBB-R sity lipoprotein receptor-related protein 8 (LRP8), low den from about 1 nM to about 100 uM. In another such aspect, sity lipoprotein receptor-related protein 1 (LRP1), and hepa the IC50 is from about 5 nM to about 100 uM. In another rin-binding epidermal growth factor-like growth factor (HB 35 such aspect, the IC50 is from about 50 nM to about 100 uM. EGF). In another such aspect, the BBB-R is a human In another such aspect, the IC50 is from about 100 nM to BBB-R. In one such aspect, the BBB-R is TfR. In another about 100 LM. In another aspect, the antibody has an affinity such aspect, the BBB-R is TfR and the antibody does not for the BBB-R from about 5 nM to about 10 uM. In another inhibit TfR activity. In another such aspect, the BBB-R is Such aspect, the antibody, when coupled to a compound, has TfR and the antibody does not inhibit the binding of TfR to 40 an affinity for the BBB-R from about 30 nM to about 1 uM. transferrin. In another aspect, the compound-coupled anti In another Such aspect, the antibody, when coupled to a body is administered at a therapeutic dose. In one Such compound, has an affinity for the BBB-R from about 50 nM aspect, the therapeutic dose is a dose that Saturates the to about 1 LM. In another Such aspect, the compound BBB-R to which the antibody specifically binds. coupled antibody specifically binds to TfR and has an In another aspect, the compound is covalently coupled to 45 affinity for TfR between those affinities observed for the the antibody. In one Such aspect, the compound is joined to anti-TfR/BACE1 antibody and the anti-TfR/BACE1 anti the antibody by a linker. In one such aspect, the linker is body. In another such aspect, the compound-coupled anti cleavable. In another such aspect, the linker is not cleavable. body specifically binds to TfR and has an affinity for TfR In another Such aspect, the compound is directly linked to between those affinities observed for the anti-TfRP/BACE1 the antibody. In one such aspect, the antibody is a multi 50 antibody and the anti-TfR'/BACE1 antibody. In another specific antibody and the compound forms one portion of the Such aspect, the compound-coupled antibody specifically multispecific antibody. In another Such aspect, the multispe binds to TfR and has an IC50 for Tfr between those IC50s cific antibody comprises a first antigen binding site which observed for the anti-TfR"/BACE1 antibody and the anti binds the BBB-R and a second antigen binding site which TfR/BACE1 antibody. In another such aspect, the com binds a brain antigen. In another Such aspect, the brain 55 pound-coupled antibody specifically binds to TfR and has an antigen is selected from the group consisting of beta IC50 for Tfr between those IC50s observed for the anti secretase 1 (BACE1), Abeta, epidermal growth factor recep TfRP/BACE1 antibody and the anti-TfR/BACE1 antibody. tor (EGFR), human epidermal growth factor receptor 2 In one aspect, the affinity of the anti-BBB-R or anti-BBB (HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, R/compound for the BBB-R is measured using scatchard CD20, huntingtin, prion protein (PrP), leucine rich repeat 60 analysis. In another aspect, the affinity of the anti-BBB-R or kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma anti-BBB-R/compound for the BBB-R is measured using secretase, death receptor 6 (DR6), amyloid precursor protein BIACORE analysis. In another aspect, the affinity of the (APP), p75 neurotrophin receptor (p75NTR), and caspase 6. anti-BBB-R or anti-BBB-R/compound for the BBB-R is In another such aspect, the multispecific antibody binds both measured using a competition ELISA. TfR and BACE1. In another such aspect, the multispecific 65 In another aspect, the BBB-R is selected from the group antibody binds both TfR and Abeta. In another such aspect, consisting of transferrin receptor (TfR), insulin receptor, the multispecific antibody is labeled. In another aspect, the insulin-like growth factor receptor (IGF receptor), low den US 9,611,323 B2 7 8 sity lipoprotein receptor-related protein 8 (LRP8), low den points after administration. In another Such aspect, the sity lipoprotein receptor-related protein 1 (LRP1), and hepa increase in CNS retention results in a ratio of greater than rin-binding epidermal growth factor-like growth factor (HB 0.1% at one or more time points after administration. In EGF). In another such aspect, the BBB-R is a human another aspect, the increase in CNS retention of the com BBB-R. In one such aspect, the BBB-R is TfR. In another 5 pound is measured relative to the CNS retention of a such aspect, the BBB-R is TfR and the antibody does not compound in the absence of a coupled antibody. In another inhibit TfR activity. In another such aspect, the BBB-R is aspect, the increase in CNS retention of the compound is TfR and the antibody does not inhibit the binding of TfR to measured by imaging. In another aspect, the increase in CNS transferrin. In another aspect, the compound-coupled anti retention of the compound is measured by an indirect body is administered at a therapeutic dose. In one Such 10 readout Such as a modification of one or more physiological aspect, the therapeutic dose is a dose that Saturates the symptoms. BBB-R to which the antibody specifically binds. In another aspect, the antibody has an IC50 for the BBB-R In another aspect, the compound is covalently coupled to from about 1 nM to about 100 uM. In another such aspect, the antibody. In one Such aspect, the compound is joined to the IC50 is from about 5 nM to about 100 uM. In another the antibody by a linker. In one such aspect, the linker is 15 such aspect, the IC50 is from about 50 nM to about 100 uM. cleavable. In another such aspect, the linker is not cleavable. In another such aspect, the IC50 is from about 100 nM to In another Such aspect, the compound is directly linked to about 100 LM. In another aspect, the antibody has an affinity the antibody. In one such aspect, the antibody is a multi for the BBB-R from about 5 nM to about 10 uM. In another specific antibody and the compound forms one portion of the Such aspect, the antibody, when coupled to a compound, has multispecific antibody. In another Such aspect, the multispe an affinity for the BBB-R from about 30 nM to about 1 uM. cific antibody comprises a first antigen binding site which In another Such aspect, the antibody, when coupled to a binds the BBB-R and a second antigen binding site which compound, has an affinity for the BBB-R from about 50 nM binds a brain antigen. In another Such aspect, the brain to about 1 LM. In another Such aspect, the compound antigen is selected from the group consisting of beta coupled antibody specifically binds to TfR and has an secretase 1 (BACE1), Abeta, epidermal growth factor recep 25 affinity for TfR between those affinities observed for the tor (EGFR), human epidermal growth factor receptor 2 anti-TfR/BACE1 antibody and the anti-TfR/BACE1 anti (HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, body. In another such aspect, the compound-coupled anti CD20, huntingtin, prion protein (PrP), leucine rich repeat body specifically binds to TfR and has an affinity for TfR kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma between those affinities observed for the anti-TfRP/BACE1 secretase, death receptor 6 (DR6), amyloid precursor protein 30 antibody and the anti-TfR/BACE1 antibody. In another (APP), p75 neurotrophin receptor (p75NTR), and caspase 6. Such aspect, the compound-coupled antibody specifically In another such aspect, the multispecific antibody binds both binds to TfR and has an IC50 for TfR between those IC50s TfR and BACE1. In another such aspect, the multispecific observed for the anti-TfR/BACE1 antibody and the anti antibody binds both TfR and Abeta. In another such aspect, TfR'/BACE1 antibody. In another such aspect, the com the multispecific antibody is labeled. In another aspect, the 35 pound-coupled antibody specifically binds to TfR and has an compound is reversibly coupled to the antibody such that the IC50 for Tfr between those IC50s observed for the anti compound is released from the antibody concurrent with or TfRP/BACE1 antibody and the anti-TfR/BACE1 antibody. after BBB transport. In one aspect, the affinity of the anti-BBB-R or anti-BBB A method of increasing retention in the CNS of a com R/compound for the BBB-R is measured using scatchard pound administered to a subject, wherein the compound is 40 analysis. In another aspect, the affinity of the anti-BBB-R or coupled to an antibody which binds with low affinity to a anti-BBB-R/compound for the BBB-R is measured using BBB-R, such that the retention in the CNS of the compound BIACORE analysis. In another aspect, the affinity of the is increased. In one aspect, the compound is a neurological anti-BBB-R or anti-BBB-R/compound for the BBB-R is disorder drug. In another aspect, the compound is an imag measured using a competition ELISA. ing agent. In another aspect, the compound is labeled. In 45 In another aspect, the BBB-R is selected from the group another aspect, the antibody is labeled. In another aspect, the consisting of transferrin receptor (TfR), insulin receptor, antibody does not impair the binding of the BBB-R to one insulin-like growth factor receptor (IGF receptor), low den or more of its native ligands. In another Such aspect, the sity lipoprotein receptor-related protein 8 (LRP8), low den antibody specifically binds to TfR in such a manner that it sity lipoprotein receptor-related protein 1 (LRP1), and hepa does not inhibit binding of the TfR to transferrin. In another 50 rin-binding epidermal growth factor-like growth factor (HB aspect, the compound is administered to a mammal. In EGF). In another such aspect, the BBB-R is a human another Such aspect, the mammal is a human. In another BBB-R. In one such aspect, the BBB-R is TfR. In another Such aspect, the mammal has a neurological disorder. In such aspect, the BBB-R is TfR and the antibody does not another Such aspect, the neurological disorder is selected inhibit TfR activity. In another such aspect, the BBB-R is from the group consisting of Alzheimer's disease (AD), 55 TfR and the antibody does not inhibit the binding of TfR to stroke, dementia, muscular dystrophy (MD), multiple scle transferrin. In another aspect, the compound-coupled anti rosis (MS), amyotrophic lateral sclerosis (ALS), cystic body is administered at a therapeutic dose. In one Such fibrosis, Angelman’s syndrome, Liddle syndrome, Parkin aspect, the therapeutic dose is a dose that saturates the son's disease, Pick's disease, Paget’s disease, cancer, and BBB-R to which the antibody specifically binds. traumatic brain injury. 60 In another aspect, the compound is covalently coupled to In another aspect, the increase in CNS retention of the the antibody. In one such aspect, the compound is joined to compound is measured relative to the CNS retention of a the antibody by a linker. In one such aspect, the linker is compound coupled with a typical antibody not having cleavable. In another such aspect, the linker is not cleavable. lowered affinity for the BBB-R. In another aspect, the In another Such aspect, the compound is directly linked to increase in CNS retention of the compound is measured as 65 the antibody. In one such aspect, the antibody is a multi a ratio of the amount of the compound found in the CNS specific antibody and the compound forms one portion of the relative to the amount found in the serum at one or more time multispecific antibody. In another Such aspect, the multispe US 9,611,323 B2 10 cific antibody comprises a first antigen binding site which to about 1 LM. In another Such aspect, the compound binds the BBB-R and a second antigen binding site which coupled antibody specifically binds to TfR and has an binds a brain antigen. In another Such aspect, the brain affinity for TfR between those affinities observed for the antigen is selected from the group consisting of beta anti-TfR/BACE1 antibody and the anti-TfR/BACE1 anti secretase 1 (BACE1), Abeta, epidermal growth factor recep body. In another such aspect, the compound-coupled anti tor (EGFR), human epidermal growth factor receptor 2 body specifically binds to TfR and has an affinity for TfR (HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, between those affinities observed for the anti-TfRP/BACE1 CD20, huntingtin, prion protein (PrP), leucine rich repeat antibody and the anti-TfR'/BACE1 antibody. In another kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma Such aspect, the compound-coupled antibody specifically secretase, death receptor 6 (DR6), amyloid precursor protein 10 binds to TfR and has an IC50 for Tfr between those IC50s (APP), p75 neurotrophin receptor (p75NTR), and caspase 6. observed for the anti-TfR"/BACE1 antibody and the anti In another such aspect, the multispecific antibody binds both TfR/BACE1 antibody. In another such aspect, the com TfR and BACE1. In another such aspect, the multispecific pound-coupled antibody specifically binds to TfR and has an antibody binds both TfR and Abeta. In another such aspect, IC50 for Tfr between those IC50s observed for the anti the multispecific antibody is labeled. In another aspect, the 15 TfRP/BACE1 antibody and the anti-TfR/BACE1 antibody. compound is reversibly coupled to the antibody such that the In one aspect, the affinity of the anti-BBB-R or anti-BBB compound is released from the antibody concurrent with or R/compound for the BBB-R is measured using scatchard after BBB transport. analysis. In another aspect, the affinity of the anti-BBB-R or In another embodiment, the invention provides a method anti-BBB-R/compound for the BBB-R is measured using of optimizing the pharmacokinetics and/or pharmacodynam BIACORE analysis. In another aspect, the affinity of the ics of a compound to be efficacious in the CNS of a subject, anti-BBB-R or anti-BBB-R/compound for the BBB-R is wherein the compound is coupled to an antibody which measured using a competition ELISA. binds with low affinity to a BBB-R, and the antibody is In another aspect, the BBB-R is selected from the group selected such that its affinity for the BBB-R after coupling consisting of transferrin receptor (TfR), insulin receptor, to the compound results in an amount of transport of the 25 insulin-like growth factor receptor (IGF receptor), low den antibody conjugated to the compound across the BBB that sity lipoprotein receptor-related protein 8 (LRP8), low den optimizes the pharmacokinetics and/or pharmacodynamics sity lipoprotein receptor-related protein 1 (LRP1), and hepa of the compound in the CNS. In one aspect, the compound rin-binding epidermal growth factor-like growth factor (HB is a neurological disorder drug. In another aspect, the EGF). In another such aspect, the BBB-R is a human compound is an imaging agent. In another aspect, the 30 BBB-R. In one such aspect, the BBB-R is TfR. In another compound is labeled. In another aspect, the antibody is such aspect, the BBB-R is TfR and the antibody does not labeled. In another aspect, the antibody does not impair the inhibit TfR activity. In another such aspect, the BBB-R is binding of the BBB-R to one or more of its native ligands. TfR and the antibody does not inhibit the binding of TfR to In another such aspect, the antibody specifically binds to transferrin. In another aspect, the compound-coupled anti TfR in such a manner that it does not inhibit binding of the 35 body is administered at a therapeutic dose. In one Such TfR to transferrin. In another aspect, the BBB is in a aspect, the therapeutic dose is a dose that saturates the mammal. In another Such aspect, the mammal is a human. In BBB-R to which the antibody specifically binds. another Such aspect, the mammal has a neurological disor In another aspect, the compound is covalently coupled to der. In another such aspect, the neurological disorder is the antibody. In one such aspect, the compound is joined to selected from the group consisting of Alzheimer's disease 40 the antibody by a linker. In one such aspect, the linker is (AD), stroke, dementia, muscular dystrophy (MD), multiple cleavable. In another such aspect, the linker is not cleavable. sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic In another Such aspect, the compound is directly linked to fibrosis, Angelman’s syndrome, Liddle syndrome, Parkin the antibody. In one such aspect, the antibody is a multi son's disease, Pick's disease, Paget’s disease, cancer, and specific antibody and the compound forms one portion of the traumatic brain injury. In another aspect, the BBB is in a 45 multispecific antibody. In another Such aspect, the multispe human. cific antibody comprises a first antigen binding site which In one aspect, the optimizing may include the generation binds the BBB-R and a second antigen binding site which of a series of antibody-compound complexes in which each binds a brain antigen. In another Such aspect, the brain antibody has a different affinity for the BBB-R, and assess antigen is selected from the group consisting of beta ing the pharmacokinetics and/or pharmacodynamics of each 50 secretase 1 (BACE1), Abeta, epidermal growth factor recep in the CNS. In another aspect, optimizing may be relative to tor (EGFR), human epidermal growth factor receptor 2 a known standard. Such as, but not limited to, the pharma (HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, cokinetics and/or pharmacodynamics of the compound CD20, huntingtin, prion protein (PrP), leucine rich repeat when directly introduced into the CNS or when introduced kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma to the subject in the absence of a coupled anti-BBB-R 55 secretase, death receptor 6 (DR6), amyloid precursor protein antibody. (APP), p75 neurotrophin receptor (p75NTR), and caspase 6. In another aspect, the antibody has an IC50 for the BBB-R In another such aspect, the multispecific antibody binds both from about 1 nM to about 100 uM. In another such aspect, TfR and BACE1. In another such aspect, the multispecific the IC50 is from about 5 nM to about 100 uM. In another antibody binds both TfR and Abeta. In another such aspect, such aspect, the IC50 is from about 50 nM to about 100 uM. 60 the multispecific antibody is labeled. In another aspect, the In another such aspect, the IC50 is from about 100 nM to compound is reversibly coupled to the antibody such that the about 100 LM. In another aspect, the antibody has an affinity compound is released from the antibody concurrent with or for the BBB-R from about 5 nM to about 10 uM. In another after BBB transport. Such aspect, the antibody, when coupled to a compound, has In another embodiment the invention provides a method an affinity for the BBB-R from about 30 nM to about 1 uM. 65 of treating a neurological disorder in a mammal comprising In another Such aspect, the antibody, when coupled to a treating the mammal with an antibody that binds a BBB-R compound, has an affinity for the BBB-R from about 50 nM and is coupled to a compound, wherein the antibody has US 9,611,323 B2 11 12 been selected to have a low affinity for the BBB-R and aspect, the therapeutic dose is a dose that saturates the thereby improves CNS uptake of the antibody and coupled BBB-R to which the antibody specifically binds. compound. In one aspect, the compound is a neurological In another aspect, the compound is covalently coupled to disorder drug. In another aspect, the compound is an imag the antibody. In one such aspect, the compound is joined to ing agent. In another aspect, the compound is labeled. In the antibody by a linker. In one such aspect, the linker is another aspect, the antibody is labeled. In another aspect, the cleavable. In another such aspect, the linker is not cleavable. antibody does not impair the binding of the BBB-R to one In another Such aspect, the compound is directly linked to or more of its native ligands. In another Such aspect, the the antibody. In one such aspect, the antibody is a multi antibody specifically binds to TfR in such a manner that it specific antibody and the compound forms one portion of the does not inhibit binding of the TfR to transferrin. In one 10 multispecific antibody. In another Such aspect, the multispe aspect, the mammal is a human. In another such aspect, the cific antibody comprises a first antigen binding site which mammal has a neurological disorder. In another such aspect, binds the BBB-R and a second antigen binding site which the neurological disorder is selected from the group con binds a brain antigen. In another Such aspect, the brain sisting of Alzheimer's disease (AD), stroke, dementia, mus antigen is selected from the group consisting of beta cular dystrophy (MD), multiple sclerosis (MS), amyotrophic 15 secretase 1 (BACE1), Abeta, epidermal growth factor recep lateral Sclerosis (ALS), cystic fibrosis, Angelman's Syn tor (EGFR), human epidermal growth factor receptor 2 drome, Liddle syndrome, Parkinson's disease, Pick's dis (HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, ease, Paget’s disease, cancer, and traumatic brain injury. CD20, huntingtin, prion protein (PrP), leucine rich repeat In one aspect, the treating results in lessening or elimi kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma nation of disorder symptoms. In another aspect, the treating secretase, death receptor 6 (DR6), amyloid precursor protein results in amelioration of the neurological disorder. (APP), p75 neurotrophin receptor (p75NTR), and caspase 6. In another aspect, the antibody has an IC50 for the BBB-R In another such aspect, the multispecific antibody binds both from about 1 nM to about 100 uM. In another such aspect, TfR and BACE1. In another such aspect, the multispecific the IC50 is from about 5 nM to about 100 uM. In another antibody binds both TfR and Abeta. In another such aspect, such aspect, the IC50 is from about 50 nM to about 100 uM. 25 the multispecific antibody is labeled. In another aspect, the In another such aspect, the IC50 is from about 100 nM to compound is reversibly coupled to the antibody such that the about 100 LM. In another aspect, the antibody has an affinity compound is released from the antibody concurrent with or for the BBB-R from about 5 nM to about 10 uM. In another after BBB transport. Such aspect, the antibody, when coupled to a compound, has In another embodiment, the invention provides a method an affinity for the BBB-R from about 30 nM to about 1 uM. 30 of making an antibody useful for transporting a compound In another Such aspect, the antibody, when coupled to a across the BBB comprising selecting an antibody specific compound, has an affinity for the BBB-R from about 50 nM for a blood-brain barrier receptor (BBB-R) because it has a to about 1 LM. In another Such aspect, the compound desirably low affinity for the BBB-R. coupled antibody specifically binds to TfR and has an In one aspect, the antibody is selected from a panel of affinity for TfR between those affinities observed for the 35 antibodies based upon the affinity of the selected antibody. anti-TfR/BACE1 antibody and the anti-TfR/BACE1 anti In another aspect, the antibody is engineered to have the body. In another such aspect, the compound-coupled anti affinity. In one such aspect, the antibody is generated using body specifically binds to TfR and has an affinity for TfR any art-known protein engineering methodology including, between those affinities observed for the anti-TfRP/BACE1 but not limited to, phage display, yeast display, random antibody and the anti-TfR/BACE1 antibody. In another 40 mutagenesis, and site-directed mutagenesis. Such aspect, the compound-coupled antibody specifically In one aspect, the compound is a neurological disorder binds to TfR and has an IC50 for TfR between those IC50s drug. In another aspect, the compound is an imaging agent. observed for the anti-TfR"/BACE1 antibody and the anti In another aspect, the compound is labeled. In another TfR'/BACE1 antibody. In another such aspect, the com aspect, the antibody is labeled. In another aspect, the anti pound-coupled antibody specifically binds to TfR and has an 45 body does not impair the binding of the BBB-R to one or IC50 for TfR between those IC50s observed for the anti more of its native ligands. In another Such aspect, the TfRP/BACE1 antibody and the anti-TfR/BACE1 antibody. antibody specifically binds to TfR in such a manner that it In one aspect, the affinity of the anti-BBB-R or anti-BBB does not inhibit binding of the TfR to transferrin. In another R/compound for the BBB-R is measured using scatchard aspect, the BBB is in a mammal. In another such aspect, the analysis. In another aspect, the affinity of the anti-BBB-R or 50 mammal is a human. In another such aspect, the mammal anti-BBB-R/compound for the BBB-R is measured using has a neurological disorder. In another such aspect, the BIACORE analysis. In another aspect, the affinity of the neurological disorder is selected from the group consisting anti-BBB-R or anti-BBB-R/compound for the BBB-R is of Alzheimer's disease (AD), stroke, dementia, muscular measured using a competition ELISA. dystrophy (MD), multiple sclerosis (MS), amyotrophic lat In another aspect, the BBB-R is selected from the group 55 eral Sclerosis (ALS), cystic fibrosis, Angelman's syndrome, consisting of transferrin receptor (TfR), insulin receptor, Liddle syndrome, Parkinson's disease, Pick's disease, Pag insulin-like growth factor receptor (IGF receptor), low den et's disease, cancer, and traumatic brain injury. In another sity lipoprotein receptor-related protein 8 (LRP8), low den aspect, the BBB is in a human. sity lipoprotein receptor-related protein 1 (LRP1), and hepa In another aspect, the antibody has an IC50 for the BBB-R rin-binding epidermal growth factor-like growth factor (HB 60 from about 1 nM to about 100 uM. In another such aspect, EGF). In another such aspect, the BBB-R is a human the IC50 is from about 5 nM to about 100 uM. In another BBB-R. In one such aspect, the BBB-R is TfR. In another such aspect, the IC50 is from about 50 nM to about 100 uM. such aspect, the BBB-R is TfR and the antibody does not In another such aspect, the IC50 is from about 100 nM to inhibit TfR activity. In another such aspect, the BBB-R is about 100 LM. In another aspect, the antibody has an affinity TfR and the antibody does not inhibit the binding of TfR to 65 for the BBB-R from about 5 nM to about 10 uM. In another transferrin. In another aspect, the compound-coupled anti Such aspect, the antibody, when coupled to a compound, has body is administered at a therapeutic dose. In one Such an affinity for the BBB-R from about 30 nM to about 1 uM. US 9,611,323 B2 13 14 In another Such aspect, the antibody, when coupled to a with low affinity. In one aspect, the affinity of the antibody compound, has an affinity for the BBB-R from about 50 nM for the BBB-R is from about 5 nM to about 10 uM. In to about 1 LM. In another Such aspect, the compound another aspect, the affinity of the antibody for the BBB-R is coupled antibody specifically binds to TfR and has an from about 20 nM to about 1 uM. In another aspect, the affinity for TfR between those affinities observed for the 5 antibody has an IC50 for the BBB-R from about 1 nM to anti-TfR/BACE1 antibody and the anti-TfR/BACE1 anti about 100 LM. In another such aspect, the IC50 is from body. In another such aspect, the compound-coupled anti about 5 nM to about 100 uM. In another such aspect, the body specifically binds to TfR and has an affinity for TfR IC50 is from about 50 nM to about 100 uM. In another such between those affinities observed for the anti-TfRP/BACE1 aspect, the IC50 is from about 100 nM to about 100 uM. In antibody and the anti-TfR'/BACE1 antibody. In another 10 another Such aspect, the antibody, when coupled to a com Such aspect, the compound-coupled antibody specifically pound, has an affinity for the BBB-R from about 50 nM to binds to TfR and has an IC50 for TfR between those IC50s about 1 LM. In another such aspect, the compound-coupled observed for the anti-TfR"/BACE1 antibody and the anti antibody specifically binds to TfR and has an affinity for TfR TfR/BACE1 antibody. In another such aspect, the com between those affinities observed for the anti-TfR/BACE1 pound-coupled antibody specifically binds to TfR and has an 15 antibody and the anti-TfR'/BACE1 antibody. In another IC50 for TfR between those IC50s observed for the anti Such aspect, the compound-coupled antibody specifically TfRP/BACE1 antibody and the anti-TfR/BACE1 antibody. binds to TfR and has an affinity for TfR between those In one aspect, the affinity of the anti-BBB-R or anti-BBB affinities observed for the anti-TfRP/BACE1 antibody and R/compound for the BBB-R is measured using scatchard the anti-TfR/BACE1 antibody. In another such aspect, the analysis. In another aspect, the affinity of the anti-BBB-R or compound-coupled antibody specifically binds to TfR and anti-BBB-R/compound for the BBB-R is measured using has an IC50 for TfR between those IC50s observed for the BIACORE analysis. In another aspect, the affinity of the anti-TfR/BACE1 antibody and the anti-TfR/BACE1 anti anti-BBB-R or anti-BBB-R/compound for the BBB-R is body. In another such aspect, the compound-coupled anti measured using a competition ELISA. body specifically binds to TfR and has an IC50 for TfR In another aspect, the BBB-R is selected from the group 25 between those IC50s observed for the anti-TfRP/BACE1 consisting of transferrin receptor (TfR), insulin receptor, antibody and the anti-TfR/BACE1 antibody. In one aspect, insulin-like growth factor receptor (IGF receptor), low den the affinity of the anti-BBB-R or anti-BBB-R/compound for sity lipoprotein receptor-related protein 8 (LRP8), low den the BBB-R is measured using scatchard analysis. In another sity lipoprotein receptor-related protein 1 (LRP1), and hepa aspect, the affinity of the anti-BBB-R or anti-BBB-R/com rin-binding epidermal growth factor-like growth factor (HB 30 pound for the BBB-R is measured using BIACORE analy EGF). In another such aspect, the BBB-R is a human sis. In another aspect, the affinity of the anti-BBB-R or BBB-R. In one such aspect, the BBB-R is TfR. In another anti-BBB-R/compound for the BBB-R is measured using a such aspect, the BBB-R is TfR and the antibody does not competition ELISA. inhibit TfR activity. In another such aspect, the BBB-R is In another aspect, the BBB-R is selected from the group TfR and the antibody does not inhibit the binding of TfR to 35 consisting of transferrin receptor (TfR), insulin receptor, transferrin. In another aspect, the compound-coupled anti insulin-like growth factor receptor (IGF receptor), low den body is administered at a therapeutic dose. In one Such sity lipoprotein receptor-related protein 8 (LRP8), low den aspect, the therapeutic dose is a dose that Saturates the sity lipoprotein receptor-related protein 1 (LRP1), and hepa BBB-R to which the antibody specifically binds. rin-binding epidermal growth factor-like growth factor (HB In another aspect, the compound is covalently coupled to 40 EGF). In one such aspect, the BBB-R is TfR. In another such the antibody. In one Such aspect, the compound is joined to aspect, the BBB-R is TfR and the antibody does not inhibit the antibody by a linker. In one such aspect, the linker is TfR activity. In another such aspect, the BBB-R is TfR and cleavable. In another such aspect, the linker is not cleavable. the antibody does not inhibit the binding of TfR to trans In another Such aspect, the compound is directly linked to ferrin. In another such aspect, the BBB-R is a human the antibody. In one such aspect, the antibody is a multi 45 BBB-R. specific antibody and the compound forms one portion of the In another aspect, the antibody is coupled to a compound. multispecific antibody. In another Such aspect, the multispe In one aspect, the compound is a neurological disorder drug. cific antibody comprises a first antigen binding site which In another aspect, the compound is an imaging agent. In binds the BBB-R and a second antigen binding site which another aspect, the compound is labeled. In another aspect, binds a brain antigen. In another Such aspect, the brain 50 the antibody is labeled. In another aspect, the antibody does antigen is selected from the group consisting of beta not impair the binding of the BBB-R to one or more of its secretase 1 (BACE1), Abeta, epidermal growth factor recep native ligands. In another such aspect, the antibody specifi tor (EGFR), human epidermal growth factor receptor 2 cally binds to TfR in such a manner that it does not inhibit (HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, binding of the TfR to transferrin. CD20, huntingtin, prion protein (PrP), leucine rich repeat 55 In another aspect, the compound is covalently coupled to kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma the antibody. In one such aspect, the compound is joined to secretase, death receptor 6 (DR6), amyloid precursor protein the antibody by a linker. In one such aspect, the linker is (APP), p75 neurotrophin receptor (p75NTR), and caspase 6. cleavable. In another such aspect, the linker is not cleavable. In another such aspect, the multispecific antibody binds both In another Such aspect, the compound is directly linked to TfR and BACE1. In another such aspect, the multispecific 60 the antibody. In one such aspect, the antibody is a multi antibody binds both TfR and Abeta. In another such aspect, specific antibody and the compound forms one portion of the the multispecific antibody is labeled. In another aspect, the multispecific antibody. In another Such aspect, the multispe compound is reversibly coupled to the antibody such that the cific antibody comprises a first antigen binding site which compound is released from the antibody concurrent with or binds the BBB-R and a second antigen binding site which after BBB transport. 65 binds a brain antigen. In another Such aspect, the brain In another embodiment, the invention provides an anti antigen is selected from the group consisting of beta body which binds to a blood-brain barrier receptor (BBB-R) secretase 1 (BACE1), Abeta, epidermal growth factor recep US 9,611,323 B2 15 16 tor (EGFR), human epidermal growth factor receptor 2 mild cognitive impairment and prodromal AD), stroke, (HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, dementia, muscular dystrophy (MD), multiple Sclerosis CD20, huntingtin, prion protein (PrP), leucine rich repeat (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma Angelman’s syndrome, Liddle syndrome, Parkinson's dis secretase, death receptor 6 (DR6), amyloid precursor protein ease, Pick's disease, Paget’s disease, cancer (e.g. cancer (APP), p75 neurotrophin receptor (p75NTR), and caspase 6. affecting the CNS or brain), and traumatic brain injury. In another such aspect, the multispecific antibody binds both The invention additionally concerns a method of making TfR and BACE1. In another such aspect, the multispecific an antibody useful for transporting a therapeutic compound antibody binds both TfR and Abeta. In another such aspect, Such as a neurological disorder drug across the blood-brain the multispecific antibody is labeled. In another aspect, the 10 barrier comprising selecting an antibody against a blood compound is reversibly coupled to the antibody such that the brain barrier receptor (BBB-R) because it has an affinity for compound is released from the antibody concurrent with or the BBB-R which is from about 5 nM to about 10 uM. In one after BBB transport. embodiment the antibody is selected from a panel of anti In another aspect, the antibody is an antibody fragment bodies because it has the desired affinity. Alternatively, or with an antigen-binding region that binds the BBB-R, 15 additionally, the antibody is engineered to have the desired including, but not limited to, Fab, Fab'. F(ab')2, and Fv. In affinity. The method optionally further comprises coupling another aspect, the antibody is a full-length antibody. the antibody with a therapeutic compound Such as a neuro In another embodiment, the invention provides the use of logical disorder drug. For example, the method can comprise an antibody that binds with low affinity to a BBB-R for the making a multispecific antibody which comprises a first manufacture of a medicament for treating a neurological antigen binding site which binds the BBB-R and a second disorder. Any of the foregoing described low-affinity anti antigen binding site which binds a brain antigen. BBB-R antibodies or any of the low-affinity anti-BBB-R The invention additionally provides a method of treating antibodies described elsewhere herein may be used in the a neurological disorder in a mammal comprising treating the method. mammal with a multispecific antibody that binds both a In another embodiment, the invention provides an anti 25 blood-brain barrier receptor (BBB-R) and a brain antigen, body that binds with low affinity to a BBB-R for use in wherein the anti-BBB-R antibody has been selected to have treating a neurological disorder. Any of the foregoing a low affinity for the BBB-R and thereby improves brain described low-affinity anti-BBB-R antibodies or any of the uptake of the anti-brain antigen antibody. Optionally, the low-affinity anti-BBB-R antibodies described elsewhere multispecific antibody binds both transferrin receptor (TfR) herein may be used in the method. Accordingly, in a first 30 and BACE1 or Abeta. aspect, the invention provides an antibody which binds to a It will be understood that any of the foregoing methods blood-brain barrier receptor (BBB-R), wherein the affinity and compositions of the invention may be combined with of the antibody for the BBB-R is from about 5 nM to about one another and/or with the further aspects of the invention 10 uM (e.g. from about 20 nM to about 1 uM). Optionally, described in the specification herein. the BBB-R is selected from the group consisting of trans 35 ferrin receptor (TfR), insulin receptor, insulin-like growth BRIEF DESCRIPTION OF THE FIGURES factor receptor (IGF receptor), low density lipoprotein receptor-related protein 1 (LRP1), low density lipoprotein FIGS. 1A-E depict significant brain vascular uptake of receptor-related protein 8 (LRP8), and heparin-binding epi systemically administered anti-TfR antibody. FIG. 1A dermal growth factor-like growth factor (HB-EGF). Option 40 shows brain uptake after IV administration of trace doses ally, the antibody is coupled with a therapeutic compound, (approximately 50 g/kg) of ''Ilanti-TfR and 'Icon Such as a neurological disorder drug. In one embodiment, trol IgG in mice and was quantified as a mean percentage of the antibody is a multispecific antibody which comprises a injected dose per gram of brain at 5, 30 min. 1, 4, 24, 48, first antigen binding site which binds the BBB-R and a and 72 hours after IV injection (n-6). Uptake of ''Ilanti second antigen binding site which binds a brain antigen, for 45 TfR was decreased by injection with 4 mg/kg unlabeled instance where the brain antigen is selected from the group anti-TfR (cold). FIG. 1B shows quantification of mean consisting of beta-secretase 1 (BACE1), amyloid beta antibody uptake in brain 1 and 24 hours after a 20 mg/kg IV (Abeta), epidermal growth factor receptor (EGFR), human injection of control IgG or anti-TfR (***p=0.0002, n=10). epidermal growth factor receptor 2 (HER2), Tau, apolipo FIG. 1C shows ratio of mean percent brain to serum con protein A3 (ApoE3), apolipoprotein E4 (ApoE4), alpha 50 centrations (**p=0.003, n=10). FIGS. 1D and 1E depict synuclein, CD20, Huntingtin, prion protein (PrP), leucine immunohistochemical staining of brain sections following rich repeat kinase 2 (LRRK2), parkin, presenilin 1, prese IV-injection with anti-TfR (FIG. 1D, upper panels), which nilin 2, gamma secretase, death receptor 6 (DR6), amyloid shows co-localization with anti-collagen IV, a vascular precursor protein (APP), p75 neurotrophin receptor marker (lower panel). IV-injection with control IgG (FIG. (p75NTR), and caspase 6. The antibody (e.g. multispecific 55 1E, upper panels) exhibits vascular distribution in brain only antibody) includes antibody fragments and full-length anti after 1 hour and an absence of antibody after 24 hours. Scale bodies. bar-50 um. In another embodiment, the invention provides a method FIGS. 2A-F show that affinity of anti-TfR antibodies and of transporting a therapeutic compound, Such as a neuro extent of brain uptake are inversely related when adminis logical disorder drug, across the blood-brain barrier com 60 tered at a therapeutically relevant dose (20 mg/kg) as prising exposing the anti-BBB-R antibody coupled with a compared to a low trace dose (approximately 50 g/kg). neurological disorder drug to the blood-brain barrier such FIG. 2A depicts a competitive binding ELISA in which that the antibody transports the neurological disorder drug increasing concentrations of anti-TfR'''P' variant anti coupled thereto across the blood-brain barrier. The blood bodies are used to compete against biotinylated TfR for brain barrier in this method may be in a mammal, e.g. one 65 binding to TfR. The anti-TfR competition ELISA was per with a neurological disorder, examples of which include: formed in Maxisorp plates (Neptune, N.J.) coated with 2.5 Alzheimer's disease (AD) (including, but not limited to, ug/ml of purified murine TfR extracellular domain in PBS at US 9,611,323 B2 17 18 4°C. overnight. Plates were washed with PBS/0.05% Tween in FIG. 3F average brain to serum ratio at 1, 12, 24, and 48 20 and blocked using Superblock blocking buffer in PBS hours after a 20 mg/kg IV injection of antibody in mice (Thermo Scientific, Hudson, N.H.). A titration of anti-TfR", (n=10). The experiments in FIGS. 3E and 3F was performed anti-TfR, anti-TfR, or anti-TfRP (1:3 serial dilution) was using the same protocol as the experiment described with combined with biotinylated anti-TfR (0.5 nM final concen regard to FIG. 1B. FIG. 3G shows immunohistochemical tration) and added to the plate for 1 hour at room tempera staining of brain sections from mice 24 hours after IV ture. Plates were washed with PBS/0.05% Tween 20, and injection with either anti-TfR/BACE 1 (left panels) or con HRP-streptavidin (Southern Biotech, Birmingham) was trol IgG (right panels). Co-localization of antibody with added to the plate and incubated for 1 hour at room tem NeuN is observed after anti-TfR/BACE 1 treatment (NeuN perature. Plates were washed with PBS/0.05% Tween 20, 10 neuronal staining coincides with pervasive antibody stain and biotinylated anti-TfR bound to the plate was detected ing) but absent in control IgG treated mice (only the NeuN using TMB substrate (BioFX Laboratories, Owings Mills) neuronal staining pattern is observed, no antibody staining). The results in FIG. 2A present data from a single experiment FIGS. 4A-E show that a single systemic dose of anti in which all five anti-TfR variants were separately assessed. TfR/BACE1 significantly reduces central and peripheral The IC50 values determined from this data are shown in 15 Af. FIGS. 4A-D show quantification of brain (A,B) and Table 2. FIG. 2B depicts quantification of mean brain uptake plasma (C.D) A? a levels after a 25 mg/kg or 50 mg/kg after IV-injection of trace doses (approximately 50 ug/kg) of IV-injection of control IgG, anti-BACE1, or anti-TfR/ the ''Ilanti-TfR'''P' variants after 5 min. 1, 4, 6, and BACE1. Briefly, for Abeta1-40 measurements, hemi-brains 24 hours (n=3). The results in FIG. 2B present data from a were homogenized in 5M guanidine hydrochloride buffer single experiment in which all five anti-TfR variants were and samples rotated for 3 hours at room temperature prior to separately assessed. FIG. 2C shows quantification of mean dilution (1:10) in 0.25% casein, 5 mM EDTA (pH 8.0) in brain uptake after 20 mg/kg IV injection of anti-TfR variants PBS containing freshly added aprotinin (20 mg/mL) and at 1 and 24 hours using the methods described with regard leupeptin (10 mg/ml). Diluted homogenates were centri to FIG. 1B. The experiment was replicated under the same fuged at 14,000 rpm for 20 min. and supernates were conditions using anti-TfR', and all results presented in FIG. 25 isolated for Abeta1-40 measurement. For antibody concen 2C. FIG. 2D is a model illustrating the inverse relationship tration measurements, the corresponding hemi-brain from between affinity and brain uptake. FIG. 2E is a comparison each mouse was homogenized in 1% NP-40 as described of immunohistochemical staining of brain sections after IV above. Whole blood was collected in EDTA microtainer injection with either the high affinity anti-TfR' or lower tubes (BD Diagnostics) prior to perfusion, centrifuged at affinity anti-TfR'' antibodies showing differences in anti 30 5,000xg for 15 minutes and the supernatant was isolated for body distribution (staining in left panels is for anti-TfR measuring plasma mouse Abeta 1-40 and anti-TfR/BACE1 alone) and extent of co-localization with NeuN (staining in concentrations. The concentrations of total mouse Abeta1 right panels is for both anti-TfR and NeuN). Scale bar=50 40 in plasma and brain were determined using a sandwich um. FIG. 2F is a representative high magnification image of ELISA following similar procedures described above. Rab anti-TfR localization in neurons (as indicated by NeuN 35 bit polyclonal antibody specific for the C-terminus of staining); this data shows that anti-TfRP and NeuN colocal Abeta1-40 (Millipore, Bedford Mass.) was coated onto ize and thus that anti-TfR traverses the BBB and interacts plates, and biotinylated anti-mouse Abeta monoclonal anti with neurons, whereas anti-TfR mainly localizes to the body M3.2 (Covance, Dedham Mass.) was used for detec vasculature as opposed to neurons. Scale bar 20 Lum. tion. The assay had lower limit of quantification values of FIGS. 3A-G show that a bispecific anti-TfR/BACE1 40 1.96 pg/ml in plasma and 39.1 pg/g in brain. Statistical antibody inhibits AB in vitro and accumulates in the brain. analysis of differences between experimental groups was FIG. 3A is a schematic model of a bispecific antibody which performed using a two-tailed unpaired t-test. * represent was engineered to bind both TfR and B-secretase (BACE1). significance compared to control IgG, while # represent FIG. 3B shows binding affinity for TfR of the parental significance compared to anti-BACE1. * p<0.05, ** p<0.01, anti-TfR and anti-TfR/BACE1 as measured by the anti 45 *** p<0.001; n=10 for all groups. FIG. 4E shows mean TfR competition ELISA assay described above for FIG. 2A. Af31-40 reduction from data in (A-D) calculated as a per FIG. 3C shows quantification of A? levels produced by centage of A? a levels relative to control IgG-injected HEK293 cells stably expressing APP after treatment with 1CC. anti-TfR/BACE1, anti-BACE1, and control IgG in a cell FIGS. 5A-B depict the light and heavy chain amino acid based assay. The ability of the antibodies to inhibit AB1-40 50 sequences of anti-BACE1 clone YW412.8 obtained from a production in HEK293 cells stably expressing wild-type naive sort of the natural diversity phage display library and human amyloid precursor protein was assessed as follows. affinity-matured forms of YW412.8. FIG. 5A depicts the HEK293-APPWT cells were seeded overnight at a density variable light (VL) sequence alignments (SEQID NOS. 1-6). of 3x10" cells/well in a 96-well plate. 50 ul of fresh media FIG. 5B depicts the variable heavy (VH) sequence align (DMEM+10% FBS) containing an anti-BACE1 antibody or 55 ments (SEQ ID Nos. 7-8). In both figures, the HVR a control IgG1 antibody was incubated with the cells for 24 sequences for each clone are indicated by the boxed regions, hours at 37° C. The cellular media was harvested and with the first box indicating HVR-L1 (FIG. 5A) or HVR-H1 assayed for the presence of AB1-40 using a AB1-40 HTRF(R) (FIG. 5B), the second box indicating HVR-L2 (FIG. 5A) or assay (CisBio) according to the manufacturers instructions. HVR-H2 (FIG. 5B), and the third box indicating HVR-L3 Af31-40 values were normalized for cell viability, as deter 60 (FIG. 5A) or HVR-H3 (FIG. 5B). mined using the CellTiter-Glo Luminescent Cell Viability FIGS. 6A-B depict the light and heavy chain amino acid Assay (Promega). Experiments were performed at least sequences of clone Fab 12 obtained from a naive sort of a three times, and each point in each experiment was repeated synthetic diversity phage display library and affinity-ma in duplicate. FIG. 3D depicts quantification of mean brain tured forms of Fab 12. FIG. 6A depicts the light chain uptake after trace doses of III-labeled antibody 30 min. 65 sequence alignments (SEQ ID NOS. 9-12). FIG. 6B depicts 6, 24, and 48 hours after IV-injection in mice (n=4). FIG.3E the heavy chain sequence alignments (SEQ ID NO. 13). In shows quantification of mean antibody uptake in brain and both figures, the HVR sequences for each clone are indicated US 9,611,323 B2 19 20 by the boxed regions, with the first box indicating HVR-L1 encompasses the blood-CSF barrier (choroid plexus) where (FIG. 6A) or HVR-H1 (FIG. 6B), the second box indicating the barrier is comprised of ependymal cells rather than HVR-L2 (FIG. 6A) or HVR-H2 (FIG. 6B), and the third box capillary endothelial cells. indicating HVR-L3 (FIG. 6A) or HVR-H3 (FIG. 6B). The “central nervous system” or “CNS’ refers to the FIGS. 7A-B depict the heavy chain (FIG. 7A: SEQ ID complex of nerve tissues that control bodily function, and NO. 14) and light chain (FIG. 7B: SEQ ID NO. 15) of an includes the brain and spinal cord. exemplary anti-Abeta antibody. A “blood-brain barrier receptor” (abbreviated “BBB-R” FIGS. 8A-B depict the quantification of anti-TfR'''''' herein) is a transmembrane receptor protein expressed on in the serum (FIG. 8A) and brain (FIG. 8B) after a single brain endothelial cells which is capable of transporting 10 molecules across the blood-brain barrier. Examples of therapeutic dose administration in mice. Six-eight week old BBB-R herein include: transferrin receptor (TfR), insulin wild type female C57B/6 mice were used for all studies. receptor, insulin-like growth factor receptor (IGF-R), low Mice were intravenously injected with 20 mg/kg of anti-TfR density lipoprotein receptors including without limitation variants or control IgG. Antibody levels in brain and serum low density lipoprotein receptor-related protein 1 (LRP1) were measured at 1 and 12 hours and 1, 2, 4, 5, 6, and 8 days 15 and low density lipoprotein receptor-related protein 8 post injection. Total injection volume did not exceed 260 uL (LRP8), and heparin-binding epidermal growth factor-like and antibodies were diluted in D-PBS (Invitrogen) when growth factor (HB-EGF). An exemplary BBB-R herein is necessary. The experiment was performed using the same transferrin receptor (TfR). protocol as the experiment whose results are shown in FIG. The “transferrin receptor” (“TfR') is a transmembrane 1B. glycoprotein (with a molecular weight of about 180,000) FIGS. 9A-E show the varying degrees to which bispecific composed of two disulphide-bonded sub-units (each of anti-TfR'''/BACE1 antibodies accumulate in the brain apparent molecular weight of about 90,000) involved in iron and inhibit AB production in vivo. FIG. 9A depicts the uptake in vertebrates. In one embodiment, the TfR herein is results of an anti-TfR competition ELISA assay using anti human TfR comprising the amino acid sequence as in TfR'''/BACE1, following the same assay procedure as 25 Schneider et al. Nature 311: 675-678 (1984), for example. that described in FIG. 2A. The IC50 values determined from A “neurological disorder” as used herein refers to a this data are shown in Table 3. FIGS. 9B and 9D quantitate disease or disorder which affects the CNS and/or which has the amount of observed antibody (9B) and the amount of an etiology in the CNS. Exemplary CNS diseases or disor Abeta1-40 (9D) observed in the plasma at 1, 2, 4, 6, 8 and ders include, but are not limited to, neuropathy, amyloidosis, 30 cancer, an ocular disease or disorder, viral or microbial 10 days after a 50 mg/kg IV injection of antibodies in mice infection, inflammation, ischemia, neurodegenerative dis (n-6). FIG.9C depicts quantification of mean brain uptake ease, seizure, behavioral disorders, and a lysosomal storage and FIG. 9E depicts the amount of Abeta1-40 observed in disease. For the purposes of this application, the CNS will be the brains of those same treated mice at 1, 2, 4, 6, 8 and 10 understood to include the eye, which is normally seques days after treatment. Six to eight week-old wild type female 35 tered from the rest of the body by the blood-retina barrier. C57B76 mice were used for all studies. Mice were intrave Specific examples of neurological disorders include, but are nously injected with 50 mg/kg anti-TfR/BACE1 variants, not limited to, neurodegenerative diseases (including, but control IgG, or anti-BACE1. After the indicated time, mice not limited to, Lewy body disease, postpoliomyelitis Syn were perfused with D-PBS, and brain and plasma antibody drome, Shy-Draeger syndrome, olivopontocerebellar atro concentration for each animal was measured as described 40 phy, Parkinson's disease, multiple system atrophy, striatoni above. The assay was performed as described in the FIG. 4 gral degeneration, tauopathies (including, but not limited to, description. Alzheimer disease and Supranuclear palsy), prion diseases FIGS. 10A-B and 11A-B show the varying degrees to (including, but not limited to, bovine spongiform encepha which bispecific anti-TfR'Abeta antibodies accumulate lopathy, Scrapie, Creutzfeldt-Jakob Syndrome, kuru, Gerst in the brain of PS2APP mice (FIG. 10) and wild type mice 45 mann-Straussler-Scheinker disease, chronic wasting disease, (FIG. 11). FIGS. 10A and 11A depict quantification of the and fatal familial insomnia), bulbar palsy, motor neuron amount of observed antibody in the plasma 1 day after a 50 disease, and nervous system heterodegenerative disorders mg/kg i.p. injection of antibodies in mice (n=4-6). FIGS. (including, but not limited to, Canavan disease, Hunting 10B and 11B quantitate mean brain uptake in the same ton's disease, neuronal ceroid-lipofuscinosis, Alexanders treated mice. 50 disease, Tourette’s syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, lafora DETAILED DESCRIPTION OF EMBODIMENTS disease, Rett syndrome, hepatolenticular degeneration, OF THE INVENTION Lesch-Nyhan syndrome, and Unverricht-Lundborg Syn drome), dementia (including, but not limited to, Pick's I. Definitions 55 disease, and spinocerebellar ataxia), cancer (e.g. of the CNS and/or brain, including brain metastases resulting from The “blood-brain barrier” or “BBB” refers to the physi cancer elsewhere in the body). ological barrier between the peripheral circulation and the A "neurological disorder drug is a drug or therapeutic brain and spinal cord which is formed by tight junctions agent that treats one or more neurological disorder(s). Neu within the brain capillary endothelial plasma membranes, 60 rological disorder drugs of the invention include, but are not creating a tight barrier that restricts the transport of mol limited to, antibodies, peptides, proteins, natural ligands of ecules into the brain, even very small molecules such as urea one or more CNS target(s), modified versions of natural (60 Daltons). The blood-brain barrier within the brain, the ligands of one or more CNS target(s), aptamers, inhibitory blood-spinal cord barrier within the spinal cord, and the nucleic acids (i.e., small inhibitory RNAs (siRNA) and short blood-retinal barrier within the retina are contiguous capil 65 hairpin RNAs (shRNA)), ribozymes, and small molecules, lary barriers within the CNS, and are herein collectively or active fragments of any of the foregoing. Exemplary referred to a the blood-brain barrier or BBB. The BBB also neurological disorder drugs of the invention are described US 9,611,323 B2 21 22 herein and include, but are not limited to: antibodies, aptam which is incorporated herein by reference in its entirety. ers, proteins, peptides, inhibitory nucleic acids and Small Several other isoforms of human BACE1 exist including molecules and active fragments of any of the foregoing that isoforms B, C and D. See UniProtKB/Swiss-Prot Entry either are themselves or specifically recognize and/or act P56817, which is incorporated herein by reference in its upon (i.e., inhibit, activate, or detect) a CNS antigen or target entirety. molecule Such as, but not limited to, amyloid precursor The terms “anti-beta-secretase antibody”, “anti-BACE1 protein or portions thereof, amyloid beta, beta-secretase, antibody”, “an antibody that binds to beta-secretase' and gamma-secretase, tau, alpha-synuclein, parkin, huntingtin, “an antibody that binds to BACE1’ refer to an antibody that DR6, presenilin, ApoE, glioma or other CNS cancer mark is capable of binding BACE1 with sufficient affinity such ers, and neurotrophins. Non-limiting examples of neurologi 10 that the antibody is useful as a diagnostic and/or therapeutic cal disorder drugs and disorders they may be used to treat are agent in targeting BACE1. In one embodiment, the extent of provided in the following Table 1: binding of an anti-BACE1 antibody to an unrelated, non BACE1 protein is less than about 10% of the binding of the TABLE 1. antibody to BACE1 as measured, e.g., by a radioimmuno 15 assay (RIA). In certain embodiments, an antibody that binds Non-limiting examples of neurological disorder drugs and to BACE1 has a dissociation constant (Kd) of s1 LM, s100 the corresponding disorders they may be used to treat nM, is 10 nM, s1 nM, s(). 1 nM, s().01 nM, or s().001 nM Drug Neurological disorder (e.g. 10M or less, e.g. from 10M to 10M, e.g., from Anti-BACE1 Antibody Alzheimers, acute and chronic 10M to 10'M). In certain embodiments, an anti-BACE1 brain injury, stroke antibody binds to an epitope of BACE 1 that is conserved Anti-Abeta Antibody Alzheimer's disease among BACE1 from different species and isoforms. In one Neurotrophin Stroke, acute brain injury, spinal embodiment, an antibody is provided that binds to the cord injury epitope on BACE1 bound by anti-BACE1 antibody Brain-derived neurotrophic Chronic brain injury (Neurogenesis) factor (BDNF), Fibroblast YW412.8.31. In other embodiments, an antibody is pro growth factor 2 (FGF-2) 25 vided that binds to an exosite within BACE1 located in the Anti-Epidermal Growth Factor Brain cancer catalytic domain of BACE1. In one embodiment an antibody Receptor (EGFR)-antibody is provided that competes with the peptides identified in Glial cell-line derived Parkinson's disease neural factor (GDNF) Kornacker et al., Biochem. 44:11567-11573 (2005), which is Brain-derived neurotrophic Amyotrophic lateral Sclerosis, incorporated herein by reference in its entirety, (i.e., Pep factor (BDNF) depression 30 tides 1, 2, 3, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 2-12, 3-12, 4-12, Lysosomal enzyme Lysosomal storage disorders of the brain 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, 4, 5, 6, 5-10, 5-9, Ciliary neurotrophic factor Amyotrophic lateral Sclerosis scrambled, Y5A, P6A, Y7A, F8A, I9A, P10A and L11A) for (CNTF) binding to BACE1. Exemplary BACE1 antibody sequences Neuregulin-1 Schizophrenia are depicted in FIGS. 5A-B and FIGS. 6A-B. One exem Anti-HER2 antibody (e.g. Brain metastasis from HER2-positive 35 plary antibody herein comprises the variable domains of the trastuzumab) C8Ce antibody YW412.8.31 (e.g. as in FIGS. 5A-B). A “native sequence' protein herein refers to a protein An "imaging agent' is a compound that has one or more comprising the amino acid sequence of a protein found in properties that permit its presence and/or location to be nature, including naturally occurring variants of the protein. detected directly or indirectly. Examples of Such imaging 40 The term as used herein includes the protein as isolated from agents include proteins and Small molecule compounds a natural source thereof or as recombinantly produced. incorporating a labeled moiety that permits detection. The term “antibody' herein is used in the broadest sense A “CNS antigen' or “brain antigen' is an antigen and specifically covers monoclonal antibodies, polyclonal expressed in the CNS, including the brain, which can be antibodies, multispecific antibodies (e.g. bispecific antibod targeted with an antibody or Small molecule. Examples of 45 ies) formed from at least two intact antibodies, and antibody Such antigens include, without limitation: beta-secretase 1 fragments so long as they exhibit the desired biological (BACE1), amyloid beta (Abeta), epidermal growth factor activity. receptor (EGFR), human epidermal growth factor receptor 2 "Antibody fragments' herein comprise a portion of an (HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, intact antibody which retains the ability to bind antigen. CD20, huntingtin, prion protein (PrP), leucine rich repeat 50 Examples of antibody fragments include Fab, Fab'. F(ab')2. kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma and FV fragments; diabodies; linear antibodies; single-chain secretase, death receptor 6 (DR6), amyloid precursor protein antibody molecules; and multispecific antibodies formed (APP), p75 neurotrophin receptor (p75NTR), and caspase 6. from antibody fragments. In one embodiment, the antigen is BACE1. The term “monoclonal antibody” as used herein refers to The term “BACE1, as used herein, refers to any native 55 an antibody obtained from a population of Substantially beta-secretase 1 (also called B-site amyloid precursor protein homogeneous antibodies, i.e., the individual antibodies cleaving enzyme 1, membrane-associated aspartic protease comprising the population are identical and/or bind the same 2, memapsin 2, aspartyl protease 2 or Asp2) from any epitope, except for possible variants that may arise during vertebrate source, including mammals such as primates (e.g. production of the monoclonal antibody. Such variants gen humans) and rodents (e.g., mice and rats), unless otherwise 60 erally being present in minor amounts. In contrast to poly indicated. The term encompasses “full-length,’ unprocessed clonal antibody preparations that typically include different BACE1 as well as any form of BACE1 which results from antibodies directed against different determinants (epitopes), processing in the cell. The term also encompasses naturally each monoclonal antibody is directed against a single deter occurring variants of BACE1, e.g., splice variants or allelic minant on the antigen. In addition to their specificity, the variants. The amino acid sequence of an exemplary BACE1 65 monoclonal antibodies are advantageous in that they are polypeptide is the sequence for human BACE1, isoform A uncontaminated by other immunoglobulins. The modifier as reported in Vassar et al., Science 286:735-741 (1999), “monoclonal indicates the character of the antibody as US 9,611,323 B2 23 24 being obtained from a substantially homogeneous popula absence of endogenous immunoglobulin production (see, tion of antibodies, and is not to be construed as requiring e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 production of the antibody by any particular method. For (1993); Jakobovits et al., Nature, 362:255-258 (1993); Brug example, the monoclonal antibodies to be used in accor germann et al., Year in Immuno., 7:33 (1993); and U.S. Pat. dance with the present invention may be made by the Nos. 5,591,669, 5,589,369 and 5,545,807)); selection from hybridoma method first described by Kohler et al., Nature, phage display libraries expressing human antibodies or 256:495 (1975), or may be made by recombinant DNA human antibody fragments (see, for example, McCafferty et methods (see, e.g., U.S. Pat. No. 4,816,567). The “mono al., Nature 348:552-553 (1990); Johnson et al., Current clonal antibodies' may also be isolated from phage antibody Opinion in Structural Biology 3:564-571 (1993); Clackson libraries using the techniques described in Clackson et al., 10 et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991); Griffith et al., EMBO J. 12:725-734 222:581-597 (1991), for example. Specific examples of (1993); U.S. Pat. Nos. 5,565,332 and 5,573,905); generation monoclonal antibodies herein include chimeric antibodies, via in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 humanized antibodies, and human antibodies, including and 5.229,275); and isolation from human antibody produc antigen-binding fragments thereof. 15 ing hybridomas. The monoclonal antibodies herein specifically include A “multispecific antibody' herein is an antibody having "chimeric' antibodies (immunoglobulins) in which a portion binding specificities for at least two different epitopes. of the heavy and/or light chain is identical with or homolo Exemplary multispecific antibodies may bind both a BBB-R gous to corresponding sequences in antibodies derived from and a brain antigen. Multispecific antibodies can be prepared a particular species or belonging to a particular antibody as full-length antibodies or antibody fragments (e.g. F(ab') class or Subclass, while the remainder of the chain(s) is bispecific antibodies). Engineered antibodies with two, three identical with or homologous to corresponding sequences in or more (e.g. four) functional antigen binding sites are also antibodies derived from another species or belonging to contemplated (see, e.g., US Appln No. US 2002/0004587 another antibody class or Subclass, as well as fragments of A1, Miller et al.). Multispecific antibodies can be prepared Such antibodies, so long as they exhibit the desired biologi 25 as full length antibodies or antibody fragments. cal activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Antibodies herein include “amino acid sequence variants' Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimericanti with altered antigen-binding or biological activity. Examples bodies of interest herein include “primatized' antibodies of Such amino acid alterations include antibodies with comprising variable domain antigen-binding sequences enhanced affinity for antigen (e.g. "affinity matured anti derived from a non-human primate (e.g. Old World Monkey, 30 bodies), and antibodies with altered Fc region, if present, Such as baboon, rhesus or cynomolgus monkey) and human e.g. with altered (increased or diminished) antibody depen constant region sequences (U.S. Pat. No. 5,693,780). dent cellular cytotoxicity (ADCC) and/or complement “Humanized forms of non-human (e.g., murine) anti dependent cytotoxicity (CDC) (see, for example, WO bodies are chimeric antibodies that contain minimal 00/42072, Presta, L. and WO 99/51642, Iduosogie et al.); sequence derived from non-human immunoglobulin. For the 35 and/or increased or diminished serum half-life (see, for most part, humanized antibodies are human immunoglobu example, WO00/42072, Presta, L.). lins (recipient antibody) in which residues from a hyper An “affinity modified variant' has one or more substituted variable region of the recipient are replaced by residues from hyperVariable region or framework residues of a parent a hyperVariable region of a non-human species (donor antibody (e.g. of a parent chimeric, humanized, or human antibody) Such as mouse, rat, rabbit or nonhuman primate 40 antibody) that alter (increase or reduce) affinity. In one having the desired specificity, affinity, and capacity. In some embodiment, the resulting variant(s) selected for further instances, framework region (FR) residues of the human development will have reduced affinity for the BBB-R immunoglobulin are replaced by corresponding non-human according to the present invention. A convenient way for residues. Furthermore, humanized antibodies may comprise generating Such substitutional variants uses phage display. residues that are not found in the recipient antibody or in the 45 Briefly, several hypervariable region sites (e.g. 6-7 sites) are donor antibody. These modifications are made to further mutated to generate all possible amino Substitutions at each refine antibody performance. In general, the humanized site. The antibody variants thus generated are displayed in a antibody will comprise substantially all of at least one, and monovalent fashion from filamentous phage particles as typically two, variable domains, in which all or substantially fusions to the gene III product of M13 packaged within each all of the hypervariable regions correspond to those of a 50 particle. The phage-displayed variants are then screened for non-human immunoglobulin and all or Substantially all of their biological activity (e.g. binding affinity). In order to the FRS are those of a human immunoglobulin sequence, identify candidate hypervariable region sites for modifica except for FR substitution(s) as noted above. The humanized tion, alanine Scanning mutagenesis can be performed to antibody optionally also will comprise at least a portion of identify hyperVariable region residues contributing signifi an immunoglobulin constant region, typically that of a 55 cantly to antigen binding. Alternatively, or additionally, it human immunoglobulin. For further details, see Jones et al., may be beneficial to analyze a crystal structure of the Nature 321:522-525 (1986); Riechmann et al., Nature 332: antigen-antibody complex to identify contact points between 323-329 (1988); and Presta, Curr: Op. Struct. Biol. 2:593 the antibody and its target. Such contact residues and 596 (1992). neighboring residues are candidates for Substitution accord A "human antibody' herein is one comprising an amino 60 ing to the techniques elaborated herein. Once Such variants acid sequence structure that corresponds with the amino acid are generated, the panel of variants is subjected to Screening sequence structure of an antibody obtainable from a human and antibodies with altered affinity may be selected for B-cell, and includes antigen-binding fragments of human further development. antibodies. Such antibodies can be identified or made by a The antibody herein may be conjugated with a "heterolo variety of techniques, including, but not limited to: produc 65 gous molecule' for example to increase half-life or stability tion by transgenic animals (e.g., mice) that are capable, upon or otherwise improve the antibody. For example, the anti immunization, of producing human antibodies in the body may be linked to one of a variety of non-proteinaceous US 9,611,323 B2 25 26 polymers, e.g., polyethylene glycol (PEG), polypropylene three-dimensional configurations of different classes of glycol, polyoxyalkylenes, or copolymers of polyethylene immunoglobulins are well known. glycol and polypropylene glycol. Antibody fragments. Such The term “recombinant antibody', as used herein, refers as Fab', linked to one or more PEG molecules are an to an antibody (e.g. a chimeric, humanized, or human exemplary embodiment of the invention. 5 antibody or antigen-binding fragment thereof) that is The antibody herein may be a “glycosylation variant” expressed by a recombinant host cell comprising nucleic such that any carbohydrate attached to the Fc region, if acid encoding the antibody. Examples of “host cells' for present, is altered. For example, antibodies with a mature producing recombinant antibodies include: (1) mammalian carbohydrate structure that lacks fucose attached to an Fc cells, for example, Chinese Hamster Ovary (CHO), COS, 10 myeloma cells (including YO and NS0 cells), baby hamster region of the antibody are described in US Pat Appl No US kidney (BHK), Hela and Vero cells; (2) insect cells, for 2003/0157108 (Presta, L.). See also US 2004/0093621 example, Sf9. Sf21 and Tn5; (3) plant cells, for example (Kyowa Hakko Kogyo Co., Ltd). Antibodies with a bisect plants belonging to the genus Nicotiana (e.g. Nicotiana ing N-acetylglucosamine (GlcNAc) in the carbohydrate tabacum); (4) yeast cells, for example, those belonging to attached to an Fc region of the antibody are referenced in 15 the genus Saccharomyces (e.g. Saccharomyces cerevisiae) WO 2003/011878, Jean-Mairet et al. and U.S. Pat. No. or the genus Aspergillus (e.g. Aspergillus niger); (5) bacte 6,602,684. Umana et al. Antibodies with at least one galac rial cells, for example Escherichia. coli cells or Bacillus tose residue in the oligosaccharide attached to an Fc region subtilis cells, etc. of the antibody are reported in WO 1997/30087, Patel et al. As used herein, “specifically binding or “binds specifi See also, WO 1998/58964 (Raju, S.) and WO 1999/22764. 20 cally to refers to an antibody selectively or preferentially (Raju, S.) concerning antibodies with altered carbohydrate binding to an antigen. The binding affinity is generally attached to the Fc region thereof. See also US 2005/0123546 determined using a standard assay, Such as Scatchard analy (Umana et al.) describing antibodies with modified glyco sis, or Surface plasmon resonance technique (e.g. using Sylation. BIACORE(R). The term “hypervariable region' when used herein refers 25 An “antibody that binds to the same epitope' as a refer to the amino acid residues of an antibody that are responsible ence antibody refers to an antibody that blocks binding of for antigen binding. The hyperVariable region comprises the reference antibody to its antigen in a competition assay amino acid residues from a “complementarity determining by 50% or more, and conversely, the reference antibody region' or “CDR (e.g. residues 24-34 (L1), 50-56 (L2) and blocks binding of the antibody to its antigen in a competition 89-97 (L3) in the light chain variable domain and 31-35 30 assay by 50% or more. In one embodiment, an anti-BACE1 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain antibody binds to the BACE1 epitope bound by variable domain; Kabat et al., Sequences of Proteins of YW412.8.31 Immunological Interest, 5th Ed. Public Health Service, The term “cytotoxic agent as used herein refers to a National Institutes of Health, Bethesda, Md. (1991)) and/or substance that inhibits or prevents a cellular function and/or those residues from a “hypervariable loop' (e.g. residues 35 causes cell death or destruction. Cytotoxic agents include, 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain but are not limited to, radioactive isotopes (e.g., At', I'', variable domain and 26-32 (H1), 53-55 (H2) and 96-101 Il 25, y90, Re86, Re88, Sm', Bi212, P32, Pb’’ and radio (H3) in the heavy chain variable domain; Chothia and Lesk active isotopes of Lu); chemotherapeutic agents or drugs J. Mol. Biol. 196:901-917 (1987)). “Framework” or “FR” (e.g., methotrexate, adriamicin, Vinca alkaloids (Vincristine, residues are those variable domain residues other than the 40 vinblastine, etoposide), doxorubicin, melphalan, mitomycin hyperVariable region residues as herein defined. C. chlorambucil, daunorubicin or other intercalating agents); A “full length antibody' is one which comprises an growth inhibitory agents; enzymes and fragments thereof antigen-binding variable region as well as a light chain Such as nucleolytic enzymes; antibiotics; toxins such as constant domain (CL) and heavy chain constant domains, Small molecule toxins or enzymatically active toxins of CH1, CH2 and CH3. The constant domains may be native 45 bacterial, fungal, plant or animal origin, including fragments sequence constant domains (e.g. human native sequence and/or variants thereof. constant domains) or amino acid sequence variants thereof. An "effective amount of an agent, e.g., a pharmaceutical A“naked antibody' is an antibody (as herein defined) that formulation, refers to an amount effective, at dosages and for is not conjugated to a heterologous molecule, such as a periods of time necessary, to achieve the desired therapeutic cytotoxic moiety, polymer, or radiolabel. 50 or prophylactic result. Antibody “effector functions’ refer to those biological The term “Fc region' herein is used to define a C-terminal activities attributable to the Fc region (a native sequence Fc region of an immunoglobulin heavy chain that contains at region or amino acid sequence variant Fc region) of an least a portion of the constant region. The term includes antibody. Examples of antibody effector functions include native sequence Fc regions and variant Fc regions. In one C1q binding, complement dependent cytotoxicity (CDC), Fc 55 embodiment, a human IgG heavy chain Fc region extends receptor binding, antibody-dependent cell-mediated cyto from Cys226, or from Pro230, to the carboxyl-terminus of toxicity (ADCC), etc. In one embodiment, the antibody the heavy chain. However, the C-terminal lysine (Lys447) of herein essentially lacks effector function. the Fc region may or may not be present. Unless otherwise Depending on the amino acid sequence of the constant specified herein, numbering of amino acid residues in the Fc domain of their heavy chains, full length antibodies can be 60 region or constant region is according to the EU numbering assigned to different “classes”. There are five major classes system, also called the EU index, as described in Kabat et of full length antibodies: IgA, Ig), IgE. IgG, and IgM, and al., Sequences of Proteins of Immunological Interest, 5th Ed. several of these may be further divided into “subclasses' Public Health Service, National Institutes of Health, (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The Bethesda, Md., 1991. heavy-chain constant domains that correspond to the differ- 65 “Framework or “FR refers to variable domain residues ent classes of antibodies are called alpha, delta, epsilon, other than hypervariable region (HVR) residues. The FR of gamma, and mu, respectively. The Subunit structures and a variable domain generally consists of four FR domains: US 9,611,323 B2 27 28 FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR II. Compositions and Methods sequences generally appear in the following sequence in VH (or VL): FR1-H1 (L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. II. Production of Anti-BBB-R Antibodies and An “immunoconjugate' is an antibody conjugated to one Conjugates Thereof or more heterologous molecule(s), including but not limited 5 to a label or cytotoxic agent. Optionally such conjugation is The methods and articles of manufacture of the present via a linker. invention use, or incorporate, an antibody that binds to A “linker as used herein is a structure that covalently or BBB-R. The BBB-Rantigen to be used for production of, or non-covalently connects the anti-BBB-R antibody to heter screening for, antibodies may be, e.g., a soluble form of or 10 a portion thereof (e.g. the extracellular domain), containing ologous molecule. In certain embodiments, a linker is a the desired epitope. Alternatively, or additionally, cells peptide. In other embodiments, a linker is a chemical linker. expressing BBB-R at their cell surface can be used to A “label' is a marker coupled with the antibody herein generate, or screen for, antibodies. Other forms of BBB-R and used for detection or imaging. Examples of Such labels useful for generating antibodies will be apparent to those include: radiolabel, a fluorophore, a chromophore, or an 15 skilled in the art. Examples of BBB-Rs herein include affinity tag. In one embodiment, the label is a radiolabel used transferrin receptor (TfR), insulin receptor, insulin-like for medical imaging, for exampletc99m or I123, or a spin growth factor receptor (IGF-R), low density lipoprotein label for nuclear magnetic resonance (NMR) imaging (also receptor-related protein 1 (LRP1) and LRP8 etc., and hepa known as magnetic resonance imaging, mri). Such as iodine rin-binding epidermal growth factor-like growth factor (HB 123 again, iodine-131, indium-111, fluorine-19, carbon-13, EGF). nitrogen-15, oxygen-17, gadolinium, manganese, iron, etc. According to the present invention, a “low affinity' anti An “individual' or “subject' is a mammal. Mammals BBB-R (e.g. anti-TfR) antibody is selected based on the data include, but are not limited to, domesticated animals (e.g., herein demonstrating that such antibodies display improved cows, sheep, cats, dogs, and horses), primates (e.g., humans CNS (for example, brain) uptake. In order to identify such and non-human primates such as monkeys), rabbits, and 25 low affinity antibodies, various assays for measuring anti rodents (e.g., mice and rats). In certain embodiments, the body affinity are available including, without limitation: individual or Subject is a human. Scatchard assay and Surface plasmon resonance technique An "isolated antibody is one which has been separated (e.g. using BIACORE(R). According to one embodiment of from a component of its natural environment. In some the invention, the antibody has an affinity for the BBB-R embodiments, an antibody is purified to greater than 95% or 30 antigen (e.g. for TfR) from about 5 nM, or from about 20 99% purity as determined by, for example, electrophoretic nM, or from about 100 nM, to about 10 uM, or to about 1 (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary elec uM, or to about 500 nM. Thus, the affinity may be in the trophoresis) or chromatographic (e.g., ion exchange or range from about 5 nM to about 10 uM, or in the range from reverse phase HPLC). For review of methods for assessment about 20 nM to about 1 uM, or in the range from about 100 of antibody purity, see, e.g., Flatman et al., J. Chromatogr: 35 nM to about 500 nM, e.g. as measured by Scatchard analysis B 848:79-87 (2007). or BIACORE(R). The term “package insert” is used to refer to instructions Thus, the invention provides a method of making an customarily included in commercial packages of therapeutic antibody useful for transporting a neurological disorder drug products, that contain information about the indications, across the blood-brain barrier comprising selecting an anti usage, dosage, administration, combination therapy, con 40 body from a panel of antibodies against a blood-brain barrier traindications and/or warnings concerning the use of Such receptor (BBB-R) because it has an affinity for the BBB-R therapeutic products. which is in the range from about 5 nM, or from about 20 nM, The term “pharmaceutical formulation” refers to a prepa or from about 100 nM, to about 10 uM, or to about 1 uM, ration which is in Such form as to permit the biological or to about 500 mM. Thus, the affinity may be in the range activity of an active ingredient contained therein to be 45 from about 5 nM to about 10 uM or in the range from about effective, and which contains no additional components 20 nM to about 1 uM, or in the range from about 100 nM to which are unacceptably toxic to a subject to which the about 500 nM, e.g. as measured by Scatchard analysis or formulation would be administered. BIACORE(R). As will be understood by one of ordinary skill A “pharmaceutically acceptable carrier refers to an in the art, conjugating a heterologous molecule/compound to ingredient in a pharmaceutical formulation, other than an 50 an antibody will often decrease the affinity of the antibody active ingredient, which is nontoxic to a subject. A pharma for its target due, e.g., to steric hindrance or even to ceutically acceptable carrier includes, but is not limited to, elimination of one binding arm if the antibody is made a buffer, excipient, stabilizer, or preservative. multispecific with one or more arms binding to a different As used herein, “treatment' (and grammatical variations antigen than the antibody's original target. In one embodi thereof such as “treat' or “treating) refers to clinical 55 ment, a low affinity antibody of the invention specific for intervention in an attempt to alter the natural course of the TfR conjugated to BACE1 had a Kd for TfR as measured by individual being treated, and can be performed either for BIACORE of about 30 nM. In another embodiment, a low prophylaxis or during the course of clinical pathology. affinity antibody of the invention specific for TfR conjugated Desirable effects of treatment include, but are not limited to, to BACE1 had a Kd for TfR as measured by BIACORE of preventing occurrence or recurrence of disease, alleviation 60 about 600 nM. of symptoms, diminishment of any direct or indirect patho One exemplary assay for evaluating antibody affinity is by logical consequences of the disease, preventing metastasis, Scatchard analysis. For example, the anti-BBB-R antibody decreasing the rate of disease progression, amelioration or of interest can be iodinated using the lactoperoxidase palliation of the disease state, and remission or improved method (Bennett and Horuk, Methods in Enzymology 288 prognosis. In some embodiments, antibodies of the inven 65 pg. 134-148 (1997)). A radiolabeled anti-BBB-R antibody is tion are used to delay development of a disease or to slow purified from free 'I-Na by gel filtration using a NAP-5 the progression of a disease. column and its specific activity measured. Competition US 9,611,323 B2 29 30 reaction mixtures of 50 LL containing a fixed concentration An exemplary BIACORE(R) analysis using the composi of iodinated antibody and decreasing concentrations of tions of the invention may be performed as follows. Kd was serially diluted unlabeled antibody are placed into 96-well measured using Surface plasmon resonance assays using a plates. Cells transiently expressing BBB-R are cultured in BIACORE(R)-2000 (BIAcore, Inc., Piscataway, N.J.) at 25° growth media, consisting of Dulbecco's modified eagle's C. using anti-human Fc kit (BiAcore Inc., Piscataway, N.J.). medium (DMEM) (Genentech) supplemented with 10% Briefly, carboxymethylated dextran biosensor chips (CMS, FBS, 2 mM L-glutamine and 1x penicillin-streptomycin at BIACORE, Inc.) were activated with N-ethyl-N'-(3-dimeth 37°C. in 5% CO. Cells are detached from the dishes using ylaminopropyl)-carbodiimide hydrochloride (EDC) and Sigma Cell Dissociation Solution and washed with binding N-hydroxysuccinimide (NHS) according to the supplier's buffer (DMEM with 1% bovine serum albumin, 50 mM 10 instructions. Anti-human Fc antibody was diluted with 10 HEPES, pH 7.2, and 0.2% sodium azide). The washed cells mM sodium acetate, pH 4.0, to 50 ug/ml before injection at are added at an approximate density of 200,000 cells in 0.2 a flow rate of 5 ul/minute to achieve approximately 10000 mL of binding buffer to the 96-well plates containing the response units (RU) of coupled protein. Following the 50-uL competition reaction mixtures. The final concentra injection of antibody, 1 M ethanolamine was injected to tion of the unlabeled antibody in the competition reaction 15 block unreacted groups. For kinetics measurements, anti with cells is varied, starting at 1000 nM and then decreasing TfR antibody variants were injected in HBS-P to reach about by 1:2 fold dilution for 10 concentrations and including a 220 RU, then two-fold serial dilutions of MuTfR-His (0.61 Zero-added, buffer-only sample. Competition reactions with nM to 157 nM) were injected in HBS-Pat 25° C. at a flow cells for each concentration of unlabeled antibody are rate of approximately 30 ul/min. Association rates (kon) and assayed in triplicate. Competition reactions with cells are dissociation rates (koff) were calculated using a simple incubated for 2 hours at room temperature. After the 2-hour one-to-one Langmuir binding model (BIACORE(R) Evalua incubation, the competition reactions are transferred to a tion Software version 3.2) by simultaneously fitting the filter plate and washed four times with binding buffer to association and dissociation sensorgrams. The equilibrium separate free from bound iodinated antibody. The filters are dissociation constant (Kd) was calculated as the ratio koff counted by gamma counter and the binding data are evalu 25 kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999) ated using the fitting algorithm of Munson and Rodbard According to another embodiment, Kd is measured using (1980) to determine the binding affinity of the antibody. surface plasmon resonance assays with a BIACORE(R)-2000 An exemplary scatchard analysis using the compositions device (BIAcore, Inc., Piscataway, N.J.) at 25° C. using of the invention may be performed as follows. Anti-TFR" anti-human Fc kit (BiAcore Inc., Piscataway, N.J.). Briefly, was iodinated using the lactoperoxidase method (Bennett 30 carboxymethylated dextran biosensor chips (CMS, BIA and Horuk, Methods in Enzymology 288 pg. 134-148 CORE, Inc.) are activated with N-ethyl-N'-(3-dimethylam (1997)). Radiolabeled anti-TFR was purified from free inopropyl)-carbodiimide hydrochloride (EDC) and N-hy 'I-Na by gel filtration using a NAP-5 column; purified droxysuccinimide (NHS) according to the supplier's anti-TFR had a specific activity of 19.82 uCi/ug. Compe instructions. Anti-human Fc antibody is diluted with 10 mM tition reaction mixtures of 50 uL containing a fixed concen 35 sodium acetate, pH 4.0, to 50 g/ml before injection at a flow tration of iodinated antibody and decreasing concentrations rate of 5ul/minute to achieve approximately 10000 response of serially diluted unlabeled antibody were placed into units (RU) of coupled protein. Following the injection of 96-well plates. The 293 cells transiently expressing murine antibody, 1 M ethanolamine is injected to block unreacted TfR were cultured in growth media, consisting of Dulbec groups. For kinetics measurements, anti-BBB-R antibody co's modified eagle's medium (DMEM) (Genentech) 40 variants are injected in HBS-P to reach about 220 RU, then supplemented with 10% FBS, 2 mM L-glutamine and 1 x two-fold serial dilutions of BBB-R-His (0.61 nM to 157 nM) penicillin-streptomycin at 37° C. in 5% CO. Cells were are injected in HBS-P at 25° C. at a flow rate of approxi detached from the dishes using Sigma Cell Dissociation mately 30 ul/min. Association rates (kon) and dissociation Solution and washed with binding buffer (DMEM with 1% rates (koff) are calculated using a simple one-to-one Lang bovine serum albumin, 50 mM HEPES, pH 7.2, and 0.2% 45 muir binding model (BIACORE(R) Evaluation Software ver Sodium azide). The washed cells were added at an approxi sion 3.2) by simultaneously fitting the association and dis mate density of 200,000 cells in 0.2 mL of binding buffer to Sociation sensorgrams. The equilibrium dissociation the 96-well plates containing the 50-uL competition reaction constant (Kd) is calculated as the ratio koff/kon. See, e.g., mixtures. The final concentration of the iodinated antibody Chen et al., J. Mol. Biol. 293:865-881 (1999). in each competition reaction with cells was 100 pM (134, 50 A Surrogate measurement for the affinity of one or more 000 cpm per 0.25 mL). The final concentration of the antibodies for the BBB-R is its half maximal inhibitory unlabeled antibody in the competition reaction with cells concentration (IC50), a measure of how much of the anti varied, starting at 1000 nM and then decreasing by 1:2 fold body is needed to inhibit the binding of a known BBB-R dilution for 10 concentrations and including a Zero-added, ligand to the BBB-R by 50%. Several methods of determin buffer-only sample. Competition reactions with cells for 55 ing the IC50 for a given compound are art-known; a com each concentration of unlabeled antibody were assayed in mon approach is to perform a competition binding assay, triplicate. Competition reactions with cells were incubated such as that described herein in the examples, i.e. with for 2 hours at room temperature. After the 2-hour incubation, regard to FIG. 2A. In general, a high IC50 indicates that the competition reactions were transferred to a Millipore more of the antibody is required to inhibit binding of the Multiscreen filter plate and washed four times with binding 60 known ligand, and thus that the antibody's affinity for that buffer to separate free from bound iodinated antibody. The ligand is relatively low. Conversely, a low IC50 indicates filters were counted on a Wallac Wizard 1470 gamma that less of the antibody is required to inhibit binding of the counter (PerkinElmer Life and Analytical Sciences: known ligand, and thus that the antibody's affinity for that Waltham, Mass.). The binding data were evaluated using ligand is relatively high. New Ligand software (Genentech), which uses the fitting 65 An exemplary competitive ELISA assay to measure IC50 algorithm of Munson and Rodbard (1980) to determine the is one in which increasing concentrations of anti-TfR or binding affinity of the antibody. anti-TfR/brain antigen (i.e., anti-TfR/BACE1, anti-TfR/ US 9,611,323 B2 31 32 Abeta and the like) variant antibodies are used to compete amino acids are selected from glycine, alanine, proline, against biotinylated TfR for binding to TfR. The anti-TfR asparagine, glutamine and lysine. The linker may be a competition ELISA was performed in Maxisorp plates (Nep “cleavable linker facilitating release of the neurological tune, N.J.) coated with 2.5 lug/ml of purified murine TfR drug upon delivery to the brain. For example, an acid-labile extracellular domain in PBS at 4°C. overnight. Plates were linker, peptidase-sensitive linker, photolabile linker, dim washed with PBS/0.05% Tween 20 and blocked using ethyl linker or disulfide-containing linker (Chari et al., Superblock blocking buffer in PBS (Thermo Scientific, Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020) Hudson, N.H.). A titration of each individual anti-TfR or may be used. anti-TfR/brain antigen (i.e., anti-TfR/BACE1 or anti-TfR/ The invention herein expressly contemplates, but is not Abeta) (1:3 serial dilution) was combined with biotinylated 10 limited to, conjugates prepared with cross-linker reagents anti-TfR (0.5 nM final concentration) and added to the plate including, but not limited to, BMPS, EMCS, GMBS, HBVS, for 1 hour at room temperature. Plates were washed with LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, PBS/0.05% Tween 20, and HRP-streptavidin (Southern Bio SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, tech, Birmingham) was added to the plate and incubated for sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, 1 hour at room temperature. Plates were washed with 15 and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which PBS/0.05% Tween 20, and biotinylated anti-TfR bound to are commercially available (e.g., from Pierce Biotechnol the plate was detected using TMB substrate (BioFX Labo ogy, Inc., Rockford, Ill., U.S.A.). ratories, Owings Mills). For a neuropathy disorder, a neurological drug may be In one embodiment, the low affinity anti-BBB-Rantibody selected that is an analgesic including, but not limited to, a herein is coupled with a label and/or neurological disorder narcotic/opioid analgesic (i.e., morphine, fentanyl, hydroco drug or imaging agent in order to more efficiently transport done, meperidine, methadone, oxymorphone, pentazocine, the label and/or drug or imaging agent across the BBB. Such propoxyphene, tramadol, codeine and oxycodone), a non coupling can be achieved by chemical cross-linkers or by steroidal anti-inflammatory drug (NSAID) (i.e., ibuprofen, generating fusion proteins etc. naproxen, diclofenac, diflunisal, etodolac, fenoprofen, flur Covalent conjugation can either be direct or via a linker. 25 biprofen, indomethacin, ketorolac, mefenamic acid, meloxi In certain embodiments, direct conjugation is by construc cam, nabumetone, Oxaprozin, piroXicam, Sulindac, and tol tion of a protein fusion (i.e., by genetic fusion of the two metin), a corticosteroid (i.e., cortisone, prednisone, genes encoding BBB-R antibody and neurological disorder prednisolone, dexamethasone, methylprednisolone and tri drug and expression as a single protein). In certain embodi amcinolone), an anti-migraine agent (i.e., Sumatriptin, ments, direct conjugation is by formation of a covalent bond 30 almotriptan, froVatriptan, Sumatriptan, rizatriptan, eletriptan, between a reactive group on one of the two portions of the Zolmitriptan, dihydroergotamine, eletriptan and ergot anti-BBB-Rantibody and a corresponding group or acceptor amine), acetaminophen, a salicylate (i.e., aspirin, choline on the neurological drug. In certain embodiments, direct salicylate, magnesium salicylate, diflunisal, and Salsalate), a conjugation is by modification (i.e., genetic modification) of anti-convulsant (i.e., carbamazepine, clonazepam, gabapen one of the two molecules to be conjugated to include a 35 tin, lamotrigine, pregabalin, tiagabine, and topiramate), an reactive group (as nonlimiting examples, a Sulfhydryl group anaesthetic (i.e., isoflurane, trichloroethylene, halothane, or a carboxyl group) that forms a covalent attachment to the sevoflurane, benzocaine, chloroprocaine, cocaine, cyclom other molecule to be conjugated under appropriate condi ethycaine, dimethocaine, propoxycaine, procaine, novo tions. As one nonlimiting example, a molecule (i.e., an caine, proparacaine, tetracaine, articaine, bupivacaine, car amino acid) with a desired reactive group (i.e., a cysteine 40 ticaine, cinchocaine, etidocaine, levobupivacaine, lidocaine, residue) may be introduced into, e.g., the anti-BBB-R anti mepivacaine, piperocaine, prilocaine, ropivacaine, trime body and a disulfide bond formed with the neurological caine, saxitoxin and tetrodotoxin), and a cox-2-inhibitor drug. Methods for covalent conjugation of nucleic acids to (i.e., celecoxib, rofecoxib, and Valdecoxib). For a neuropa proteins are also known in the art (i.e., photocrosslinking, thy disorder with vertigo involvement, a neurological drug see, e.g., Zatsepin et al. Russ. Chem. Rev. 74: 77-95 (2005)) 45 may be selected that is an anti-vertigo agent including, but Non-covalent conjugation can be by any noncovalent attach not limited to, meclizine, diphenhydramine, promethazine ment means, including hydrophobic bonds, ionic bonds, and diazepam. For a neuropathy disorder with nausea electrostatic interactions, and the like, as will be readily involvement, a neurological drug may be selected that is an understood by one of ordinary skill in the art. Conjugation anti-nausea agent including, but not limited to, promethaz may also be performed using a variety of linkers. For 50 ine, chlorpromazine, prochlorperazine, trimethobenzamide, example, an anti-BBB-R antibody and a neurological drug and metoclopramide. For a neurodegenerative disease, a may be conjugated using a variety of bifunctional protein neurological drug may be selected that is a growth hormone coupling agents such as N-Succinimidyl-3-(2-pyridyldithio) or neurotrophic factor; examples include but are not limited propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) to brain-derived neurotrophic factor (BDNF), nerve growth cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), 55 factor (NGF), neurotrophin-4/5, fibroblast growth factor bifunctional derivatives of imidoesters (such as dimethyl (FGF)-2 and other FGFs, neurotrophin (NT)-3, erythropoi adipimidate HCl), active esters (such as disuccinimidyl etin (EPO), hepatocyte growth factor (HGF), epidermal Suberate), aldehydes (such as glutaraldehyde), bis-azido growth factor (EGF), transforming growth factor (TGF)- compounds (such as bis (p-azidobenzoyl) hexanediamine), alpha, TGF-beta, vascular endothelial growth factor bis-diazonium derivatives (such as bis-(p-diazoniumben 60 (VEGF), interleukin-1 receptor antagonist (IL-1 ra), ciliary Zoyl)-ethylenediamine), diisocyanates (such as toluene 2,6- neurotrophic factor (CNTF), glial-derived neurotrophic fac diisocyanate), and bis-active fluorine compounds (such as tor (GDNF), neurturin, platelet-derived growth factor 1,5-difluoro-2,4-dinitrobenzene). Peptide linkers, comprised (PDGF), heregulin, neuregulin, artemin, persephin, interleu of from one to twenty amino acids joined by peptide bonds, kins, glial cell line derived neurotrophic factor (GFR), may also be used. In certain such embodiments, the amino 65 granulocyte-colony Stimulating factor (CSF), granulocyte acids are selected from the twenty naturally-occurring amino macrophage-CSF, netrins, cardiotrophin-1, hedgehogs, leu acids. In certain other Such embodiments, one or more of the kemia inhibitory factor (LW), midkine, pleiotrophin, bone US 9,611,323 B2 33 34 morphogenetic proteins (BMPs), metrins, saposins, sema ecenes (especially T-2 toxin, Verracurin A, roridin A and phorins, and stem cell factor (SCF). anguidine); urethan; vindesine (ELDISINE(R), For cancer, a neurological drug may be selected that is a FILDESINR); dacarbazine; mannomustine; mitobronitol; chemotherapeutic agent. Examples of chemotherapeutic mitolactol; pipobroman, gacytosine; arabinoside ("Ara-C); agents include alkylating agents such as thiotepa and 5 thiotepa; taxoids, e.g., TAXOL(R) paclitaxel (Bristol-Myers CYTOXANR) cyclosphosphamide: alkyl sulfonates such as Squibb Oncology, Princeton, N.J.), ABRAXANETM Cremo buSulfan, improSulfan and piposulfan; aziridines such as phor-free, albumin-engineered nanoparticle formulation of benzodopa, carboquone, meturedopa, and uredopa; ethylen paclitaxel (American Pharmaceutical Partners, Schaumberg, imines and methylamelamines including altretamine, trieth Ill.), and TAXOTERE(R) doxetaxel (Rhone-Poulenc Rorer, ylenemelamine, trietylenephosphoramide, triethiylenethio 10 phosphor-amide and trimethylolomelamine; acetogenins Antony, France); chlorambucil; gemcitabine (GEMZAR(R): (especially bullatacin and bullatacinone); delta-9-tetrahy 6-thioguanine; mercaptopurine; methotrexate; platinum ana drocannabinol (dronabinol, MARINOL(R); beta-lapachone: logs such as cisplatin and carboplatin: vinblastine (VEL lapachol; colchicines; betulinic acid; a camptothecin (in BANR); platinum: etoposide (VP-16); ifosfamide; mitox cluding the synthetic analogue topotecan (HYCAMTINR). 15 antrone; Vincristine (ONCOVINR); oxaliplatin; leucovovin; CPT-11 (irinotecan, CAMPTOSAR(R), acetylcamptothecin, vinorelbine (NAVELBINE(R); novantrone; edatrexate: Scopollectin, and 9-aminocamptothecin), bryostatin: callys daunomycin; aminopterin; ibandronate; topoisomerase tatin: CC-1065 (including its adozelesin, carzelesin and inhibitor RFS 2000; difluoromethylornithine (DMFO): ret bizelesin synthetic analogues); podophyllotoxin: podoph inoids such as retinoic acid; capecitabine (XELODAR); yllinic acid; teniposide; cryptophycins (particularly crypto pharmaceutically acceptable salts, acids or derivatives of phycin 1 and cryptophycin 8); dolastatin; duocarmycin any of the above; as well as combinations of two or more of (including the synthetic analogues, KW-2189 and CB1 the above such as CHOP, an abbreviation for a combined TM1); eleutherobin, pancratistatin; a sarcodictyin; spong therapy of cyclophosphamide, doxorubicin, Vincristine, and istatin: nitrogen mustards such as chlorambucil, chlornap prednisolone, and FOLFOX, an abbreviation for a treatment hazine, cholophosphamide, estramustine, ifosfamide, 25 regimen with oxaliplatin (ELOXATINTM) combined with mechlorethamine, mechlorethamine oxide hydrochloride, 5-FU and leucovovin. melphalan, novembichin, phenesterine, prednimustine, tro Also included in this definition of chemotherapeutic fosfamide, uracil mustard; nitroSureas Such as carmustine, agents are anti-hormonal agents that act to regulate, reduce, chlorozotocin, fotemustine, lomustine, nimustine, and ran block, or inhibit the effects of hormones that can promote the imnustine; antibiotics Such as the enediyne antibiotics (e.g., 30 growth of cancer, and are often in the form of systemic, or calicheamicin, especially calicheamicin gammal I and cali whole-body treatment. They may be hormones themselves. cheamicin omegal (see, e.g., Agnew, Chem Intl. Ed. Engl. Examples include anti-estrogens and selective estrogen 33: 183-186 (1994)); dynemicin, including dynemicin A; an receptor modulators (SERMs), including, for example, esperamicin; as well as neocarzinostatin chromophore and tamoxifen (including NOLVADEX(R) tamoxifen), EVISTAR related chromoprotein enediyne antibiotic chromophores), 35 raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, aclacinomysins, actinomycin, authramycin, aZaserine, bleo keoxifene, LY117018, onapristone, and FARESTONR tore mycins, cactinomycin, carabicin, carminomycin, carzino mifene; anti-progesterones; estrogen receptor down-regula philin, chromomycinis, dactinomycin, daunorubicin, deto tors (ERDs); agents that function to Suppress or shut down rubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCINR) the ovaries, for example, leutinizing hormone-releasing hor doxorubicin (including morpholino-doxorubicin, cyanomor 40 mone (LHRH) agonists such as LUPRONR) and ELI pholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy GARDR) leuprolide acetate, goserelin acetate, buserelin doxorubicin), epirubicin, esorubicin, idarubicin, marcello acetate and tripterelin; other anti-androgens Such as fluta mycin, mitomycins such as mitomycin C, mycophenolic mide, nilutamide and bicalutamide; and aromatase inhibitors acid, nogalamycin, olivomycins, peplomycin, potfiromycin, that inhibit the enzyme aromatase, which regulates estrogen puromycin, quelamycin, rodorubicin, Streptonigrin, Strepto 45 production in the adrenal glands, such as, for example, Zocin, tubercidin, ubenimex, Zinostatin, Zorubicin; anti-me 4(5)-imidazoles, aminoglutethimide, MEGASE(R) megestrol tabolites such as methotrexate and 5-fluorouracil (5-FU); acetate, AROMASINR exemestane, formestanie, fadrozole, folic acid analogues such as denopterin, methotrexate, RIVISOR(R) vorozole, FEMARAR) letrozole, and ARIMI pteropterin, trimetrexate; purine analogs such as fludarabine, DEXR anastrozole. In addition, such definition of chemo 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine 50 therapeutic agents includes bisphosphonates Such as clodro analogs such as ancitabine, azacitidine, 6-azauridine, car nate (for example, BONEFOS(R) or OSTAC(R), mofur, cytarabine, dideoxyuridine, doxifluridine, enocit DIDROCAL(R) etidronate, NE-58095, ZOMETAR) Zole abine, floXuridine; androgens Such as calusterone, dromo dronic acid/Zoledronate, FOSAMAX(R) alendronate, ARE stanolone propionate, epitiostanol, mepitioStane, DIAR pamidronate, SKELIDR tiludronate, or ACTONEL(R) testolactone; anti-adrenals such as aminoglutethimide, mito 55 risedronate; as well as troxacitabine (a 1,3-dioxolane tane, triloStane; folic acid replenisher such as frolinic acid; nucleoside cytosine analog); antisense oligonucleotides, par aceglatone; aldophosphamide glycoside; aminolevulinic ticularly those that inhibit expression of genes in signaling acid; eniluracil; amsacrine; bestrabucil; bisantrene; ediatraX pathways implicated in aberrant cell proliferation, Such as, ate; defofamine; demecolcine; diaziquone; eflornithine; for example, PKC-alpha, Raf, H-Ras, and epidermal growth elliptinium acetate; an epothilone; etoglucid, gallium nitrate; 60 factor receptor (EGF-R); vaccines such as THERATOPE(R) hydroxyurea; lentinan; lonidainine; maytansinoids Such as vaccine and gene therapy vaccines, for example, ALL maytansine and ansamitocins; mitoguaZone; mitoxantrone; OVECTINR vaccine, LEUVECTINR vaccine, and mopidanmol; nitraerine; pentostatin: phenamet, pirarubicin; VAXIDR vaccine; LURTOTECANR) topoisomerase 1 losoxantrone: 2-ethylhydrazide; procarbazine; PSKR poly inhibitor; ABARELIX(R) rmRH; lapatinib ditosylate (an saccharide complex (JHS Natural Products, Eugene, Oreg.); 65 ErbB-2 and EGFR dual tyrosine kinase small-molecule razoxane, rhizoxin; sizofiran; Spirogermanium; tenuaZonic inhibitor also known as GW572016); and pharmaceutically acid; triaziquone; 2.2.2"-trichlorotriethylamine; trichoth acceptable salts, acids or derivatives of any of the above. US 9,611,323 B2 35 36 Another group of compounds that may be selected as tase, alpha-N-acetylglucosaminidase, N-acetyl-galac neurological drugs for cancer treatment or prevention are tosamine-6-sulfatase, beta-galactosidase, arylsulphatase B. anti-cancer immunoglobulins (including, but not limited to, beta-glucuronidase, acid alpha-glucosidase, glucocerebrosi trastuzumab, bevacizumab, alemtuXumab, cetuximab, gem dase, alpha-galactosidase A, hexosaminidase A, acid sphin tuZumab ozogamicin, ibritumomab tiuxetan, panitumumab gomyelinase, beta-galactocerebrosidase, beta-galactosidase, and rituximab). In some instances, antibodies in conjunction arylsulfatase A, acid ceramidase, aspartoacylase, palmitoyl with a toxic label may be used to target and kill desired cells protein thioesterase 1 and tripeptidyl amino peptidase 1). (i.e., cancer cells), including, but not limited to, to situ For amyloidosis, a neurological drug may be selected that momab with a '.' I radiolabel. includes, but is not limited to, an antibody or other binding For an ocular disease or disorder, a neurological drug may 10 molecule (including, but not limited to a small molecule, a be selected that is an anti-angiogenic ophthalmic agent (i.e., peptide, an aptamer, or other protein binder) that specifically bevacizumab, ranibizumab and pegaptainib), an ophthalmic binds to a target selected from: beta secretase, tau, preseni glaucoma agent (i.e., carbachol, epinephrine, demecarium lin, amyloid precursor protein or portions thereof, amyloid bromide, apraclonidine, brimonidine, brinzolamide, beta peptide or oligomers or fibrils thereof, death receptor 6 levobunolol, timolol, betaxolol, dorzolamide, bimatoprost, 15 (DR6), receptor for advanced glycation endproducts carteolol, metipranolol, dipivefrin, travoprost and latano (RAGE), parkin, and huntingtin, a cholinesterase inhibitor prost), a carbonic anhydrase inhibitor (i.e., methazolamide (i.e., galantamine, donepezil, rivastigmine and tacrine); an and acetazolamide), an ophthalmic antihistamine (i.e., nap NMDA receptor antagonist (i.e., memantine), a monoamine haZoline, phenylephrine and tetrahydrozoline), an ocular depletor (i.e., tetrabenazine); an ergoloid mesylate; an anti lubricant, an ophthalmic steroid (i.e., fluorometholone, pred cholinergic antiparkinsonism agent (i.e., procyclidine, nisolone, loteprednol, dexamethasone, difluprednate, rimex diphenhydramine, trihexylphenidyl, benztropine, biperiden olone, fluocinolone, medrysone and triamcinolone), an oph and trihexyphenidyl); a dopaminergic antiparkinsonism thalmic anesthetic (i.e., lidocaine, proparacaine and agent (i.e., entacapone, Selegiline, pramipexole, bromocrip tetracaine), an ophthalmic anti-infective (i.e., levofloxacin, tine, rotigotine, selegiline, ropinirole, rasagiline, apomor gatifloxacin, ciprofloxacin, moxifloxacin, chloramphenicol, 25 phine, carbidopa, levodopa, pergolide, tolcapone and aman bacitracin/polymyxin b, Sulfacetamide, tobramycin, azithro tadine); a tetrabenazine; an anti-inflammatory (including, mycin, besifloxacin, norfloxacin, Sulfisoxazole, gentamicin, but not limited to, a nonsteroidal anti-inflammatory drug idoxuridine, erythromycin, natamycin, gramicidin, neomy (i.e., indomethicin and other compounds listed above); a cin, ofloxacin, trifluridine, ganciclovir, Vidarabine), an oph hormone (i.e., estrogen, progesterone and leuprolide); a thalmic anti-inflammatory agent (i.e., nepafenac, ketorolac, 30 Vitamin (i.e., folate and nicotinamide); a dimebolin; a homo flurbiprofen, Suprofen, cyclosporine, triamcinolone, taurine (i.e., 3-aminopropanesulfonic acid; 3APS); a sero diclofenac and bromfenac), and an ophthalmic antihistamine tonin receptor activity modulator (i.e., xaliproden); an, an or decongestant (i.e., ketotifen, olopatadine, epinastine, nap interferon, and a glucocorticoid. haZoline, cromolyn, tetrahydrozoline, pemirolast, bepotast For a viral or microbial disease, a neurological drug may ine, naphazoline, phenylephrine, nedocromil, lodoxamide, 35 be selected that includes, but is not limited to, an antiviral phenylephrine, emedastine and azelastine). compound (including, but not limited to, an adamantane For a seizure disorder, a neurological drug may be antiviral (i.e., rimantadine and amantadine), an antiviral selected that is an anticonvulsant or antiepileptic including, interferon (i.e., peginterferon alfa-2b), a chemokine receptor but not limited to, barbiturate anticonvulsants (i.e., primi antagonist (i.e., maraviroc), an integrase Strand transfer done, metharbital, mephobarbital, allobarbital, amobarbital, 40 inhibitor (i.e., raltegravir), a neuraminidase inhibitor (i.e., aprobarbital, alphenal, barbital, brallobarbital and phenobar oseltamivir and Zanamivir), a non-nucleoside reverse tran bital), benzodiazepine anticonvulsants (i.e., diazepam, clon Scriptase inhibitor (i.e., efavirenz, etravirine, delavirdine and azepam, and lorazepam), carbamate anticonvulsants (i.e. nevirapine), a nucleoside reverse transcriptase inhibitors felbamate), carbonic anhydrase inhibitor anticonvulsants (tenofovir, abacavir, lamivudine, Zidovudine, stavudine, (i.e., acetazolamide, topiramate and Zonisamide), diben 45 entecavir, emtricitabine, adefovir, Zalcitabine, telbivudine Zazepine anticonvulsants (i.e., rufinamide, carbamazepine, and didanosine), a protease inhibitor (i.e., darunavir, ataza and oXcarbazepine), fatty acid derivative anticonvulsants navir, fosamprenavir, tipranavir, ritonavir, nelfinavir, ampre (i.e., divalproex and valproic acid), gamma-aminobutyric navir, indinavir and saquinavir), a purine nucleoside (i.e., acid analogs (i.e., pregabalin, gabapentin and vigabatrin), Valacyclovir, famciclovir, acyclovir, ribavirin, ganciclovir, gamma-aminobutyric acid reuptake inhibitors (i.e., 50 valganciclovir and cidofovir), and a miscellaneous antiviral tiagabine), gamma-aminobutyric acid transaminase inhibi (i.e., enfuvirtide, foscarnet, palivizumab and fomivirsen)), tors (i.e., vigabatrin), hydantoin anticonvulsants (i.e. phe an antibiotic (including, but not limited to, an aminopeni nytoin, ethotoin, fosphenytoin and mephenytoin), miscella cillin (i.e., amoxicillin, amplicillin, oxacillin, nafcillin, cloxa neous anticonvulsants (i.e., lacosamide and magnesium cillin, dicloxacillin, flucoxacillin, temocillin, azlocillin, car Sulfate), progestins (i.e., progesterone), oxazolidinedione 55 benicillin, ticarcillin, mezlocillin, piperacillin and anticonvulsants (i.e., paramethadione and trimethadione), bacampicillin), a cephalosporin (i.e., cefazolin, cephalexin, pyrrolidine anticonvulsants (i.e., levetiracetam), Succinim cephalothin, cefamandole, ceftriaxone, cefotaxime, cefpo ide anticonvulsants (i.e., ethoSuximide and methSuximide), doXime, ceftazidime, cefadroxil, cephradine, loracarbef, triazine anticonvulsants (i.e., lamotrigine), and urea anticon cefotetan, cefuroxime, cefprozil, cefaclor, and cefoxitin), a Vulsants (i.e., phenacemide and pheneturide). 60 carbapenem/penem (i.e., imipenem, meropenem, For a lysosomal storage disease, a neurological drug may ertapenem, faropenem and doripenem), a monobactam (i.e., be selected that is itself or otherwise mimics the activity of aztreonam, tigemonam, norcardicin A and tabtoxinine-beta the enzyme that is impaired in the disease. Exemplary lactam, a beta-lactamase inhibitor (i.e., clavulanic acid, recombinant enzymes for the treatment of lysosomal storage taZobactam and Sulbactam) in conjunction with another disorders include, but are not limited to those set forth in 65 beta-lactam antibiotic, an aminoglycoside (i.e., amikacin, e.g., U.S. Patent Application publication no. 2005/0142141 gentamicin, kanamycin, neomycin, netilmicin, Streptomy (i.e., alpha-L-iduronidase, iduronate-2-Sulphatase, N-sulfa cin, tobramycin, and paromomycin), an ansamycin (i.e., US 9,611,323 B2 37 38 geldanamycin and herbimycin), a carbacephem (i.e., lorac described above), and a miscellaneous anxiolytic/sedative/ arbef), a glycopeptides (i.e., teicoplanin and Vancomycin), a hypnotic (i.e. diphenhydramine, Sodium oxybate, Zaleplon, macrollide (i.e., azithromycin, clarithromycin, dirithromy hydroxy Zine, chloral hydrate, aolpidem, buspirone, doxepin, cin, erythromycin, roXithromycin, troleandomycin, tellithro eSZopiclone, ramelteon, meprobamate and ethclorvynol)), a mycin and spectinomycin), a monobactam (i.e., aztreonam), secretin (see, e.g., Ratliff-Schaub et al. Autism 9: 256-265 a quinolone (i.e., ciprofloxacin, enoxacin, gatifloxacin, levo (2005)), an opioid peptide (see, e.g., Cowen et al., J. floxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxa Neurochem. 89:273-285 (2004)), and a neuropeptide (see, cin, trovafloxacin, grepafloxacin, sparfloxacin and tema e.g., Hethwa et al. Am. J. Physiol. 289: E301-305 (2005)). floxacin), a Sulfonamide (i.e., mafenide, For CNS inflammation, a neurological drug may be Sulfonamidochrysoidine, Sulfacetamide, Sulfadiazine, Sul 10 selected that addresses the inflammation itself (i.e., a non famethizole, Sulfanilamide, SulfaSalazine, Sulfisoxazole, steroidal anti-inflammatory agent Such as ibuprofen or trimethoprim, trimethoprim and Sulfamethoxazole), a tetra naproxen), or one which treats the underlying cause of the cycline (i.e., tetracycline, demeclocycline, doxycycline, inflammation (i.e., an anti-viral or anti-cancer agent). minocycline and oxytetracycline), an antineoplastic or cyto According to one embodiment of the invention, the “cou toxic antibiotic (i.e., doxorubicin, mitoxantrone, bleomycin, 15 pling' is achieved by generating a multispecific antibody daunorubicin, dactinomycin, epirubicin, idarubicin, pli (e.g. a bispecific antibody). Multispecific antibodies are camycin, mitomycin, pentostatin and valrubicin) and a mis monoclonal antibodies that have binding specificities for at cellaneous antibacterial compound (i.e., bacitracin, colistin least two different sites. In one embodiment, the multispe and polymyxin B)), an antifungal (i.e., metronidazole, nita cific antibody comprises a first antigen binding site which Zoxanide, tinidazole, chloroquine, iodoquinol and paromo binds the BBB-R and a second antigen binding site which mycin), and an antiparasitic (including, but not limited to, binds a brain antigen, such as beta-secretase 1 (BACE1) or quinine, chloroquine, amodiaquine, pyrimethamine, Sulpha Abeta, and the other brain antigens disclosed herein. doxine, proguanil, mefloquine, atovaquone, primaquine, An exemplary brain antigen bound by Such multispecific/ artemesinin, halofantrine, doxycycline, clindamycin, bispecific antibody is BACE1, and an exemplary antibody mebendazole, pyrantel pamoate, thiabendazole, diethylcar 25 binding thereto is the YW412.8.31 antibody in FIGS. 5a-b bamazine, ivermectin, rifampin, amphotericin B. melarso herein. prol, efornithine and albendazole). In another embodiment, the brain antigen is Abeta, exem For ischemia, a neurological drug may be selected that plary such antibodies being described in WO20070684-12, includes, but is not limited to, a thrombolytic (i.e., uroki WO2008011348, WO20080156622, and WO2008156621, nase, alteplase, reteplase and tenecteplase), a platelet aggre 30 expressly incorporated herein by reference, with an exem gation inhibitor (i.e., aspirin, cilostaZol, clopidogrel, prasu plary Abeta antibody comprising IgG4 MABT5102A anti grel and dipyridamole), a statin (i.e., lovastatin, pravastatin, body comprising the heavy and light chain amino acid fluvastatin, rosuvastatin, atorvastatin, simvastatin, cerivas sequences in FIGS. 7a and 7b, respectively. tatin and pitavastatin), and a compound to improve blood Techniques for making multispecific antibodies include, flow or vascular flexibility, including, e.g., blood pressure 35 but are not limited to, recombinant co-expression of two medications. immunoglobulin heavy chain-light chain pairs having dif For a behavioral disorder, a neurological drug may be ferent specificities (see Milstein and Cuello, Nature 305:537 selected from a behavior-modifying compound including, (1983)), WO 93/08829, and Traunecker et al., EMBO.J. 10: but not limited to, an atypical antipsychotic (i.e., risperi 3655 (1991)), and “knob-in-hole' engineering (see, e.g., done, olanzapine, apripiprazole, quetiapine, paliperidone, 40 U.S. Pat. No. 5,731,168). Multi-specific antibodies may also asenapine, clozapine, illoperidone and Ziprasidone), a phe be made by engineering electrostatic steering effects for nothiazine antipsychotic (i.e., prochlorperazine, chlorprom making antibody Fc-heterodimeric molecules (WO 2009/ azine, fluiphenazine, perphenazine, trifluoperazine, thior 089004A1); cross-linking two or more antibodies or frag idazine and mesoridazine), a thioxanthene (i.e., thiothixene), ments (see, e.g., U.S. Pat. No. 4,676.980, and Brennan et al., a miscellaneous antipsychotic (i.e., pimozide, lithium, 45 Science, 229: 81 (1985)); using leucine Zippers to produce molindone, haloperidol and loxapine), a selective serotonin bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., reuptake inhibitor (i.e., citalopram, escitalopram, paroX 148(5):1547-1553 (1992)); using “diabody” technology for etine, fluoxetine and Sertraline), a serotonin-norepinephrine making bispecific antibody fragments (see, e.g., Hollinger et reuptake inhibitor (i.e., duloxetine, Venlafaxine, desvenla al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and faxine, a tricyclic antidepressant (i.e., doxepin, clomip 50 using single-chain FV (SFV) dimers (see, e.g. Gruber et al., ramine, amoxapine, nortriptyline, amitriptyline, trimip J. Immunol., 152:5368 (1994)); and preparing trispecific ramine, imipramine, protriptyline and desipramine), a antibodies as described, e.g., in Tutt et al. J. Immunol. 147: tetracyclic antidepressant (i.e., mirtazapine and maproti 60 (1991). line), a phenylpiperazine antidepressant (i.e., traZodone and Engineered antibodies with three or more functional anti nefazodone), a monoamine oxidase inhibitor (i.e., isocar 55 gen binding sites, including "Octopus antibodies' or "dual boxazid, phenelzine, Selegiline and tranylcypromine), a ben variable domain immunoglobulins' (DVDs) are also Zodiazepine (i.e., alprazolam, estazolam, fluraZeptam, clon included herein (see, e.g. US 2006/0025576A1, and Wu et azepam, lorazepam and diazepam), a norepinephrine al. Nature Biotechnology (2007)). dopamine reuptake inhibitor (i.e., bupropion), a CNS The antibody or fragment herein also includes a “Dual stimulant (i.e., phentermine, diethylpropion, methamphet 60 Acting FAb’ or “DAF comprising an antigen binding site amine, dextroamphetamine, amphetamine, methylpheni that binds to the BBB-R (e.g. TfR) as well as the brain date, dexmethylphenidate, lisdexamfetamine, modafinil, antigen (e.g. BACE1) (see, US 2008/0069820, for example). pemoline, phendimetrazine, benzphetamine, phendimetra In one embodiment, the antibody is an antibody fragment, Zine, armodafinil, diethylpropion, caffeine, atomoxetine, various Such fragments being disclosed above. doxapram, and mazindol), an anxiolytic/sedative/hypnotic 65 In another embodiment, the antibody is an intact or (including, but not limited to, a barbiturate (i.e., secobarbi full-length antibody. Depending on the amino acid sequence tal, phenobarbital and mephobarbital), a benzodiazepine (as of the constant domain of their heavy chains, intact anti US 9,611,323 B2 39 40 bodies can be assigned to different classes. There are five In one aspect, assays are provided for identifying anti major classes of intact antibodies: IgA, Ig|D, IgE. IgG, and BACE1 antibodies thereof having biological activity. Bio IgM, and several of these may be further divided into logical activity may include, e.g., inhibition of BACE1 Subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, aspartyl protease activity. Antibodies having such biological and IgA2. The heavy chain constant domains that corre activity in vivo and/or in vitro are also provided, e.g. as spond to the different classes of antibodies are called C, 6. evaluated by homogeneous time-resolved fluorescence e, Y, and L, respectively. The subunit structures and three HTRF assay or a microfluidic capillary electrophoretic dimensional configurations of different classes of immuno (MCE) assay using synthetic Substrate peptides, or in vivo in globulins are well known. In one embodiment, the intact cell lines which express BACE 1 substrates such as APP. 10 The antibody (including the multispecific antibody) antibody lacks effector function. herein is optionally recombinantly produced in a host cell Techniques for generating antibodies are known and transformed with nucleic acid sequences encoding its heavy examples provided above in the definitions section of this and light chains (e.g. where the host cell has been trans document. In one embodiment, the antibody is a chimeric, formed by one or more vectors with the nucleic acid humanized, or human antibody or antigen-binding fragment 15 thereof. therein). The host cell is optionally a mammalian cell, for Various techniques are available for determining binding example a Chinese Hamster Ovary (CHO) cell. of the antibody to the BBB-R. One such assay is an enzyme III. Pharmaceutical Formulations linked immunosorbent assay (ELISA) for confirming an ability to bind to human BBB-R (and brain antigen). Accord Therapeutic formulations of the antibodies used in accor ing to this assay, plates coated with antigen (e.g. recombi dance with the present invention are prepared for storage by nant sRBB-R) are incubated with a sample comprising the mixing an antibody having the desired degree of purity with anti-BBB-R antibody and binding of the antibody to the optional pharmaceutically acceptable carriers, excipients or antigen of interest is determined. stabilizers (Remington's Pharmaceutical Sciences 16th edi In one aspect, an antibody of the invention is tested for its 25 tion, Osol, A. Ed. (1980)), in the form of lyophilized antigen binding activity, e.g., by known methods such as formulations or aqueous solutions. Acceptable carriers, ELISA, Western blot, etc. excipients, or stabilizers are nontoxic to recipients at the Assays for evaluating uptake of systemically adminis dosages and concentrations employed, and include buffers tered antibody and other biological activity of the antibody Such as phosphate, citrate, and other organic acids; antioxi can be performed as disclosed in the examples or as known 30 dants including ascorbic acid and methionine; preservatives for the anti-brain antigen antibody of interest. (such as octadecyldimethylbenzyl ammonium chloride; hex Exemplary assays where the multispecific antibody binds amethonium chloride; benzalkonium chloride, benzetho BACE1 shall now be described. nium chloride; phenol, butyl or benzyl alcohol; alkyl para Competition assays may be used to identify an antibody bens Such as methyl or propyl paraben; catechol; resorcinol; that competes with any of the antibodies or Fabs descried 35 cyclohexanol: 3-pentanol; and m-cresol); low molecular herein, for example, YW412.8, YW412.8.31, YW412.8.30, weight (less than about 10 residues) polypeptides; proteins, YW412.8.2, YW412.8.29, YW412.8.51, Fab 12, LC6, LC9, Such as serum albumin, gelatin, or immunoglobulins; hydro LC10 for binding to BACE1. In certain embodiments, such philic polymers such as polyvinylpyrrolidone; amino acids a competing antibody binds to the same epitope (e.g., a Such as glycine, glutamine, asparagine, histidine, arginine, linear or a conformational epitope) that is bound by any of 40 or lysine; monosaccharides, disaccharides, and other carbo the antibodies or Fabs descried herein, for example, hydrates including glucose, mannose, or dextrins; chelating YW412.8, YW412.8.31, YW412.8.30, YW412.8.2, agents such as EDTA; Sugars such as Sucrose, mannitol, YW412.8.29, YW412.8.51, Fab 12, LC6, LC9, LC10. trehalose or Sorbitol; salt-forming counter-ions such as Detailed exemplary methods for mapping an epitope to Sodium; metal complexes (e.g. Zn-protein complexes); and/ which an antibody binds are provided in Morris (1996) 45 or non-ionic surfactants such as TWEENTM, PLURON "Epitope Mapping Protocols.” in Methods in Molecular ICSTM or polyethylene glycol (PEG). Biology vol. 66 (Humana Press, Totowa, N.J.). The formulation herein may also contain more than one In an exemplary competition assay, immobilized BACE1 active compound as necessary, optionally those with is incubated in a solution comprising a first labeled antibody complementary activities that do not adversely affect each that binds to BACE1 (e.g., YW412.8, YW412.8.31, 50 other. The type and effective amounts of such medicaments YW412.8.30, YW412.8.2, YW412.8.29, YW412.8.51, depend, for example, on the amount of antibody present in Fab12, LC6, LC9, LC10) and a second unlabeled antibody the formulation, and clinical parameters of the Subjects. that is being tested for its ability to compete with the first Exemplary Such medicaments are discussed below. antibody for binding to BACE1. The second antibody may The active ingredients may also be entrapped in micro be present in a hybridoma Supernatant. As a control, immo 55 capsules prepared, for example, by coacervation techniques bilized BACE1 is incubated in a solution comprising the first or by interfacial polymerization, for example, hydroxym labeled antibody but not the second unlabeled antibody. ethylcellulose or gelatin-microcapsules and poly-(methyl After incubation under conditions permissive for binding of methacylate) microcapsules, respectively, in colloidal drug the first antibody to BACE1, excess unbound antibody is delivery systems (for example, liposomes, albumin micro removed, and the amount of label associated with immobi 60 spheres, microemulsions, nano-particles and nanocapsules) lized BACE1 is measured. If the amount of label associated or in macroemulsions. Such techniques are disclosed in with immobilized BACE1 is substantially reduced in the test Remington's Pharmaceutical Sciences 16th edition, Osol, A. sample relative to the control sample, then that indicates that Ed. (1980). the second antibody is competing with the first antibody for Sustained-release preparations may be prepared. Suitable binding to BACE1. See Harlow and Lane (1988) Antibod 65 examples of Sustained-release preparations include semi ies: A Laboratory Manual ch. 14 (Cold Spring Harbor permeable matrices of Solid hydrophobic polymers contain Laboratory, Cold Spring Harbor, N.Y.). ing the antibody, which matrices are in the form of shaped US 9,611,323 B2 41 42 articles, e.g. films, or microcapsules. Examples of Sustained encephalopathy, HIV-related dementia, amyotropic lateral release matrices include polyesters, hydrogels (for example, sclerosis (ALS), inclusion-body myositis (IBM), and ocular poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), diseases relating to beta-amyloid deposition (i.e., macular polylactides (U.S. Pat. No. 3,773.919), copolymers of L-glu degeneration, drusen-related optic neuropathy, and cataract). tamic acid and Y ethyl-L-glutamate, non-degradable ethyl Cancers of the CNS are characterized by aberrant prolif ene-vinyl acetate, degradable lactic acid-glycolic acid copo eration of one or more CNS cell (i.e., a neural cell) and lymers such as the LUPRON DEPOTTM (injectable include, but are not limited to, glioma, glioblastoma multi microspheres composed of lactic acid-glycolic acid copoly forme, meningioma, astrocytoma, acoustic neuroma, chon mer and leuprolide acetate), and poly-D-(-)-3-hydroxybu droma, oligodendroglioma, medulloblastomas, ganglio tyric acid. 10 glioma, Schwannoma, neurofibroma, neuroblastoma, and The formulations to be used for in vivo administration extradural, intramedullary or intradural tumors. must be sterile. This is readily accomplished by filtration Ocular diseases or disorders are diseases or disorders of through sterile filtration membranes. the eye, which for the purposes herein is considered a CNS In one embodiment the formulation is isotonic. organ subject to the BBB. Ocular diseases or disorders 15 include, but are not limited to, disorders of Sclera, cornea, IV. Therapeutic Uses of Anti-BBB-R Antibodies iris and ciliary body (i.e., Scleritis, keratitis, corneal ulcer, corneal abrasion, Snow blindness, arc eye, Thygeson's The anti-BBB-R antibodies (including multispecific anti Superficial punctate keratopathy, corneal neovascularisation, bodies comprising them) may be utilized in a variety of in Fuchs' dystrophy, keratoconus, keratoconjunctivitis sicca, vivo methods. For example, the invention provides a method iritis and uveitis), disorders of the lens (i.e., cataract), of transporting a therapeutic compound across the blood disorders of choroid and retina (i.e., retinal detachment, brain barrier comprising exposing the anti-BBB-R antibody retinoschisis, hypertensive retinopathy, diabetic retinopathy, coupled to a therapeutic compound (e.g. a multispecific retinopathy, retinopathy of prematurity, age-related macular antibody which binds both the BBB-R and a brain antigen) degeneration, macular degeneration (wet or dry), epiretinal to the BBB such that the antibody transports the therapeutic 25 membrane, retinitis pigmentosa and macular edema), glau compound coupled thereto across the BBB. In another coma, floaters, disorders of optic nerve and visual pathways example, the invention provides a method of transporting a (i.e., Leber's hereditary optic neuropathy and optic disc neurological disorder drug across the blood-brain barrier drusen), disorders of ocular muscles/binocular movement comprising exposing the anti-BBB-R antibody coupled to a accommodation/refraction (i.e., Strabismus, ophthalmopare brain disorder drug (e.g. a multispecific antibody which 30 sis, progressive external opthalmoplegia, esotropia, exotro binds both the BBB-R and a brain antigen) to the BBB such pia, hypermetropia, myopia, astigmatism, anisometropia, that the antibody transports the neurological disorder drug presbyopia and ophthalmoplegia), visual disturbances and coupled thereto across the BBB. In one embodiment, the blindness (i.e., amblyopia, Lever's congenital amaurosis, BBB here is in a mammal (e.g. a human), e.g. one which has Scotoma, color blindness, achromatopsia, nyctalopia, blind a neurological disorder, including, without limitation: 35 ness, river blindness and micro-opthalmia/coloboma), red Alzheimer's disease (AD), stroke, dementia, muscular dys eye, Argyll Robertson pupil, keratomycosis, Xerophthalmia trophy (MD), multiple sclerosis (MS), amyotrophic lateral and andaniridia. Sclerosis (ALS), cystic fibrosis, Angelman's syndrome, Viral or microbial infections of the CNS include, but are Liddle syndrome, Parkinson's disease, Pick's disease, Pag not limited to, infections by viruses (i.e., influenza, HIV. et's disease, cancer, traumatic brain injury, etc. 40 poliovirus, rubella), bacteria (i.e., Neisseria sp., Streptococ In one embodiment, neurological disorder is selected cus sp., Pseudomonas sp., Proteus sp., E. coli, S. aureus, from: a neuropathy, amyloidosis, cancer (e.g. involving the Pneumococcus sp., Meningococcus sp., Haemophilus sp., CNS or brain), an ocular disease or disorder, a viral or and Mycobacterium tuberculosis) and other microorganisms microbial infection, inflammation (e.g. of the CNS or brain), Such as fungi (i.e., yeast, Cryptococcus neoformans), para ischemia, neurodegenerative disease, seizure, behavioral 45 sites (i.e., toxoplasma gondii) or amoebas resulting in CNS disorder, lysosomal storage disease, etc. pathophysiologies including, but not limited to, meningitis, Neuropathy disorders are diseases or abnormalities of the encephalitis, myelitis, vasculitis and abscess, which can be nervous system characterized by inappropriate or uncon acute or chronic. trolled nerve signaling or lack thereof, and include, but are Inflammation of the CNS is inflammation that is caused not limited to, chronic pain (including nociceptive pain), 50 by an injury to the CNS, which can be a physical injury (i.e., pain caused by an injury to body tissues, including cancer due to accident, Surgery, brain trauma, spinal cord injury, related pain, neuropathic pain (pain caused by abnormalities concussion) or an injury due to or related to one or more in the nerves, spinal cord, or brain), and psychogenic pain other diseases or disorders of the CNS (i.e., abscess, cancer, (entirely or mostly related to a psychological disorder), viral or microbial infection). headache, migraine, neuropathy, and symptoms and Syn 55 Ischemia of the CNS, as used herein, refers to a group of dromes often accompanying Such neuropathy disorders such disorders relating to aberrant blood flow or vascular behav as Vertigo or nausea. ior in the brain or the causes therefor, and includes, but is not Amyloidoses are a group of diseases and disorders asso limited to: focal brain ischemia, global brain ischemia, ciated with extracellular proteinaceous deposits in the CNS, stroke (i.e., Subarachnoid hemorrhage and intracerebral including, but not limited to, secondary amyloidosis, age 60 hemorrhage), and aneurysm. related amyloidosis, Alzheimer's Disease (AD), mild cog Neurodegenerative diseases are a group of diseases and nitive impairment (MCI), Lewy body dementia, Downs disorders associated with neural cell loss of function or syndrome, hereditary cerebral hemorrhage with amyloidosis death in the CNS, and include, but are not limited to: (Dutch type); the Guam Parkinson-Dementia complex, cere adrenoleukodystrophy, Alexander's disease, Alper's disease, bral amyloid angiopathy, Huntington's disease, progressive 65 amyotrophic lateral Sclerosis, ataxia telangiectasia, Batten Supranuclear palsy, multiple Sclerosis; Creutzfeld Jacob dis disease, cockayne syndrome, corticobasal degeneration, ease, Parkinson's disease, transmissible spongiform degeneration caused by or associated with an amyloidosis, US 9,611,323 B2 43 44 Friedreich's ataxia, frontotemporal lobar degeneration, Ken individual having a neurological disease or disorder com nedy's disease, multiple system atrophy, multiple Sclerosis, prising administering to the individual an effective amount primary lateral Sclerosis, progressive Supranuclear palsy, of the anti-BBB-R antibody (optionally coupled to a neu spinal muscular atrophy, transverse myelitis, Refsum’s dis rological disorder drug). In one Such embodiment, the ease, and spinocerebellar ataxia. method further comprises administering to the individual an Seizure diseases and disorders of the CNS involve inap effective amount of at least one additional therapeutic agent. propriate and/or abnormal electrical conduction in the CNS, In further embodiments, the invention provides an anti and include, but are not limited to: epilepsy (i.e., absence BBB-R antibody for use in reducing or inhibiting amyloid seizures, atonic seizures, benign Rolandic epilepsy, child plaque formation in a patient at risk or Suffering from a hood absence, clonic seizures, complex partial seizures, 10 neurological disease or disorder (e.g., Alzheimer's disease). frontal lobe epilepsy, febrile seizures, infantile spasms, An “individual according to any of the above embodiments juvenile myoclonic epilepsy, juvenile absence epilepsy, Len is optionally a human. In certain aspect, the anti-BBB-R nox-Gastaut syndrome, Landau-Kleffner Syndrome, antibody for use in the methods of the invention improves Dravet's syndrome, Otahara syndrome, West syndrome, uptake of the neurological disorder drug with which it is myoclonic seizures, mitochondrial disorders, progressive 15 coupled. myoclonic epilepsies, psychogenic seizures, reflex epilepsy, In a further aspect, the invention provides for the use of Rasmussen's Syndrome, simple partial seizures, secondarily a low affinity anti-BBB-R antibody in the manufacture or generalized seizures, temporal lobe epilepsy, toniclonic sei preparation of a medicament. In one embodiment, the medi Zures, tonic seizures, psychomotor seizures, limbic epilepsy, cament is for treatment of neurological disease or disorder. partial-onset seizures, generalized-onset seizures, status epi In a further embodiment, the medicament is for use in a lepticus, abdominal epilepsy, akinetic seizures, autonomic method of treating neurological disease or disorder com seizures, massive bilateral myoclonus, catamenial epilepsy, prising administering to an individual having neurological drop seizures, emotional seizures, focal seizures, gelastic disease or disorder an effective amount of the medicament. seizures, Jacksonian March, Lafora Disease, motor seizures, In one such embodiment, the method further comprises multifocal seizures, nocturnal seizures, photosensitive sei 25 administering to the individual an effective amount of at Zure, pseudo seizures, sensory seizures, subtle seizures, least one additional therapeutic agent. Sylvan seizures, withdrawal seizures, and visual reflex Sei In a further aspect, the invention provides a method for Zures). treating Alzheimer's disease. In one embodiment, the Behavioral disorders are disorders of the CNS character method comprises administering to an individual having ized by aberrant behavior on the part of the afflicted subject 30 Alzheimer's disease an effective amount of a multispecific and include, but are not limited to: Sleep disorders (i.e., antibody which binds both BACE1 and TfR. In one such insomnia, parasomnias, night terrors, circadian rhythm sleep embodiment, the method further comprises administering to disorders, and narcolepsy), mood disorders (i.e., depression, the individual an effective amount of at least one additional Suicidal depression, anxiety, chronic affective disorders, therapeutic agent. An "individual' according to any of the phobias, panic attacks, obsessive-compulsive disorder, 35 above embodiments may be a human. attention deficit hyperactivity disorder (ADHD), attention The anti-BBB-R antibodies of the invention can be used deficit disorder (ADD), chronic fatigue syndrome, agora either alone or in combination with other agents in a therapy. phobia, post-traumatic stress disorder, bipolar disorder), For instance, the anti-BBB-R antibody of the invention may eating disorders (i.e., anorexia or bulimia), psychoses, be co-administered with at least one additional therapeutic developmental behavioral disorders (i.e., autism, Rett's syn 40 agent. In certain embodiments, an additional therapeutic drome, Aspberger's syndrome), personality disorders and agent is a therapeutic agent effective to treat the same or a psychotic disorders (i.e., Schizophrenia, delusional disorder, different neurological disorder as the anti-BBB-R antibody and the like). is being employed to treat. Exemplary additional therapeutic Lysosomal storage disorders are metabolic disorders agents include, but are not limited to: the various neurologi which are in some cases associated with the CNS or have 45 cal drugs described above, cholinesterase inhibitors (such as CNS-specific symptoms; such disorders include, but are not donepezil, galantamine, rovastigmine, and tacrine), NMDA limited to: Tay-Sachs disease, Gaucher's disease, Fabry receptor antagonists (such as memantine), amyloid beta disease, mucopolysaccharidosis (types I, II, III, IV, V, VI and peptide aggregation inhibitors, antioxidants, Y-secretase VII), glycogen storage disease, GM1-gangliosidosis, modulators, nerve growth factor (NGF) mimics or NGF metachromatic leukodystrophy, Farber's disease, Canavan's 50 gene therapy, PPARY agonists, HMS-CoA reductase inhibi leukodystrophy, and neuronal ceroid lipofuscinoses types 1 tors (statins), ampakines, calcium channel blockers, GABA and 2, Niemann-Pick disease, Pompe disease, and Krabbe's receptor antagonists, glycogen synthase kinase inhibitors, disease. intravenous immunoglobulin, muscarinic receptor agonists, In one aspect, the antibody is used to detect a neurological nicrotinic receptor modulators, active or passive amyloid disorder before the onset of symptoms and/or to assess the 55 beta peptide immunization, phosphodiesterase inhibitors, severity or duration of the disease or disorder. In one aspect, serotonin receptor antagonists and anti-amyloid beta peptide the antibody permits detection and/or imaging of the neu antibodies. In certain embodiments, the at least one addi rological disorder, including imaging by radiography, tional therapeutic agent is selected for its ability to mitigate tomography, or magnetic resonance imaging (MRI). one or more side effects of the neurological drug. In one aspect, a low affinity anti-BBB-Rantibody for use 60 Such combination therapies noted above encompass com as a medicament is provided. In further aspects, a low bined administration (where two or more therapeutic agents affinity anti-BBB-R antibody for use in treating a neurologi are included in the same or separate formulations), and cal disease or disorder is provided (e.g., Alzheimer's dis separate administration, in which case, administration of the ease). In certain embodiments, a low affinity anti-BBB-R antibody of the invention can occur prior to, simultaneously, antibody for use in a method of treatment is provided. In 65 and/or following, administration of the additional therapeu certain embodiments, the invention provides a low affinity tic agent and/or adjuvant. Antibodies of the invention can anti-BBB-R antibody for use in a method of treating an also be used in combination with other interventional thera US 9,611,323 B2 45 46 pies such as, but not limited to, radiation therapy, behavioral one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg therapy, or other therapies known in the art and appropriate or 10 mg/kg (or any combination thereof) may be adminis for the neurological disorder to be treated or prevented. tered to the patient. Such doses may be administered inter The anti-BBB-R antibody of the invention (and any mittently, e.g. every week or every three weeks (e.g. Such additional therapeutic agent) can be administered by any that the patient receives from about two to about twenty, or Suitable means, including parenteral, intrapulmonary, and e.g. about six doses of the antibody). An initial higher intranasal, and, if desired for local treatment, intralesional loading dose, followed by one or more lower doses may be administration. Parenteral infusions include intramuscular, administered. However, other dosage regimens may be intravenous, intraarterial, intraperitoneal, or Subcutaneous useful. The progress of this therapy is easily monitored by administration. Dosing can be by any Suitable route, e.g. by 10 conventional techniques and assays. injections. Such as intravenous or Subcutaneous injections, It is understood that any of the above formulations or depending in part on whether the administration is brief or therapeutic methods may be carried out using an immuno chronic. Various dosing schedules including but not limited conjugate of the invention in place of or in addition to an to single or multiple administrations over various time anti-BBB-R antibody. points, bolus administration, and pulse infusion are contem 15 plated herein. V. Articles of Manufacture Lipid-based methods of transporting the antibody or frag ment thereof across the blood-brain barrier include, but are In another aspect of the invention, an article of manufac not limited to, encapsulating the antibody or fragment ture containing materials useful for the treatment, prevention thereof in liposomes that are coupled to antibody binding and/or diagnosis of the disorders described above is pro fragments that bind to receptors on the vascular endothelium vided. The article of manufacture comprises a container and of the blood-brain barrier (see e.g., U.S. Patent Application a label or package insert on or associated with the container. Publication No. 20020025313), and coating the antibody or Suitable containers include, for example, bottles, vials, active fragment thereof in low-density lipoprotein particles Syringes, IV solution bags, etc. The containers may be (see e.g., U.S. Patent Application Publication No. 25 formed from a variety of materials such as glass or plastic. 20040204354) or apolipoprotein E (see e.g., U.S. Patent The container holds a composition which is by itself or Application Publication No. 20040131692). combined with another composition effective for treating, Antibodies of the invention would be formulated, dosed, preventing and/or diagnosing the condition and may have a and administered in a fashion consistent with good medical sterile access port (for example the container may be an practice. Factors for consideration in this context include the 30 intravenous solution bag or a vial having a stopper pierce particular disorder being treated, the particular mammal able by a hypodermic injection needle). At least one active being treated, the clinical condition of the individual patient, agent in the composition is an antibody of the invention. The the cause of the disorder, the site of delivery of the agent, the label or package insert indicates that the composition is used method of administration, the scheduling of administration, for treating the condition of choice. Moreover, the article of and other factors known to medical practitioners. The anti 35 manufacture may comprise (a) a first container with a body need not be, but is optionally formulated with one or composition contained therein, wherein the composition more agents currently used to prevent or treat the disorder in comprises an antibody of the invention; and (b) a second question. The effective amount of Such other agents depends container with a composition contained therein, wherein the on the amount of antibody present in the formulation, the composition comprises a further cytotoxic or otherwise type of disorder or treatment, and other factors discussed 40 therapeutic agent. The article of manufacture in this embodi above. These are generally used in the same dosages and ment of the invention may further comprise a package insert with administration routes as described herein, or about indicating that the compositions can be used to treat a from 1 to 99% of the dosages described herein, or in any particular condition. Alternatively, or additionally, the article dosage and by any route that is empirically/clinically deter of manufacture may further comprise a second (or third) mined to be appropriate. 45 container comprising a pharmaceutically-acceptable buffer, For the prevention or treatment of disease, the appropriate such as bacteriostatic water for injection (BWFI), phos dosage of an antibody of the invention (when used alone or phate-buffered saline, Ringer's solution and dextrose solu in combination with one or more other additional therapeutic tion. It may further include other materials desirable from a agents) will depend on the type of disease to be treated, the commercial and user standpoint, including other buffers, type of antibody, the severity and course of the disease, 50 diluents, filters, needles, and Syringes. whether the antibody is administered for preventive or It is understood that any of the above articles of manu therapeutic purposes, previous therapy, the patient's clinical facture may include an immunoconjugate of the invention in history and response to the antibody, and the discretion of place of or in addition to an anti-BBB-R antibody. the attending physician. The antibody is Suitably adminis The article of manufacture optionally further comprises a tered to the patient at one time or over a series of treatments. 55 package insert with instructions for treating a neurological Depending on the type and severity of the disease, about 1 disorder in a Subject, wherein the instructions indicate that ug/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody treatment with the antibody as disclosed herein treats the can be an initial candidate dosage for administration to the neurological disorder, and optionally indicates that the anti patient, whether, for example, by one or more separate body has improved uptake across the BBB due to its low administrations, or by continuous infusion. One typical daily 60 affinity for the BBB-R. dosage might range from about 1 lug/kg to 100 mg/kg or more, depending on the factors mentioned above. For III. Examples repeated administrations over several days or longer, depending on the condition, the treatment would generally This example evaluated the transferrin receptor (TfR), be sustained until a desired Suppression of disease symptoms 65 which mediates iron transport into the brain via the holo occurs. One exemplary dosage of the antibody would be in transferrin complex (Skarlatos et al. Brain Res 683: 164-171 the range from about 0.05 mg/kg to about 10 mg/kg. Thus, (1995)). A human chimeric anti-murine transferrin receptor US 9,611,323 B2 47 48 (anti-TfR) antibody that does not compete with endog radiolabeled trace data, these results indicate that systemi enous transferrin binding to TfR was compared to a human cally administered anti-TfR can accumulate in the brain, control IgG in a double-labeled experiment for uptake in however the tissue distribution of antibody in brain wild type mouse brain. A single trace dose (approximately remained to be understood. 50 ug/kg) of ''Ilanti-TfR and 'Icontrol IgG was 5 To address the distribution of systemically administered injected into wild type mice intravenously (i.v.) and brain antibodies in brain, wild type mice were injected 20 mg/kg uptake was measured at 5 min., 0.5, 1, 4, 24, 48, and 72 i.v. with either anti-TfR or control IgG, perfused with PBS hours. A significant increase in ''Ilanti-TfR uptake in the to flush out any remaining circulating antibody, and brain brain, measured as a percentage of injected dose per gram of sections were stained with fluorescent anti-human secondary brain, was observed at all time points (FIG. 1A). At its peak, 10 IgG to determine antibody localization. After 1 hour of 1 hour after injection, there was a >11-fold difference in circulation, anti-TfR had a pronounced vascular distribu ''Ilanti-TfR brain accumulation as compared to 'I tion, as indicated by its co-localization with the basement control IgG (n-6). If unlabeled anti-TfR (4 mg/kg body membrane marker anti-collagen IV (FIG. 1D, left column). weight) was also co-administered, brain accumulation of Although less pronounced, control IgG also localized to the ''Ilanti-TfR was nearly reduced to the level of control 15 IgG, indicating specific, target-driven uptake. vasculature, indicating that after 1 hour of exposure, thera To evaluate whether significant antibody uptake in brain peutic dose levels of systemically administered IgG main also occurs at therapeutic dose levels, wild type mice were tains a vascular distribution (FIG. 1E, left column). How administered either anti-TfR or control IgG at 20 mg/kg ever, there was a marked difference in antibody localization intravenously (i.v.). Human antibody concentration in the 24 hours after injection. Anti-TfR distribution was no cortex and serum was determined 1 and 24 hours after longer exclusively vascular, but instead, exhibited modest injection using a human Fc sandwich ELISA. Briefly, after parenchymal staining (FIG. 1D, right columns). In contrast, the indicated time of administration, mice were perfused control IgG antibody was largely absent in brain tissue 24 with D-PBS at a rate of 2 ml/min. for 8 minutes. Brains were hours after injection (FIG. 1E, right columns). These results extracted and the cortex and hippocampus were isolated, 25 indicate that when dosed at therapeutically relevant levels, homogenized in 1% NP-40 (Cal-Biochem) in PBS contain anti-TfR may penetrate the BBB as evidenced by modest ing Complete Mini EDTA-free protease inhibitor cocktail parenchymal staining, however, the bulk of the brain-accu tablets (Roche Diagnostics). Homogenized brain samples mulated antibody was largely confined to endothelial cells of were rotated at 4° C. for 1 hour before centrifugation at the BBB. 14,000 rpm for 20 minutes. The Supernatant was isolated for 30 Accumulation in the parenchyma requires binding to brain antibody measurement. Whole blood was collected surface TfRs expressed on brain endothelial cells as well as prior to perfusion in serum separator microcontainer tubes dissociation from the receptor following RMT. Without (BD Diagnostics), allowed to clot for at least 30 minutes, being bound by any theory, it was hypothesized that reduced and spun down at 5,000xg for 90 seconds. The supernatant affinity for TfR may facilitate dissociation after RMT and was isolated for serum antibody measurements. Antibody 35 allow enhanced accumulation in the parenchyma. Further, concentrations in mouse serum and brain samples were an anti-TfR with reduced affinity would be less efficiently measured by ELISA. NUNC 384-well Maxisorp immuno captured and transported in a concentration-limited environ plates (Neptune, N.J.) were coated with F(ab')2 fragment of ment, such as in the brain, where anti-TfR concentrations are donkey anti-human IgG, Fc fragment-specific polyclonal low. In a clinical setting, however, serum levels of an antibody (Jackson ImmunoResearch, West Grove, Pa.) over 40 anti-TfR therapeutic would still be sufficiently high to night at 4°C. Plates were blocked with PBS containing 0.5% maintain Saturation of the receptor in the vascular lumen. BSA for 1 hour at 25° C. Each antibody was used as an To test this prediction, variants of anti-TfR were gener internal standard to quantify respective antibody concentra ated that vary in their binding affinity for TfR. These variants tion. Plates were washed with PBS containing 0.05% were tested in a competition ELISA assay (FIG. 2A); Tween-20 using a microplate washer (Bio-Tek Instruments, 45 anti-TfR had the strongest affinity and lowest IC50 of any Inc., Winooski, Vt.). Standards and samples were diluted in of the tested antibodies for TfR, and each of anti-TfR'' PBS containing 0.5% BSA, 0.35M NaCl, 0.25% CHAPS, 5 had successively lower affinity and higher IC50. Later, mM EDTA, 0.05% Tween-20 and 15 ppm Proclin, and were variant anti-TfR' was generated and tested in the same assay added to the microplate for two hours at 25° C. Bound along with anti-TfR'''P variants; as shown in FIG. 2A, it antibody was detected with horseradish peroxidase-conju 50 was substantially less able to compete for binding to TfR gated F(ab')2 goat anti-human IgG, Fc-specific polyclonal than any of the other tested anti-TfR antibodies, and it had antibody (Jackson ImmunoResearch), developed using 3.3', a corresponding high IC50 value (Table 2). 5.5'-tetramethylbenzidine (TMB) (KPL, Inc., Gaithersburg, Md.) and absorbance measured at 450 nm on a Multiskan TABLE 2 Ascent reader (Thermo Scientific, Hudson, N.H.). Concen 55 IC50 measurements for anti-TfR antibodies trations were determined from the standard curve using a four-parameter non-linear regression program. The assay Antibody IC50 (nM) Standard deviation had lower limit of quantification (LLOQ) values of 3.12 Anti-TfR- 1.7 O.1 ng/ml in serum and 15.6 ng/g in brain. Statistical analysis of Anti-TfR 6.9 0.4 differences between experimental groups was performed 60 Anti-TfR 65 12 using a two-tailed unpaired t-test. Anti-TfRP 111 16 Compared to control IgG, concentration of anti-TfR was Anti-TfR >5 x 10' significantly higher in the brain 24 hours after antibody administration (FIG. 1B, p=0.0002, n=10). Additionally, These variants were tested in both a non-TfR saturating human IgG concentration in brain was >2.5-fold higher 65 (trace dosing) and TfR saturating (therapeutic dosing) envi compared to serum for anti-TfR compared to control IgG at ronment. Trace levels of ''Ilanti-TfR', ''Ilanti 'I 24 hours (FIG. 1C, p=0.003, n=10). Together with the anti-TfR. ''Ilanti-TfRP and ''Ilanti-TfR (which vary US 9,611,323 B2 49 50 in affinity for TfR, with the affinity of anti-TfR">affinity of half-antibodies were purified separately and annealed to anti-TfR>affinity of anti-TfR>affinity of anti generate an aglycosylated bispecific IgG in vitro. The bind TfReaffinity of anti-TfR) were injected i.v. into mice and ing affinity of the anti-TfR/BACE1 antibody to TfR was brain uptake was measured 1, 4, or 24 hours after injection. considerably reduced compared to the parental anti-TfR' This assay was performed originally with 'Ilanti-TfR', due to the loss of bivalent binding (FIG. 3B). BACE1 is 'Ilanti-TfR', ''Ilanti-TfR, and ''Ilanti-TfRP, and expressed primarily on neurons in the CNS and is consid later repeated upon the construction of ''Ilanti-TfR', the ered to be the primary contributor of beta amyloid (A13140) results of which are shown in FIG. 2B. Consistent with the formation via APP cleavage (Vassar et al., Science 286:735 proposed model, trace dose levels of lower affinity anti-TfR 741 (1999)). An antibody to BACE1 has been described as 10 an effective means to inhibit BACE 1 activity, and may antibodies resulted in less uptake in brain compared to reduce AB production in vivo. Inhibition of BACE1 by higher affinity variants (FIG. 2B). In striking contrast to anti-TfR/BACE1 was examined in a HEK293 cell line trace dosing, however, brain uptake of these same lower stably expressing APP. Compared to anti-BACE1, the bispe affinity variants at therapeutic levels (20 mg/kg assessed at cific antibody had both similar efficacy and potency in 1 and 24 hours) exhibited increased brain uptake at 24 hours 15 inhibiting AB production, Suggesting that the anti-TfR/ as affinity was lowered, while no significant difference in BACE1 is a fully functional large molecule inhibitor of uptake was observed at 1 hour (FIG. 2C). These data support BACE1 activity (FIG. 3C). the hypothesis that a lower affinity RMT antibody would Based on this model, this lower affinity bispecific would exhibit decreased transport under limiting concentrations be expected to be a more favorable candidate for increased while transport under Saturating conditions would be unaf uptake compared to the anti-TfR alone. To investigate the fected. brain accumulation of the bispecific antibody, trace doses of Thus, the following model is proposed: compared to a 'Ilanti-TfR"/BACE1 were compared to 'Ilanti-TfR' high affinity antibody, fewer low affinity antibodies bind to and 'Ilanti-BACE1 and brain uptake was evaluated at 30 receptors on the luminal side of the vasculature under min. 6, 24, and 48 hours after i.v. injection. Significantly non-Saturating concentrations, leading to lower endothelial higher brain uptake was observed with ''Ilanti-TfR/ uptake (FIG. 2D, left panels). At a higher therapeutic dose, 25 BACE1 compared to 'Ilanti-BACE1 at all time points however, luminal receptors would be saturated regardless of (FIG. 3d, n=4). Consistent with the affinity hypothesis, brain affinity resulting in similar endothelial uptake (FIG. 2D, uptake of this non-saturating trace dose of ''Ilanti-TfR right panels). Under these conditions, lower affinity RMT was much greater than that of the lower affinity ''Ilanti antibodies can achieve greater brain accumulation by 1) TfR/BACE1. To assess antibody accumulation at therapeu maximizing dissociation from the RMT target facilitating tic dose levels, mice were injected i.v. with anti-TfR"/ release into the brain, and 2) reducing the likelihood of BACE1 or anti-BACE 1 at 20 mg/kg and brain uptake of efflux out of the brain as concentrations are limited on the antibody was determined after 1, 12, 24, and 48 hours. parenchymal side of the BBB. Thus in a therapeutic setting, Compared to the monospecific anti-BACE1, administration a lower affinity antibody for an RMT target is surprisingly of the bispecificanti-TfR"/BACE1 resulted in a significantly 35 higher brain uptake at all time points (FIG.3E). As predicted advantageous for parenchymal accumulation. by the affinity model, the extent of uptake was significantly To evaluate the localization of these variants exhibiting greater than the higher affinity anti-TfR alone (compare increased brain uptake, mice were dosed i.v. with 20 mg/kg FIG.3E to 1B). Peak accumulation was achieved at 24 hours of either the high affinity anti-TfR or the lower affinity after injection, reaching concentrations of ~20 nM and variants anti-TfR''. After 24 hours, animals were PBS 40 remained elevated 48 hours after injection, even as periph perfused and brain sections were co-stained for human IgG eral levels of antibody cleared to ~12% of its concentration and the neuronal marker NeuN (FIG. 2E). As observed at 1 hour. Enhanced uptake by the bispecific is dramatically before, high affinity anti-TfR treated animals had mostly apparent when comparing the average percent of antibody in vascular staining with low levels of parenchymal signal the brain versus the serum (FIG. 3F). (FIG. 2E, top row). However, the lower affinity anti To determine localization of anti-TfR/BACE1 after sys TfR had noticeably more pronounced cellular staining 45 temic administration, mice were PBS-perfused 24 hours not depicting cortical blood vessels (FIG. 2E, data for after injection, and antibody distribution was visualized with anti-TfR'' ''). Furthermore, co-localized staining with anti-human fluorescent secondary (FIG. 3G). Similar to the NeuN indicated a redistribution of antibody from the vas lower affinity anti-TfR antibody localization, there was culature to neurons. This is especially pronounced in a Substantial staining of the parenchyma in addition to vas representative higher magnification image of anti-TfR vari 50 cular staining. Parenchymal co-localization with NeuN indi ant (FIG. 2F). Together with the brain uptake data, these cated that these antibodies were localized to the neuronal results indicate that a significantly higher brain accumula population. In contrast, animals injected with control IgG tion of antibody can be achieved by lowering the affinity of showed a complete lack of both vascular and parenchymal anti-TfR for TfR, and that lower affinity antibodies such as staining. Together, these data indicate that the bispecific anti-TfR' selectively distributed to neurons. 55 anti-TfR"/BACE1 traverses across the BBB and can signifi Transport of anti-TfR antibodies across the BBB was cantly accumulate in the brain parenchyma. further established when evaluating a bispecific antibody To assess the efficacy of anti-TfR"/BACE1 on AB (anti-TfR/BACE1) that binds both TfR and the amyloid production in Vivo, wild type mice were administered a precursor protein (APP) cleavage enzyme, beta secretase single 25 mg/kg or 50 mg/kg dose of control IgG, anti (BACE1) (FIG. 3A). The high affinity anti-TfR was used to 60 BACE1, or anti-TfR/BACE1. Based on the observation that engineer the TfR binding arm of the bispecific using stan brain antibody uptake peaks 24 hours after injection (see dard knob in hole bispecific antibody construction tech FIG.3E), brain and plasma AB levels were determined at nology (see, e.g., Ridgway et al., Protein Eng. (1996) 9(7): 24 and 48 hours after i.v. antibody administration. At 25 617-621). In addition to the knob and hole mutations in the mg/kg, anti-TfR/BACE1 was able to significantly reduce Fc for anti-TfR (hole) and anti-BACE1 (knob), the anti-TfR 65 brain AB a levels compared to control IgG both after 24 arm of the antibody comprised a mutation in the Fc region (p=0.001, n=10) and 48 (p=0.0003, n=10) hours post-injec that abrogated glycosylation (N297G). The knob and hole tion, while anti-BACE1 had no effect on AB reduction US 9,611,323 B2 51 52 (FIG. 4A). At 50 mg/kg, anti-TfR"/BACE1 had a even more brain exposure may reduce the frequency of dosing in the dramatic effect on reducing brain Aflao at both time points clinic, which would have potential benefit not only for compared to control IgG (FIG. 3B, p<0.0001, n=10 for both patient compliance and convenience but also in lessening 24 and 48 hr). Administration of anti-BACE 1 at this dose any potential side effects or off-target effects of the antibody also significantly reduced brain AB a levels compared to and/or of a therapeutic compound coupled thereto. control (p<0.0001, n=10 for 24 hr, p=0.006, n=10 for 48 hr), Further studies were performed to assess whether further though to a significantly lesser extent than the bispecific lessening the affinity of the bispecific anti-TfR"/BACE1 anti-TfR/BACE1 (p<0.0001, n=10 for both 24 and 48 antibody could further improve its BBB and parenchymal hours). Notably, the ability of the bispecific to reduce Afa penetrance. Two further bispecific antibodies were con was 2- to 3-fold greater than anti-BACE1 for all time points 10 structed: anti-TfRP/BACE1 and anti-TfR/BACE1, using and doses measured. The maximal effect of anti-TfR"/ the same construction methodology employed for the anti BACE1 was achieved 48 hours after injection at 50 mg/kg, TfR/BACE1 antibody. Competition ELISA assays were with a 50.0+1.9% reduction in brain AB, a compared to performed (FIG. 9A) and the resulting IC50s were as control IgG (FIG. 4E). Significant reductions in peripheral follows: A? a was also observed at both doses and time points for 15 anti-TfR/BACE1 (FIG. 4C-D). Treatment with anti TABLE 3 BACE1 resulted in a reduction in peripheral AB ao only at the 24 hour time point (p=0.01 for 25 mg/kg, p=0.002 for 50 ICSO/afinity measurements for anti-TTR/BACEl antibodies mg/kg. n=10 for each). These data confirm that antibodies Antibody IC50 Kd (Biacore) (nM) engineered to cross the BBB can be pharmacodynamically efficacious. Furthermore, the increase in brain penetrance of Anti-TfR-/BACE1 15 nM 33.3 1.7 the bispecific renders it more potent BACE 1 inhibitor drug Anti-TfRP/BACE1 1.6M 630 SO by significantly reducing brain Aflao levels. Anti-TfR/BACE1 >50 M N.D. At therapeutic doses of 20 mg/kg, however, BBB pen etrance and entry into the non-vascular portions of the CNS Surface plasmon resonance measurements of Kd were was enhanced in the anti-TfR'-treated animals relative to 25 also made for the binding between each bispecific antibody the anti-TfR or anti-TfRP-treated animals (FIG. 8B). Nota and TfR. The BIACORE(R) analysis in Table 3 was per bly, anti-TfR'' achieved a higher initial concentration in the formed as follows. Kd was measured using Surface plasmon brain, which steadily decreased after day 2; anti-TfR, on resonance assays using a BIACORE(R)-T-100 (BIAcore, Inc., the other hand, retained a consistently high level of brain Piscataway, N.J.) at 25° C. using penta-His Ab capture exposure over the tested 8 day period. Relatedly, anti-TfR 30 (Qiagen, Valencia, Calif.). Briefly, carboxymethylated dex concentration in the serum decreased the least of all of the tran biosensor chips (CMS, BIACORE, Inc.) were activated anti-TfR antibodies over the assessed period (FIG. 8A). In with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide all, this data indicates that generally a lower affinity for TfR hydrochloride (EDC) and N-hydroxysuccinimide (NHS) Surprisingly reduces serum clearance and increases brain according to the Supplier's instructions. Penta-His antibody exposure, but that at some threshold the lower affinity begins was diluted with 100 mM sodium acetate, pH 4.0, to 50 to impair the maximum brain exposure obtainable with the 35 ug/ml before injection at a flow rate of 5 ul/minute to antibody. In this example, an optimum would seem to be achieve approximately 10000 response units (RU) of found between the affinities of antibodies anti-TfRP and coupled protein. Following the injection of antibody, 1 M anti-TfR for transferrin receptor. ethanolamine was injected to block unreacted groups. For Importantly, these data highlight several causative mecha kinetics measurements, MuTfR-His was injected in HBS-P nisms behind increasing uptake of an antibody into the CNS 40 to reach about 50 RU, then two-fold serial dilutions of using a lower-affinity antibody approach. First, high affinity anti-TfR"/BACE1 (1.95 nM to 1000 nM) or anti-TfRP/ anti-TfR antibodies (e.g., anti-TfR', FIG. 1D) limit brain BACE1 (9.75 nM to 5000 nM) were injected in HBS-Pat uptake by quickly Saturating the TfR in the brain vascula 25°C. at a flow rate of approximately 30 ul/min. Association ture, thus reducing the total amount of antibody taken up rates (k) and dissociation rates (kit) were calculated using into the brain and also restricting its distribution to the 45 a simple one-to-one Langmuir binding model (BIACORER) vasculature. Strikingly, lowering affinity (e.g., anti-TfR. Evaluation Software version 3.2) by simultaneously fitting and anti-TfR''P/BACE1, FIGS. 2C, 2E, 2F, 3E-G and 9C) the association and dissociation sensorgrams. The equilib improves brain uptake and distribution, with a robust shift rium dissociation constant (Kd) was calculated as the ratio observed in localization from the vasculature to neurons and koff/kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 associated neuropil. Second, the lower affinity of the anti 50 (1999). The affinity of anti-TfR/BACE1 was too weak to be body for TfR is proposed to impair the ability of the determined by Surface plasmon resonance. antibody to return to the vascular side of the BBB via TfR As shown in FIG. 9A and Tables 2 and 3, the bispecific from the CNS side of the membrane because the overall anti-TfR/BACE1 and anti-TfRP/BACE1 antibodies bound affinity of the antibody for TfR is low and the local con markedly less well to TfR than the corresponding monospe centration of the antibody on the CNS side of the BBB is cific anti-TfR and anti-TfRP (compare FIG. 9A to FIG. non-Saturating due to the rapid dispersal of the antibody into 55 2A). For anti-TfR'/BACE1, the partial binding curves for the CNS compartment (see, e.g., FIGS. 1D, 2E and 2F). the bispecific and monospecific also suggest that the bispe Third, in vivo, antibodies with less affinity for TfR are not cific anti-TfR/BACE1 has a much higher IC50 than the cleared from the system as efficiently as those with greater corresponding monospecific anti-TfR. affinity for TfR (see FIGS. 8A and 9B), and thus remain at These antibodies were tested in the same in vivo AB higher circulating concentrations than their higher-affinity 60 production assay described above. Briefly, 6-8 week old counterparts. This is advantageous because the circulating wild type C57B 1/6 mice were administered via i.V. tail vein antibody levels of the lower-affinity antibody are sustained injection a single 50 mg/kg dose of control IgG, monospe at therapeutic levels for a longer period of time than the cific anti-BACE1, or anti-TfR/BACE1, anti-TfRP/BACE1, higher-affinity antibody, which Subsequently improves or anti-TfR/BACE1, 6 mice per antibody treatment per uptake of antibody in brain for longer period of time 65 timepoint, for a total of 180 mice treated. Brain and plasma (compare anti-TfR/BACE1 to anti-TfRP/BACE1 in FIG. A? a levels were determined at 1, 2, 4, 6, 8, and 10 days 9C). Furthermore, this improvement in both plasma and after i.v. antibody administration (FIGS. 9B-9E). The con US 9,611,323 B2 53 54 centration of bispecific antibodies found in the brain (FIG. sion of AD. The pharmacokinetic properties of each of these 9C) at the earliest time point was greatest with anti-TfR"/ bispecific antibodies were assessed. BACE1 and anti-TfRP/BACE1, each of which had concen A single 50 mg/kg dose of control IgG, monospecific trations of more than twice the concentration achieved by anti-Abeta antibody, or each bispecific antibody was anti-TfR/BACE1 at the 1 day time point. However, the injected i.p. into 8-16 week old wild type C57BL/6J mice or anti-TfR"/BACE1 brain concentration levels returned to mice expressing both human presenilin 2 and human amy control levels by day 6, and the anti-TfRP/BACE1 levels did loid precursor protein (PS2APP). Due to a limited number of so by day 10. In contrast, the lowest affinity bispecific transgenic animals, only two of the bispecific variants (anti antibody anti-TfR/BACE1 had a much lower relative TfRP/Abeta and anti-TfR/Abeta) were assayed in the dropoff in brain antibody concentration as compared to 10 anti-TfR/BACE1 and anti-TfRP/BACE1, in keeping with PS2APP mice. Four to six replicate mice were dosed in each the proposed model that a lower affinity for anti-TfR leads treatment group. The mice were sacrificed after 24 hours and to a reduced ability for the antibody to be exported from the drug levels measured in both the brain and plasma, as with parenchymal space. The levels of Abeta1-40 in the brain the anti-TfR/BACE1 bispecific studies. Prior to sacrifice, (FIG. 9E) were reduced in roughly the same proportion 15 blood was also collected 6 hours post-dose for an early expected by the observed concentrations of bispecific anti evaluation of plasma antibody concentrations. Plasma mea body in the brain: anti-TfR/BACE1 and anti-TfRP/BACE1 surements of antibody concentration (FIGS. 10A and 11A) had similarly reduced levels of observed brain Abeta1-40 at showed that all antibodies were present at similar levels at the earliest time points (days 1-2), which either rapidly 6 hours post-dose. However, control monospecific anti increased at subsequent time points (anti-TfR"/BACE1) or Abeta levels were reduced compared to control IgG by 24 more moderately increased at Subsequent time points (anti hours. This is similar to previous observations for this TfRP/BACE1), consistent with the observed decreases in anti-Abeta molecule. At 24 hours, anti-TfR"/Abeta showed brain concentration of each of these antibodies (FIG. 9C). similar antibody levels in the periphery as anti-Abeta, Notably, while the anti-TfR/BACE1 antibody treatment whereas anti-TfRP/Abeta and anti-TfR/Abeta showed resulted in a relatively more modest reduction in brain 25 slightly elevated levels, intermediate between anti-Abeta Abeta1-40 levels than that observed with the other bispecific and control IgG. antibodies, this reduction was consistent across all time The concentration of bispecific antibodies found in the points (FIG. 9E). brain was increased compared to both control IgG and Plasma measurements (FIG. 9B) showed that anti-TfR"/ anti-Abeta (FIGS. 10B and 11B). As compared to anti BACE1 was cleared by day 4, while anti-TfRP/BACE1 30 Abeta, anti-TfR"/Abeta had concentrations 12-fold higher, persisted at relatively low levels across all time points, and anti-TfRP/Abeta had concentrations 8- to 15-fold higher, levels of anti-TfR/BACE1 remained similar to controls and anti-TfR/Abeta had concentrations 4- to 5-fold higher. across all time points. Consistent with this finding, observed The increase in brain uptake of the anti-TfR'''/Abeta plasma Abeta1-40 levels (FIG. 9D) were similarly reduced antibodies compared to anti-Abeta was even greater than the from control anti-gld levels at all time points with each of increases seen for the anti-TfR'''/BACE bispecific anti anti-TfRP/BACE1, anti-TfR/BACE1 and anti-BACE1. bodies compared to anti-BACE1. This is likely due to the Anti-TfR/BACE1 showed similar reductions at the 1, 2 and decreased peripheral exposure of anti-Abeta compared to 4-day time points, rapidly returning to control levels at later control IgG 24 hours after dosing, which resulted in lower time points, in keeping with the observed disappearance of 40 levels of anti-Abeta in brain as compared to control IgG. the antibody from the plasma. These findings are the first demonstration that large mol These results again demonstrate that bispecific anti-TfR/ ecule antibodies administered at therapeutically relevant BACE1 antibodies effectively cross the BBB and inhibit doses can traverse the BBB and produce significant and BACE 1 activity in a mammalian in vivo system. They also Sustained brain uptake. Furthermore, these results demon suggest that an affinity for TfR between that of the anti 45 strating an inverse relationship between antibody affinity TFRP/BACE1 and the anti-TfR/BACE1 may provide an and extent of brain uptake furthers understanding of RMT optimal combination of persistence and activity in the paren dynamics. This novel insight can be applied to a variety of chyma/brain. It is noted, however that for each brain target, other potential RMT targets to provide a more effective the potency of the bispecific arm specific for that target will strategy for antibody drug delivery into the CNS. Addition dictate how much of the anti-TfR/target bispecific antibody 50 ally, these in vivo results demonstrate that a bispecific must be present in the CNS side of the BBB in order to antibody can greatly improve the potency of a promising achieve the desired results, and thus what degree of affinity anti-amyloidogenic therapeutic by significantly increasing of the anti-TfR for TfR must be used in the bispecific to brain penetrance of the targeting antibody drug. This could obtain that concentration. This invention provides a means be highly advantageous, as enhanced drug delivery would to determine and design the bispecific antibody to achieve 55 such target levels in the CNS after administration on the translate to less potential side effects due to lower therapeu nonprivileged side of the BBB. tic dosing required. More generally, this technology has vast These results were confirmed and extended using another potential to be applied to therapeutics for a wide range of anti-TfRbispecific antibody, anti-TfR/Abeta, that binds both CNS diseases and represents an improved approach to TfR and amyloid beta. Three bispecific variants were pre 60 provide safer antibody drugs. pared: anti-TfR"/Abeta, anti-TfRP/Abeta and anti-TfR / Although the foregoing invention has been described in Abeta, using the same methods as used for the preparation Some detail by way of illustration and example for purposes of the anti-TfR/BACE1 bispecific above. Abeta is the main of clarity of understanding, the descriptions and examples substituent of amyloid plaques, which are believed to be should not be construed as limiting the scope of the inven involved in the development of AD. Inhibition of plaque 65 tion. The disclosures of all patent and scientific literature formation by Abeta binding and removal, in either its free or cited herein are expressly incorporated in their entirety by oligomerized State, may inhibit the development or progres reference. US 9,611,323 B2 55
SEQUENCE LISTING
<16O is NUMBER OF SEO ID NOS : 15
<210s, SEQ ID NO 1 &211s LENGTH: 108 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: sequence is synthesized
<4 OOs, SEQUENCE: 1 Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val 1. 5 1O 15 Gly Asp Arg Val Thir Ile Thr Cys Arg Ala Ser Glin Asp Val Ser 2O 25 3 O Thir Ala Val Ala Trp Tyr Glin Glin Llys Pro Gly Lys Ala Pro Llys 35 4 O 45 Lieu. Lieu. Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser SO 55 6 O Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile 65 70 7s Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Glin 8O 85 9 O Ser Tyr Thr Thr Pro Pro Thr Phe Gly Glin Gly Thr Lys Val Glu 95 1OO 105 Ile Lys Arg
<210s, SEQ ID NO 2 &211s LENGTH: 108 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: sequence is synthesized
<4 OOs, SEQUENCE: 2 Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val 1. 5 1O 15 Gly Asp Arg Val Thir Ile Thr Cys Arg Ala Ser Glin Asp Val Ser 2O 25 3 O Thir Ala Val Ala Trp Tyr Glin Glin Llys Pro Gly Lys Ala Pro Llys 35 4 O 45 Lieu. Lieu. Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser SO 55 6 O Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile 65 70 7s Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Glin 8O 85 9 O Phe Pro Thr Tyr Lieu Pro Thr Phe Gly Glin Gly Thr Lys Val Glu 95 1OO 105 Ile Lys Arg
<210s, SEQ ID NO 3 &211s LENGTH: 108 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: sequence is synthesized
<4 OOs, SEQUENCE: 3 Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val US 9,611,323 B2 57 58 - Continued
15
Gly Asp Arg Val Thir Ile Thr Cys Arg Ala Ser Glin Asp Wall Ser 2O 25 3 O
Thir Ala Val Ala Trp Tyr Glin Glin Llys Pro Gly Lys Ala Pro Lys 35 4 O 45
Lell Lieu. Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser SO 55 6 O
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile 65 70 7s
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Glin 8O 85 9 O
Gly Tyr Asn Asp Pro Pro Thr Phe Gly Glin Gly. Thir Lys Wall Glu 95 1OO 105
Ile Lys Arg
<210s, SEQ ID NO 4 &211s LENGTH: 108 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: sequence is synthesized
<4 OOs, SEQUENCE: 4
Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Lieu. Ser Ala Ser Wall 1. 5 1O 15
Gly Asp Arg Val Thir Ile Thr Cys Arg Ala Ser Glin Asp Wall Ser 2O 25 3 O
Thir Ala Val Ala Trp Tyr Glin Glin Llys Pro Gly Lys Ala Pro Lys 35 4 O 45
Lell Lieu. Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser SO 55 6 O
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile 65 70 7s
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Glin 8O 85 9 O
Ser Ser Thr Asp Pro Thr Thr Phe Gly Glin Gly. Thir Lys Wall Glu 95 1OO 105
Ile Lys Arg
<210s, SEQ ID NO 5 &211s LENGTH: 108 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: sequence is synthesized
<4 OOs, SEQUENCE: 5
Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Lieu. Ser Ala Ser Wall 1. 5 1O 15
Gly Asp Arg Val Thir Ile Thr Cys Arg Ala Ser Glin Val Wall Ala 2O 25 3 O
Asn Ser Lieu Ala Trp Tyr Glin Glin Llys Pro Gly Lys Ala Pro Lys 35 4 O 45
Lell Lieu. Ile Tyr Lieu Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser SO 55 6 O
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile 65 70 7s US 9,611,323 B2 59 - Continued
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Glin 8O 85 9 O Asp Ala Thir Ser Pro Pro Thr Phe Gly Glin Gly. Thir Lys Val Glu 95 1OO 105 Ile Lys Arg
<210s, SEQ ID NO 6 &211s LENGTH: 108 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: sequence is synthesized
<4 OOs, SEQUENCE: 6 Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val 1. 5 1O 15 Gly Asp Arg Val Thir Ile Thr Cys Arg Ala Ser Glin Asp Val Ser 2O 25 3 O Thir Ala Val Ala Trp Tyr Glin Glin Llys Pro Gly Lys Ala Pro Llys 35 4 O 45 Lieu. Lieu. Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser SO 55 6 O Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile 65 70 7s Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Glin 8O 85 9 O Tyr Ala Thr Asp Pro Pro Thr Phe Gly Glin Gly Thr Lys Val Glu 95 1OO 105 Ile Lys Arg
<210s, SEQ ID NO 7 &211s LENGTH: 119 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: sequence is synthesized
<4 OO > SEQUENCE: 7 Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly 1. 5 1O 15 Gly Ser Lieu. Arg Lieu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 2O 25 3 O Gly Tyr Ala Ile His Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. 35 4 O 45 Glu Trp Val Gly Trp Ile Ser Pro Ala Gly Gly Ser Thr Asp Tyr SO 55 6 O Ala Asp Ser Val Lys Gly Arg Phe Thir Ile Ser Ala Asp Thir Ser 65 70 7s
Lys Asn. Thir Ala Tyr Lieu Gln Met Asn. Ser Lieu. Arg Ala Glu Asp 8O 85 9 O
Thr Ala Val Tyr Tyr Cys Ala Arg Gly Pro Phe Ser Pro Trp Val 95 1OO 105
Met Asp Tyr Trp Gly Glin Gly Thr Lieu Val Thr Val Ser Ser 11O 115
<210s, SEQ ID NO 8 &211s LENGTH: 119 US 9,611,323 B2 61 - Continued
212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: sequence is synthesized
<4 OOs, SEQUENCE: 8 Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly 1. 5 1O 15 Gly Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Phe Thr Phe Lieu. 2O 25 3 O Gly Tyr Gly Ile His Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. 35 4 O 45 Glu Trp Val Gly Trp Ile Ser Pro Ala Gly Gly Ser Thr Asp Tyr SO 55 6 O Ala Asp Ser Val Lys Gly Arg Phe Thir Ile Ser Ala Asp Thir Ser 65 70 7s Lys Asn. Thir Ala Tyr Lieu Gln Met Asn. Ser Lieu. Arg Ala Glu Asp 8O 85 9 O Thr Ala Val Tyr Tyr Cys Ala Arg Gly Pro Phe Ser Pro Trp Val 95 1OO 105 Met Asp Tyr Trp Gly Glin Gly Thr Lieu Val Thr Val Ser Ser 11O 115
<210s, SEQ ID NO 9 &211s LENGTH: 108 212. TYPE: PRT <213> ORGANISM: Artificial sequence & 22 O FEATURE; <223> OTHER INFORMATION: sequence is synthesized
<4 OOs, SEQUENCE: 9 Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val 1. 5 1O 15 Gly Asp Arg Val Thir Ile Thr Cys Arg Ala Ser Glin Ser Val Ser 2O 25 3 O Ser Ala Val Ala Trp Tyr Glin Glin Llys Pro Gly Lys Ala Pro Llys 35 4 O 45 Lieu. Lieu. Ile Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser SO 55 6 O Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile 65 70 7s Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Glin 8O 85 9 O Tyr Ser Tyr Ser Pro Phe Thr Phe Gly Glin Gly Thr Lys Val Glu 95 1OO 105 Ile Lys Arg
<210s, SEQ ID NO 10 &211s LENGTH: 108 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: sequence is synthesized
<4 OOs, SEQUENCE: 10 Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val 1. 5 1O 15
Gly Asp Arg Val Thir Ile Thr Cys Arg Ala Ser Glin Ser Val Ser 2O 25 3 O US 9,611,323 B2 63 - Continued
Ser Ala Val Ala Trp Tyr Glin Glin Llys Pro Gly Lys Ala Pro Llys 35 4 O 45 Lieu. Lieu. Ile Ser Trp Ala Ser Trp Leu Tyr Ser Gly Val Pro Ser SO 55 6 O Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile 65 70 7s Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Glin 8O 85 9 O Tyr Ser Tyr Ser Pro Phe Thr Phe Gly Glin Gly Thr Lys Val Glu 95 1OO 105 Ile Lys Arg
<210s, SEQ ID NO 11 &211s LENGTH: 108 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: sequence is synthesized
<4 OOs, SEQUENCE: 11 Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val 1. 5 1O 15 Gly Asp Arg Val Thir Ile Thr Cys Arg Ala Ser Glin Ser Val Ser 2O 25 3 O Ser Ala Val Ala Trp Tyr Glin Glin Llys Pro Gly Lys Ala Pro Llys 35 4 O 45 Lieu. Lieu. Ile Trp Tyr Ala Ser Trp Leu Tyr Ser Gly Val Pro Ser SO 55 6 O Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile 65 70 7s Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Glin 8O 85 9 O Tyr Ser Tyr Ser Pro Phe Thr Phe Gly Glin Gly Thr Lys Val Glu 95 1OO 105 Ile Lys Arg
<210s, SEQ ID NO 12 &211s LENGTH: 108 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: sequence is synthesized
<4 OOs, SEQUENCE: 12 Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val 1. 5 1O 15 Gly Asp Arg Val Thir Ile Thr Cys Arg Ala Ser Glin Ser Val Ser 2O 25 3 O
Ser Ala Val Ala Trp Tyr Glin Glin Llys Pro Gly Lys Ala Pro Llys 35 4 O 45
Lieu. Lieu. Ile Trp Trp Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser SO 55 6 O
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile 65 70 7s
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Glin 8O 85 9 O US 9,611,323 B2 65 - Continued Tyr Ser Tyr Ser Pro Phe Thr Phe Gly Glin Gly Thr Lys Val Glu 95 1OO 105 Ile Lys Arg
<210s, SEQ ID NO 13 &211s LENGTH: 126 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: sequence is synthesized
<4 OOs, SEQUENCE: 13 Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly 1. 5 1O 15 Gly Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Phe Asn. Phe Tyr 2O 25 3 O Tyr Ser Ser Ile His Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. 35 4 O 45 Glu Trp Val Ala Ser Ile Ser Pro Tyr Ser Gly Tyr Thr Ser Tyr SO 55 6 O Ala Asp Ser Val Lys Gly Arg Phe Thir Ile Ser Ala Asp Thir Ser 65 70 7s Lys Asn. Thir Ala Tyr Lieu Gln Met Asn. Ser Lieu. Arg Ala Glu Asp 8O 85 9 O Thr Ala Val Tyr Tyr Cys Ala Arg Gln Pro Thr His Tyr Tyr Tyr 95 1OO 105 Tyr Ala Lys Gly Tyr Lys Ala Met Asp Tyr Trp Gly Glin Gly Thr 11O 115 12 O
Leul Wall. Thir Wal Ser Ser 125
<210s, SEQ ID NO 14 &211s LENGTH: 438 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: sequence is synthesized
<4 OOs, SEQUENCE: 14 Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly 1. 5 1O 15 Gly Ser Lieu. Arg Lieu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 2O 25 3 O Ser Tyr Gly Met Ser Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. 35 4 O 45 Glu Lieu Val Ala Ser Ile Asn Ser Asn Gly Gly Ser Thr Tyr Tyr SO 55 6 O Pro Asp Ser Wall Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala 65 70 7s
Lys Asn. Ser Lieu. Tyr Lieu Gln Met Asn. Ser Lieu. Arg Ala Glu Asp 8O 85 9 O Thr Ala Val Tyr Tyr Cys Ala Ser Gly Asp Tyr Trp Gly Glin Gly 95 1OO 105
Thir Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 11O 115 12 O
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 125 13 O 135 US 9,611,323 B2 67 - Continued Ala Leu Gly Cys Lieu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 14 O 145 15 O Val Ser Trp Asn Ser Gly Ala Lieu. Thir Ser Gly Val His Thr Phe 155 160 1.65 Pro Ala Val Lieu. Glin Ser Ser Gly Lieu. Tyr Ser Leu Ser Ser Val 17O 17s 18O Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 185 190 195 Asn Val Asp His Llys Pro Ser Asn. Thir Lys Val Asp Lys Arg Val 2 OO 2O5 21 O Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu 215 22O 225 Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Llys Pro Llys 23 O 235 24 O Asp Thr Lieu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255 Val Asp Val Ser Glin Glu Asp Pro Glu Val Glin Phe Asn Trp Tyr 26 O 265 27 O Val Asp Gly Val Glu Val His Asn Ala Lys Thr Llys Pro Arg Glu 27s 28O 285 Glu Glin Phe Asin Ser Thr Tyr Arg Val Val Ser Val Lieu. Thr Val 290 295 3OO Lieu. His Glin Asp Trp Lieu. Asn Gly Lys Glu Tyr Lys Cys Llys Val 3. OS 310 315 Ser ASn Lys Gly Lieu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 32O 3.25 33 O Ala Lys Gly Glin Pro Arg Glu Pro Glin Val Tyr Thr Lieu Pro Pro 335 34 O 345 Ser Glin Glu Glu Met Thr Lys Asn Glin Val Ser Lieu. Thr Cys Lieu. 350 355 360 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 365 37O 375 Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Lieu 38O 385 390 Asp Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Arg Lieu. Thr Val Asp 395 4 OO 405 Lys Ser Arg Trp Glin Glu Gly Asn Val Phe Ser Cys Ser Val Met 410 415 42O His Glu Ala Lieu. His Asn His Tyr Thr Glin Llys Ser Lieu. Ser Lieu. 425 43 O 435 Ser Lieu. Gly
<210s, SEQ ID NO 15 &211s LENGTH: 219 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: sequence is synthesized
<4 OOs, SEQUENCE: 15 Asp Ile Val Met Thr Glin Ser Pro Leu Ser Leu Pro Val Thr Pro 1. 5 1O 15
Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Glin Ser Leu Val 2O 25 3 O Tyr Ser Asn Gly Asp Thr Tyr Lieu. His Trp Tyr Lieu Gln Llys Pro US 9,611,323 B2
- Continued
35 4 O 45 Gly Glin Ser Pro Glin Lieu. Lieu. Ile Tyr Llys Val Ser Asn Arg Phe SO 55 6 O Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 65 70 7s Phe Thr Lieu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val 8O 85 9 O Tyr Tyr Cys Ser Glin Ser Thr His Val Pro Trp Thr Phe Gly Glin 95 OO O5 Gly. Thir Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val 1O 15 2O Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala 25 3O 35 Ser Val Val Cys Lieu. Lieu. Asn. Asn. Phe Tyr Pro Arg Glu Ala Lys 4 O 45 SO Val Glin Trp Llys Val Asp Asn Ala Lieu. Glin Ser Gly Asn. Ser Glin 55 60 65 Glu Ser Val Thr Glu Glin Asp Ser Lys Asp Ser Thr Tyr Ser Leu 70 7s 8O Ser Ser Thr Lieu. Thir Lieu. Ser Lys Ala Asp Tyr Glu Lys His Lys 85 90 95 Val Tyr Ala Cys Glu Val Thr His Glin Gly Leu Ser Ser Pro Val 2 OO 2O5 21 O Thir Lys Ser Phe Asn Arg Gly Glu. Cys 215
What is claimed is: 35 10. The method of claim 1, wherein the antibody coupled 1. A method of transporting a compound across the to the compound is administered at a therapeutic dose. blood-brain barrier comprising exposing the blood-brain 11. The method of claim 10, wherein the therapeutic dose barrier to an antibody which binds with an affinity from is BBB-R-saturating. about 30 nM to about 10 uM to a blood-brain barrier 12. The method of claim 1, wherein the antibody is a receptor (BBB-R) coupled to the compound such that the 40 multispecific antibody and the compound optionally forms antibody transports the compound coupled thereto across the one portion of the multispecific antibody. blood-brain barrier, wherein the BBB-R is a transferrin 13. The method of claim 12 wherein the multispecific receptor (TfR). antibody comprises a first antigen binding site which binds 2. The method of claim 1, wherein the compound is a the BBB-R and a second antigen binding site which binds a neurological disorder drug or an imaging agent. 45 brain antigen. 3. The method of claim 1, wherein the antibody does not 14. The method of claim 13, wherein the brain antigen is impair the binding of the BBB-R to one or more of its native selected from the group consisting of beta-secretase 1 ligands. (BACE1), Abeta, epidermal growth factor receptor (EGFR), 4. The method of claim 1, wherein the blood-brain barrier human epidermal growth factor receptor 2 (HER2), Tau, is in a mammal. 50 apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, hun 5. The method of claim 4, wherein the mammal has a tingtin, prion protein (PrP), leucine rich repeat kinase 2 neurological disorder. (LRRK2), parkin, presenilin 1, presenilin 2, gamma secre 6. The method of claim 5, wherein the neurological tase, death receptor 6 (DR6), amyloid precursor protein disorder is selected from the group consisting of Alzheim (APP), p75 neurotrophin receptor (p75NTR), and caspase 6. er's disease (AD), stroke, dementia, muscular dystrophy 55 15. The method of claim 13, wherein the multispecific (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis antibody binds both TfR and BACE1. (ALS), cystic fibrosis, Angelman’s syndrome, Liddle Syn 16. The method of claim 13, wherein the multispecific drome, Parkinson's disease, Pick's disease, Paget’s disease, antibody binds both TfR and Abeta. cancer, and traumatic brain injury. 17. A method of treating a neurological disorder in a 7. The method of claim 4 wherein the mammal is a 60 mammal comprising treating the mammal with an antibody human. that binds a BBB-R and is coupled to a compound, wherein 8. The method of claim 1, wherein the antibody coupled the antibody has been selected to have an affinity from about to the compound has an affinity for the BBB-R from about 30 nM to about 10 uM for the BBB-R and thereby improves 30 nM to about 1 uM. CNS uptake of the antibody and coupled compound, 9. The method of claim 1, wherein the antibody does not 65 wherein the BBB-R is a transferrin receptor (TfR). inhibit the binding of TfR to transferrin. k k k k k